Supercritical Fluid Chromatography: Volume 2 9783110618983, 9783110618938

Table of contents :
Preface
Contents
List of Contributors
1. Chiral preparative SFC
2. Supercritical fluid chromatography in bioanalysis
3. Ultrafast supercritical fluid chromatography
4. SFC applications for active pharmaceutical ingredient analysis
5. Analysis of terpenes (mono-, sesqui-, di-, and triterpenes) by SFE and SFC-MS
6. Synthesis in supercritical carbon dioxide
7. Applications of supercritical fluid chromatography in the field of edible lipids
Index

Citation preview

Gérard Rossé (Ed.) Supercritical Fluid Chromatography

Also of Interest Supercritical Fluid Chromatography. Volume 1 Rossé (Ed.), 2018 ISBN 978-3-11-050075-2, e-ISBN 978-3-11-050077-6

Electrophoresis. Theory and Practice Michov, 2019 ISBN 978-3-11-033071-7, e-ISBN 978-3-11-033075-5

Inorganic Trace Analytics. Trace Element Analysis and Speciation Matusiewicz, Bulska (Eds.), 2017 ISBN 978-3-11-037194-9, e-ISBN  978-3-11-036673-0

Organic Trace Analysis. Nießner, Schäffer, 2017 ISBN 978-3-11-044114-7, e-ISBN 978-3-11-044115-4

Reviews in Analytical Chemistry. Editor-in-Chief: Israel Schechter e-ISSN 2191-0189

Supercritical Fluid Chromatography Volume 2 Edited by Gérard Rossé

Editor Dr. Gérard Rossé Department of Pharmacology and Physiology College of Medicine, Drexel University New College Building, 245 North 15th Street Philadelphia, PA 19102 USA [email protected]

ISBN 978-3-11-061893-8 e-ISBN (PDF) 978-3-11-061898-3 e-ISBN (EPUB) 978-3-11-061906-5 Library of Congress Control Number: 2018031531 Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at http://dnb.dnb.de. © 2019 Walter de Gruyter GmbH, Berlin/Boston Typesetting: Integra Software Services Pvt. Ltd. Printing and binding: CPI books GmbH, Leck Cover image: sumos / iStock / Getty Images Plus www.degruyter.com

Preface When Oleg Lebedev from De Gruyter reached out to me about writing a book on supercritical fluid chromatography (SFC), I immediately replied “Yes.” In the last few years, the field of SFC has seen developing rapidly and a special edition on SFC appears to be particularly timely. I am most grateful to not only the pioneers in the field of SFC but also the renowned experts from industry and academia for contributing 15 chapters and allowing us to assemble a collection of truly exciting ideas and reports. I am equally grateful to my colleagues and friends who agreed to act as reviewers. To reveal the broad impact of modern SFC in the life sciences, I decided to cover applications of the technique in the pharmaceutical industry, the food industry, the fragrance and perfume industry, natural products, and substance abuse (doping). The 15 chapters are divided into two volumes dedicated to the concepts, potentials, and limitations of the relevant SFC applications. In some cases, I retained some redundancy of content to represent the authors’ individual views on a topic. Each volume has a balanced number of chapters and can be read independently. This two-volume series opens with an overview of the history and expectant future of SFC and continues with recent applications in the pharmaceutical industry and other fascinating areas of science. The two volumes are not a comprehensive treatise on the subject, nor are they a repackaging of what others have pioneered and written. Their intention is to serve as a source of inspiration and stimulation for readers to continue exploring the possibilities of chromatography and synthesis with a supercritical fluid. For me, a simple way to describe SFC is “today’s technology enabling tomorrow’s innovation.” SFC is not new but went through different phases of acceptance, development, and success. In the 1980s it was considered science-fiction technology and received a burst of interest in the 1990s. In 2010, the efforts of two major manufacturers in developing analytical instruments to meet industry standards reawakened the scientific community. SFC bridges the gap between gas chromatography (GC) and liquid chromatography (LC), and its broader impact on numerous frontier areas of industrial and environmental analytical chemistry is being studied. The successful coupling of SFC to mass spectrometers (SFC-MS) is now straightforward and p ­ rovides highly reliable and robust SFC-MS instruments with applications in the sample analysis and mass-triggered purification of complex mixtures. In the last few years, multiple studies have demonstrated SFC/SFC-MS to be as robust, reliable, and precise as LC/LC-MS. Scientists should see modern SFC as another form of liquid chromatography that happens to use CO2 as the mobile phase. The main advantages of SFC are compelling. It is faster than LC, it significantly reduces solvent consumption and waste. For sample, purification SFC fraction dry-down time is 15-fold faster than for aqueous fractions obtained in LC. SFC is also a “green” technology using CO2 produced in existing fermentation plants, and it limits or eliminates the use of most toxic organic solvents. https://doi.org/10.1515/9783110618983-201

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 Preface

SFC has found its place in the pharmaceutical industry with an increasing body of applications for chiral and achiral molecules in both the research and ­development phases of the drug discovery process. As illustrated in this two-volume series, the current interest in SFC extends well beyond the pharmaceutical industry. Chapters encompassing applications for polar and nonpolar mixtures of importance are ­covering widely disparate areas in substance abuse, natural products including cannabinoids, bioactive lipids, flavor, and fragrance. At a time dominated by aggressive project timelines, the need to deliver products faster and in a world seeking renewable technologies for solving the waste and disposal problems, SFC can contribute to the more expedient delivery of better compounds while minimizing the impact on the environment in which we live. The initiation of this book coincided with my involvement with Dart NeuroScience in building and using one of the largest operation-based SFC-MS systems in the pharmaceutical industry. During this time I had the opportunity to measure the impact of SFC-MS on productivity and cost reduction. The closure of our R&D operations led me to reflect on the achievements and value of SFC. SFC is truly enabling future innovations and could revolutionize the field of chromatography. However, the full potential of SFC has yet to be realized. Understanding the fundamentals of SFC, setting even higher requirements on system quality and cost-effectiveness, and building innovative systems will continue to elevate interest, expand the breadth of applications, and grow the market for SFC-/SFC-MS-based technologies. I want to thank Oleg Lebedev, Lena Stoll, and Sabina Dabrowski from De Gruyter for supporting me in the editing process. I dedicate this book to the Dart NeuroScience family. Special thanks go to the team working on implementing SFC-MS with me and to Tim Tully, company founder, who enable me to work in this fascinating area of science. Steven de Belle did a great job in providing advice and reviewing my grammars. John van Antwerp, Ronan Cleary, and many great friends at Waters Corporation helped solve challenges and break new frontiers. I am very grateful to my wife, Frédérique, and my beautiful daughters, Alyssa and Noanne. San Diego, CA September 2018

Gérard Rossé

Contents Preface 

 V

List of Contributors 

 IX

Emmanuelle Lipka and David Speybrouck 1 Chiral preparative SFC   1 Lucie Nováková, Kateřina Plachká, Maria Khalikova, František Švec 2 Supercritical fluid chromatography in bioanalysis   33 Chandan L. Barhate and Philip A. Searle 3 Ultrafast supercritical fluid chromatography 

 77

Lu Zeng and Paddi Ekhlassi 4 SFC applications for active pharmaceutical ingredient assay  Cyrille Santerre, David Touboul 5 Analysis of terpenes (mono-, sesqui-, di-, and triterpenes) by SFE and SFC-MS   113 Alexander Marziale 6 Synthesis in supercritical carbon dioxide 

 127

Paola Donato, Danilo Sciarrone, Paola Dugo, Luigi Mondello 7 Applications of supercritical fluid chromatography in the field of edible lipids   163 Index 

 189

 93

List of Contributors Chandan L. Barhate Discovery Chemistry and Technology AbbVie Inc. 1 North Waukegan Rd Dept. R467, Bldg. AP9 North Chicago, IL 60064-6114 USA Paola Donato Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali University of Messina Polo Annunziata viale Annunziata 98168 Messina Italy Paola Dugo Dipartimento di Scienze Biomediche, Odontoiatriche e delle Immagini Morfologiche e Funzionali University of Messina via Consolare Valeria 98125 Messina Italy Paddi Ekhlassi Takeda Pharmaceuticals 10275 Science Center Dr San Diego CA 92121 USA [email protected] Maria Khalikova Charles University Faculty of Pharmacy Department of Analytical Chemistry Akademika Heyrovského 1203 500 05 Hradec Králové Czech Republic

https://doi.org/10.1515/9783110618983-202

Emmanuelle Lipka Univ. Lille Inserm, U995 LIRIC - Lille Inflammation Research International Center 59000 Lille France and UFR Pharmacie Laboratoire de Chimie Analytique BP 83 59006 Lille France [email protected] Alexander Marziale Novartis Pharma AG Novartis Institutes for Biomedical Research (NIBR) Klybeckstrasse 141 4057 Basel Switzerland [email protected] Luigi Mondello Dipartimento di Scienze Biomediche, Odontoiatriche e delle Immagini Morfologiche e Funzionali University of Messina via Consolare Valeria 98125 Messina Italy and Chromaleont S.r.l. via Leonardo Sciascia Coop. Fede Pal. B 98168 Messina Italy and University Campus Bio-Medico of Rome via Álvaro del Portillo 21 00128 Rome Italy

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 List of Contributors

Lucie Nováková Charles University Faculty of Pharmacy Department of Analytical Chemistry Akademika Heyrovského 1203 500 05 Hradec Králové Czech Republic [email protected]

Philip A. Searle Discovery Chemistry and Technology AbbVie Inc. 1 North Waukegan Rd Dept. R467, Bldg. AP9 North Chicago, IL 60064-6114 USA [email protected]

Kateřina Plachká Charles University Faculty of Pharmacy Department of Analytical Chemistry Akademika Heyrovského 1203 500 05 Hradec Králové Czech Republic

David Speybrouck Analytical Sciences - Discovery Sciences Janssen Research & Development, a division of Janssen-Cilag Campus de Maigremont, CS10615 27106 Val de Reuil Cedex France [email protected]

Cyrille Santerre Institut Supérieur International Parfum Cosmétique Arômes Plateforme scientifique (ISIPCA) 34-36 rue du parc de Clagny 78000 Versailles France and Institut de Chimie des Substances Naturelles CNRS UPR2301 Université Paris-Sud Université Paris-Saclay Avenue de la Terrasse 91198 Gif-sur-Yvette France [email protected] Danilo Sciarrone Dipartimento di Scienze Biomediche, Odontoiatriche e delle Immagini Morfologiche e Funzionali University of Messina via Consolare Valeria 98125 Messina Italy

František Švec Charles University Faculty of Pharmacy Department of Analytical Chemistry Akademika Heyrovského 1203 500 05 Hradec Králové Czech Republic David Touboul Institut de Chimie des Substances Naturelles CNRS UPR2301 Université Paris-Sud Université Paris-Saclay Avenue de la Terrasse 91198 Gif-sur-Yvette France [email protected] Lu Zeng Takeda Pharmaceuticals 10275 Science Center Dr San Diego CA 92121 USA [email protected]

Emmanuelle Lipka and David Speybrouck

1 Chiral preparative SFC

Abstract: Since 1992, the US Food & Drug Administration (FDA) has requested the assessment of each enantiomer pharmacological activity? for racemic drugs and has promoted the development of new chiral drugs as single enantiomers, resulting in the increase of the development of pure ones. Thus in 2017, 3 of 4 top-selling small-­ molecule drugs were chiral compounds and all of them were pure enantiomers. ­Consequently, the preparation of pure enantiomer is now a key step for the development of a new drug. Up until recently, preparative liquid chromatography was the method of choice for chiral separation both during the drug discovery phase, to get a few grams, and for the clinical trials requiring kilograms of pure enantiomers. However, the emergence of the Supercritical Fluid Chromatography (SFC) in the 1990s has  changed things. Indeed, the preparative SFC offers many advantages including  high productivity thanks to high flow rate and short equilibration time as well as low solvent consumption compared to liquid chromatography. Nowadays, SFC is becoming the primary method for preparative chiral chromatography. Keywords: This work covers recent developments in preparative SFC for the separation of chiral compounds, displaying several aspects like instrumentation, scale up, mobile phases and chiral stationary phases, and environmental considerations compared to liquid chromatography.

1.1 Introduction Among the 30 top-selling drugs on the market in September 2014, ten pharmaceutical blockbusters were pure stereoisomers. This is also the case of Gilead’s Sovaldi (Sofosbuvir), a hepatitis C treatment now set to rake in as much as $10 billion in its first year on the market. Those numbers clearly show that separating racemates is now a key step in the development of a new drug [1]. But drug discovery (meaning to target identification until late lead optimization) is also a long and very uncertain business. From this point of view, the development of a highly rapid and economic method with an elevated productivity was required; in addition environmental and safety issues are important concerns. Supercritical fluid chromatography (SFC) ­perfectly fulfilled Emmanuelle Lipka, Univ. Lille, Inserm, U995 – LIRIC – Lille Inflammation Research International Center, F-59000 Lille, France; UFR Pharmacie, Laboratoire de Chimie Analytique, BP 83, F-59006 Lille, France David Speybrouck, Analytical Sciences – Discovery Sciences, Janssen Research & Development, a division of Janssen-Cilag, Campus de Maigremont, CS10615, F-27106 Val de Reuil Cedex, France https://doi.org/10.1515/9783110618983-001

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 Emmanuelle Lipka and David Speybrouck

these requirements and has found applications in many areas: food-­related applications, natural products, fossil fuels, polymers, bioactive compounds, and achiral pharmaceutics. But it remains the most active in chiral applications particularly in the pharmaceutical field. When Berger started his own SFC company in the mid1990s, the eponymous Berger Instruments (which was later bought by M ­ ettler-Toledo, Thar Instruments, and ultimately Waters) focused its efforts “on the one niche where it was absolutely obvious that SFC just killed LC: chiral s­ eparations,” Berger says [2]. Strangely SFC is as old as high-performance liquid chromatography (HPLC), but as explained by M. Saito, its development took much more time due, among other reasons, to the ambiguous position of SFC between gas chromatography and liquid chromatography (LC) [3]. In 1989, Macaudiere and Caude published results of chiral SFC separation on polysaccharide phases (Chiralcel® OB) [4]. Since the 1990s, the interest in chiral SFC has not stopped to increase and reaches out to become the method of choice for the isolation of enantiomers, particularly in a preparative scale because of its intrinsic properties and green features [5]. All these reasons have contributed to making SFC a sustainable chromatography technique, which can be characterized by green metrics such as environmental factors. They also make SFC a technique of choice for chiral separations spanning a range from small preparative (a few milligrams to hundreds of grams) to larger enantioseparation scale. Thanks to further upgrades in instrumentation and chiral column design, SFC is now outperforming HPLC.

1.2 Instrumentation 1.2.1 Preparative SFC devices Nowadays, the preparative SFC is exclusively used with packed column and the mobile phase is composed of a mixture of supercritical carbon dioxide (scCO2) and modifier. As explained previously, the mobile phase used for the first SFC separations was composed of 100% of supercritical fluid. A preparative SFC device (Figure 1.1) looks like a preparative high-pressure gradient HPLC system. Indeed, the preparative SFC system consists of two pumps (one for CO2 and one for the cosolvent), an injector (designed either to inject a large quantity of a ­compound or to inject several compounds thanks to an autosampler), an oven to control the temperature of the column, one or several detectors (UV detector is the most suitable for preparative SFC, which will be discussed in Section 2.3), and a fraction collector. Thus, an SFC prep system seems to be as “simple” as an HPLC system; however, due to the utilization of CO2 some specificities are required. First, the system must be supplied with CO2 to work several hours with high flow rate. The system can be either connected to one or several cylinders with deep tubes



 Chiral preparative SFC 

 3

Oven and column

UV detector High-pressure flow cell

Modifier pump

Back pressure Regulator Sample loop

Feed solution CO2 pump

Event or Recycling Cyclones

Chiller head pump CO2 cylinder Figure 1.1: Supercritical fluid chromatography apparatus.

to provide liquid CO2 or to a bulk CO2 storage. For the second option, the vapor phase of CO2 is withdrawn and then liquefied under elevated pressure, thanks to a booster pump. Some SFC prep systems can recycle CO2 to reduce its consumption. To do that, once CO2 is separated from the modifier in the cyclone to collect the target, the ­pressure is held constant at 55  bars and the CO2 is cooled at 0–3  °C to liquefy it. This  requires a good design to get a good yield of recycling without any cross-­ contamination. 2 pumps are used to deliver the mobile phase. Most of the preparative systems have one pump dedicated to the modifier and one pump to deliver CO2. Thereby, the modifier and CO2 are mixed in a mixing chamber located past the pumps. However, some devices have another configuration with a mixing point located between the outlet of the modifier pump and the inlet of the second pump (CO2 pump). Thereby, the modifier and CO2 are mixed before the second pump, which delivers the whole mobile phase. This configuration allows to use a “low”-pressure pump for the modifier because the applied pressure on the pump will be the pressure of the CO2 supply (around 55 bars) and not the inlet pressure of the column (up to 300 bars). A second requirement for the pump is linked to the compressibility of CO2. Even though CO2 is liquid, its compressibility is higher than a solvent. It means that the

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 Emmanuelle Lipka and David Speybrouck

pump must be able to manage this compressibility to deliver an accurate and reliable flow rate. Some systems are equipped with a mass flow meter to measure on line the flowrate and thus tune the frequency of the pump to get the right flow rate. ­Moreover, the pump heads of the CO2 pump are chilled to maintain CO2 liquid and to avoid bubbles that could impact the flowrate. The Diode array detectors used are the same as those used in HPLC; the only difference is the flow cell that is designed to be compatible with high pressure. In preparative SFC, the collection is not as easy as in HPLC mode because of scCO2. Indeed, the depressurization of the mobile phase downstream the column generates both a significant expansion in the volume (500 times higher) and aerosol ­phenomenon. This expansion may result in a remixing of compounds causing a difference between the separation observed and the fraction purity check afterward [6]. As for the aerosol, it can cause loss of product, resulting in a low recovery or worse a contamination of the area around the device by the product. Thus, it is challenging to use an open-bed collection (meaning collection in a tube or a flask without any process or option to collect under high pressure as with a cyclone) system as used in LC. Several strategies developed to overcome this issue are reported by Berger and Perrut [7] in a paper published in 1990 with fraction collection either at atmospheric pressure or high pressure. The cyclone uses the centrifugal force generated by tangential injection of the mobile phase to separate the droplets of a modifier (same technology used in the Dyson bagless vacuum cleaner to separate air and the dust). Once a cyclone is full, it is drained in a flask. This collection technique provides good recovery but the main drawback of the cyclones is the limitation of the number of fractions. Usually chiral separation means two enantiomers, so three fractions are necessary: one for each enantiomer and one for the volume between both peaks. However, the chiral SFC technique is so efficient; the isomers of a molecule having two asymmetric centers (four isomers) can be separated in one shot as shown in Figure 1.2. Hence, five or six fraction collections are not much. Another process was developed by T. Berger based on a phase separator designed to control the depressurization of CO2 from the Supercritical Fluid (SF) state to a gas phase without the formation of “fog”. This collection technique was one of the reasons of success of Multigram II™ and Multigram III™ (from Berger) devices for batch purifications. Lastly, a few suppliers provide systems with open-bed collection such as Waters with the system prep SFC100 or Pic Solution. These systems are mainly used for the purification of compound libraries. Finally, an additional module called back-pressure regulator (BPR) is required to control the outlet pressure and to maintain the whole system under elevated pressure (100–150 bars). This BPR is usually connected online between the UV detector and the collection vessel. The BPR must have a very low dead volume and can be manual or automated. The injector is quite the same as those used in LC, but only requires a step of loop depressurization before loading. However, in a preparative scale, the injection is a



0

 Chiral preparative SFC 

1

2

3

4

 5

5

Figure 1.2: Separation of the four isomers of a proprietary compound. Column: Chiralpak AD 5 µm 100 × 4.6 mm, mobile phase: CO2/ETOH 70/30 v/v, flow rate 3.5 mL/min, λ: 250 nm, temperature: 35 °C, outlet pressure: 10.5 MPa.

key step and can have a considerable impact on the peak shape and the separation. That’s why several injection modes were developed and discussed in the following sections.

1.2.2 Injection mode As explained previously, the injection in preparative chiral SFC is the key step to obtain a good resolution. Once the method is developed, different parameters must be considered to get a good preparative separation such as the solvent used to dissolve the sample, the concentration and the volume of the solution injected, and the injection mode used. In preparative SFC, both the injected volume and the feed solution concentration are much higher than that in analytical SFC with an obvious impact on the peak shape because the distribution isotherm is not linear anymore. Regarding the feed solution, a high sample concentration is preferable to inject a small volume than a large volume of a diluted solution (Figure 1.3). Hence the solvent used to dissolve the sample is the key factor. Usually (in HPLC), the solvent used to dissolve the sample is the solvent used as the mobile phase or a solvent with a weaker elution strength to minimize peak broadening and peak distortion [8, 9]. However, in the SFC mode, due to the nature of the mobile phase, it is not possible to dissolve the solute in the mobile phase [10] unless a highly sophisticated injection process is applied. Often, the solvent used to dissolve the sample is the cosolvent used in the mobile phase. However, to enhance the concentration of the sample and thus reduce the injection volume [11], it will be helpful to utilize a dissolution solvent different from the cosolvent. However, only a few solvents (alcohol, alkane, and acetonitrile [ACN])

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14 mg (Vinj = 500 µL of 28 mg/mL feed solution) 14 mg (Vinj = 700 µL of 20 mg/mL feed solution) 14 mg (Vinj = 1,000 µL of 14 mg/mL feed solution)

14 mg (Vinj = 2,500 µL of 5.6 mg/mL feed solution) 14 mg (Vinj = 5,000 µL of 2.8 mg/mL feed solution)

Figure 1.3: Separation of 14 mg of a mixture of 2-amino benzophenone + (4-amino-3-nitrophenyl) phenyl-methanone) with different concentrations and different injected volumes. Column: 2-ethylpyridine 150 × 21 mm, mobile phase: CO2/MeOH 80/20, flow rate: 50 mL/min, temperature: 35 °C.

are compatible with the chiral stationary phases (CSPs) based on polysaccharide-derived selector coated on silica. The polymer coated on the silica can swell or dissolve with other solvents such as methylene chloride and tetrahydrofuran (THF) even in a very low quantity. Fortunately, with the new generation of polysaccharide-based CSPs immobilized on the silica gel or with the brush-type stationary phase, the choice of a solvent is wider. These phases are compatible with all solvents that can be used either as a mobile phase or just as a solvent to dissolve the compound [12]. The nontraditional modifiers can have a huge positive impact on the productivity, thanks to the better solubility of the solute [13] even if the selectivity is low. This topic is addressed in the following sections.

1.2.2.1 Mixed stream, modifier stream, and dry loading injection modes To optimize the injection step in prep SFC, three different injection modes were developed. The first one is called mixed stream injection mode. It means that CO2



 Chiral preparative SFC 

 7

and cosolvent are mixed upstream of the injection loop, and the sample is pushed into the column by the total mobile phase (Figure 1.4(A)). Since the sample is dissolved in the cosolvent, more polar than the mobile phase, the mixed stream injec-

Oven and column

Oven and column

Modifier pump

Modifier Sample loop pump Feed solution

Sample loop CO2 pump

Feed solution CO2 pump

CO2 cylinder

CO2 cylinder

(A)

(B) Oven and column

Modifier pump Extractor CO2 pump

CO2 cylinder

(C)

Figure 1.4: Different injection modes. (A) Mixed stream; (B) modifier stream; (C) dry loading.

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 Emmanuelle Lipka and David Speybrouck

tion mode first generates a change in the composition of the mobile phase during the injection step, resulting in a change in a peak distortion with a negative impact on the resolution [14], and second, the difference of solvent between the sample and the mobile phase can cause sample precipitation which may result in peak tailing or clogging. The second injection mode was developed by Berger [15] and is called the modifier stream injection mode (Figure 1.4(B)). In this injection mode, the sample loop is connected to the modifier flow stream, before the mixing point of the CO2/ modifier stream. The sample is pushed by the modifier stream prior to mixing with CO2. The advantage of the modifier stream is the reduced impact of the injection on the separation because unlike the mixed stream injection mode, the ratio of CO2/ modifier remains constant before, during, and after the injection step. A drawback of the modifier stream injection mode is the peak broadening resulting from the time taken to inject the sample. For a low percentage of modifier, the flow rate of the modifier pump is low and the injection time is high. Miller and Sebastian [11] and Dai et al. [16] compared mixed stream and modifier stream injection modes and concluded that for most of the compounds studied, the modifier stream gave better resolutions. Some suppliers such as Waters implement the modifier stream injection [17], whereas others use the mixed stream injection (Novasep and PicSolution) and one supplier proposes both for the European market (Sepiatec). Our experience with both has shown that the modifier stream injection mode is more efficient unless the percentage of the modifier is low (below 5%). By analogy with dry loading injection used in flash chromatography for poor soluble compounds, an injection by extraction was developed (Figure 1.4(C)) [18]. An extractor containing the dry sample adsorbed on silica or not is connected instead of the sample loop and flushed with the mobile phase for a few seconds to extract the compound. This mode combines supercritical fluid extraction and SFC.

1.2.2.2 Stacked injection Due to the low loadability of CSPs, the purification of several hundred milligrams or grams of a racemate would require a column with hundred grams of packing to do the separation at once. Because of the cost of CSPs, in drug discovery, 20 or 30  mm id columns are usually used with a loading capacity of a few milligrams. Thus, several injections of a few milliliters of the feed solution are required to purify the whole sample. To improve the productivity of the preparative chiral separation, an injection strategy called stacked injection is often used. The stacked injection (Figure 1.5) includes performing the second injection before the end of the elution of the second enantiomer from the first injection (even sometimes the first enantiomer).



 Chiral preparative SFC 

Injection n+2

Injection n+1

 9

Injection n

Figure 1.5: Stacked injection: three injections in the column.

The injection frequency is based on the time between the beginning of the first peak and the end of the second peak. Obviously, this mode cannot be used efficiently in the gradient mode. As shown in the example of Figure 1.6, the run time is 6 min and the injection frequency is 3.8 min, resulting in 145 min to inject 38 times instead of 228 min with a standard injection mode, improving the productivity considerably. The stacked injection is often used in preparative chiral SFC because most of the time there are only two peaks to be separated with close retention times and the isocratic mode is used.

0 (a)

5

10

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 (b)

Figure 1.6: Column: Chiralcel OD-H (250 × 30 mm id) 5 µm, mobile phase: 60/40 CO2/MeOH (0.3% iPrNH2), flow rate: 120 mL/min, outlet pressure: 10 MPa, temperature: 35 °C, λ: 230 nm, injection: 82 mg dissolved in 2,500 µL MeOH. (a) An injection and (b) stacked injection (38 injections).

Stacked injection is one of the most efficient strategies to improve the productivity. The productivity is a key point in chiral preparative SFC and is expressed in kkd (kilogram of product isolated/kilogram of CSP/day). Other metrics such as production rate (kg of product/day) or throughput (kg of feed/day) can be used. At the discovery stage, the challenge is not to get a high productivity but to reach a very short cycle time between the chiral separation submission and the delivery of the pure ­enantiomers because the number of requests is high and a productivity of 0.1 kkd is often good enough. Unlike the discovery stage, for the production in development of a non-GMP (good manufacturing practice) batch or a GMP batch involving the separation of several kilograms of racemate, the productivity must be as high as possible and can be around 1 or 2 kkd. In 2002, Cox separated carboxylic acid enantiomers on

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a ­Chiralpak®AD-H (250 × 21 mm id) 5 µm column, with a sample dissolved in pure MeOH at 160  mg/mL. The separation of flurbiprofen was achieved through staked injections with a productivity of 2 kkd [19].

1.2.3 Detectors In a preparative scale, the peak collection is based on peak polarity and amplitude rather than on slope or threshold which has proved to be a robust collect mode, dynamically adapted to variations in loading and retention time [20]. Detectors play a key role here. Among the available conventional detectors, diode array detectors, polarimetric, circular dichroism [21], evaporative light-scattering detector (ELSD) [22], and mass spectrometry (MS) [23] could be coupled to the packed columns mentioned earlier. The most common detection mode reported in the preparative SFC is diode array detection , mainly because of the low absorbance of carbon dioxide in the short wavelength range. As the mobile phase in SFC is composed of the most part by carbon dioxide, separations are typically performed by collecting fractions at detector wavelengths between 200 and 220 nm. The signal allows either an a ­ bsorbance-based collection or a timebased collection of the peak. ELSD is a preferred detector in SFC rather than in HPLC because of the ease of vaporization of the mobile phase after its nebulization [22]. MS detection is recognized as a specific tool not only to distinguish the targeted enantiomers from other achiral impurities (e.g., b ­ y-product or excess of starting material) but also for the identification of ­diastereoisomers. As for ELSD, SFC is particularly suitable to be coupled to MS and results in a flexible and robust ­instrument. Indeed, as the mobile phase rapidly expands, thus assisting n ­ ebulization, when leaving the end of a capillary, SFC is amenable to electrospray, atmospheric pressure photoionization, and atmospheric pressure chemical ionization MS source integration [23]. Above all, it should be kept in mind that in the case of preparative separations, the detector sensitivity is no longer an issue due to the high concentration injected.

1.3 Theoretical aspects of preparative separation Before carrying out the preparative separation, an analytical method must be developed to select the best CSP–modifier combination. Based on the capacity of the analytical device, the expertise, and the data collected from literature, a set of columns and modifier are selected. Usually this screening is performed in the gradient mode and the purification is done in the isocratic mode. Based on the analytical retention time, the percentage of the modifier is selected. Hamman et al. [24] developed a model to determine the percentage of the modifier from the retention time in the gradient mode. Once the method has been developed on the analytical system, different



 Chiral preparative SFC 

 11

strategies can be applied to scale-up on the preparative system. The choice depends on the quantity of racemate to be purified.

1.3.1 Overloading To get a few hundred milligrams of pure enantiomers with a short cycle time such as in drug discovery, a large overloading study is not required. Based on the separation obtained on the analytical unit, a scale-up is handled straight away on the prep system. Usually the total amount of compound is dissolved with a concentration depending on the quality of the separation and on the solubility of the racemate. However, whatever the solubility and the separation, the quantity injected is much higher than the quantity injected on the analytical unit with an obvious impact on the peak shape. Hence, as shown in Figure 1.7, the strategy is to inject a few hundred microliters (e.g., 500 µL) to check the resolution, then additional injections are done with increasing volumes until the touching band resolution level, meaning that two peaks are just baseline resolved as in this example with 1,500 µL. This strategy requires a few injections to determine the suitable injection volume. Then the stacked injection is carried out. When the quantity of racemate to be purified is in the range of several hundred grams or kilograms or even tons, the strategy displayed earlier is not recommended 620 600 580 560 540 520 500 480 460 440 420 400 380 360 340 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0 –20

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19 20 21 22 23

Figure 1.7: Separation of a mixture of 2-aminobenzophenone + (4-amino-3-nitrophenyl)phenyl-methanone) sample concentration: 60 mg/mL. Column: 2-ethylpyridine 150 × 21 mm, mobile phase: CO2/MeOH 80/20, flow rate: 50 mL/min, temperature: 35 °C. Injected volume: black chromatogram 500 µL, green chromatogram 1,500 µL.

12 

 Emmanuelle Lipka and David Speybrouck

[25]. A comprehensive study of overloading and upscaling must be carried out to get the best productivity. The method must be efficient and robust [25] and in line with the request of the submitter. It means that the method will not be the same if either both enantiomers must be isolated with a low enantiomeric excess (e.g., ee 95) or if only the eutomer must be collected with a high enantiomeric excess. First, a study of the equilibrium isotherm must be carried out. Indeed, in preparative chromatography, the adsorption isotherms of the solute govern the separation. The adsorption isotherm is the plot of the amount of solute adsorbed on the stationary phase as a function of the quantity of solute in the mobile phase. For very low quantity injected, like on the analytical unit, irrespective of this quantity, the amount on both phases is proportional resulting in a linear adsorption isotherm (the slope is equivalent to the Henry constant of the isotherm). This takes the form of a Gaussian peak whose intensity is proportional to the amount injected. If the quantity injected is much higher, the isotherm becomes nonlinear and two cases arise. Most of the time, the isotherm has a convex shape. It means that the amount adsorbed is not much proportional to the quantity injected. This isotherm is called Langmuir isotherm and results in an asymmetric peak with the front of the peak corresponding to higher concentration. The retention time of the end of the peak is constant but the front of the peak depends on the quantity injected. The higher the quantity injected, the lower the retention time of the beginning of the peak. In the second case, the isotherm observed is concave and called Freundlich isotherm. For this isotherm, the peak is not symmetric as that for Langmuir isotherm but the retention time of the front of the peak is constant irrespective of the quantity injected and the retention time of the end of the peak is proportional to the quantity injected. As explained by Guiochon and Tarafder [26], the determination of ­parameters controlling the phase equilibria is important to predict the elution band profile. Different methods such as the frontal analysis method [27, 28] and the inverse method [29, 30] have been developed. The overloading can be studied on the analytical unit before transferring on the preparative unit when the quantity of material available is low or straightaway on the preparative system.

1.3.2 Scale-up In both HPLC and SFC, when the method and the overloading are developed on the analytical unit using the same CSP with the same particle size as used on the prep system, the scale-up is easy to handle on the preparative unit. For example, if the dimension of the analytical column is 250 × 4.6 mm (5 µm), the flow rate is 3 mL/min, and the quantity injected is 1 mg, the peak shape and the retention times obtained on the prep system will be the same for a column 250 × 21 mm (5 µm) with a flow rate of 62.5 mL/min (Figure 1.8).



 Chiral preparative SFC 

3.40

100

 13

TIC 7.2407

%

3.00

–1 0.00

1.00

2.00

3.00

1

2

3

4.00

5.00

Time

6.00

(a) 640 620 600 580 560 540 520 500 480 460 440 420 400 380 360 340 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0 –20 0

(b)

4

5

6

7

Figure 1.8: Analysis and purification of a proprietary compound. Column: Chiralpak AD-H, mobile phase: CO2/EtOH 75/25. (a) Column 250 × 4.6 mm, flow rate: 3 mL/min, sample volume: 10 µL and (b) column: 250 × 21 mm, flow rate: 62.5 mL/min, sample volume: 200 µL.

However, with the introduction of columns packed with 3 µm particle size or with the 2.5 µm introduced by Waters with the trefoil columns or the 1.6 µm from Daicel, the utilization of short columns packed with small particle sizes is widespread in analytical scale, allowing shorter retention times and high efficiency. Until now, preparative columns are not packed with sub-2 µm particle, making the transposition not so easy. However, if the column length and the particle size were changed prop-

14 

 Emmanuelle Lipka and David Speybrouck

erly to keep the same ratio L/dp (where L is the column length and dp the particle diameter) constant between the analytical column and the preparative column, the column efficiency would remain the same and the separation would be the same in both the analytical unit and the prep unit. For example, a column 100 × 4.6 mm packed with 2 µm particle size would give the same efficiency, retention times, and separation, with 60% more pressure drop when compared to a column 250 × 21 mm packed with 5 µm particle size (if the quantity injected and the flow rate were proportional). This assumption is true in HPLC; indeed, the additional pressure drop due to the smaller particle would have little impact on retention times and the separation. However, in SFC, this kind of scale-up is more challenging due to the compressibility of CO2. Indeed, a modification in pressure drop impacts the density of CO2 resulting in a modification of the polarity of the mobile and a modification of the retention time [14]. To overcome this issue, the outlet pressure on the preparative system can be tuned (usually increased) to get the same inlet pressure on both the analytical unit and the preparative unit [31]. Finally, the last challenge regarding the scale-up from the analytical unit to the preparative unit is the control of the flow rate. On the analytical devices, the flow rate measurement is based on volume control, while for most of the preparative devices a mass flow rate calculator is used. This difference of configuration can result in time shift between the analytical scale and the preparative scale. In conclusion, if you had to separate 100 mg or 10 kg of racemate, the strategy and the time spent to determine the right parameters to scale-up the separation would not be the same. In the first case, the scale-up would require a few preparative injections before the production, while for 10  kg, it would be interesting to spend more time to measure different physical parameters to develop the most efficient and robust method.

1.4 Packed columns SFC The capillary approach in SFC using open tubular columns was introduced in 1984 and has been successfully used until the mid-1990s. Those columns are now almost abandoned, and consequently the term “packed-column” or “p-SFC” is taken as being implicit and is typically not mentioned specifically in the literature. Regarding column geometries, for the laboratory preparative scale on SFC-CSP, one would find, in almost all catalogs, 5 or 10 µm columns of 250 mm length, available in 10, 20, 21.2, 30, or 50 mm internal diameter. It is worth mentioning that, although having the same SP chemistry, some manufacturers develop custom-made SFC columns, whereas others market LC-columns that can easily be adapted for use in SFC after appropriate conditioning. In this case, some 10 or 20 µm columns of 250, 500, or even 800 mm length, available in 10, 20, 21, 30, 50, 100, and 150 mm internal diameter can be found



 Chiral preparative SFC 

 15

from many suppliers and can be used under flow rates up to 1,000 mL/min. Shorter preparative 5 µm columns in 150 mm length recently reached the market, promising a reduced analysis frame, thanks to higher flow rates that could be used without the fear of generating excessive pressure drop. But those columns must be implemented with a preparative SFC device equipped with pumps able to deliver flow rates much higher than 100 mL/min. From a theoretical point of view, direct separation of enantiomers by chromatography on CSPs requires to form transient diastereoisomers between the solute and the SP.

1.4.1 Chiral stationary phases Since the 2000s, polysaccharide-based CSPs appear, by far, as the most successful and widely used phases in preparative enantioseparations. Pirkle-type CSPs have also been reported but these are lagging behind cellulose and amylose CSPs because of their low capacity of loading. Indeed, the loading ability of a stationary phase is an important parameter when preparative separations have to be performed. Nevertheless, it must be underlined that the optimal phase in terms of loading capacity may vary from one racemate to another, depending on the structure of the racemic compound to be separated, as it is a function of the number of accessible interaction sites per mass unit of phase. Both brush-type and polysaccharide CSPs have high loadability with saturation capacity ranging between 1 and 40 mg/g CSP and 7.5 to more than 100 mg/g CSP for the former and latter phases, respectively [32]. Considering brushtype stationary phase, the enantioseparation is produced through the bonded phase ligand’s rigid structure and accessibility for π-bond interaction with solutes near its chiral center, whereas cellulose and amylose stationary phases developed by Okamoto et al. achieve chiral discrimination due to their raised number of chiral centers and a highly ordered secondary structure [33]. The tris(3,5-­dimethylphenylcarbamate) of cellulose and amylose is extremely popular (with a little higher rate of success for the second one). Together with the cellulose tris(4-methylbenzoate) and the amylose tris[(S)-α-methylbenzylcarbamate], these columns form the so-called golden four. While it may be commonly accepted that there is no “universal” stationary phase (comparable to a C18 in reversed-phase LC), these four ones cover more than 80% of enantioseparations. Chlorinated polysaccharides have also reached the market. Some of them have already proven their complementary selectivity to nonchlorinated CSPs in the preparative setting [34]. However, these phases have a major drawback: they are more or less soluble in many organic solvents, such as chlorinated solvents, ethyl acetate, THF, dioxane, and toluene, reducing the choice of an organic modifier. The main bottleneck, like the limited resistance of these coated phases, was overcome by an approach aiming to immobilize polysaccharide derivatives, in order to enlarge the choice of cosolvent. Today, the immobilized versions of the existing coated ones are

16 

 Emmanuelle Lipka and David Speybrouck

of particular importance in the preparative area for two reasons: first, new cosolvents may improve resolution through improved efficiency, and second, may in particular cases improve the solubility of the sample in order to gain in productivity. Four different types of chiral selectors are commercially available today: the amylose and cellulose tris(3,5-dimethylphenylcarbamate), the amylose and cellulose tris(3,5-­ dichlorophenylcarbamate), the amylose tris(3-chlorophenylcarbamate), and lastly the amylose tris(3-chloro-4-methylphenylcarbamate) patented by Daicel Industries and developed based on a collaboration with E.R. Francotte. Just a few months ago, Phenomenex in collaboration with B. Chankvetadze patented its first immobilized columns (amylose tris(3,5-dimethylphenylcarbamate) Lux i-Amylose-1, and cellulose tris(3,5-dichlorophenylcarbamate) Lux i-Cellulose-5), showing that manufacturers are very dynamic in this area. It must be emphasized that the same CSP coated and immobilized can exhibit different enantioselectivities as the conformation of the polymer is influenced by the linkage between the stationary phase and the packing material [35]. Preparative applications of these immobilized CSPs have been yet reported [36–38] and particularly in two reviews by Miller [39, 40]. At Jansen Discovery, the Chiralpak IC has joined the Chiralpak AD-H and Chiralcel OD-H which are the top three for the resolution of enantiomers. Indeed among 4,000 separations, AD-H is successful for half of them (46.9% success rate), IC (15.1% success rate), and lastly Chiralcel OD-H (10.8% success rate) (Figure 1.9). A similarity between these immobilized phases and the brush-type CPSs can be noted, as the latter are bonded to the silica support allowing the use of solvents with high eluting strength. Another interesting property of the Pirkle-type CPSs is that most of them are available in both enantiomeric and diastereoisomeric forms, opening up the possibility of reversing the elution order of the separated enantiomers by switching the chiral selector. This is a great advantage in peak collection, as ideally the enantiomer of interest should be the first eluting.

4.8

3.3

2.5

0.8

0.5

0.4

Chiralpak AD

5.4

Chiralpak IC 46.9

9.7

Chiralcel OD Chiralcel OJ Whelk 01

10.8 15.1

Chiralpak IA Chiralpak AS Lux cellulose-2

Based on 4,062 chiral separations

Lux cellulose-4

Figure 1.9: Utilization ratio of chiral stationary phases at Janssen Discovery France from 2005 to 2017.



 Chiral preparative SFC 

 17

1.4.2 A novel and original chiral selector: coupled columns First, the properties of scCO2 such as high diffusivity and lower viscosity are particularly suitable to couple two (or more) columns in series. Second, there is no distinction in SFC between normal-phase and reversed-phase separations. Those two features allow to connect in series achiral and chiral column combinations or columns with different polarities to create a unique selectivity. Hence, the number of combinations, comprising same chiral–chiral stationary phases (SPs) tuning, different chiral–chiral SPs tuning, and achiral–chiral SPs tuning, is countless, giving rise each time to new selectivity properties. Preparative separations were successfully reported on different tandems [22, 41–43]. All these columns were used with their analytical dimensions and particle size, thanks to their high loading ability, except in the last case [43].

1.5 Mobile phase As explained previously, the main constituent of the mobile phase is a supercritical fluid and CO2 is the most appropriate one because its critical point is easily accessible and is compatible with the compounds analyzed and because of its miscibility with all the organic solvents. Moreover, CO2 is nontoxic, cheap, and “green.” However, CO2 is not as easy as an organic solvent to handle, and it must be stored in cylinders for semipreparative system or in a large tank for the preparative scale. For a flow rate of 50 mL/min the duration of use of a cylinder B50 is less than 15 h except if the SFC device is equipped with a CO2 recycling module. To enhance the elution of a larger range of compounds and to counterbalance the low polarity of CO2 that is equivalent to hexane polarity, a modifier (or cosolvent) is added to the mobile phase. The proportion of cosolvent can go up to 70% for some separations.

1.5.1 Modifiers The modifier is usually an alcohol and the first objective of the addition of a modifier is to increase the elution strength of the mobile phase and thus to decrease the retention times of the solutes. For example, the retention time of 1-(1-naphtyl) ethylamine is 10.67 with 7.5% of ethanol in the mobile phase and 1.06 with 30% (Figure 1.10). By adding a cosolvent, the mobile phase is more often subcritical than supercritical, however, without any disruption in the physical properties of the fluids (viscosity, density, and diffusivity). According to T. Berger, above 16% of cosolvent in the mobile phase, the fluid is in subcritical state [44], and the term supercritical fluid is used

18 

 Emmanuelle Lipka and David Speybrouck

CO2/EtOH 92.5/7.5

CO2/EtOH 90/10

CO2/EtOH 70/30

CO2/EtOH 85/15

(D)

(C)

(B)

(A) 0

2

4

6

8

10

12

14

16

Figure 1.10: Analysis of 1-(1-naphthyl) ethylamine. Column: Chiralpak IA 100 × 4.6 mm with 3 µm, mobile phase: CO2/MeOH, flow rate: 3.5 mL/min. (A) 7.5% EtOH; (B) 10% EtOH; (C) 15% EtOH; and (D) 30% EtOH.

more to describe the technique than the state of the fluid. Moreover, unlike achiral SFC, for which the modification of the nature of cosolvent doesn’t impact considerably on the selectivity, in chiral SFC on polysaccharide-based stationary phase, switching from one cosolvent to another one can change drastically the enantioselectivity [45]. This example (Figure 1.11) shows an analysis of a racemic mixture on Chiralpak IC with a good resolution using isopropanol as cosolvent, a baseline resolution with ethanol and no separation with methanol. The same observation was done by White showing that it was interesting to use methanol, ethanol, and isopropanol for a chiral screening to optimize the method development [46]. The change of a modifier can also result in enantiomers reversal elution order as shown in Figure 1.12. Last, as shown in Figure 1.13, mixture of alcohols can be an option to enhance the enantioselectivity. The resolution was better with a mixture methanol/isopropanol than with methanol or isopropanol. According to Wang [47] and Wenslow [48], the d ­ ifferent chiral selectivities of CSPs associated with the use of different alcohol modifiers are due to different alterations of the steric environment of the chiral cavities in the CSP by the different mobile-phase modifiers. Thus, in chiral separation, the nature of the modifier is as important as the choice of the chiral selector [49, 50]. That’s why a screening with different CSPs and different modifiers is required to find the best ­separation, especially as there is no algorithm to determine the best couple stationary phase/ mobile phase.



 Chiral preparative SFC 

 19

AU

5.03

(a)

6.0e–2 4.0e–2 2.0e–2 0.27 0.42 0.0 –0.00

5.88 3.38 1.00

2.00

3.00

4.00

5.00

6.00

7.00

5.00

6.00

7.00

6.00

Time 7.00

AU

4.18 4.38

(b)

5.0e–2 0.0

0.43

–0.00

1.00

2.00

3.00

4.00

4.69

AU

1.0e–1

(c)

5.0e–2

0.00 0.0 –0.00

1.00

2.00

3.00

4.00

5.00

Figure 1.11: Analysis of a proprietary compound. Column: Chiralpak IC (100 × 4.6 mm id) 3 µm, flow rate: 3.5 mL/min, mobile phase: CO2/alcohol from 95/5 to 35/65. (a) Isopropanol; (b) ethanol; and (c) methanol.

As shown in the above-presented examples, the nature of the modifier can impact the selectivity. The most popular are methanol, ethanol, and isopropanol but a few applications of ACN as a cosolvent (pure or in mixture with alcohol) have been described. Even though the results seem to be quite disappointing with a lower success rate, it may be a choice in only a second screening [51]. For the immobilized polysaccharide-based CSPs and for the brush-type CSPs such as the column Whelk O1, the selection of solvent is larger since there is no restriction. Solvents such as dioxane, dichloromethane, THF, ethyl acetate, ethyl ether, and ethyl tert-butyl ether can be used, leading to different selectivities [52–54]. However, these solvents are usually not polar enough to elute all compounds and thus must be associated with an alcohol [51]. In preparative chiral SFC, even if the selectivity is worse, the nonstandard solvents can offer better solubility of the solute leading to better productivity, thanks to a gain of feed concentration [40]. This solvent can be used as pure or in mixture with an alcohol as modifier, or only as a solvent to dissolve the sample. Miller showed that the productivity for the separation of a racemate can be improved by using dichloromethane in the mobile phase. A selectivity of 2.33 was achieved on Chiralpak AD by using methanol as a modifier. The scale-up was not efficient due to a low solubility of the solute in the mobile phase, resulting in poor peak shape and bad separation. With a mixed modifier methanol/dichloromethane 1:1 on Chiralpak IA (the same chiral selector as Chiralpak AD but immobilized on the silica), the productivity on the scale-up increased to fivefold and the consumption of solvent was reduced to eightfold despite a lower selectivity (1.59) [13].

0.89

0.89 2.0e–1 –1 1.75e 1.5e–1 1.73 1.25e–1 –1 10e 7.5 0.45 5.0 2.5 0.72 0.34 0.0 –0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

10e 7.5 5.0 2.5 0.36 0.0 –0.000.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

2.0e–1 1.75e–1 1.5e–1 1.25e–1 –1

AU

1.5e–1

2.5 0.00 0.0 –0.00

5.0

1.0e–1 7.5

1.25e–1

1.75e–1

(b)

0.00

–0.00

0.0

2.0

4.0

6.0

8.0

1.0e–1

0.50

0.51

0.50

1.00

0.88

1.00

0.76 0.88

1.19

1.50

1.57

1.50

1.57

2.00

2.00

2.50

2.50

3.00

3.00

Figure 1.12: Analysis of proprietary compound column: Chiralpak AD-3 100 × 4.6 mm, mobile phase: (a) CO2/iPrOH 75/25 and (b) CO2/EtOH 75/25. Top: Analysis of the R enantiomer. Bottom: Analysis of the racemate.

(a)

AU

AU AU

20   Emmanuelle Lipka and David Speybrouck

 Chiral preparative SFC 

AU



AU

(a) 4.0e–1 2.0e–1 0.0 –2.0e–1

AU

(b)

1.00

2.00

10.19

2: Diode array Range: 5.884e–1

3.00

4.00

5.00 4.78

1.25 1.00

6.00

7.00

2.0e–1 0.0

0.83 1.00

8.00

9.00

10.00

5.29

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

7.46

3.00

4.00

5.00

12.00

6.00

7.00

8.00

11.00

12.00

2: Diode array Range: 5.892e–1

1.64 2.00

11.00

2: Diode array Range: 7.008e–1

2.06

5.96

–2.0e–1

(c)

6.10

2.0e–1 0.65 0.22 1.32 0.0

–2.0e–1

 21

9.00

10.00

11.00

Time 12.00

Figure 1.13: Analysis of proprietary compound column: Chiralpak AD-H (150 × 4.6 mm id) 5 µm, mobile phase: CO2/alcohol from 95/5 to 35/65. Alcohol: (a) methanol/isopropanol 50/50; (b) methanol; and (c) isopropanol.

1.5.2 Additives Based on the nature of the solute, a third component of low volatility known as additive may be added to the mobile phase to minimize peak distortion arising from unwanted interaction between polar solutes and the stationary phase. These additives are generally present at 0.1–2.0 vol.% of the modifier. Usually a basic additive is used to elute a basic compound [55] and an acidic additive for an acidic compound [56]. The additive can be triethylamine (TEA), diethylamine, isopropylamine (iPrNH2), or ammonia for the basic additive, or trifluoroacetic acid (TFA) or formic acid (FA) for the acidic additive. The addition of acidic additive is not always required to achieve elution of acidic additive, due to the intrinsic acidity of the mobile phase resulting in the reaction of the alcohol used as a modifier and CO2. The impact of the additive on the retention times and the plate number has been already demonstrated. However, the impact of the additive on the enantioselectivity is a controversial topic. Some works published concluded that there was no clear impact [57], while at the same time other authors showed that the additive can influence the enantioselectivity with increase [55], decrease, disappearance, and appearance of the separation and even the reversal of the order of elution of the enantiomers by changing the additive concentration [58]. The assumption regarding the role of the additive is multiple and not fully elucidated. As explained earlier, the general rules are to use the basic additive for basic analytes and the acidic additive for acidic analytes; however, the use of a basic additive with an acidic compound [59] or vice versa [60] can have a positive effect on the selectivity. Figure 1.14 shows the separation of atropoisomers containing an acidic moiety.

(b)

(c)

Figure 1.14: Analysis of a proprietary compound. Column: Chiralpak AD-H (150 × 4.6 mm id) 5 µm. Mobile phase: CO2/iPrOH. Top: racemate; bottom: one isomer (a) without additive, (b) 0.3% TFA, and (c) 0.3% iPrNH2.

(a)

22   Emmanuelle Lipka and David Speybrouck



 Chiral preparative SFC 

 23

From Figure 1.14, it is shown that there is no separation without an additive due to the peak broadening; a baseline resolution appears by the addition of 0.3% of TFA with a very good selectivity, thanks to the addition of iPrNH2 in the mobile phase. By ­injecting an isomer, we can highlight a reversal elution order between acidic ­conditions  and basic conditions. In preparative chiral SFC, it is often better to elute first the eutomer due to the distribution isotherm, so the substitution of an additive by another one can be beneficial. However, if a basic additive is used to separate an acidic racemate, the pure enantiomer isolated might be a salt after evaporation, requiring an extraction step to obtain the acidic form. Other parameters must be taken into consideration for chiral preparative SFC, when an additive must be selected. First, it must have a low boiling point to get rid of it easily. Some users replace the organic basic additive such as TEA or DEA by ammonia because it is easier to evaporate [61, 62]. It should also be borne that at times the additive can also promote degradation or racemization of the solute during the purification or the evaporation step.

1.5.3 Solubility and stability of the sample in preparative separation The resolution one might achieve on the analytical device is not the only argument for selecting the conditions for the scale-up. Once the method is developed, both solubility and stability must be validated before the purification. Irrespective of the quality of the separation, if the solubility of the compound in the mobile phase is very low, the productivity will be impacted. Moreover, the concentration of the feed solution will be low with the risk of a potential crystallization of the compound in the tubbing or in the column. With coated polysaccharide-based CSPs the choice of solvent is limited, that’s why the substitution by the immobilized polysaccharide-based CSPs can be a good alternative. With these phases, solvents such as dichloromethane or ethyl acetate can be used as a mobile phase or a solvent to dissolve the solute, resulting in a higher concentration and productivity. Even if the immobilized phases are usually less enantioselective, the enhancement of the solubility with the “exotic solvent” may generate a better productivity. These solvents can be used as a modifier, pure or mixed with a standard solvent, leading to better peak shape and thus a better productivity [34]. Another solution is to use dichloromethane or THF to dissolve the compounds (pure and mixed with alcohol) while standard solvent such as methanol or ethanol will be utilized for the mobile phase. Another alternative to improve the solubility is to generate the formation of a salt with the solute. For example, the addition of a base such as trimethylamine with an acidic solute generates a salt with a better solubility in an alcohol. In a preparative chiral SFC, the stability of the solute must be taken into consideration to choose the modifier. Degradation can occur during the separation or during the evaporation step. Indeed, some compounds containing, for example, carboxylic

24 

 Emmanuelle Lipka and David Speybrouck

acid moiety can react with the alcohol used as a modifier to give an ester moiety. In this case, if the selectivity is good enough, it is better to use isopropanol than ­methanol. In the same way, Byrne et al. [63] demonstrated that 2,2,2-trifluoroethanol can be a good modifier to analyze the alcohol-sensitive product. Finally, the pH of the mobile phase must be considered to avoid any racemization of epimerization and the pH is more impacted by the additive in the mobile phase than by the cosolvent. That’s why we would recommend not to use an additive except if it is necessary. Bajpai et al. [64] suggested to connect a make-up pump on the preparative SFC to neutralize the acid or the base used as an additive during the purification. The aim of this postcolumn addition of solvent containing base or acid is to form a salt with the additive to neutralize it.

1.6 Preparative SFC: a greener and safer chromatography? The comparison of HPLC and SFC from environmental and safety points of view remains a hot and vast question. In 1998, Anastas and Warner defined 12 p ­ rinciples of green chemistry [65]. Not all, but some, can be directly applied to analytical ­chemistry: prevent waste, use safer solvents and processes, minimize use of energy, avoid ­chemical derivatives, analyze in real time to prevent pollution, and lastly increase the safety of the operator [66]. Some papers have addressed this topic of green chromatography in practice, but essentially at the analytical level [67–69]. However, three of the principles, called the three Rs (reduce, replace, and recycle) are considered to be most relevant for greening the large-scale preparative separation technologies and can be applied on a daily basis in each laboratory [67]. SFC has established itself as an ecological method, in particular at the small-scale preparative level, mainly because of the numerous advantages of CO2, and reduced amount of hazardous solvents used and evaporated (energy), but also with regard to HPLC, which consumes large volumes of water (in reversed phase) or alkane (in normal phase) and organic solvents. A few years ago, green metrics have appeared in order to evaluate the environmental impact of a separation process: in particular solvent usage (L/g racemate) corresponding to the volume of solvent consumed to purify a known amount of ­racemate.

1.6.1 Reduce solvent and energy consumption It is known that an SFC equipment is around twofold more expensive than an HPLC one for the equivalent analytical and preparative performances [70]. But, on the other hand, the whole solvent volume required for preparative SFC is between two and ten times lower than for the preparative HPLC [71]. As a result, there is savings in time to evaporate



 Chiral preparative SFC 

 25

and to evacuate solvents. Besides, one will find very pure CO2 inexpensive, but energy consumption needed to heat and cool is unavoidable and must not be neglected [72]. According to an estimation from E.R. Francotte, water removal needs seven times more energy compared with the energy required to evaporate small amounts of modifier in SFC [73]. Nonetheless, to get a more precise idea, the power requirement for a preparative SFC system (pumping 350 g/min of MeOH/CO2 10:90, at 35 °C, at 100 bar) is less than 5 kW and for heating and cooling solvent 2 kW is necessary, while the power requirement for solvent evaporation with a comparable HPLC unit would be in the order of 20 kW, showing that even in terms of energy consumption SFC is cheaper than HPLC [74]. The lower solvent consumption is attested in two examples (among others): in 2009, an article by Tognarelli from Jasco reported that for 24 h of experiments the HPLC approach consumed 910  mL of ACN and produced 1,430  mL waste, whereas the SFC approach consumed 1,080 mL of MeOH and produced the same volume of waste [75]. The second example described that RP-HPLC purification used in their company produced in a year, on average 4,800 L of solvent waste (ACN and H2O mixture) while for SFC only a part (around 35 %) of waste MeOH is produced in a year for the same number of injections [76]. On the kilogram scale, Welch et al. presented the example of two processes for the same separation: an HPLC process run on a 30 cm internal diameter chiral column that consumes 36,000 L of solvent (10,000 L evaporated) in comparison to an SFC process run on a 5 cm internal diameter chiral column that consumes 900 L of solvent (215 L evaporated) [74]. With a relevant optimization of the SFC methodology, the solvent usage can be also reduced. As reported by Miller, changing a chromatographic process (MeOH method to DCM/MeOH method) led to a reduction from 0.68 to 0.31 L/g racemate [13]. Many other examples are shown in his other article, where dichloromethane is extensively used as a dissolution solvent and an exclusive modifier [77].

1.6.2 Replace organic solvent Healthy, safety, and environmental considerations are a major concern for all the academic and industrial actors. Moreover, particularly in a preparative scale, flammability and toxicity are extremely high as users are handling very large volumes of solvents. In any case, whatever the approach, HPLC or SFC, the question of the extensive use of either ACN, or methanol for the first one, or dichloromethane and THF for the second, needs to be carefully reviewed for safer solvent purpose. Following this trend, n-hexane which is reprotoxic and neurotoxic was replaced by n-heptane; second, ethanol (EtOH), a renewable resource, is now recognized as an appropriate substitute for MeOH and ACN. Those two latter have been ranked as hazardous solvents due to their inherent toxicity and the great importance of safe detoxification of their waste [78, 79]. Recently, cyclopentyl methyl ether and 2-methyl THF were successfully chosen to incorporate greener alternatives to THF (following a note from Sigma-Aldrich Inc.) [80]. DCM is associated with both acute and chronic toxicities for humans, and persists in the environment with a half-life

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 Emmanuelle Lipka and David Speybrouck

of 18 months in water. ­Therefore, Taygerly et al. have developed a green chromatography solvent selection in order to replace DCM/MeOH mixtures [81]. Lastly, the replacement of hazardous organic solvent by a large amount of carbon dioxide is desirable from an ecofriendly point of view. It is nontoxic, nonflammable, and a renewable resource as the SFC installation uses carbon dioxide that is condensed from the atmosphere delivered into the chromatographic device and then returned to the atmosphere (or recycled). It is important to indicate that preparative SFC is not a net generator of carbon dioxide. In addition, carbon dioxide is also a recovered industrial waste byproduct (e.g., from the production of metals such as iron and steel, and the production of chemicals) or comes from natural processes such as beverage fermentation, whereas incineration of organic solvents results in the net generation of carbon dioxide [74].

1.6.3 Recycle solvents CO2 can be recycled along the purification process by applying suitable pressure and temperature. A process to recycle online carbon dioxide was developed by Perrut in 1984 and nowadays several SFC suppliers propose this option [82]. Depending on the percentage of CO2 used in the mobile phase and the SFC suppliers, up to 95% of the CO2 can be recycled, reducing once again the cost of CO2. Additionally, in the case of binary mixtures as mobile phase (CO2 and a cosolvent), the pure enantiomers are collected and dissolved in the modifier, which can be easily reused after evaporation. All these advantages clearly show that greenness is not the icing on the cake, but the cake itself particularly as the scale is increasing.

1.7 Conclusion In 1902, the first LC implemented by Tsvet was a preparative separation. This is also the case of SFC when Klepser performed his preparative one. After a relatively difficult initial development during the second half of the twentieth century, SFC has now become a well-established technique in both academic and industrial laboratories for pharmaceutical research and development [83]. Since the introduction of this technology, preparative SFC has been the main field of application. The low consumption of solvent compared to HPLC, the advantages of scCO2 as mobile phase, and the amenability of the technique to the separations of enantiomers, which is a critical issue for the development of active pharmaceutical ingredients, are the main reasons explaining this phenomenon. The preparative SFC applications are numerous and were reviewed in 2016 in a comprehensive article that could be consulted for a better insight by interested readers [84]. In 2017, Leek and Andersson from AstraZeneca published two challenging case studies of up to kilogram chiral separations [1]. For example, during the development of a first-line anti-inflammatory therapy for



 Chiral preparative SFC 

 27

asthma patients, the synthesis of the racemic Active pharmaceutical ingredient (API), compound B, an organic carboxylic acid, was outsourced and the first large batch (kg amount) required in the project was for early-stage toxicology testing. A 250 × 50 mm Chiralpak IC column, using 20% ethanol in CO2 at 120 bar and 40 °C with a flow rate of 450 g/min, was used for the large-scale batch. As a result, 3.4 g racemate was resolved in 155 s, leading to a product fraction of less than 70 mL. A total of 1.1 kg of the API was obtained in an enantiomeric excess of 99.9%, a yield of 96%, and a chromatographic throughput of 5.7 kg racemate/kg CSP/day, for this step. Besides, most striking is the apparition of patent claiming the separation of chiral isomers by SFC [85]. Even though many tools are available today to users, facilitating the task, it must be kept in mind that the preparative scale is a long road. It requires a previous analytical development, a transposition study to a preparative scale as shown figure 1.15, and hereafter an enantiomeric verification step [86]. 600 500 400 300 200 100 0 –100

0

5

Minutes

10

15

(a) 1800 1600 1400 1200 1000 800 600 400 200 0 –200

Fraction 1 collected Fraction 2 collected

5 Injection

(b)

10

15

20

25

Injection Minutes

Figure 1.15: Analysis and purification of compound 1: (a) Analytical-scale chromatograms, 0.5 mM in SFC on Chiralpak AS-H; CO2/EtOH 80/20 v:v, 3 mL/min; 150 bars outlet pressure; 40 °C. (b) Preparative scale chromatograms, 20 mM in SFC on Chiralpak AS-H; CO2/EtOH 85/15 v:v, 3 mL/min; 150 bars outlet pressure; 40 °C.

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Lucie Nováková, Kateřina Plachká, Maria Khalikova, František Švec

2 Supercritical fluid chromatography in bioanalysis

Abstract: Bioanalysis requires use of various methods for monitoring of both endogenous species and xenobiotics in biological materials. Sample complexity, high diversity of analytes, and often low concentrations call for methods with high selectivity and sensitivity. Supercritical fluid chromatography coupled to mass spectrometry (SFC-MS) has established itself as an important alternative technique in addition to more traditional high-performance liquid chromatography and gas chromatography. A significant increase in interest for SFC-MS applied in bioanalytical field is observed particularly since the introduction of novel SFC instrumentation that provides robust platform, meeting the rigorous criteria of method validation in strictly regulated environment. SFC-MS has demonstrated many benefits in a wide range of specific applications such as analysis of drugs and their metabolites, analysis of chiral drugs, doping screening and analysis of drugs of abuse, analysis of fat-soluble vitamins and carotenoids, and, finally, in metabolomics concerning mostly lipidomic analyses. Keywords: biological matrices, chiral separations, drugs, drugs of abuse, lipidomics, mass spectrometry, metabolites, method validation, matrix effects, supercritical fluid chromatography, vitamins

2.1 Introduction Bioanalysis can be considered as a subdiscipline of analytical chemistry covering the measurements of compounds in biological fluids including blood, plasma, serum, saliva, and urine, as well as other biological specimens such as feces, skin, hair, and organ tissue. Generally, two types of the analytes can be considered: (i) endogenous biological compounds such as proteins, lipids, and various small-molecule biomarkers, and (ii) xenobiotics, that is, compounds that are not naturally present in biological organisms. The xenobiotics involve not only drugs and their metabolites but also all the chemicals to which an organism is exposed and are extrinsic to its metabolism. The need for analysis of biological samples is an interdisciplinary task [1–3]. Bioanalytical methods are needed during drug discovery process, targeting new drug candidates for the evaluation of toxicokinetic [4], pharmacokinetic [5], and pharmacodynamic studies including particularly drug metabolism. They are used not only during the whole drug Lucie Nováková, Kateřina Plachká, Maria Khalikova, František Švec, Department of Analytical ­Chemistry, Faculty of Pharmacy, Charles University, Hradec Králové, Czech Republic https://doi.org/10.1515/9783110618983-002

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 Lucie Nováková, Kateřina Plachká, Maria Khalikova, František Švec

development process, for clinical studies, but also to compare pharmacokinetic profiles of generic drug formulations, for therapeutic drug monitoring [6, 7], as well as in forensic [8], ­toxicology [9], and doping control laboratories [10]. Moreover, bioanalytical methods can enable identification of large molecules such as proteins and peptides, which make them useful for the discovery of biomarkers for diagnostics of various diseases [11]. Bioanalytical method includes several steps necessary to cover each phase from sample collection to sample preparation, separation, detection, data processing, method validation, and interpretation of results [1–3]. Sample preparation is a crucial part of a bioanalytical method due to high complexity of the biological matrices and high content of interfering compounds. An appropriately designed sample preparation technique is necessary to remove interferences and to isolate, clean-up, and/or preconcentrate the analytes [3]. Sample preparation is usually carried out using conventional methods such as protein precipitation (PP), liquid–liquid extraction (LLE), and solid phase extraction (SPE). Despite extensive research and development in the field of sample preparation resulting in variety of miniaturized forms of both SPE and LLE, these approaches prevail only in research domains, while conventional methods are preferred in routine laboratories [2]. Efficient and fast chromatographic methods with sensitive and selective detection are needed in the steps following sample preparation [3, 12, 13]. High-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) is a method of choice in bioanalytical applications [1, 2]. The analysis of biological samples in clinical research places special requirements on analytical methods including high throughput of samples as well as small sample and solvent consumptions. Therefore, ultrahigh-­ performance liquid chromatography (UHPLC) has received a considerable attention. However, the request for certain selectivity, wide application range, and complementary techniques opens a space for the introduction of new techniques. Indeed, many benefits of SFC methods in terms of different selectivity, speed, and efficiency, the development of SFC-specific stationary phases, and the design of robust instrumentation allow SFC to establish itself in bioanalytical research and routine laboratories. The new SFC platforms adapted for working with sub-2 µm particles were then designated as ultrahigh-performance supercritical fluid chromatography (UHPSFC). Method validation has to be always carried out to ensure that bioanalytical method provides consistent, reliable, reproducible, and accurate data [14]. Two guidelines regulate the procedure of validation of bioanalytical methods: (i) Guideline on Bioanalytical Method Validation issued by European Medicine Agency [15] and (ii) Guideline for Industry, Bioanalytical Method Validation issued by Food and Drug Administration [16]. The parameters of selectivity, sensitivity lower limit of quantitation (LLOQ), calibration curve, accuracy, precision, stability, dilution integrity, and matrix effects are requested by both authorities to be evaluated [15, 16]. Limits of acceptance for method precision and method accuracy are also the same in both guidelines. Method precision expressed as relative standard deviation must be less than 15% and less than 20% for quality control (QC) samples and LLOQ concentration, respectively. Method accuracy should be ±15% and ±20% of the nominal value for QC samples and LLOQ, respectively.



 Supercritical fluid chromatography in bioanalysis 

 35

However, the two guidelines differ in the requested numbers of concentration levels and/or replicates. Evaluation of stability is necessary to guarantee that the conditions during sample preparation, storage, and analysis do not affect obtained results. Several stability tests are required including freeze and thaw stability, short-term stability, longterm stability, stability of the stock solution, stability of the processed sample under storage conditions, and on-instrument/autosampler stability. Besides the aforementioned parameters, European Medicine Agency requires carry-over to be investigated [15]. The reader is referred to these specific guidelines for further details [15, 16].

2.2 Important aspects of SFC in bioanalysis 2.2.1 Typical SFC conditions in bioanalysis While the choice of generic conditions for reversed-phase HPLC-MS/MS (RP-HPLC-MS/ MS) bioanalytical method can be straightforward, these conditions are more difficult to define in SFC-MS/MS. Indeed, typical initial conditions in RP-HPLC-MS/MS include the use of C18 stationary phase, acetonitrile or methanol as an organic modifier, 0.1% formic acid as an aqueous component, and gradient elution from 5% to 95% of an organic modifier in the mobile phase. When using conventional sample preparation methods, such as PP, LLE, and SPE, evaporation of sample extract and reconstitution in the a solvent with the composition close to that of the initial mobile phase is often required to prevent peak distortion. Determination of such generic conditions in SFC-MS is not that easy and straightforward. Although SFC separations are carried out with the same type of CO2/organic modifier-based mobile phase, the generic stationary phase, such as C18 in RP-HPLC, does not exist in SFC. Figure 2.1 was drawn to depict the current state of the art of SFC conditions in bioanalytical methods based on 69 published papers addressing a variety of applications of SFC in bioanalysis. These papers will be discussed in detail in Section 2.3, while general features will be discussed below. In Figure 2.1, the variety of sample preparation methods used in SFC is shown in the inner circle, typical mobile phases are shown in the second circle, stationary phases are represented with the third circle, and detection approaches are depicted in the outer cycle. The larger is the part of a circle for a particular method parameter, the more frequent was the use of this specific condition in published SFC bioanalytical methods. SFC offers an important benefit in the sample preparation step. Polarity of CO2-based mobile phase is close to that of hexane. Therefore, organic solvents such as ACN usually used in PP and SPE, and nonpolar solvents, such as hexane, heptane, and ethyl acetate used in LLE, are compatible with the SFC mobile phase and can be injected directly. This compatibility allows omission of evaporation and reconstitution steps, thus enabling a decrease in sample preparation time. Such approach is now possible due to the ultimate sensitivity of current mass spectrometers despite actual sample dilution during

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 Lucie Nováková, Kateřina Plachká, Maria Khalikova, František Švec

Figure 2.1: Representation of the variability of chromatographic conditions used in SFC methods in bioanalysis. LLE, liquid–liquid extraction; LLE+E, LLE + evaporation; PP, protein precipitation; SPE, solid phase extraction; DS, “dilute and shoot”; SLE, supported liquid extraction; SE, solvent extraction; MeOH, methanol; AmF, ammonium formate; AmAc, ammonium acetate; EtOH, ethanol; BEH, bridged ethyl hybrid; 2-EP, 2–ethylpyridine; ODS, octadecylsilica; 2-PIC, 2-picolylamine; 1-AA, 1-aminoantracene; HILIC, hydrophilic interaction liquid chromatography; ESI, electrospray ionization; MS, mass spectrometry; MS/MS, tandem mass spectrometry; UV, ultraviolet; APCI, atmospheric pressure chemical ionization; CI, chemical ionization; FID, flame ionization detector; ECD, electron capture detector.

some procedures typical of sample preparation. On the other hand, such approach is impossible when sample preconcentration is needed to achieve desirable method sensitivity. Indeed, Figure 2.1 reveals that LLE with included evaporation was the most widely used sample preparation step (36%) in SFC bioanalytical methods despite longer time required for this procedure. LLE without the evaporation was applied only in 8% of the reported methods. PP is very popular due to its simplicity in both method development



 Supercritical fluid chromatography in bioanalysis 

 37

and sample preparation itself and is also widely used in SFC bioanalytical methods (19%). In this case, direct injection of supernatant was mostly preferred without the sample evaporation and reconstitution. SPE is a multistep procedure that is more demanding in terms of method development and sample preparation time. However, it enables more selective extraction and clean-up. Therefore, it was used to a similar extent (17%) as PP. Simple and fast “dilute and shoot approach”, which is indispensable in high-throughput screening procedures with urine, was used to a lesser extent in SFC bioanalysis (7%). Indeed, the selection of dilution solvent is very challenging due to the character of urine and SFC mobile phase and will be discussed in Section 3.3. The choice of the stationary phase in SFC is strictly application dependent as no generic phase is available so far. Therefore, a wide range of different SFC-dedicated, RP-HPLC, NP-HPLC, and chiral stationary phases is used in bioanalytical SFC. Hybrid silica modified with 2-ethylpyridine functionalities (2-EP), diol, and plain hybrid silica belong among three of the most widely used stationary phases in bioanalytical SFC applications (Figure 2.1). Despite normal phase-like separation character of SFC, C18 provides an interesting alternative for nonpolar analytes in bioanalysis. However, all these stationary phases can be used with the mobile phase of the same composition including CO2, organic modifier, and, eventually, an additive in case of analytes with acid–base activity. Simple and generic conditions enabling compatibility of SFC with MS are favored. Therefore, CO2/MeOH with or without the addition of ammonium formate were used in a majority of over 60% of SFC bioanalytical applications. Make-up solvent or compensation solvent is often used to facilitate coupling SFC and MS and to enhance MS response, particularly when pure CO2/MeOH is used. Make-up solvent usually comprises methanol or ethanol, and is often completed with ionization supporting additives such as formic acid, ammonium hydroxide, ammonium acetate, and water. Temperature and back-pressure regulator (BPR), at which the value of pressure is set, are further two important parameters affecting density of mobile phase in SFC. Although their optimization is helpful only for fine method tuning under CO2/modifier conditions, their selection must be wise. A combination of a higher pressure exceeding 120 bar and lower temperature in a range of 40–50 °C are recommended as reasonable starting points for the method development [17]. SFC is compatible with a wide range of both HPLC and GC detectors. In the past, most of the SFC separations were carried out using GC detectors [18, 19]. Consequently, the detection in SFC progressed to HPLC detectors such as UV and evaporative light-scattering detector [19, 20]. Similarly, a trend toward SFC-MS hyphenation has emerged to ensure sensitive and selective measurements particularly including complex matrices [19]. In SFC, like in HPLC bioanalytical methods, MS/MS detection is the method of choice. It was used in 57% of developed methods as also shown in Figure 2.1. Despite lower selectivity, simple MS detection is also quite popular and was applied in 23% of methods. ESI ionization was preferred with both MS and MS/MS detection. Remaining SFC bioanalytical methods used UV (16%) and GC-type detection such as flame ionization and electron capture detectors (3%).

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2.2.2 Coupling of SFC-MS Several interfacing approaches have been developed for hyphenation of SFC and MS. Their suitability depends on the type of application [21]. Early interfacing approaches for online coupling of open-tubular SFC-MS involved direct fluid introduction into chemical ionization [22, 23] and electron ionization sources [24, 25]. The high volatility of CO2 can enhance nebulization during the ionization process. This was found beneficial for hyphenations of SFC and MS [19]. Thermospray and particle–beam interface were used in early interfacing approaches with packed-column SFC-MS [19, 21]. The thermospray source was able to accommodate the high flow rates of CO2-based mobile phase and, subsequently, reagent gases were added to this source in order to better control the ionization mechanism [26–28]. The particle–beam interface receives the analytes carried as a fine stream of particles [21, 29]. The reproducibility and sensitivity of this interface could be improved when extra particle-forming solvent was added [30]. The atmospheric pressure chemical ionization (APCI) [31] and electrospray ionization (ESI) [32, 33] quickly became the most popular atmospheric pressure ionization (API) sources that followed the development in HPLC-MS and GC-MS [19] (see also Figure 2.1). Yet, neither of these ionization sources is as universal as electron ionization. However, both API ionizations are useful in SFC-MS with their suitability varying as a function of analytes physicochemical properties and chromatographic conditions [21]. APCI is a gas-phase process that requires a protic solvent or modifier, and is particularly suitable for detection of thermally stable, less polar, low-molecular weight compounds. Therefore, it was initially considered to be more universal for SFC separations and was widely used in SFC-MS methods [31, 34, 35]. Nevertheless, successful applications of ESI emerged in the 1990s [36, 37], and this perception was diminished [20]. ESI requires a droplet formation in a liquid state. It is particularly suitable for the ionization of more polar, medium-to-high molecular weight analytes. Even its application to nonpolar analytes was also successful [37]. Recently, UHPSFC-MS/MS was compared to UHPLC-MS/MS using ESI with quite similar results for the ion source settings parameters. The beneficial effect of high desolvation temperatures, high gas flow, and low capillary voltage on method sensitivity was shown [38, 39]. The same effect was also demonstrated for APCI sources settings in both HPLC-MS and SFC-MS systems [40]. Contradictory results were achieved by Fujito et al. [41] who observed different capillary voltage settings needed for LC-MS and SFC-MS. Several reports confirmed the applicability of API sources used in HPLC-MS to SFC-MS systems without any modifications [42, 43, 44]. However, some modifications are usually required to ensure uniform pulse free flow, chromatographic integrity, and ionization of wide range of analytes [21]. In contrast to HPLC-MS, the transfer of the eluent from the end of column to the API source is critical due to the compressibility and behavior of SFC mobile phase [21]. The supercritical and/or near-critical state of the mobile phase in SFC is the result of pressure maintained typically above 100 bar via BPR [20]. However, both nebulization and evaporation of analytes in APCI and ESI sources



 Supercritical fluid chromatography in bioanalysis 

 39

occur at atmospheric pressure [20], which is constant and therefore nearly independent of nature and flow of the mobile phase [21]. The CO2 is decompressed by the pressure drop between column end and the ion source, and aerosol is formed, which helps the nebulization of the analyte. The mobile phase decompression is also highly endothermic. Therefore, SFC requires higher temperature of nebulizer and source [20]. Among other conditions, the pressure affects the solvating power of SFC mobile phase. As a result, the density drop after exit from the column can cause the decrease in solvating power to the point at which analytes may precipitate. This precipitation on capillary walls can lead to significant carry-over effects. The loss of chromatographic performance, degradation of peak shape, deterioration of mass transfer, and decrease in detector sensitivity can also occur [45]. Moreover, the pressure drop below a certain pressure and temperature leads to the boiling of the mobile phase when modifier is present. Hence, CO2-rich phase and modifier-rich phase would be produced, causing peak shape distortion as well as loss of mass transfer efficiency and detector response [21]. Thus, the interface is a critical part of SFC-MS system. Several interfaces are now commonly used: (i) pre-BPR flow splitting; (ii) total-­flow-­introduction–pressure-regulating fluid interface; (iii) total flow introduction with mechanical BPR; and (iv) total flow introduction with passive BPR [21]. Pre-BPR flow splitting is based on the division of the mobile phase when 1–20% of it is directed to the API source through a tee union with close to zero dead volume and a restrictor/transfer line. The rest of the mobile phase continues to BPR to ensure adequate regulation of pressure. This interface is straightforward, easily operated, and provides good chromatographic fidelity. However, the dimensions of the transfer line have to be chosen carefully to prevent the precipitation of analytes and boiling of the mobile phase [21]. In addition, the sensitivity can be affected by the reduction of eluent volume directed to the mass spectrometer, and the split ratio can vary depending on BPR pressure [46]. These drawbacks are mostly negligible. However, total-flow-introduction interfaces are more convenient when a wider dynamic range or better sensitivity is required, particularly when mass-sensitive ionization method is applied. Three types of total-flow-introduction interfaces are available. In the first, CO2 miscible fluid is used as a pressure-regulating fluid. Its pump is under pressure control and this fluid is mixed with chromatographic eluent in a single chromatographic tee. Therefore, mechanical BPR and pressure transducer are not needed [47]. This interface has several advantages. The tee controlling pressure is close to mass spectrometer, which minimizes phase transition of the mobile phase and poor transfer of analytes. The dead volume and extra-column broadening are also reduced. Above all, better sensitivity and greater dynamic range can be achieved when entire sample is delivered to the ionization source. Moreover, the pressure-regulating fluid can be used to enhance the ionization mechanism. Among a few drawbacks, let us name the user-friendliness that is reduced by the necessity of independent pump. The dimensions of the transfer line as well as the increase in volume of solvent loaded to the API source dictate the range of compatible chromatographic conditions, particularly postcolumn pressure as well as flow rates of mobile phase and pressure-regulating

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 Lucie Nováková, Kateřina Plachká, Maria Khalikova, František Švec

fluid [21]. Total-flow-introduction interface with mechanical BPR should compensate drawbacks of the previous interface, but it also includes several problems, particularly large dead volume of components. The full downstream pressure is controlled but the pressure transducer is required. Therefore, the dead volume in the mechanical BPR system and in the transfer lines must be minimized as much as possible and the conditions must be carefully set to avoid analytes precipitation and mobile phase boiling [21]. Total flow introduction with passive BPR represents the simplest interface. It works sufficiently only under specific conditions because there is no active downstream pressure control as the column is coupled directly to the mass spectrometer. Thus, the conditions must be set in a way that the mobile phase is close to liquid-like state at which the effect of variations in pressure on the strength of the mobile phase is small [21]. Recently, new geometries of pre-BPR splitting with or without a make-up pump were studied by Grand-Guillaume Perrenoud et al. [39]. They optimized parameters such as mobile phase flow rate and composition, make-up solvent flow rate, BPR pressure, and split ratio. Moreover, the effect of these parameters on chromatographic performance, sensitivity, and user friendliness was also evaluated. The addition of make-up solvent improved robustness, efficiency, and sensitivity. It also helped to avoid analyte precipitation [39]. With respect to growing popularity of SFC-MS methods, several studies focused on optimization of the parameters such as composition of the mobile phase and make-up solvent, ion source set-up, temperature, and BPR pressure [10, 48, 49]. In most studies, a positive effect of an additive on sensitivity was confirmed [10, 48, 50–52], even though the recommendations for this additive differed. Volatile additives such as ammonium formate, ammonium acetate, and ammonium hydroxide were advantageous in the most studies [53–55], but the benefits of small amount of water (2–5%) were also noted [10, 38, 56, 57].

2.2.3 Matrix effects in SFC-MS Matrix effects are a bottleneck of all chromatographic methods when using MS as a detection technique. The matrix coextracted with the analytes or the compounds of another origin can alter the process of ionization, resulting in changes in signal response of mass spectrometry. This change can be both ion suppression and ion enhancement, called positive and negative matrix effects. As a consequence of matrix effects, poor accuracy, linearity, and precision of the method are observed. Moreover, in the case of negative matrix effects, the method sensitivity can also be compromised, resulting in false negatives. The exact mechanism of matrix effects remains unknown. They probably originate from the competition between the analytes and the coeluting undetected, that is, not shown in the selected reaction monitoring channel, matrix components in ionization process [58–60]. The phenomenon of matrix effects has been widely discussed in HPLC-MS, while in SFC-MS it has received less attention so far. The studies



 Supercritical fluid chromatography in bioanalysis 

 41

focused on fundamental aspects of matrix effects in SFC are missing. Provided that completely different mechanism is used for the separation in SFC, differences in matrix effects related to the separation step of a bioanalytical method might be expected. In the early SFC works, the evaluation of matrix effects was often missing, although their determination is now mandatory based on bioanalytical guidelines recommendations [15, 16]. The evaluation of matrix effects in SFC-MS bioanalytical method was presented for the first time in 2006 [61]. Postextraction addition approach revealed an ion suppression in the range −24% to −33% for R/S warfarin in human plasma. Systematic works focused on evaluation of matrix effect in various matrices and comparing UHPSFC with other chromatographic approaches were published later [10, 50, 62–65]. These works led to similar conclusions demonstrating an important advantage of UHPSFC over UHPLC and GC in terms of matrix effects incidence. Systematically lower matrix effects were observed under UHPSFC-MS conditions for large groups of doping agents using triple quadrupole analyzers [10, 50, 62] and in analysis of oxylipins using quadrupole time-of-flight MS [64]. In comprehensive lipidomic analysis, the matrix effects in UHPSFC-HRMS (high-resolution mass spectrometry) were demonstrated with a signal enhancement of +21%, while signal suppression of −16% to −26% was observed in UHPLCHRMS and in direct infusion (from −10% to −37%) [63]. The comparison between UHPLC-MS and UHPSFC-MS revealed substantial differences in matrix effects as a function of the sample matrix [65]. Generally, signal enhancement was observed in the case of UHPLC-MS, while signal suppression was observed in UHPSFC-MS. This agrees with previous study focused on analysis of doping agents in urine [50]. More important matrix effects were observed for UHPLC-MS/MS with triple quadrupole when analyzing a set of 11 drugs in urine after “dilute and shoot,” while in case of plasma after PP pretreatment, the matrix effects were similar for both methods. Fewer interferences were identified in urine than in plasma using HRMS. Alkali metal and Mg2+ clusters were responsible for suppression in both matrices and resulted in more extensive sample-to-sample variation in case of urine [65]. Further fundamental studies and systematic comparison with other techniques are still needed.

2.3 Applications of SFC in bioanalysis SFC is relatively new technique in the bioanalytical field. Only a few tens of bioanalytical papers were published so far using SFC, while hundreds to thousands papers can be found describing bioanalytical applications of GC and HPLC. Indeed, only a few review articles concerning SFC applications in clinical analysis [66, 67], biological applications [68], and metabolite analysis [69, 70] have been published so far, at a time of writing this chapter. However, this has changed recently. With

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only a couple of SFC bioanalytical methods published before 2014, this number has rapidly grown in the past few years. The distribution of individual reports across the bioanalytical applications of SFC is shown in Figure 2.2. Analysis of drugs and their metabolites in biological samples constituted the most important part (38%) of these applications. Other important areas included chiral drugs separations (17%) and lipidomic analysis (14%). Analysis of drugs of abuse and doping screening (9%), analysis of vitamins and carotenoids (8%), metabolomics (7%), and review articles (7%) were represented about equally.

Lipidomics 14%

Review articles 7%

Drugs and their metabolites 38%

Vitamins and carotenoids 8%

Chiral drugs 17% Metabolomics 7%

Drugs of abuse and doping 9%

Figure 2.2: SFC used in various application fields of bioanalysis. The literature search was carried out in the Web of Science database in October 2017. No restrictions for publication date were needed because the first bioanalytical applications of SFC were dated in 1990s and their appearance was very scarce in that period.

2.3.1 SFC in analysis of drugs and their metabolites Analysis of drugs and their metabolites is required for different reasons including evaluation of pharmacokinetic profile, quantitative determination of a drug and its metabolites, evaluation of bioavailability of a drug from the drug formulation, and evaluation of in vitro metabolism. SFC has established itself as an alternative technique in this applications field, particularly in recent years. Selected applications are listed in Table 2.1.

Sample type

Prostate cancer cells, tissue, plasma

Dried plasma spots

Human liver microsomes

Fetal bovine serum

Beagle dog plasma

Human serum

Rat plasma

Compounds

Adrenal C19 steroids + metabolites

Beta blockers, ­steroids, polar ­metabolites HIV protease ­inhibitors

Ten substrates of enzymes UGTs and glucuronides

Nineteen endogenous androgenic steroids

Probucol

Fifteen sulfonamides and metabolites

Coenzyme Q10

PP no evaporation

PP no evaporation

PP, LLE no evaporation

LLE evaporation

LLE evaporation

NA

LLE evaporation

Sample preparation

BEH 2-EP (100 × 3 mm, 1.7 μm)

BEH (100 × 3 mm, 1.7 μm)

BEH 2-EP (100 × 3 mm, 1.7 μm)

BEH 2-EP (100 × 3 mm, 1.7 μm)

C18 SB , diol 2-EP, 2-PIC (100 × 3 mm, 1.7 μm)

Kromasil diol (150 × 3 mm, 2.5 μm)

BEH BEH 2-EP (100 × 3 mm, 1.7 μm)

Stationary phase

PDA

ESI MS/MS

CO2/EtOH gradient elution, 10 min CO2/MeOH (85:15) 3 min

ESI MS/MS

ESI MS/MS

CO2/MeOH + 10 mM AmF + 5% water gradient elution

CO2/MeOH (95:5) 1.5 min

ESI MS/MS

CO2/MeOH (80:20) 10 min

ESI MS/MS

ESI MS/MS

CO2/MeOH gradient elution, 5 min

CO2/MeOH gradient elution, 4 min

Detection

Mobile phase elution conditions

Table 2.1: Selected SFC methods used for analysis of drugs and their metabolites in biological fluids.

[75] 2016

[74] 2016

[73] 2016

[72] 2017

[71] 2017

Reference

LLOQ 2 ng/mL

 Supercritical fluid chromatography in bioanalysis 

(continued)

[76] 2016

LLOQ [49] 0.5–1.0 µg/mL 2016

LLOQ 5 ng/mL

LLOQ 0.01–10 ng/ mL

NA

NA

LLOQ 1–10 ng/mL

Sensitivity

  43

Rat plasma

Dog plasma

In vitro metabolism study

Mouse plasma

Derivatized standards

Ginkgolides + hydrolyzed metabolites

Lacidipine IS=nimodipine

Eight substrates and eight CYP-specific metabolites

Cytarabine

Fifteen estrogen metabolites

Derivatization with dansylchloride

PP 96-well plate

PP no evaporation

LLE evaporation

PP no evaporation

Sample preparation

Betasil diol 100 cyanopropyl (150 × 2.1 mm, 5 μm)

Silica (100 × 4.6 mm, 10 μm)

BEH 2-EP (100 × 3 mm, 1.7 μm)

HSS C18 SB (100 × 3 mm, 1.8 μm)

NA

Stationary phase

ESI MS

APCI MS/MS

UV APCI-MS/MS

CO2/MeOH (60:40) + 11 mM AmAc + 1% water 2.5 min CO2/MeOH gradient elution, 9 min

ESI MS/MS

CO2/MeOH (98:2) 1.5 min CO2/MeOH + 10 mM AmF + 2% water gradient elution, 7 min

ESI MS/MS

Detection

CO2/MeOH + 10 mM AmAc + 5% water gradient elution, 6.5 min

Mobile phase elution conditions

LLOQ 5 pg

LLOQ 50 ng/mL

LLOQ 2–200 ng/mL

LLOQ 0.1 ng/mL

LLOQ 0.2–1 µg/mL

Sensitivity

[80] 2006

[79] 2007

[78] 2014

[77] 2014

[56] 2015

Reference

Note: LLE, liquid–liquid extraction; PP, protein precipitation; MeOH, methanol; EtOH, ethanol; AmF, ammonium formate; AmAc, ammonium acetate; NA, not available; ESI, electrospray ionization; APCI, atmospheric pressure chemical ionization; MS/MS, tandem mass spectrometry; MS, mass spectrometry; PDA, photodiode array; UV, ultraviolet; LLOQ, lower limit of quantitation.

Sample type

Compounds

Table 2.1 (continued)

44   Lucie Nováková, Kateřina Plachká, Maria Khalikova, František Švec



 Supercritical fluid chromatography in bioanalysis 

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Remarkable contribution of SFC can be noted, namely, in analysis of steroids. One of the first SFC-MS methods focused on analysis of steroids was published in 2006 by Xu et al. [80]. However, only derivatized standards were addressed in this work. In comparison with HPLC-MS, similar LOQs were obtained in the analysis of estrogen steroids and their metabolites, while SFC analysis was seven times faster. Analysis of steroid compounds is important in bioanalysis due to its relation to various types of cancer and other diseases. For example, 11β hydroxyandrostenedione metabolites and 11-oxygenated steroids, such as 11-ketotestosterone and 11-ketodihydrotestosterone, appear to be related to prostate cancer [71, 74]. The analysis of steroids is particularly challenging due to their structural similarity and presence of many isomers having the same molecular weight providing the same fragments, although these fragments might have a different abundance in MS/MS spectra. Efficient separation is crucial to reliably distinguish between individual species. UHPSFC offers very powerful tool to solve this task. Very efficient separation of 19 steroid species shown in Figure 2.3 could be obtained using the simple gradient mobile phase of MeOH in CO2, typically from 2% to 9% in 4 [74] or 5  min [71]. 2-EP stationary phase was used in these separations. Indeed, an improved chromatographic efficiency, superior selectivity, and a 5  –50 times improved sensitivity was observed for UHPSFC in the former work when compared to UHPLC [74].

Figure 2.3: UHPSFC-MS/MS chromatogram showing the separation of C19 steroids. Chromatographic conditions: ACQUITY UPC2 BEH 2-EP (100 × 3.0 mm i.d., 1.7 μm) column, 4 min gradient elution 2–9.5% of methanol as organic modifier, flow rate 2 mL/min, column temperature 60 °C, BPR pressure 138 bar. Peak assignment: 5α-dione (1), A4 (2), 11K-5α-dione (3), 11KA4 (4), DHT (5), epiAST (6), 11OH-5α-dione (7), T (8), 11KAST (9), 11OHA4 (10), 11KepiAST (11), 11KDHT (12), 3α-adiol (13), 11OHAST (14), 11KT (15), 11OHepiAST (16), 11OHDHT (17), 11K-3α-adiol (18), and 11OHT (19). Reprinted from [74] with permission.

Pharmakokinetic profiles of gingkolides [56], coenzyme Q [76], probucol [75], l­acidipine [77], and cytarabine [79] were determined using SFC-MS/MS in plasma

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 Lucie Nováková, Kateřina Plachká, Maria Khalikova, František Švec

of laboratory animals. Simple CO2/MeOH-based mobile phases were generally used, enabling very fast chromatographic runs, as listed in Table 2.1. The addition of ammonium acetate and a small percentage of water in the SFC mobile phase was favored only in the case of ginkgolides and cytarabine. However, the postcolumn compensation solvents were added to those simple CO2/MeOH mobile phases to enhance the MS response and method sensitivity. MeOH with 2 mmol/L ammonium acetate [76] was used in analysis of coenzyme Q, while 2% formic acid in MeOH and 0.5% ammonium hydroxide in MeOH were applied in analysis of lacidipine [77] and ­probucol [75], respectively. Fast and efficient UHPSFC-PDA method was developed for the determination of 15 sulfonamides and their metabolites in human serum [49]. Detailed ­optimization of chromatographic conditions involved the stationary phase, mobile phase modifier and additive, column temperature, BPR pressure, and flow rate. Despite using PDA detection, the method was successfully validated for serum samples. UHPSFC coupled to single quadrupole mass analyzer was used for the study of in vitro cytochrome P450 (CYP) inhibition assay using eight different CYP substrates and eight CYP-specific metabolites [78]. Acetaminophen, coumarin, bupropion, flurbiprofen, omeprazole, dextromethorphan, chlorzoxazone, midazolam, and their hydroxy metabolites belong among these CYP substrates. Optimization of screening procedure resulted in a combination of 2-EP stationary phase and CO2/MeOH mobile phase with the addition of 10  mmol/L ammonium formate and 2% water. EtOH was used as a make-up solvent. The ionization was already enhanced through the additives in the mobile phase. Thus, they were not needed in the make-up solvent any more. UHPSFC separation of 16 compounds is shown in Figure 2.4. More challenging method development was requested in the case of ten ­substrates of uridine diphosphate glucuronosyltransferase enzyme and their respective glucuronides [73]. The substrates involved azidothymidine, codeine, chenodeoxycholic acid, dimethylsulfoxide, etoposide, 4hydroxy-3-methoxyamthamphetamine, isoferulic acid, levomedetomidine, morphine, serotonin, testosterone, trifluoperazine, and their glucuronides. Therefore, four different chromatographic approaches including RP-HPLC, hydrophilic interaction chromatography (HILIC), UHPSFC, and aqueous normal phase chromatography were compared. The polar metabolites were very strongly retained in UHPSFC and very high percentage of MeOH reaching to 80% together with addition of 5% water was required for their elution. The latter was also important for good peak shapes. For morphine glucuronides, the best selectivity was achieved in RP-HPLC conditions while the best results for testosterone and epitestosterone diastereomeric glucuronides were  obtained in  HILIC and UHPSFC. These results illustrate the complementarity of ­different strategies and importance of availability of orthogonal methods.



 47

 Supercritical fluid chromatography in bioanalysis 

13

7.5x106 UHPSFC-MS Peak intensity

12 7 15 10

0.0

1.0

2.0

6

1

16

0.0 3.0

min

14

2 4.0

5.0

6.0

10

16

2 14

0.0 0.0

7.0

ESI– overlaid SIR

Peak intensity

6.5x105 UHPSFC-MS

4 9

3

8 5

11

ESI+/ESI– overlaid SIR

1.0

2.0

3.0

min

4.0

5.0

6.0

7.0

Figure 2.4: UHPSFC-MS separation of cytochrome P450 substrates on Acquity UPC2 BEH 2-EP (100 × 3.0 mm i.d., 1.7 μm), CO2/methanol/water (98:2) with 10 mmol/L ammonium formate, gradient elution (1% for 0.5 min, 1–9.5% between 0.5 and 5 min, 9.5–30% between 5 and 6.5 min), flow rate: 2 mL/min, temperature: 40 °C, back pressure: 150 bar, make-up solvent: ethanol, injection volume: 2 μL. Peak assignment: (1) acetaminophen, (2) 6-hydroxychlorzoxazone, (3) 7-hydroxycoumarin, (4) dextrorphan, (5) coumarin, (6) hydroxybupropion, (7) phenacetin, (8) bupropion, (9) 5-hydroxyomeprazole, (10) chlorzoxazone, (11) dextromethorphan, (12) omeprazole, (13) midazolam, (14) 4′-hydroxyflurbiprofen, (15) 1′-hydroxymidazolam, and (16) flurbiprofen. Reprinted from [78] with permission.

2.3.2 SFC in analysis of chiral drugs Chiral separations have always been the key application field of SFC. Indeed, enantioselectivity of SFC has been found to be superior to that of HPLC in several published works [81–83]. Moreover, analysis time is often significantly shorter, making the chiral screening and method development of SFC faster and more straightforward [84–86]. Although chiral SFC separation was mainly used in drug discovery, drug development, and pharmaceutical QC [82, 86], it has also recently become important in the field of bioanalysis (Table 2.2). These separations are required to monitor both drug enantiomers due to the large differences in pharmacological effect of R- and S- enantiomers and their metabolites. For example, S-ketamine [87], S-citalopram [88], R-rabeprazole [89], levocetirizine (R-form) [90], S-atenolol [93], S-metoprolol [93], S-warfarin [61], S-propranolol

Sample type

Human urine

Human serum

Beagle dog plasma

Human plasma

Beagle dog plasma

Human plasma

Compounds

Ketamine metabolites

Citalopram

(R)-, (S)-rabeprazole

Racemic cetirizine

Oxcarbazepine and chiral metabolites

GSK 1278863 and ­fourteen metabolites

SPE, 96 well-plate + evaporation

LLE + evaporation

SPE, Oasis HLB + evaporation

PP no evaporation

PP+PR, 96 well plate no evaporation

LLE + evaporation

Sample preparation

Chiralpak AD-H (150 × 4.6 mm, 5 μm)

Trefoil Cel2 (150 × 3.0 mm, 2.5 μm)

Chiralpak IE (150 × 2.1 mm, 5 μm)

Trefoil CEL2 (150 × 3.0 mm, 2.5 μm)

ESI MS/MS

ESI MS/MS ESI MS/MS

ESI MS/MS

CO2/MeOH + 5% water (55:45), 5 min CO2/MeOH (60:40) 3 min

CO2/isopropanol + 20% ethylacetate + 0.5% formic acid gradient elution, 17 min

ESI MS/MS

ESI MS

Detection

CO2/MeOH (70:30) 5 min

CO2/MeOH/ACN (70:30) + 10 mM AmAc gradient elution, 4 min

CO2/isopropanol + 0.075% NH4OH gradient elution, 15 min

Lux Amylose-2 (150 × 4.6 mm, 5 μm) Trefoil CEL2 (150 × 3.0 mm, 2.5 μm)

Mobile phase elution conditions

Stationary phase

Table 2.2: Overview of SFC methods used for chiral drug analysis in biological fluids.

NA

LLOQ 0.5–5 ng/mL

LLOQ 0.2 ng/mL

LLOQ 1 ng/mL

LLOQ 2 nM

LLOQ 5 ng/mL

Sensitivity

[92] 2016

[91] 2016

[90] 2016

[89] 2016

[88] 2017

[87] 2017

Reference

48   Lucie Nováková, Kateřina Plachká, Maria Khalikova, František Švec

Mouse urine

Human plasma

Mouse blood

Human liver microsomes

Human Plasma

Nobiletin and ­metabolites

R/S warfarin

R-/S-propranolol R-/S-pindolol

Clopidogrel and metabolites

R-/S-ketoprofen

SPE, Oasis HLB 96 well-plate

NA

PP

LLE, 96 well-plate + evaporation

NA

SPE, Oasis HLB + evaporation

Chirex 3005 (250 × 4.0 mm, 5 μm)

Chiralpak AD (250 × 4.6 mm, 5 μm)

Chiracel OD-H (250 × 4.6 mm, 5 μm)

Chiralpak AD (250 × 4.6 mm, 5 μm)

Chiralpak AD (250 × 4.6 mm, 5 μm)

Chiralpack IB-3 + BEH 2-EP (100 × 4.6 mm, 3 μm)

ESI MS/MS

PDA UV 220 nm

CO2/isopropanol + 0.4% TFA + 0.4% TEA (90:10), 20 min CO2/MeOH (45:55) 2 min

APCI MS/MS

APCI MS/MS

CO2/EtOH (70:30) 3 min CO2/MeOH + 0.2% IPA 3 min

UV 220 nm

ESI MS/MS

CO2/MeOH (80:20) 20 min

CO2/MeOH + 0.5% TFA/NH4OH (2:1) (82:18), 8.5 min

LLOQ 50 pg/mL

NA

LLOQ 5 ng/mL

LLOQ 13.6 ng/mL

NA

LOD 9.2–81 ng/L

[95] 2000

[94] 2002

[43] 2006

[61] 2006

[83] 2006

[93] 2015

Note: LLE, liquid–liquid extraction; PP, protein precipitation; PR, phospholipid removal; SPE, solid phase extraction; MeOH, methanol; EtOH, ethanol; ACN, ­acetonitrile; AmAc, ammonium acetate; TFA, trifluoracetic acid; IPA, isopropyl amine; TEA, triethylamine; ESI, electrospray ionization; APCI, atmospheric pressure chemical ionization; MS/MS, tandem mass spectrometry; PDA, photodiode array; LLOQ, lower limit of quantitation; NA, not available.

Wetland microsoms

Atenolol, metoprolol propranolol, metoprolol acid

  Supercritical fluid chromatography in bioanalysis   49

50 

 Lucie Nováková, Kateřina Plachká, Maria Khalikova, František Švec

[43, 93], S-clopidogrel [94], and S-ketoprofen [95], as well as other drugs single enantiomers were reported to have substantially higher potency compared to their racemates and the opposite enantiomers. Evaluation of the differences in metabolism of the enantiomers is also important to support the theories of decreased or devoid pharmacological effects and/or potential side-effects [83, 87, 91, 92, 94]. Important differences in pharmacokinetic profiles of both enantiomers were often reported [43, 89, 91, 95]. Figure 2.5 shows an example with propranolol enantiomers.

Intensity (cps)

12,000

m/z 260 > 116

0 0.0

0.8

Intensity (cps)

(A) 200,000

Blood concentration (ng/mL)

(B)

(C)

1.6 Time (min)

2.5 m/z 267 > 116

0 0.0

0.8

300

1.6 Time (min)

2.5

250 R-form

200

S-form

150 100 50 0

0

6

12 Time (h)

18

24

Figure 2.5: SFC separation with APCI MS/MS detection of racemic propranolol (A) and racemic D7 propanolol in mouse blood plasma (B). Differences in pharmacokinetic profile for the enantiomers of propranolol (C). Chromatographic conditions: chiral OD-H (250 × 4.6 mm i.d., 5 µm), column temperature 45 °C, methanol with isopropylamine as modifier, BPR pressure 100 bar. Adapted from Ref. [43] with permission.

Nowadays, chiral SFC separations are carried out mainly using polysaccharide amylose and cellulose-based chiral stationary phases. Many papers have pointed out exceptional enantioselectivity of amylose and cellulose tris(3,5-dimethylphenylcarbamate) and



 Supercritical fluid chromatography in bioanalysis 

 51

tris(chloro-methylphenylcarbamate) [85, 96, 97]. Mobile phase composition is selected as a function of physicochemical properties of the target analytes. Distinguished a ­ nalytical procedures use CO2/organic modifier mobile phase for ­analysis of neutrals and weak acids [61, 95], while CO2/organic modifier with an addition of basic [83, 93, 61] and acidic additive is used for analysis of bases and acids, respectively [98, 99]. However, generic approaches using combined additives [93, 94, 100–102] or buffers [88] have become more popular recently. These generic ­approaches facilitate the method optimization regardless acid–base properties of the analytes, and they also improve separation efficiency and enatioselectivity [100–102]. Volatile mobile phases are of key importance for SFC-MS coupling. Therefore, ammonia [55, 87, 103] and volatile buffers [88] are the additives of choice despite compromised enantioselectivity in some cases. I­ nitially, isocratic elution was preferred in chiral ­separations [61, 83, 93–95]. However, more flexibility, higher speed, and sample throughput are attained with gradient elution. These conditions also enable a column wash to remove strongly retained compounds between individual analytical runs, which is important when complex biological samples are analyzed. Therefore, gradient elution is currently preferred in chiral SFC bioanalytical methods [87–89]. Nevertheless, all reports demonstrate high potential of chiral SFC for bioanalysis, showing separation of an enantiomeric pair in 5 min and in up to 20 min when metabolites were also included in the same analytical run (see Table 2.2).

2.3.3 SFC in analysis of drugs of abuse and doping screening Routine doping screening is very challenging due to the high number of compounds to be monitored, high number of samples to be analyzed, and finally due to the demanding minimum required performance limits of the methods [104]. Therefore, the most important features of the doping screening analysis are high speed and high sensitivity. The specifics of these analyses are discussed in more details in chapter 2 of this book. Table 2.3 presents only a brief overview of SFC bioanalytical methods to complete applications in bioanalysis. SFC has a great potential in doping analysis since it enables screening of hundreds of different classes of analytes in 6–7 min [10, 50]. Fast gradients running from 2% to 40% of organic modifier with the addition of ammonium formate and small amount of water were applied to find generic conditions. These conditions then enabled elution of all target doping agents as well as enhanced sensitivity of MS detection. Torus diol and hybrid silica (bridged ethyl hybrid – BEH) stationary phases exhibited the greatest potential to tackle complex separations of large numbers often structurally close compounds [10, 50, 62]. Doping screening is usually carried out with urine. This fact can make the sample preparation challenging because injection of aqueous solutions in SFC usually compromises peak shapes, while dilution with organic solvents can lead to sample precipitation. A mixture of 75% acetonitrile in water was found to be a good compromise avoiding sample precipitation [50]. Dilution with tetrahydrofuran

Human urine

Human urine

Human urine

Human urine

Forty-three doping agents anabolic steroids

One hundred and ten doping agents stimulants, narcotics, diuretics, β2-agonists

Cannabinoids + metabolites

Two synthetic ­cannabinoids + eleven metabolites

BEH (100 × 3.0 mm, 1.7 μm)

Zorbax Rx-Sil (150 × 4.6 mm, 5 μm)

BEH (100 × 3.0 mm, 1.7 μm)

Torus diol (100 × 3.0 mm, 1.7 μm)

Torus diol (100 × 3.0 mm, 1.7 μm)

ESI MS/MS ESI MS/MS

UV

ESI MS/MS

CO2/MeOH + 10 Mm AmF + 2% water gradient elution, 6 min CO2/MeOH + 10 Mm AmF + 2% water gradient elution, 7 min CO2/ACN (93:7) 14 min CO2/MeOH + 0.3% NH4OH gradient elution, 6 min

ESI MS/MS

ESI MS/MS

CO2/MeOH + 5 mM AmAc + 3.5% H2O gradient elution, 15 min CO2/MeOH + 10 Mm AmF + 2% water gradient elution, 6 min

Detection

Mobile phase elution conditions

LLOQ 0.038– 0.12 µg/mL

LLOQ 0.5– 1.73 µg/mL

LOD 0.002– 15 ng/mL

LOD 0.1 ng/mL

LOD 0.1 ng/mL

LOD 0.2 – 50 ng/mL

Sensitivity

[107] 2015

[106] 2015

[50] 2015

[62] 2016

[10] 2016

[105] 2016

Reference

Note: SLE, supported liquid–liquid extraction; LLE, liquid–liquid extraction; SPE, solid phase extraction; SALLE, salting-out liquid–liquid extraction; MeOH, methanol; ACN, acetonitrile; AmF, ammonium formate; AmAc, ammonium acetate; ESI, electrospray ionization; UV, ultraviolet; MS/MS, tandem mass detection; LLOQ, lower limit of quantitation.

LLE + evaporation

SPE LLE SALLE

dilute and shoot

SLE + evaporation

SLE + evaporation

Human urine

Hundred doping agents anabolic steroids, hormones and metabolic modulators, glucocorticoids, synthetic cannabinoids

BEH 2-EP (100 × 3.0 mm, 1.7 μm)

Sample preparation Stationary phase Dilute and shoot

Sample type

Polar doping agents Human urine and metabolites, stimulants, β2-agonists

Compounds

Table 2.3: Overview of SFC methods applied for doping screening and analysis of drugs of abuse in biological fluids.

52   Lucie Nováková, Kateřina Plachká, Maria Khalikova, František Švec



 Supercritical fluid chromatography in bioanalysis 

 53

before centrifugation was used in another study [105]. However, this approach can be risky due to the adsorption of analytes on the precipitate formed with tetrahydrofuran due to the presence of high concentration of salts in urine. SFC was found to be useful alternative, enabling to solve problematic separations that could not be achieved using LC and vice versa [10, 50, 62, 105]. This makes SFC a complementary approach for doping analysis. Moreover, SFC was also reported to decrease matrix effects substantially compared to LC and GC [10, 50, 62]; see also Section 2.3. Figure 2.6 presents matrix effects as a function of retention time for UHPLC-MS/ MS and for UHPSFC-MS/MS after the extraction of 100 doping agents from urine using supported liquid extraction with five times preconcentration factor. A signi­ficant difference exists between the two techniques. While most of the points representing individual analytes at different concentration levels in the range of 0.1–10 ng/mL were located within the acceptance limits ±20% in the case of UHPSFC-MS/MS, more important matrix effects were observed for UHPLC-MS/MS with some values exceeding ±100% at the lowest concentration levels (these data are not included in the graphical presentation). Synthetic cannabinoids are among very popular drugs of abuse and were identified as “legal high” products in Europe [107]. These new derivatives are intended as legal

Figure 2.6: Matrix effects expressed as a function of retention time obtained with UHPSFC-MS/ MS (A) and UHPLC-MS/MS (B). The measurement was carried out at 0.1 (red), 0.5 (blue), 1 (green), 5 (yellow), and 10 ng/mL (grey) concentration levels. This unpublished figure was created based on the data published in [10]. See this reference and Table 2.3 for experimental details.

(a)

100

100

(b)

0

%

2.60

2

2.70

1.40

UHPLC

1.20

1

UHPSFC

2.80

13

1.60

4

2.90

12

2.00

5

3.00

2.20

7 8

3.10

3.20

342.2 > 244.1

372.2 > 155.1

6

358.1 > 155.1

1.80

3

3.30

11

2.40

3.40

2.60

10

9

2.80

3.50

10

3.60

3.70

3.00

376.2 > 155.1 5

11

3.80

4

3.20

6

3.90

3.60 9

4.00

338.2 > 135.1

7

3

3.40

12

4.10

4.20

1

3.80

13

(min)

328.2 > 125.1 4.30 (min)

344.2 > 155.1

358.2 > 155.1

374.2 > 169.1 372.2 > 169.1

459.1 > 112.1 390.2 > 169.1

4.00

Figure 2.7: Chromatographic separation of synthetic cannabinoids and their metabolites using UHPSFC-MS/MS and UHPLC-MS/MS. UHPSFC conditions: ACQUITY UPC2 BEH (100 × 3.0 mm i.d., 1.7 μm), CO2/methanol with 0.3% ammonia, 6 min gradient elution from 2% to 40% organic modifier, flow rate 2 mL/min, column temperature 40 °C, injection volume 2 µL. UHPLC conditions: ACQUITY UPLC BEH C18 (50 ×  2.1 mm  i.d., 1.7 μm), gradient elution in 6.8 min with 5 mmol/L ammonium formate and methanol, flow rate 0.4 mL/min, column temperature 60  °C, injection volume 2 µL. Peak assignment: (1) MAM-2201, (2) UR-144 N-4-OHpentyl, (3) MAM-2201 N-4-OH-pentyl, (4)  AM-2201 N-4-OH-pentyl, (5) RSC-4 N-5OH-pentyl, (6) JWH-122 N-5-OH-pentyl, (7) JWH-018 N-5-OH-pentyl, (8) JWH-073 N-4-OH-buty, (9) AM-2233, (10) UR-144 N-pentanoic acid, (11) UR-144 degradant N-pentanoic acid, and (12) JWH-018 N-pentanoic acid, (13) JWH-073 N-butanoic acid. The figure was reprinted from [107] with permission. See the reference for details on chromatographic conditions and metabolite structures.

0

%

54   Lucie Nováková, Kateřina Plachká, Maria Khalikova, František Švec



 Supercritical fluid chromatography in bioanalysis 

 55

replacement of cannabis and are often not covered by the law restrictions. Moreover, they are misleadingly marketed as “herbal incenses,” “room deodorizers,” “bath salts,” and “air fresheners.”. The analysis of synthetic cannabinoids is challenging because new compounds emerge very rapidly and reference materials are often unavailable. They are also rapidly metabolized via hydroxylation and carboxylation to a number of different metabolites. UHPSFC-MS/MS was found to be an adequate alternative to UHPLC-MS/MS for the analysis of these synthetic cannabinoids providing similar analysis times and sensitivity of 0.04–0.1 ng/mL at a completely different separation selectivity and better efficiency demonstrated with a substantially narrower peak width shown in Figure 2.7 [107]. Ultrafast UHPSFC-MS/MS analysis of designer drugs from the group of cathinones and phenylethylamines was proposed recently, although biological materials were not analyzed in this study [108]. Separation potential of BEH stationary phase with CO2/MeOH mobile phase containing volatile additives such as ammonium formate or ammonium hydroxide was pointed out. These conditions enabled quick separation of 15 cathinones and phenylethylamines within 2 min, including the challenging separation of several critical pairs of isomers.

2.3.4 SFC in analysis of fat-soluble vitamins and carotenoids Analysis of very lipophilic compounds with a log P > 9, such as fat-soluble vitamins and carotenoids, can represent significant challenges when using conventional NP-HPLC and RP-HPLC. Such analyses are feasible, but these studies often report very long retention times needed to separate individual compounds, particularly when isomeric species have to be resolved [48, 109, 110]. SFC was found very useful for the separation of these classes of compounds because it is accomplished in substantially shorter analytical runs, mostly in less than 13 min (Table 2.4). Nonpolar stationary phases C18 and 1-aminoanthracene (1-AA) exhibited great potential for the separation of these compounds due to their very lipophilic character. Isocratic elution at very low percentage of modifier (2–5%) in the mobile phase [48, 113] and elution with shallow gradients of up to 25% modifier [111, 112] were needed to enable retention and sufficient selectivity. Volatile additives, especially ammonium formate, were often added to the SFC mobile phase, although these compounds display rather neutral acid–base properties. The addition resulted in an enhanced response in MS detection. Lipophilic character further defines the procedure of sample preparation, making LLE with highly nonpolar solvents the most convenient approach. Considering the lipophilic character of CO2-based mobile phase, LLE extracts of fat-soluble vitamins and carotenoids are directly compatible with SFC. Thus, evaporation and reconstitution steps can be omitted and direct injection used. However, these steps are inevitable when sample preconcentrations is needed as is the case in most of reported studies summarized in Table 2.4.

Human plasma

Human serum

Human plasma

Solutions in MeOH

Human serum LDL

Fourteen fat-­soluble vitamins and ­carotenoids

α, β, γ, δ – tocopherols α, β, γ, δ – tocotrienols

Vitamin D2, D3 + eight metabolites

Seventeen water- and fat-soluble vitamins

Carotenoids epoxidized products

LLE + evaporation

–

PP, LLE + evaporation

PP, LLE no evaporation

PP, SLE + evaporation

Sample prep

Purosphere RP 18e (250 × 4.6 mm, 5 μm)

HSS C18 SB (100 × 3.0 mm, 1.8 μm)

Torus 1-AA (100 × 3.0 mm, 1.7 μm)

BEH 2-EP (100 × 3.0 mm, 1.7 μm)

Viridis HSS C18 SB (100 × 3.0 mm, 1.7 μm)

Stationary phase

ESI MS/MS

ESI MS/MS

CO2/MeOH + 0.2% AmF + 5% water gradient elution, 4 min CO2/MeOH + 0.1% AmF gradient elution, 20 min

ESI/APCI HRMS

CO2/MeOH gradient elution, 13 min

ESI MS

ESI MS/MS

CO2/MeOH + 20 mM AmF + 2% water gradient elution, 8 min CO2/MeOH + 10 mM AmF (95:5 and 98:2) 2.5 and 4 min

Detection

Mobile phase elution conditions

Sub-fmol concentrations

NA

LOD 0.39– 5.98 ng/mL

LLOQ 0.05– 0.75 µg/mL

LLOQ 0.2– 1,600 ng/ mL

Sensitivity

[113] 2012

[57] 2014

[112] 2016

[48] 2016

[111] 2017

Reference

Note: SLE, supported liquid–liquid extraction; LLE, liquid–liquid extraction; PP, protein precipitation; MeOH, methanol; AmF, ammonium formate; ESI, electrospray ionization; APCI, atmospheric pressure chemical ionization; MS/MS, tandem mass spectrometry; MS, mass spectrometry; HRMS, high-resolution mass spectrometry; LLOQ, lower limit of quantitation; LOD, limit of detection; NA, not available.

Sample type

Compounds

Table 2.4: Overview of SFC separation methods used for analysis of fat-soluble vitamins and carotenoids in biological fluids.

56   Lucie Nováková, Kateřina Plachká, Maria Khalikova, František Švec



 57

 Supercritical fluid chromatography in bioanalysis 

Successful separation of vitamin D and its 8 metabolites using Torus 1-AA column within 13 min is shown in Figure 2.8 [112]. Figure 2.9 shows the baseline separation of all derivatives of vitamin E, that is, α, β, γ, and δ-tocopherols and tocotrienols, using BEH 2-EP stationary phase under UHPSFC-MS

Figure 2.8: UHPSFC-PDA chromatogram of vitamin D metabolites. Chromatographic conditions: Torus 1-AA column (100 × 3.0 mm i.d., 1.7 µm), CO2/MeOH, methanol gradient from 3% to 15% in 10 min, flow rate 2 mL/min, BRP pressure 200 bar, column temperature 35 °C, UV detection at 265 nm. Peak assignment: (1) vitamin D3, (2) D2, (3) 25-OHD2, (4) 25-OHD3, (5) 1-OHD3, (6) 1-OHD2, (7) 24, 25–(OH)2D3, (8) 1,25(OH)2D2, and (9) 1,25-(OH)2D3. Adapted from Ref. [112] with permission.

3.40

α-Tocopherol %

δ-Tocotrienol

2.26 2.70 2.89

γ-Tocotrienol

γ-Tocopherol

β-Tocotrienol δ-Tocopherol

β-Tocopherol

α-Tocotrienol

α-Tocopherol %

1.80

1.50

1.60

3.96

1.76

2.03

γ-Tocotrienol

1.21

δ-Tocotrienol

(b) High-speed method

1.46

α-Tocotrienol β-Tocopherol γ-Tocopherol δ-Tocopherol β-Tocotrienol

(a) High-resolution method

2.21

2.36

1.86

3.09

0.00

1.00

2.00

3.00

4.00 0.00

1.00

2.00

Figure 2.9: UHPSFC-MS separations of eight derivatives of vitamin E. High-resolution method: ACQUITY UPC2 BEH 2-EP (100 × 3.0 mm i.d., 1.7 μm), CO2/methanol with 10 mmol/L ammonium formate (98/2), flow rate 1.5 mL/min, column temperature 40 °C, BPR pressure 234 bar. Highspeed method: ACQUITY UPC2 BEH 2-EP (100 × 3.0 mm i.d., 1.7 μm), CO2/methanol with 10 mmol/L ammonium formate (95/5), column temperature 50 °C, flow rate 1.5 mL/min, and BPR pressure 130 bar. The figure was reproduced from [48] with permission.

 

58 

 Lucie Nováková, Kateřina Plachká, Maria Khalikova, František Švec

conditions [48]. The UHPSFC analytical runs took 2.5 and 4 min while applying high-speed and high-resolution method, respectively, despite challenging separation of β- and γ- derivatives, which are positional isomers and very difficult to separate with HPLC methods. Although biological samples were not analyzed in the Taguchi et al. study [57], we ­included the method in Table 2.4 due to the original approach to the analysis of both fat- and water-soluble vitamins in a single analytical run. In this study, the authors used the gradient from 98% CO2 to 100% of methanol that enabled elution of both groups of vitamins within 4 min (Figure 2.10). Their approach confirmed feasibility of analysis transferring from SFC to LC conditions and wide applicability range of SFC technique. FSV

8

WSV

17 16

7 15

6

14

5

13 12

4

11

3

0

1

2

100

10 9

%

%

100

0.50 1.00 1.50 2.00 Time (min)

2.50

0

2.50 3.00 3.50 4.00 Time (min)

4.50 5.00

Figure 2.10: Separation of 17 vitamins using gradient 98% CO2–100% organic modifier. Column: HSS C18 SB (100 × 3.0 mm i.d., 1.7 µm), organic modifier: methanol/water (95:5) with 0.2% ammonium formate, flow rate 1.2 mL/min, temperature 40 ⁰C. Further details are given in [57]. Adapted from this reference with permission.

2.3.5 SFC in metabolomics Among bioanalytical applications, SFC is probably the newest technique introduced in the metabolomics field, provided lipidomic is addressed separately. In its original form, SFC used pure CO2 as the mobile phase, which enabled only elution of nonpolar species. Addition of organic modifiers and additives substantially extended the application range of SFC. Current SFC approaches are now also applied in profiling of metabolites with a wide-range of polarities including analyses of very polar species such as amino acids [114] and polar urinary metabolites [115]. The applications of SFC in metabolomics are summarized in Table 2.5. These methods generally require use

Dilution PP no evaporation

Human urine

Human serum

Dog bile rat bile Rat serum

Sixty polar urinary metabolites

Melatonin, ­N-acetylserotonin

Metabolite ­phenotyping –fifteen bile acids

Twenty-five bile acids and conjugates

BEH Amide (100 × 3 mm, 1.7 μm)

BEH C18 (150 × 2.1 mm, 1.7 μm)

BEH (150 × 4.6 mm, 1.7 μm)

Torus diol BEH 2-EP (100 × 3 mm, 1.7 μm)

Phenomenex Luna HILIC (150 × 3 mm, 3 μm)

Stationary phase ESI, APCI MS/MS

CO2/MeOH + water + AmF CO2/MeOH + AmAc gradient elution, 8.5 min

ESI MS/MS

ESI HRMS

CO2/MeOH gradient elution, 12 min CO2/MeOH + 0.2% AmF gradient elution, 15 min

ESI MS/MS

CO2/MeOH (90:10) + 0.1% FA + 0.05% AmF 9 min

ESI CO2/MeOH + water MS/MS +AmF PDA gradient elution, 7.35 min

Detection

Mobile phase elution ­conditions

[117] 2013

[116] 2014

[51] 2016

[115] 2016

[114] 2017

Reference

Note: LLE, liquid–liquid extraction; PP, protein precipitation; MeOH, methanol; AmF, ammonium formate; AmAc, ammonium acetate; FA, formic acid; ESI, electrospray ionization; APCI, atmospheric pressure chemical ionization; MS/MS, tandem mass spectrometry; HRMS, high-resolution mass spectrometry; PDA, photodiode array.

LLE + evaporation

Dilution MeOH

PP no evaporation

Human serum

Metabolites of the tryptophan pathway (Ser, N-acetyl-Ser, melatonin, niacin, kynurenine), and other AA

Sample ­preparation

Sample type

Compounds

Table 2.5: Overview of SFC methods used for metabolomic analysis.

  Supercritical fluid chromatography in bioanalysis   59

60 

 Lucie Nováková, Kateřina Plachká, Maria Khalikova, František Švec

of polar additives, such as ammonium formate, ammonium acetate, and water, relatively steep gradient upto 45–50% of organic modifier, and a polar stationary phase. Using silica HILIC stationary phase, 26 amino acids were successfully separated as shown in Figure 2.11, and allowed comparison of ESI and APCI ionization modes. As expected, ESI was prone to significant suppression matrix effects ranging from 29% to 85% when using complex matrix, such as human serum, while APCI produced a signal enhancement of 114–142% [114]. x106 6 5.8 5.6 5.4 5.2 5 4.8 4.6 4.4 4.2 4 3.8 3.6 3.4 3.2 3 2.3 2.6 2.4 2.2 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

Mel

NAS

Serotonin Niacin

Pro Gln Phe Val Thr Leu/lle Met Tau

0.5

1

1.5

2

Trp Lys Kynurenine Glu Arg Tvr Asn

2.5 3 3.5 4 4.5 5 5.5 Counts vs acquisition time (min)

6

6.5

7

7.5

Figure 2.11: Chromatogram of a standard mixture of amino acids using SFC APCI-MS/MS in positive ion mode. Chromatographic conditions: Phenomenex Luna HILIC column (150 × 3.0 mm, 3 µm), mobile phase: CO2/MeOH with ammonium formate and water, gradient elution from 15% to 45% in 6 min with additional isocratic step for 1 min, flow rate 2 mL/min, column temperature 40 °C, BPR pressure 150 bar. Figure reprinted from [114] with permission.



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 61

Very detailed optimization was carried out to design conditions for the separation of polar urinary metabolites [115]. The optimization process included 12 stationary phases, 9 additives, and 3 temperatures. The authors emphasized the beneficial properties of the new generation SFC columns with diol and 2-­picolylamin chemistries. Also in this case, the separation of highly polar metabolites required addition of polar additives such as ammonium formate, water, or ­ammonium hydroxide in methanol [115]. All these additives were found applicable. A few applications of SFC in metabolite profiling focused on analysis of bile acids [116, 117] are listed in Table 2.5 together with the method for monitoring of endogenous melatonin and N-acetylserotonine [51]. While C18 column was preferred in the study focused on the profiling of 15 bile acids [116], a larger spectrum of bile acids together with their conjugates was determined using hybrid silica HILIC stationary phase BEH Amide [117], as demonstrated in Figure 2.12.

Figure 2.12: Chromatographic separation of the mixture of 25 bile acids. Analytes labeled with U, T, and G indicate unconjugated and taurine and glycine conjugated bile acids, respectively. Chromatographic conditions: column ACQUITY UPLC BEH Amide (100 × 3.0 mm i.d., 1.7 µm), modifier gradient: 5–25% (4.5 min), 25% (1.5 min), 25–37.5% (2.5 min), 37.5% (1 min), 37.5–40% (1 min), 40% (1.5 min), 40–50% (0.5 min), 50% (1 min), 50–5% (0.5 min), 5% (1 min). Flow rate 2.0 mL/min, column temperature 70 °C, back pressure of 138 bar. Peak assignment: U1: LCA, U2: DCA, U3: CDCA, U4: UDCA, U5: HDCA, U6: 7-oxo-DCA, U7: βMCA, U8: ωMCA, U9: CA, U10: αMCA, G1: GLCA, G2: GDCA, G3: GCDCA, G4: GUDCA, G5: GHDCA, G6: GCA, T1: TLCA, T2: TDCA, T3: TCDCA, T4: TUDCA, T5: THDCA, T6: TβMCA, T7: TωMCA, T8: TCA, and T9: TαMCA. Figure reprinted from [117] with permission.

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 Lucie Nováková, Kateřina Plachká, Maria Khalikova, František Švec

2.3.6 SFC in lipidomics Lipids are a broad class of hydrophobic metabolites playing an important role in numerous biochemical processes including energy storage, cellular signaling, and cells interactions. On the basis of variety of the chemical structure and biosynthetic pathways, lipids are classified in eight main categories: fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides [118]. The main challenge in analysis of lipids is associated with their extremely high diversity emerging from the complexity of combination of hydrophobic acyl chains and a wide range of polarities. These result from the different types of attached hydrophilic moieties such as phosphoric acid and sugars. Simultaneous analysis of complex lipids is difficult with respect to their separation, detection, and identification. Moreover, analysis of lipid isomers and lipids with very similar masses is also challenging and makes the chromatographic separation very important. Therefore, lipidomic analysis involves different analytical techniques that have to be used for complete description of “lipidomic profile” [119–121]. Several separation mechanisms based on different interaction were used in HPLC lipidomic analysis including HILIC for the separation of polar lipids in lipid classes [122–125], NP-HPLC for nonpolar lipid classes [125–128], and RP-HPLC for the separation controlled by fatty acyl moieties, enabling separation of isomers in the same lipid class when coeluting of isomers in different lipid classes was possible [52, 122, 129, 130]. Lipidomics is a large subset of metabolomics where the applicability of SFC is without a doubt. UHPSFC was not widely applied in the lipidomic analysis until 2015, and only SFC using conventional HPLC columns with 5-μm particles was used at that time (Table 2.6). Many papers showing a great potential of UHPSFC as the comprehensive and high-throughput screening method for the large number of lipid classes have been published after that time [136]. SFC technique enables the combination of different separation mechanisms by changing the mobile phase composition. This option confirms the great potential of SFC for lipidomics [139]. Different stationary phases including both nonmodified and bonded silica such as cyanopropyl, phenyl, C8, C18 were used. The cyanopropyl phase was found suitable for the analysis of a wide range of lipid classes, while C18 columns are useful for SFC separations, targeting individual lipid species according to the fatty acyl composition, such as the lipidomic analysis of intact [131] and methylated [132] species, and the separation of oxidized phosphatidylcholine [133]. BEH and BEH 2-EP columns packed with sub-2 μm particles were used for the high-throughput and comprehensive UHPSFC lipidomic analysis, covering a broader range of polarity. The former enabled the class separation of nonpolar and polar lipids in a single run analysis [136]. The coupling of RP and NP stationary phases was used for the separation of phosphatidylcholines and their oxidized products in mouse liver tissues [133]. SFC is perfectly suited for lipid analysis because it is fully compatible with the injection of extracts in nonpolar organic solvents. SFC coupled with MS leads to improvement of the chromatographic separation and identification of lipids, and

Sample type

Mouse plasma

Mouse liver

Mouse liver

Dried blood spots

Sheep plasma

Porcine brain

Tissue, plasma, ­erythrocytes

Human milk

Compound

Twelve lipid classes of four category lipids

Polar lipids ­(phospholipids, lysophospholipids, sphingolipids)

Isomer ­profiling of oxidized ­phosphatidylcholine

Phospholipids

Polar lipid ­profiling

Twenty-four lipid classes of six lipid categories

Polar and ­nonpolar lipids

­Triacylglycerols and ­diacyldlycerols

LLE no evaporation

LLE + evaporation

LLE + evaporation

PP + derivatization

LLE + evaporation

SE no evaporation

SE + derivatization

LLE no evaporation

Sample ­preparation

Table 2.6: Overview of SFC methods used for lipidomic analysis.

BEH-2EP (100 × 3.0 mm, 1.7 μm)

BEH (100 × 3.0 mm, 1.7 μm)

BEH (100 × 3.0 mm, 1.7 μm)

Inertsil ODS-EP (250 × 4.6 mm, 5 μm)

PC HILIC, ODS-4, 2-EP (250 × 4.6 mm, 5 µm)

ProC18, 2-EP (250 × 4.6 mm 5 µm)

Inertsil ODS-4 (250 × 4.6 mm, 5 µm)

Inertsil ODS-4 (250 × 4.6 mm, 5μm)

Stationary phase

ESI MS/MS ESI MS/MS ESI MS ESI HRMS ESI HRMS

CO2/MeOH + 0.1% AmF Isocratic elution 85:15 CO2/MeOH + 0.1% AmF gradient elution CO2/MeOH + 0.1% AmF gradient elution, 10 and 15 min CO2/MeOH + 1% H2O + 30 mM AmAc, gradient elution, 6 min CO2/MeOH + 1% H2O + 30 mM AmAc, gradient elution, 6 min

ESI HRMS

ESI MS/MS

CO2/MeOH + 0.1% AmF gradient elution, 6 min and 11 min

CO2/MeOH/ACN + 0.1% FA gradient elution, 25 min

ESI HRMS

[137] 2017

[63] 2017

[136] 2015

[135] 2011

[134] 2012

[133] 2012

[132] 2013

[131] 2013

Detection Reference

CO2/MeOH + 0.1% AmF gradient elution, 20 min

Mobile phase elution ­conditions

  Supercritical fluid chromatography in bioanalysis   63

SPE + evaporation

Human plasma

Oxylipins ­(eucosanoids)

Torus 1-AA (100 × 3.0 mm, 1.7 μm)

BEH (50 × 2.1 mm, 1.7 μm) HSS C18 SB (2.1 × 50 mm, 1.8 μm).

Stationary phase

CO2/ MeOH + 0.1% acetic acid gradient elution, 15 min

ESI HRMS

ESI HRMS

CO2/MeOH/ACN + 15 mM AmF gradient elution, 20 min

[64] 2017

[138] 2017

Detection Reference

Mobile phase elution ­conditions

Note: LLE, liquid–liquid extraction; PP, protein precipitation; SE, solvent extraction; SPE, solid phase extraction; MeOH, methanol; ACN, acetonitrile; AmF, ammonium formate; AmAc, ammonium acetate; FA, formic acid; ESI, electrospray ionization; MS/MS, tandem mass spectrometry; MS, mass ­spectrometry; HRMS, high-resolution mass spectrometry.

LLE + evaporation

Mouse lung tissue

Lung lipids with focus on ­ceramides

Sample ­preparation

Sample type

Compound

Table 2.6 (continued)

64   Lucie Nováková, Kateřina Plachká, Maria Khalikova, František Švec

 Supercritical fluid chromatography in bioanalysis 

LPS

PC+pPC ePC PS LPA+S1P SM LPG

LPI LPI

PA

Pl+LPE CL

PG LacCer PE LPG

Sphingosine Sphinganine GlcCer

Pl+LPE CL

(B)

Desmosterol 1-MG 2-MG DHEA

CE FA Cholesterol

0

0.8

1.2

1.6

2.0

2.4

2.8 3.2 3.6 Time (min)

4.4

4.8

LPC

SM

4.0

PS

PG Sulfat ides Sulfatides(OH) PE

Sphinganine Sphinganine HexCer HexCer(OH)

CE

50

FA Coenzyme Q10 1,3-DG 1,2-DG+Cholesterol 1-MG+Fatty amides 2-MG Cer

PC

100

TG

Relative abundance (%)

50

Cer

(A)

1,3-DG 1,2-DG

100

 65

TG



5.2

5.6

Figure 2.13: Positive-ion UHPSFC/ESI-MS chromatograms of the mixture of lipid class standards (A) and the total lipid extract of porcine brain (B). Chromatographic conditions: Acquity UPC2 BEH column (100 × 3.0 mm i.d., 1.7 µm), flow rate 1.9 mL/min, injection volume 1 µL, column temperature 60 °C, BPR pressure 124 psi, gradient of CO2 and methanol/water (99/1) with 30 mmol/L ammonium acetate (1–51% in 6 min). Peak annotation: CE, cholesteryl esters; TG, triacylglycerols; FA, fatty acids; DG, diacylglycerols; MG, monoacylglycerols; DHEA, dehydroepiandrosterone; Cer, ceramides; GlcCer, glucosylceramides; HexCer, hexosylceramides; PG, phosphatidylglycerols; LacCer, lactosylceramides; pPE, 1-alkenyl-2-acyl phosphatidylethanolamines (plasmalogens); ePE, 1-alkyl-2-acyl phosphatidylethanolamines (ethers); PE, phosphatidylethanolamines; LPG, lysophosphatidylglycerols; PI, phosphatidylinositols; LPE, lysophosphatidylethanolamines; CL, cardiolipins; LPI, lysophosphatidylinositols; PA, phosphatidic acids; PC, phosphatidylcholines; pPC, 1-alkenyl-2acyl phosphatidylcholines; ePC, 1-alkyl-2-acyl phosphatidylcholines; PS, phosphatidylserines; LPA, lysophosphatidic acids; S1P, sphingosine-1-phosphate; SM, sphingomyelins; LPC, lysophosphatidylcholines; LPS, lysophosphatidylserines. Reprinted from [136] with permission.

represents a practical platform for lipidomics. Triple quadrupole mass analyzer is the most common MS technique for lipid profiling via comprehensive analysis based on selected reaction monitoring, precursor ion scanning, and neutral loss scanning. However, exact masses cannot be obtained and precursor ions with identical masses cannot be discriminated [134]. In contrast, HRMS methods using Orbitrap Fourier transformation and a time-of-flight MS convey the advantage of simultaneous analysis of diverse lipids in a single chromatographic run with high mass accuracy [63,

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 Lucie Nováková, Kateřina Plachká, Maria Khalikova, František Švec

131]. ESI, supported by organic make-up solvent, is the most frequently used ionization technique in SFC-MS lipidomic analysis due to the ionization capability of compounds with a wide range of polarities and structures [52, 132, 136]. An example application is shown in Figure 2.13 to demonstrate SFC power in lipidomics. It presents chemical profiling of broad lipid classes in porcine brain extract [136]. Lipids were separated to the lipid classes according to their polarities, while species within the class differing only in fatty acyl chain composition were not separated and were eluted in a single chromatographic peak including lipid class internal standard. As a result, the matrix effects were similar for particular lipid classes and for lipid class internal standards. A total of 24 lipid classes containing 436 lipid species were identified in this complex matrix covering six main lipid categories: fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols (Figure 2.13).

2.4 Conclusion Advanced SFC instrumental platforms have enabled SFC-MS to establish itself as an important analytical method in bioanalytical laboratories even in strictly regulated environments. Important benefits, such as high resolution, high efficiency, and speed of analysis, particularly in case of UHPSFC that uses columns packed with sub-2 µm stationary phases, opened a space for SFC applications in both research and practical routine laboratories. Indeed, substantial increase in developed SFC-MS methods can be noted in the field of bioanalysis since 2014 and this trend is expected to further grow. It is likely that SFC becomes a powerful tool in many bioanalytical applications where conventional HPLC and GC approaches may fail or when a complementary orthogonal method is needed. These applications involve particularly chiral separations, analysis of isomeric structures, such as isoforms of lipophilic vitamins and steroids, and lipidomics. SFC in its modern form uses CO2-based mobile phase with organic modifier and polar additives, which enlarge the applicability range.

2.5 Acknowledgement The authors gratefully acknowledge the STARSS project (Reg. No. CZ.02.1.01/0.0/0.0 /15_003/0000465) cofunded by ERDF and the project of specific research SVV 260412.

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Chandan L. Barhate and Philip A. Searle

3 Ultrafast supercritical fluid chromatography Abstract: Supercritical fluid chromatography (SFC) has made a rapid progress in recent years. Advances in particle technology have reduced the conventional analysis time from 5-20 min to a few seconds. In the domain of ultrafast SFC unexpected results are seen which are absent in ultrafast liquid chromatography. In this chapter, we review the instrumental parameters which affect ultrafast separations in SFC. Experimentally, it was found that column packing, the connection tubing diameter, digital filters, sampling frequency and backpressure regulator settings needed to be optimized. Illustrative examples of how ultrafast methods can be routinely developed and used for high throughput screening using MISER analysis are demonstrated Keywords: Ultrafast SFC, Digital filters , tubing effects , MISER, Short column SFC, Screening

3.1 Introduction Recent years have seen a rapid growth in the speed of chiral and achiral chromatography [1–9]. Chiral chromatography has evolved from typical run times of 20–40 min in the 1980s and 1990s to 5–10  min in the 2000s, to subsecond separations [1]. By current standards, subminute separations can be termed as ultrafast chromatography, although its meaning is rapidly developing with advances in instrument and particle technology. Recently, subsecond separations were demonstrated in liquid chromatography using 0.5–1 cm columns [1, 3]. Packed column supercritical fluid chromatography (SFC) is rapidly following the progress taking place in liquid chromatography [2, 4, 7]. As early as 2010, it was shown that steroids, profens, xanthines, nucleic acids, and sulfonamides could be baseline separated in under a minute on a 10 cm column packed with 1.8 µm bare silica particles with low column head pressures (80 MPa with reversed-phase mobile phases. Figure 3.5 shows representative ­chromatograms of ultrafast chiral separations on SFC (5–30  s separations). These are among the fastest enantiomeric separations reported in the literature. ­Teicoplanin bonded with 1.9 µm NPSD silica particles were packed into 2 or 5 cm columns in 3 or 4.6 mm i.d. formats and were used to achieve these ultrafast separations. This speed exceeds the cycling time of most autosamplers (~1 min). Even with such short columns and very high flow rates (4−19 mL/min), the efficiencies range

19 mL/min 5-methyl-5-phenylhydantoin

19 mL/min N-(3,5-Dinitro-2-pyridinyl) leucine O –O N+

2 x 0.46 cm

O

HN O

0

2

(A)

4

α-Methyl-α-phenylsuccinimide 10 mL/min

O

H N

N

2 x 0.46 cm

OH

10

6 8 Time (s)

HN

O

NH

O N+ – O

0

(B)

2

4

6 Time (s)

8

10

N-carbobenzoxy-dl-norvaline

O

O O

4.3 mL/min

O

N H

2 x 0.46 cm

OH

5 x 0.3 cm

Solvent saver 0

(C)

5

10 15 20 25 30 35 40 Time (s)

0

(D)

5

10

15 20 Time (s)

25

30

Figure 3.5: Representative ultrafast chiral separations on SFC with optimized setup. Column: teicoplanin bonded 1.9 μm NPSD silica. The back-pressure regulator was maintained at 8 MPa. Column temperature: ambient; (A) 55:45 CO2/MeOH; column dimensions: 2 × 0.46 cm; flow rate: 19 mL/min; (B) 60:40:0.1 CO2/ MeOH/TEA; column dimensions: 2 × 0.46 cm; flow rate: 19 mL/min; (C) 96:04 CO2/MeOH; column dimensions: 2 × 0.46 cm; flow rate: 10 mL/ min; (D) 80:20:0.1 CO2/ MeOH/TEA; column dimensions: 5 × 0.30 cm; flow rate: 4.3 mL/min. Reprinted from Ref. [2].



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Table 3.3: Modifications performed on Jasco Semiprep SFC for ultrafast separations. Source

Ultrafast SFC modifications

Injector-to-column connection tubing

i.d.: 254 µm, length: 11.5 cm

Column-to-injector connection tubing

i.d.: 254 µm, length: 20 cm

Column oven

Bypassed

Safety valve 34 MPa

Bypassed

Detector flow cell

4 μL

Sampling frequency

100 Hz

BPR pressure

8 MPa

from 11,000 to 30,000 plates/m. The working pressures ranged from 150 to 410 bar. The viscosity of supercritical CO2 is nine times lower than methanol (at 110 bar) and 18 times lower than ethanol. As a result, the pressure drops can be as low as 1/15 the pressure drop in LC regardless of the particle size. Table 3.3 lists the modifications performed on Jasco semiprep SFC to reduce the extra column volume and obtain rapid separations. After bypassing the column oven, total length of connection from an injector to a detector was 31.5 cm (PEEK tubing 254 µm i.d., the shortest plumbing that could be utilized on Jasco SFC using 2 or 5 cm column). Moreover, the maximum available sampling frequency 100 Hz and lowest possible response time of 0.05 s were used.

3.9 MISER SFC In 2010 Welch et al. first demonstrated the MISER (“multiple injections in a single experimental run”) chromatographic analysis technique [26]. MISER is predominantly a flow injection analysis with the major difference being that the sample is passed through a chromatographic column to resolve the peak of interest before it enters in the detector [27]. MISER analysis has been applied to SFC and is a simple approach to performing high-throughput analysis [7, 28, 29], with the advantage being that the entire experiment resides in a single chromatogram, thereby simplifying data analysis and interpretation. Any chromatographic analysis system can carry out MISER analysis if the instrument control software allows for multiple sample injections within a single experimental run. Figure 3.6(A) demonstrates the separation of enantiomers of Tröger’s base within 20  s. A portion of the injector program specifying injection of an entire 96 well microplate in a MISER experiment is shown in Figure 3.6(B). Figure 3.6(C) shows an example of the MISER analysis of the enantiomers of Tröger’s base using the Agilent SFC system. The shortest possible injection cycle of 21 s was used for the MISER analysis. It is important to note here that even if analyte elution time is of 30 s and injection

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Open external contact B

Wait 0.1 min

Close external contact B

Parameter Eject default volume to seat with maximum speed using default offset

Figure 3.6: (A) (±)-Tröger’s base on Agilent Infinity SFC, AD-3, 4.0 × 10 mm, 3 mm, 4% MeOH, 4 mL/min, λ = 220 nm. (B) Excerpt of corresponding injector programming. (C) Twelve injections of Tröger’s base (0.4 mg/mL) with ee’s 100(+), 80, 60, 40, 20, 0, 0, 20, 40, 60, 80, 100 (−). Reprinted from Ref. [29].

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 Ultrafast supercritical fluid chromatography 

cycle time is of 21 s then analyte will simply elute in the second time window. Therefore it is not an absolute requirement to have analyte elute in the given time window for MISER analysis. Figure 3.7 demonstrates how ultrafast analysis of enantiomeric purity can be swiftly integrated into standard workflows for catalyst identification and process optimization. A high throughput analysis method was required to enable screening of the enzymatic ketoreductase-catalyzed reduction of a prochiral ketone to afford the corresponding alcohol in high enantiomeric purity. CSP screening followed by method development optimization provided the 50 s ultrafast chiral SFC assay, with

Well A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12

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Enzyme P1B02 P1B10 P1D3 P1D05 P1H09 P1H10 P2B11 P2C02 P2D11 P3C3 P3D1 P3D11 P3H2 MIF-20 KRED -208 KRED 101 KRED 108 KRED 112 KRED 119 KRED 124 KRED 130 KRED 134 KRED NADH 101 KRED NADH 102

Selectivity R R R R R R R R R S S S S R R R R R R R R R S S

Conversion >99.9% >99.9% >99.9% >99.9% >99.9% >99.9% >99.9% >99.9% >99.9% >99.9% >99.9% >99.9% >99.9% >99.9% >99.9% 98.60% 31.60% 44.70% 22.90% 29.10% 32.80% >99.9% >99.9% >99.9%

ee >99 >99 77 56 –12 –53 64 24 –36 < –99 < –99 < –99 < –99 88 91 63 –81 91 –3 –84 –63 88 < –99 < –99

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Figure 3.7: MISER chiral SFC for high-throughput enantiopurity analysis. Separations were performed on a 4.6 mm × 100 mm, 3 µm AD-3 column at a flow rate of 3.25 mL/min and an eluent composition of 65% CO2 and 35% MeOH (25 mM IBA). The column and samples were maintained at a temperature of 40 °C and 20 °C, respectively. Reprinted from Ref. [7].

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co-injection of the ketone starting material showing early elution well away from the desired enantiomer pair. Fast MISER SFC chromatographic analysis using an injection interval of 50 s afforded a high throughput analysis method with a total analysis time of only 80 min for a 96-well microplate. The results for two rows of 12 samples are shown in Figure 3.7, with a number of enzymes identified that afforded not only good conversion but also high enantioselectivity for the formation of either the (R) or the (S) enantiomer. The ultrafast chiral chromatographic analysis is well suited for firstround screening in a high-throughput mode, with conventional chromatographic analysis often being used as a confirmatory assay.

3.10 Conclusion The low viscosity of sub/supercritical fluid mobile phases containing carbon dioxide does not impose pressure limitations encountered in UHPLC on short columns. A careful choice and understanding of the plumbing of the chromatography system is needed to achieve the highest efficiency and to maximize resolution. With a low-density and viscosity eluents encountered in SFC, turbulence exists in the connection tubing, but the primary contributor to noise in SFC comes from back-pressure regulators. Researchers using SFC need to be aware of the nature of digital filters embedded in the software. The ultrafast chiral separations can enable rapid MISER SFC analysis for high-throughput enantiopurity and broader adoption of high-throughput experimentation approaches in steriochemical research.

3.11 Acknowledgments The authors thank Dr Daniel W. Armstrong , Dr M. Farooq Wahab (University of Texas at Arlington), and Dr Jon D. Williams at AbbVie Inc. for helpful discussions.

References [1] [2]

[3]

M.F. Wahab, R.M. Wimalasinghe, Y. Wang, C.L. Barhate, D.C. Patel, D.W. Armstrong, Salient sub-Second separations, Analytical Chemistry, 88 (2016) 8821–8826. C.L. Barhate, M.F. Wahab, D. Tognarelli, T.A. Berger, D.W. Armstrong, Instrumental idiosyncrasies affecting the performance of ultrafast chiral and achiral sub/supercritical fluid chromatography, Analytical Chemistry, 88 (2016) 8664–8672. O.H. Ismail, L. Pasti, A. Ciogli, C. Villani, J. Kocergin, S. Anderson, F. Gasparrini, A. Cavazzini, M. Catani, Pirkle-type chiral stationary phase on core–shell and fully porous particles: Are superficially porous particles always the better choice toward ultrafast high-performance enantioseparations? Journal of Chromatography A, 1466 (2016) 96–104.



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E.L. Regalado, C.J. Welch, Pushing the speed limit in enantioselective supercritical fluid chromatography, Journal of Separation Science, 38 (2015) 2826–2832. C.J. Welch, Are we approaching a speed limit for the chromatographic separation of enantiomers? ACS Central Science, 3 (2017) 823–829. C.L. Barhate, Fundamental studies on ultrafast chiral and achiral separations in liquid chromatography and Sub/Supercritical fluid chromatography, 2017. C.L. Barhate, L.A. Joyce, A.A. Makarov, K. Zawatzky, F. Bernardoni, W.A. Schafer, D.W. Armstrong, C.J. Welch, E.L. Regalado, Ultrafast chiral separations for high throughput enantiopurity analysis, Chemical Communications, 53 (2017) 509–512. D.C. Patel, Z.S. Breitbach, M.F. Wahab, C.L. Barhate, D.W. Armstrong, Gone in seconds: Praxis, performance, and peculiarities of ultrafast chiral liquid chromatography with superficially porous particles, Analytical Chemistry, 87 (2015) 9137–9148. C.L. Barhate, M.F. Wahab, Z.S. Breitbach, D.S. Bell, D.W. Armstrong, High efficiency, narrow particle size distribution, sub-2 μm based macrocyclic glycopeptide chiral stationary phases in HPLC and SFC, Analytica Chimica Acta, 898 (2015) 128–137. T.A. Berger, Demonstration of high speeds with low pressure drops using 1.8 μm particles in SFC, Chromatographia, 72 (2010) 597–602. O.H. Ismail, M. Antonelli, A. Ciogli, C. Villani, A. Cavazzini, M. Catani, S. Felletti, D.S. Bell, F. Gasparrini, Future perspectives in high efficient and ultrafast chiral liquid chromatography through zwitterionic teicoplanin-based 2–μm superficially porous particles, Journal of Chromatography A, 1520 (2017) 91–102. D. Kotoni, A. Ciogli, C. Molinaro, I. D’Acquarica, J. Kocergin, T. Szczerba, H. Ritchie, C. Villani, F. Gasparrini, Introducing enantioselective ultrahigh-pressure liquid chromatography (eUHPLC): Theoretical inspections and ultrafast separations on a new sub-2–μm Whelk-O1 stationary phase, Analytical Chemistry, 84 (2012) 6805–6813. T.A. Berger, Kinetic performance of a 50 mm long 1.8 μm chiral column in supercritical fluid chromatography, Journal of Chromatography A, 1459 (2016) 136–144. M.F. Wahab, P.K. Dasgupta, A.F. Kadjo, D.W. Armstrong, Sampling frequency, response times and embedded signal filtration in fast, high efficiency liquid chromatography: A tutorial, Analytica Chimica Acta, 907 (2016) 31–44. M.F. Wahab, D.C. Patel, R.M. Wimalasinghe, D.W. Armstrong, Fundamental and practical insights on the packing of modern high-efficiency analytical and capillary columns, Analytical Chemistry, 89 (2017) 8177–8191. F.G.M.F. Wahab, Understanding the science behind packing high-efficiency columns and capillaries: Facts, fundamentals, challenges, and future directions (2018). L. Sciascera, O. Ismail, A. Ciogli, D. Kotoni, A. Cavazzini, L. Botta, T. Szczerba, J. Kocergin, C. Villani, F. Gasparrini, Expanding the potential of chiral chromatography for high-throughput screening of large compound libraries by means of sub–2 μm Whelk-O 1 stationary phase in supercritical fluid conditions, Journal of Chromatography A, 1383 (2015) 160–168. R. De Pauw, K. Choikhet, G. Desmet, K. Broeckhoven, Occurrence of turbulent flow conditions in supercritical fluid chromatography, Journal of Chromatography A, 1361 (2014) 277–285. R. De Pauw, K. Shoykhet, G. Desmet, K. Broeckhoven, Understanding and diminishing the extra-column band broadening effects in supercritical fluid chromatography, Journal of Chromatography A, 1403 (2015) 132–137. K. Hupe, R. Jonker, G. Rozing, Determination of band-spreading effects in high-performance liquid chromatographic instruments, Journal of Chromatography A, 285 (1984) 253–265. C. Wang, Y. Zhang, Effects of column back pressure on supercritical fluid chromatography separations of enantiomers using binary mobile phases on 10 chiral stationary phases, Journal of Chromatography A, 1281 (2013) 127–134.

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[22] P.A. Peaden, M. Lee, Theoretical treatment of resolving power in open tubular column supercritical fluid chromatography, Journal of Chromatography A, 259 (1983) 1–16. [23] J.C. Giddings, Dynamics of chromatography: Principles and theory, CRC Press, 2002. [24] A. Tarafder, K. Kaczmarski, D.P. Poe, G. Guiochon, Use of the isopycnic plots in designing operations of supercritical fluid chromatography. V. Pressure and density drops using mixtures of carbon dioxide and methanol as the mobile phase, Journal of Chromatography A, 1258 (2012) 136–151. [25] T.A. Berger, B.K. Berger, Minimizing UV noise in supercritical fluid chromatography. I. Improving back pressure regulator pressure noise, Journal of Chromatography A, 1218 (2011) 2320–2326. [26] C.J. Welch, X. Gong, W. Schafer, E.C. Pratt, T. Brkovic, Z. Pirzada, J.F. Cuff, B. Kosjek, MISER chromatography (multiple injections in a single experimental run): The chromatogram is the graph, Tetrahedron: Asymmetry, 21 (2010) 1674–1681. [27] C.J. Welch, E.L. Regalado, C. Kraml, E.C. Welch, M.J. Welch, H. Semmelhack, D. Almstead, A. Kress, N.A. Hidalgo, M.H. Kress, MISER LC-MS analysis of teas, soft drinks and energy drinks, LCGC North America, 33 (2015) 262–269. [28] K. Zawatzky, C.L. Barhate, E.L. Regalado, B.F. Mann, N. Marshall, J.C. Moore, C.J. Welch, Overcoming “speed limits” in high throughput chromatographic analysis, Journal of Chromatography A, 1499 (2017) 211–216. [29] K. Zawatzky, M. Biba, E.L. Regalado, C.J. Welch, MISER chiral supercritical fluid chromatography for high throughput analysis of enantiopurity, Journal of Chromatography A, 1429 (2016) 374–379.

Lu Zeng and Paddi Ekhlassi

4 SFC applications for active pharmaceutical ingredient analysis Abstract: This chapter focuses on utilizing supercritical fluid chromatography (SFC) and its applications in drug development for the analysis of active pharmaceutical ingredient (API) in the manufacturing process. Various screening approaches such as sequential, parallel or tandem column chromatography offers a unique and useful tool for separations of impurities and optimizing the overall selectivity. With the new age of technology, modern SFC instruments can be qualified and chiral and achiral SFC methods can be validated for the GMP API release and/or cleaning testing under the regulatory guidelines. Thus, given the advantageous proprieties of CO2-based mobile phase and low consumption of solvents, we believe that this technology has become an equally valuable primary option for method development and validation in pharmaceutical industry analytical laboratories. Keywords: Supercritical fluid chromatography (SFC), active pharmaceutical ingredient (API), method development, method validation, parallel SFC

4.1 Introduction Drug development is a very well-regulated process to ensure the safety and efficacy of drug product to the market place. Most commonly used analytical procedures are from International Conference for Harmonization (ICH) Q2 (R1) [1], which provide guidelines for the identification tests and quantitative tests for impurity content, limit tests for the control of impurities, and quantitative tests (assay) for the active pharmaceutical ingredient (API) known as the active component in a drug product. Supercritical fluid chromatography (SFC) instrumentation and methodology have been greatly advanced in terms of the performance and functionality during the recent years. Modern SFC instruments are able to be qualified, and SFC methods are able to be validated to meet the regulatory requirements and guidelines, in the same way as high-performance liquid chromatography (HPLC). Although HPLC has been the dominant technique in pharmaceutical industries, SFC offers advantages of having faster analysis speed, less solvent consumption, lower cost, different selectivity, and

Lu Zeng, Takeda Pharmaceuticals, 10410 Science Center Dr, San Diego CA 92121, USA, lu.zeng@­takeda.com Paddi Ekhlassi, Takeda Pharmaceuticals, 10410 Science Center Dr, San Diego CA 92121, USA, [email protected] https://doi.org/10.1515/9783110618983-004

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better separations. These advantages justify that SFC can also be an excellent choice of instrument for the API assay and related substances analysis for drug development and quality control in the manufacturing process. API manufacturing process starts from drug development at preclinical and clinical studies. SFC method development initiates for the separations of API and major impurities to the preliminary validation and validation to support good-­manufacturing practice (GMP) manufacturing. SFC method development for APIs is typically similar to HPLC method development. The information of compound properties (e.g., structure of molecule containing the chiral center(s), number of nitrogen atoms, solubility in the organic solvents, values of pKa and log D or log P, and UV chromophore), and structures of degradation products and impurities are key factors to consider before scouting the trails. The targeted SFC method is intended to separate both known and unknown impurities from API. The applications of SFC methods for GMP API in pharmaceutical industry generally can be used to perform release and stability testing, and/or cleaning testing. In order to develop a suitable method for API, the downstream method validation and v ­ erification requirements have to be considered. SFC method development provides the most promising conditions of stationary and mobile phase for the separations of API with related substances and process-related impurities, and then followed by the optimization of conditions with fine tuning the parameters in order to meet the required criteria toward validation. Prevalidation of the method is important since it reassures the method is suitable for the intended purpose. The final validated SFC assay method has to meet the criteria of accuracy, precision, linearity, range, and selectivity.

4.2 SFC purity and impurity method for the API release testing and stability indicating assay With the improvement of instrumentation and methodology, SFC is able to meet the high requirements of sensitivity, accuracy, and precision for GMP release testing under the regulatory guidelines. Among the ICH-defined types of analytical procedures to be validated, SFC can apply to the identification test, quantitation tests for impurities content, limit test for the control of impurities, and quantitative tests of the API or other main components of the drug. For the commercialization of drug products containing chiral API, the enantio­ selectivity identification and quantitative methods are required per government agency guidelines. Chromatographic methods are the most efficient techniques to provide the resolutions for enantioselectivity and quantitative analysis, and thus, they are commonly used in the pharmaceutical industry from the discovery to development laboratories and commercial manufacturers. Although HPLC/UHPLC methods for release testing are still the primary tools for API analysis, SFC method is slowly becoming prevalent as well [2].



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4.3 Chiral SFC method for API purity and impurity The basic parameters to consider for chiral SFC method development are chiral stationary phases (CSPs), mobile phases, and additives [3]. The commonly used CSPs for SFC are polysaccharide derivatives (amylose based, e.g., Chirapak AD, AS, IA, or cellulose based, e.g., Chiracel OD, OJ, IB, etc.), which are either physically coated or chemically bonded on the silica particles. Although polysaccharide types of CSPs are most successful in achieving separation for enantiomers, macrocyclic types of CSPs (macrocyclic antibiotics, e.g., Chirobiotic T, V, and c­ yclodextrins, e.g., Cyclobond I 2000) and small molecular types of CSPs (Pirkle-type, e.g., Whelk O) are also preferable in many cases. The commonly used mobile phase for chiral SFC is CO2 mixed with an organic solvent (or a mixture of organic solvents), in which the organic solvent functions as the modifier to increase the eluting power. Polar, short-chained alcohols, such as methanol, ethanol, and isopropanol, are suitable as modifiers for chiral separation with or without additives based on the property of the chiral molecules. Other organic solvents or mixture of organic solvents can also be used for the improvement of selectivity or productivity in chiral separations. The use of dichloromethane or a mixture of dichloromethane with methanol as a modifier can increase the enatioselectivity [4]. Using nonpolar organic solvents (heptane and hexane, mixed with ethanol or isopropanol) as the modifier for chiral separations are also reported to have improved the enantioseparation and enantioresolution [5]. Using additives, in general, can improve peak shape and resolution [6]. The commonly used additives for chiral SFC are formic acid, trifluroacetic acid, diethyl amine, triethyl amine, and ammonium acetate. Incorporating a strong acid, like ethanesulfonic acid, into the sample diluent and mobile phase modifier provides a significant improvement of separation on the polysaccharide stationary phases for amine compounds. The strong acid acts as a counter ion to a wide range of amine-containing enantiomers, forming ion pairs to enable the enatioresolution [7]. The application of cyclic amines as the additive into the modifier was also reported to have increased retention and selectivity of amine-containing enantiomers, particularly, the primary amine on the polysaccharide stationary phases. For example, racemic 2-amino-3-phenyl-1-propanol showed no enatioresolution using 20% of 2-propanol as a modifier, whereas by adding 1% of cyclohexylamine as an additive into the modifier, an enatioresolution (Rs, 1.7) was able to be achieved [8]. Using the additives benefits the enatioresolution and improves the retention of the API enatiomers; however, the analysts have to pay attention to the chromatographic behavior of columns (usually polysaccharides CSPs) that are exposed before and after using the additives. The retention time and peak shape may not perform as expected, which is referred to as “memory effect,” similar to that occurring in chiral LC separation [9, 10]. During achiral separation, when switching from using acidic to basic additive, or vice versa, the “memory effect” has also been observed [11].

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The chiral screening strategies applied routinely in the laboratories are either sequential (or serial) column approach or parallel column approach. Sequential screening is more applicable, in which commercially available instrument can be utilized to screen selected CSPs, one at a time, with defined mobile phases. Parallel screening methods, as reported in literature, either use commercial instruments or customized instruments to conduct screening over multiple columns (four or more columns) at one time. Due to the parallel status, parallel column approach provides higher efficiency to achieve the desired enantioresolution. Tandem or coupled column screening approach could be applied when analyzing chiral APIs mixed with impurities, APIs containing multiple chiral centers, or multiple APIs in the drugs products in order to acquire stronger separation power.

4.3.1 Sequential screening approach The sequential screening approach for SFC chiral separation usually starts with polysaccharide-based CSPs, such as Chiralpak AD-H, AS-H, Chiralcel OD-H and OJ-H, and so on, applied on a conventional SFC-UV instrument. The common mobile phases are methanol, ethanol, or isopropanol, while the additives can be applied based on the property of the test molecules [12]. High-throughput sequential screening approach, using CSPs with smaller particle size (3  µm) and faster gradient chromatographic separation, provides more efficient way to achieve enatioresolution [13]. The sequential screening approach has been verified by using a test set of compounds, which are usually marketed drug products providing very valuable references for analysts. Besides using SFC-UV for sequential chiral screening, SFC-MS implements additional perspectives for the chromatographers. Chiral SFC-MS screening approach, which takes advantage of the detection capability of mass spectrometry and the analysis of mixtures of multiple racemic compounds, offers the capability of higher throughput and higher sensitivity while uses one column and one mobile phase at a time. It is  reported that six racemic compounds were analyzed in one run using chiral SFC-MS method [14]. SFC-MS screening is particularly valuable for evaluating chiral API and its precursor impurities, which are required to be controlled in the manufacturing process.

4.3.2 Parallel screening approach Parallel chiral screening is advantageous over sequential approach because more than one CSPs (usually four or more) can be screened over one mobile phase in a given time. It was reported in the literature that successful enantioresolution can be achieved by parallel screening over four CSPs (e.g., Chiralpak AD-H and AS-H and



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Chiralcel OD-H and OJ-H) while using a parallel SFC system (SFC-MS), as shown in Figure 4.1 [15, 16]. The reported procedure also used the intelligent software function to facilitate data processing, interpretation, and method optimization for enantioresolution. It is also reported that the fast screening was achieved by using parallel SFC-UV instrument to screen eight chiral columns over a four-­solvent system. Using this high-throughput approach to screen a racemic compound, it takes only 80 min acquiring 32 chromatograms [17]. These successful cases have demonstrated the removal of the chiral screening bottleneck from the development process. Due to the complex nature of API molecules and coexisting impurities, the combination of parallel chiral SFC and parallel chiral LC screening provides a powerful tool to find the suitable method for API analysis. The combination approaches are very useful for method transfer as well. Currently, application of HPLC in chiral API assay is the dominant method and transferring SFC method to the HPLC method is a common practice for the development of chiral API method because the SFC method development for chiral separation is much more efficient. The capability of performing a parallel chiral SFC and LC screening in an instrument is demonstrated in Figure 4.2. For example, application of the parallel SFC and parallel LC approach is shown in Figure 4.3. The parallel chiral screening of chiral API was performed in SFC (CO2 with methanol and ethanol). The parallel chiral screening was also performed in LC with the modes of normal phase (ethanol–hexane, or 2-propanol–hexane), polar organic phase (methanol, ethanol, and 2-propanol), and reversed phase (methanol–

Manifold to split flow to four columns

Tee to split flow to MUX ChiralPak AD

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MeOH/H2O 90/10 (0.05% FA)

Figure 4.1: Chiral SFC-MS method for high-throughput screening of enantioseparation for pharmaceutical samples [with permissions from Zeng et al. [15]].

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Figure 4.2: The customized parallel LC and SFC system, in which the SFC and LC method scouting in the same instrument for both chiral and achiral screening [Zeng [18]].

CO2

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CTC autosampler (4 injectors)

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 SFC applications for active pharmaceutical ingredient analysis 

 99

Figure 4.3: Chiral screening of a test chiral API compound using parallel SFC and parallel LC [Zeng [18]]. P1, P2, and Rs stand for peak 1, peak 2, and resolution, respectively. MP3 is in CO2–2-propanol (containing 0.1% DEA) 70/30. MP7 is in methanol (containing 0.1% DEA). MP13 is in acetonitrile– water 40/60 with 0.1% formic acid.

water, acetonitrile–water, and 2-propanol–water). This example has demonstrated the power of combination approach of using parallel SFC and LC to identify the suitable separations for an API and particularly applicable to the chiral molecule with complex purity profile.

4.3.3 Tandem method (column coupling) Tandem chromatography, also known as column coupling or coupled column chromatography, is one of the most commonly used separation techniques to improve selectivity and increase the number of theoretical plates. The chromatographer can simply connect two columns together to perform the chromatographic separation. Column coupling is more advantageous on a SFC system due to the lower ­viscosity of mobile phase and lower pressure drop through the column. Column coupling is also very useful for chiral method development to separate enantiomers, par­ticularly when the impurities coexist with API or multiple chiral APIs coexist in the same drug product. Coupling columns for enantiomeric separations can be performed in several ways, such as chiral–chiral column coupling with the same CSP, chiral– chiral column coupling with different CSPs, achiral–chiral column coupling, and chiral–achiral column coupling. An example of achiral–chiral column coupling,

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reported in the literature, characterized the fully enantioresolution of four pairs (eight individual enantiomers) of chiral drug molecules, such as, alprenolol, atenolol, oxprenolol, and propranolol. The chiral separation was achieved by coupling an achiral column (cyano-bonded phase) in the front and a chiral column (polysaccharides, Chiracel-OD phase) in the back [19]. Automated tandem SFC column screening tools have also implemented in the pharmaceutical research and development laboratories to facilitate the process and improve the efficiency of trial and error for the separations of API from complex impurities [20, 21]. For tandem SFC applications, changing the column dimensions either in length or i.d. can result in changing the overall selectivity since SFC back pressure plays a critical role in tandem column SFC method. Tandem column SFC method offers a unique and useful tool for optimizing the overall selectivity of complex m ­ olecules. An example in Figure 4.4 shows the tandem ­chromatographic ­separation of ­enantiomer–­diasteromer sample by using different CSPs for the ­separation of enantiomers and/or diastereomers. This example demonstrates that changing the columns will affect the retention time of peaks, and thus, impact the separations of desired critical pairs [22].

4.4 Achiral SFC method for API purity and impurity analysis During the development of API, analytical methods are needed for the determination of assay and impurities for quality control. Speed and efficiency in method development and analysis are very important. Thus, utilizing SFC for faster analysis, less solvent consumption, lower cost, and better separation efficiency can be advantageous compared to HPLC or UHPLC, which is the conventional primary method in pharmaceutical industries. While application of SFC for chiral separations has become popular over years, the use of SFC for achiral separations has been limited due to challenges with sensitivity, robustness, and reproducibility. One of the few literature examples describes development of achiral SFC impurity method to enable detection and quantification of low level impurities down to 0.05% [23]. Another recent example is the work published by a group of European scientists, which developed and validated salbutamol sulfate impurities using achiral SFC as an alternative to ion-pairing LC [24].

4.4.1 SFC API purity method Our analytical chemistry laboratory developed and qualified an achiral method for API assay/impurities assessment, thereby expanding the typical use of SFC



 SFC applications for active pharmaceutical ingredient analysis 

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Figure 4.4: Using IB and AS column coupling for the separations of stereoisomers under four different scenarios: (A) IB column (25 × 4.6 mm) coupled with AS column (250 × 4.6 mm); (B) AS column (250 × 4.6 mm) coupled with IB column (250 × 4.6 mm); (C) IB column (250 × 4.6 mm) coupled with AS column (150 × 2.1 mm); (D) AS column (150 × 2.1 mm) coupled with IB column (250 × 4.6 mm). Shift in retention time of the stereoisomers was observed by simply switching the coupling order of IB and AS columns in the case of (A) and (B), as well as switching the coupling order and column length and size, in the case of (C) and (D)[with permissions from Wang et al. [22]].

beyond chiral analysis. As a demonstration, two original UHPLC assay and related substances methods were converted successfully to a single SFC method with significantly shortened run time, improved performance, and reduced cost.

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All in all, it is evident that this technology can easily become an equally valuable primary option for API method development and release testing in analytical laboratories. Specifically, mobile phase with various modifiers, gradient conditions, column temperature, and stationary phases were evaluated with detailed descriptions in the following sections. One of the main challenges encountered was the lower sensitivity of SFC compared to UHPLC. The lower sensitivity can be explained by higher background noise of UV detector. However, the desired results were achieved by increasing sample concentration and injection volume. The other problem observed during the development was coelution of the two known impurity peaks, which was resolved by selecting a stationary phase suitable for the compounds tested in the study.

4.4.1.1 SFC Instrumentation There are several well-known instrument manufacturing companies, such as Agilent Technologies, Water Corp., and Shimadzu Scientific Instruments, that produce SFC instrumentations for analytical and preparative purposes. With the dawn of new and robust instruments, the advantageous properties of CO2-based mobile phase, and low consumption of solvents, baseline noise and back pressure issues have significantly improved. In the case study, Waters ACQUITY Ultra Performance Convergence Chromatography (UPC2®) with Waters ACQUITY QDa® Detector was utilized to carry out the SFC experiments. The UPC2®analytical instrument, which was introduced to the market in 2012, uses cosolvents in a subcritical state, and thus, the term “convergence chromatography” is used by Waters to describe this technique. The uniqueness of the technique is the ability to combine the power of normal phase with the ease of reversed phase, allowing separation of a much wider variety of compounds with one chromatographic system [25]. Therefore, unlike reversedphase chromatography, this technique allows polar compounds to be retained and elute last. To make this technique more powerful, the addition of mass spectrometer (Waters ACQUITY QDa® Detector) aids in making the method development more efficient by using mass detection for tracking and troubleshooting. Additionally, in the case study, the use of MS was found to be highly beneficial to detect an impurity coexisting in the API, but which was not initially observed in UV trace due to low concentration.

4.4.1.2 Column screen, mobile phase, and modifier For this case study, large set of columns types, such as polar, polar basic, benzyl, substituted benzyl, and chiral phases, covering various dimensions and particle



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Table 4.1: Column screen for a given API sample. Column Type

Brand and Size

Polar

DIOL, 1.8 µm, 2.1 × 50 mm Waters ACQUITY Torus Diol, 1.7 µm, 3.0 × 150 mm Princeton Chromatography Silica Column, 3 µm, 3.0 × 50 mm Phenomenex Kinetex HILIC, 1.7 µm, 2.1 × 100 mm

Polar basic

Waters ACQUITY UPC2 BEH 2 EP, 1.7 µm, 3.0 × 50 mm Waters ACQUITY Torus 2-PIC, 1.7 µm, 3.0 × 50 mm and 3.0 × 150 mm

Benzyl

Waters ACQUITY Torus 1-AA, 1.7 µm, 3.0 x150 mm

Substituted benzyl

Waters ACQUITY UPC2 CSH Fluoro-phenyl, 1.7 µm, 3.0 × 50 mm

Chiral

Chiralpak AD-H, 5 µm, 2.1 × 150 mm Chiralpak AD-3, 3 µm, 2.1 × 150 mm Chiralcel OD-H, 5 µm, 2.1 × 150 mm Chiralcel OD-3, 3 µm, 2.1 × 150 mm

Chiral + achiral coupling Achiral + chiral coupling

Chiralcel OD-H, 5 µm, 2.1 × 150 mm + Torus 2-PIC, 1.7 µm, 3.0 × 50 mm Torus 2-PIC, 1.7 µm, 3.0 × 50 mm + Chiralcel OD-H, 5 µm, 2.1 × 150 mm

sizes (detailed descriptions in Table 4.1), were evaluated to evaluate selectivity, sensitivity, and detection properties. Change in column parameters (i.e., length, particle size) and conditions such as flow rate can cause a change in pressure. Varying these parameters has a direct impact on run time, resolution, and column pressure. One of primary goals in the study was to reduce the run time. During the method development, flow rate was set to operate at a higher rate without compromising the resolution between the two critical pairs while controlling the column pressure so that it did not exceed column manufacture limit. Consequently, the run time can be reduced usually by using smaller particle size and/or shorter column. Nowadays, in order to prevent damage to stationary phase and instrument, all chromatographic instruments are capable of shutting down the analysis when column pressure limit is exceeded. The automated shut down function provides scientists the opportunity to push the limits without causing any damage to the column or instrument. For method scouting, a test sample (mixture of four components) was dissolved in methanol. Since one of the known impurities was polar and eluted early in the UHPLC method, polar and polar basic columns (listed in Table 4.1) were evaluated. None of these columns provided a desired separation of the main API peak from the

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impurities. As for the other types of columns, with exception of one, the API peak coeluted with one particular impurity or two of the same impurities coeluted with one other. Coupling Chiralcel OD-H and Waters Acquity UPC2 Torus 2-PIC columns in tandem c­ hromatography led to improved separation between the peaks. However, when the order of these two columns was switched, two of the impurities were found to be coeluting. The order of the column can impact the overall separation due to potential change of pressure at the head of the individual column causing shift in retention time and resolution. The similar phenomenon was observed and reported in the literature [22]. Ultimately, the single column approach was advantageous due to performance and cost, despite the fact that column coupling provided good separation and selectivity. Using the new ACQUITY UPC2 Torus™ 1-aminoanthracene column simplified the method development process and resulted in desired separation with baseline resolution achieved in less than 4 minutes (Figure 4.5). Given the advantageous proprieties of CO2-based mobile phase, polar and nonpolar substances can easily be separated. In addition cosolvents such as alcohols (methanol, ethanol, and isopropanol) and acetonitrile may be required to enable the

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3.27

Imp-4 3.80

0 –0.00

4.00

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Time 4.50

5.00

Figure 4.5: Chromatogram of the separation of an API from known impurities using the ACQUITY UPC2 Torus™ 1-aminoanthracene 1.7 µm, 3.0 × 150 mm column.



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elution of more polar compounds. In our case study, we focused on cosolvents that are MS favorable, and methanol provided better separation and better peak shape than acetonitrile.

4.4.1.3 Influence of additive and temperature in SFC method Additive can impact peak shape, resolution, retention time, and order of elution. For acidic compounds, small amount of trifluroacetic acid, formic acid, or acetic acid could be used, while for basic compounds, triethylamine, ammonium acetate, ammonium hydroxide, or diethylamine are preferred. We evaluated several types of additives and 40 mM ammonium hydroxide in methanol gave the best results with enhanced peak shape when compared to 20 mM ammonium acetate methanol. Typically, increasing the buffer strength can improve peak shape but may affect selectivity. However, in our case study, increasing the buffer strength from 20 mM ammonium hydroxide to 40 mM ammonium hydroxide improved peak shapes without impacting selectivity. Most of the time HPLC/UPLC analysis is done at ambient column temperature; however, heating the column can improve peak sharpness and decrease elution time resulting to shorter run time. Chromatography at high column temperature can lead to stationary phase degradation. This issue is minimized with new generation of column chemistries. In our case study, column temperatures in the range of 50–60 °C were assessed and 60 °C was determined as the optimal temperature, resulting in the best peak shape without compromising resolution. Some chromatography instruments have the capability to control sample tempera­ ture by maintaining the temperature of sample compartment at lower than ambient temperature. This feature is useful especially for compounds that can degrade at room temperature. Since we had a concern with the API stability at room temperature over time, sample compartment was set at 10 °C. Sample concentration and injection volume can effect resolution, retention, and peak shape. To avoid the solvent strength between mobile phase and sample diluent, it is preferable that the sample is dissolved in the mobile phase. During the study, to increase the signal response on SFC, the sample concentration and injection volume were increased from 0.25 to 0.8 mg/mL and 2 to 6 µL, respectively, to compensate for the sensitivity.

4.4.1.4 Additional perspectives for an SFC achiral API method The instrument employed for this study was not qualified because it is operated in a non-GMP environment. The validation parameters evaluated in the study included linearity, accuracy, injection precision, quantitation limit (QL), and detection ­

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limit. ­Linearity was evaluated at both high and low concentration ranges. The high ­concentration range was evaluated from 80% to 120% of the nominal concentration (0.8 mg/mL) with five data points. The correlation coefficient (R2) was greater than 0.99. The low-level linearity was evaluated from 1% (QL) to 5% of the nominal concentration with five data points. The correlation coefficient (R2) was greater than 0.99. ­Relative standard deviation in retention time for nominal concentration and QL level were less than 1 and 8%, respectively. Signal-to-noise (S/N) evaluation of detection limit met the 3:1 S/N requirement and QL met the 10:1 S/N requirement. All in all, the results indicated that this SFC method was validated and met ICH validation ­guidelines.

4.4.2 SFC API impurity profiling Controlling the impurities of API during drug development and manufacturing process is a challenging task. Identification, characterization, and quantitative determination of impurities in the APIs are regulated according to ICH Q3B (R2) guidelines [26], which state that the impurities acceptable for drug products with a dose of less than 2  g/day require the identification of impurities at 0.1% levels and above. Currently, HPLC method is the standard approach for impurity profiling and quantitative assay in pharmaceutical laboratories. HPLC provides sufficient specificity and sensitivity to perform separation, identification, and quantitative analyses of the impurities. The ­combination of the orthogonal chromatographic methods, such as LC with LC (reversed-­reversed phase, reversed-normal phase), SFC with SFC (same stationary phase and different stationary phase), and LC with SFC (reversed-phase LC, or normal phase LC with SFC), provides useful tools for the separation of the impurities from API or one impurity from another. Although the majority of published methods for impurity profiling of APIs use the combination of LC and LC [27], recent studies show that the combination of HPLC and SFC methods offers better chance to separate and identify most of the impurities in the APIs, since SFC method provides orthogonal selectivity to the well-established reversed-phase HPLC method. The truly efficient way to combine two methods of impurity analysis into one is through two-dimensional (2D) chromatography. The 2D chromatography is more powerful in increasing the peak capability, which is the resolution of maximum number of peaks in a given chromatographic time. The 2D LC-LC method provides the assurance of peak purity, and therefore, the specificity of the method toward quantitative analysis. The 2D HPLC-UHPLC application for forced degradation API profiling, in which the first dimension was the SB-CN column and the second dimension was the C18 column, reported to have sufficient orthogonality to profile screening in pharmaceutical degradation studies [28]. Similarly, 2D SFC-SFC also offers the orthogonality and selectivity for impurity analysis. In Figure 4.6, 2D SFC-SFC method, in which the first dimension is an achiral



 SFC applications for active pharmaceutical ingredient analysis 

(A) UV chromatogram of achiral separation

2.83

%

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2

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1.72 2.02 1.60 1.89

An1 1.00e6

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0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 (B) UV chromatogram of 1st dimension achiral separation (arrow showing the peak-cut)

%

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API (enantiomer 2)

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 107

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2.50

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Figure 4.6: The chromatograms of 2D SFC-SFC for the analysis of an API (racemic sample) using heart-cutting mode from first dimension with achiral column to second dimension with chiral column for chiral analysis. (A) The first dimension SFC-UV chromatogram showed that the API peak was separated from other impurities. (B) The first dimension SFC-MS chromatogram showed that the API peak was cut and transferred to the second column. (C) The second dimension SFC-MS ­chromatogram presented the chiral analysis of API (enantiomer 2) and enantiomeric impurity (enantiomer 1) [with permissions from Zeng, et al. [29]].

column and the second dimension is a chiral column, was used to analyze a test ­compound (API in this case) [29]. The achiral column in the first dimension separated the test compound (API) from the coexisting impurities, whereas the chiral column in the second dimension resolved the API from its enantiomeric impurity. The 2D LC-SFC have been utilized for the analysis of API, in which the first dimension of achiral reversed-phase LC column resolves the process-related impurities from the API and provides achiral purity and the second dimension SFC separation provides the chiral purity (enantiomeric excess) [30]. With spiked sample of 0.1% and 0.5% of enantiomer, 2D LC-SFC method showed the capability to quantify the undesired enantiomer at 0.1% level (Figure 4.7). The 2D RPLC-SFC instrumentation requires transfer of water containing samples from the first dimension RPLC to the second dimension SFC; therefore, the interface uses trap columns to hold the trapping fractions for the second dimension sample loading. In the case of 2D SFC-SFC, fractions of effluents from the first ­dimension can be ­transferred directly to the second dimension without a trapping device. Both 2D RPLC-SFC and 2D SFC-SFC are potentially the new tools for the impurity profiling and quantitative analysis of pharmaceutical APIs in the near future.

Detector response (mAU)

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1st dimension (RPLC-UV)

15 10

1D - Achiral purity: 99.0%

5 0 10

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2D–Chiral purity: 100% ee

400 200

Unspiked sample 0

1.0

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Secondary column retention time (min) Figure 4.7: The chromatograms of 2D RPLC-SFC for the analysis of a drug substance (racemic API) using heart-cutting mode from first dimension using achiral RPLC column (top chromatogram) to second dimension using chiral SFC column for chiral analysis (bottom chromatogram). The spiked samples of 0.1% and 0.5% were analyzed to show the quantitative capability of the method reached to 0.1% level [with permissions from Venkatramani, et al. [30]].

4.5 SFC instrument qualification and API method validation ICH, United States Pharmacopeia Convention, FDA, and other regulatory agencies require analytical hardware and software to be qualified and methods to be validated to show that they are suitable for their intended use in order to ensure quality, safety, and efficacy of drug. Specifically, ICH Q2 (R1) [1] and United States ­Pharmacopeia Convention chapters [31], [32], [33], and [34] provide framework for validation of analytical methods and procedures, qualification of analytical instruments, transfer of analytical procedures, validation of compendial methods, and verification of compendial methods. Thus, pharmaceutical companies set up systems to meet this requirement in order to be in compliance. To do so, understanding drug life cycle is very important. On average, it takes about 10–12 years from drug discovery, development to marketing. However, setting up method validation criteria is challenging sometimes



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and needs to be appropriate for the phase of drug develop­ment. For example, for Phase I clinical studies, specification criteria for a given parameter cannot be so stringent that future batches of API will not be able meet the criteria, especially when the synthetic route of API is not well defined and could be modified or changed later. A planned protocol should be laid out with acceptance criteria in advance of performing the SFC instrument qualification and method validation. In a primer published by Agilent called “Qualification and Validation for Supercritical Fluid Chromatography” [35], Dr. Huber describes all the background information and necessary steps to qualify and validate SFC instrument and analytical method. To minimize any potential issues, preliminary method validation experiments shall be performed prior to actual validation experiments. Depending on the results, further method development or optimization maybe necessary to successfully meet all the criteria and to avoid out-of-specification results. Upon completion of equipment qualification and method validation, a report shall be written to summarize all the results. Feasibility studies have demonstrated that SFC can replace HPLC as the primary tool for chiral purity method for API release and stability testing, as reported by the laboratory of a major pharmaceutical company [2]. By comparing the key validation parameters (accuracy, precision, specificity, detection limit, quantitation limit, linearity, and range), the results demonstrated that chiral SFC methods can meet the requirements as that of HPLC methods. Although HPLC provides better sensitivity, SFC still fulfills the sensitivity requirement for the intended use. In addition, chiral SFC improves the efficiency of method development without compromising the method performance; thus, it should be considered as the method of choice for API release testing and stability studies.

4.6 Conclusion Compared to HPLC method, SFC method provides better resolution and peak capacity, along with shorter chromatographic analysis time. The applicability of SFC has a great potential in supporting API development and manufacture in pharmaceutical industry, particularly considering the costs of saving and high-throughput process. As the SFC methods currently have been routinely applied in GMP chiral QC method for API release and stability testing for chiral molecules, it is expected in the near future to reach the same routine applications for achiral API release and stability testing. For impurity profiling of APIs, SFC has been demonstrated to be orthogonal and complementary to HPLC for the separations of an API from the achiral and chiral impurities. Although publications have been ­reported using SFC method for API purity and impurity assay, there are no reports of the use of SFC for API stability-indicating methods for drug substances and drug products, as well as for genotoxic impurity (and/or potential genotoxic impurity). Thus, the application of SFC technique in these challenging areas is expected to be the the topic of future research.

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Acknowledgment The assistance of Ms Karen Yuan in the preparation of this manuscript is gratefully appreciated.

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[17] Ning J. Parallel SFC method development screening for enhanced speed and quality, paper presented at SFC 2011 International Conference, New York. [18] Zeng L. Advanced SFC/MS Technologies to support drug discovery and development, paper presented at SFC 2012 International Conference, Brussels, Belgium. [19] Phinney KW, Sander LC, Wise SA. Coupled chiral/chiral column techniques in subcritical fluid chromatography for the separation of chiral and nonchiral compounds. Analytical Chemistry 1998, 70, 2331–5. [20] Welch CJ, Biba M, Gouker JR, Kath G, Augustine P, Hosek P. Solving multicomponent chiral separation challenges using a new SFC tandem column screening tool. Chirality 2007, 19, 184–9. [21] Ventura M. Use of achiral columns coupled with chiral columns in SFC separations to simplify isolation of chemically pure enantiomer product. American Pharmaceutical Review 2013, 90–95. [22] Wang C, Tymiak AA, Zhang Y. Optimization and simulation of tandem column supercritical fluid chromatography separations using column back pressure as a unique parameter. Analytical Chemistry 2014, 86, 4033–40. [23] Dunkle MN, Pereira AS, David F, Sandra P. Sensitive determination of impurities in achiral pharmaceutical by supercritical Fluid Chromatography Using the 1260 Infinity analytical SFC System Agilent Application Note. Agilent Technologies Inc., 2010. (Accessed October 12, 2017, at https://www.agilent.com/cs/library/applications/5990–6413EN.pdf.) [24] Dispas A, Desfontaine V, Andri B, et al. Quantitative determination of salbutamol sulfate impurities using achiral supercritical fluid chromatography. Journal of Pharmaceutical and Biomedical Analysis 2017, 134, 170–80. [25] Assay of the drug substance anthralin using Acquity UPC2 system. Waters Corporation, 2012. (Accessed October 6, 2017, at http://www.waters.com/webassets/cms/library/ docs/720004236en.pdf.) [26] ICH Q3B(R2), Impurities in new drug products. Geneva, 2006. (Accessed October 13, 2017, at https://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q3B_R2/ Step4/Q3B_R2__Guideline.pdf.) [27] Holm R, Elder DP. Analytical advances in pharmaceutical impurity profiling. European Journal of Pharmaceutical Sciences 2016, 87, 118–35. [28] Huidobro A, Pruim P, Schoenmakers P, Barbas C. Ultra rapid liquid chromatography as second dimension in a comprehensive two-dimensional method for the screening of pharmaceutical samples in stability and stress studies. Journal of Chromatography A 2008, 1190, 182–90. [29] Zeng L, Xu R, Zhang Y, Kassel DB. Two-dimensional supercritical fluid chromatography/mass spectrometry for the enantiomeric analysis and purification of pharmaceutical samples. Journal of Chromatography A 2011, 1217, 3080–8. [30] Venkatramani CJ, Al-Sayah M, Li G, et al. Simultaneous achiral-chiral analysis of pharmaceutical compounds using two-dimensional reversed phase liquid chromatographysupercritical fluid chromatography. Talanta 2016, 148, 548–55. [31] Chapter : Analytical Instrument Qualification. In: Unites States Pharmacopeia. Rockville, MD, USA, 2008. [32] Chapter : Transfer of Analytical Procedures. In: United States Pharmacopeia. Rockville, MD, USA, 2011. [33] Chapter : Validation of Compendial Methods. In: United States Pharmacopeia. Rockville, MD, USA, 2009. [34] Chapter : Verification of Compendial Methods. In: United States Pharmacopeia. Rockville, MD, USA, 2009. [35] Qualification and validation for supercritical fluid chromatography. Agilent Technologies Inc., 2011. (Accessed October 12, 2017, at https://www.agilent.com/cs/library/primers/ Public/5990-9148EN.pdf.)

Cyrille Santerre, David Touboul

5 Analysis of terpenes (mono-, sesqui-, di-, and triterpenes) by SFE and SFC-MS Abstract: This chapter introduces various applications of supercritical fluid extraction (SFE) and supercritical fluid chromatography hyphenated with mass spectrometry (SFC-MS) focused on terpenes and terpenoids. SFE and SFC offer unique advantages compared to conventional techniques such as efficiency, faster process and low environmental impact. Additionally, the drastic effects of sample heating in hydrodistillation for example can be avoided. Both theoretical and practical aspects will be discussed and are illustrated with academic and industrial ­applications. Keywords: Supercrical fluids, chromatography, extraction, terpenes, terpenoids, mass spectrometry Terpenes originate from the activated form of isoprene, that is, isopentenyl pyrophosphate (also isopentenyl diphosphate) and dimethylallyl pyrophosphate (also dimethylallyl diphosphate), present in plants and some insects. Their general molecular formulas are multiples of (C5H8)n leading to the following classification: ­hemiterpenes (n = 1), monoterpenes (n = 2), sesquiterpenes (n = 3), diterpenes (n = 4), sesterpenes (n = 5), triterpenes (n = 6), and tetraterpenes (n = 8) (Figure 5.1). Terpenoids are defined as terpenes bearing additional functional groups such as hydroxyl, aldehyde, ketone, and so on. Depending on their chemical structures, terpenoids show a large variety of natural activities such as protection against predators and parasites [1], ­precursor of steroids (squalene) [2], or antioxidant (vitamin A) [3]. Among them, monoterpenoids are the major constituents of essential oils used in cosmetic and aromatherapy. Terpenoids are classified in the prenol category from LIPID MAPS [4] bearing similar physical chemical properties with other lipids, that is, a high solubility in organic solvent and a very low solubility in water. Nowadays, innovation in the extraction of natural products field is a real challenge. “Take care of our planet and leave a clean earth in inheritance” is a true concern

Cyrille Santerre, Institut Supérieur International Parfum Cosmétique Arômes, Plateforme ­scientifique, ISIPCA, 34–36 rue du parc de Clagny, 78000 Versailles, France; Institut de Chimie des Substances Naturelles, CNRS UPR2301, Université Paris-Sud, Université Paris-Saclay, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France. David Touboul, Institut de Chimie des Substances Naturelles, CNRS UPR2301, Université Paris-Sud, Université Paris-Saclay, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France. https://doi.org/10.1515/9783110618983-005

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Figure 5.1: Examples of different classes of terpenoids [1].

and a real awareness after having used more or less aggressive solvents for the environment for years. Therefore, new rules and philosophies have appeared such as ecoextraction and green chemistry. Thanks to our expertise and our feedback, terpenoids are of high importance in the family of raw material for cosmetics, perfume, food, and other industrial fields. For ages civilization has tried to innovate in terms of extraction techniques. We can list a lot of different techniques. For essential oils [5], steam or hydrodistillation could be used for different parts of plants; pressing processing could be used more specifically for orange peels and other citrus fruits. Other extracts are obtained using different solvents from less polar to very polar. Water (polar) can be used for decoction, infusion, percolation, and extraction with or without cosolvent (e.g., glycerin or propylene glycol). Soxhlet extraction used volatile nonpolar solvents (e.g., petroleum ether, hexane) [6]. Note that the choice of the solvent has a real impact on the composition of the extracts and all its characteristics. Older techniques such as enfleurage [6] used fats (e.g., tallows), but today they are almost not in use for production. Apolar solvent like benzene or hexane can also be used to produce concretes, but they were replaced by greener techniques [7] and solvents such as O2. Thus, developments of technological innovations are mandatory, breaking away from the past rather than continuing what was done before. New technologies were created with this aim: being quicker, using less energy, and trying to replace conventional solvent with greener compounds. For example, we can mention microwaves [8] and ultrasounds associated with SFE (Figure 5.2) [9] or alone [10] to set time and energy saving. Moreover, a solvent like CO2 for extraction and/or fractionation of terpenoids gives very interesting results.



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Figure 5.2: CO2 SFE system hyphenated with ultrasounds transductor. From [9].

5.1 Theoretical approach Extraction with supercritical (SC) fluids is a method frequently applied for the extraction and/or purification of essential oils. A theoretical approach is necessary to better understand and to be able to master some important parameters, in particular, the thermodynamical aspect. For citrus oils, a common practice to study terpenes and derivatives extraction is to consider extracts as a binary synthetic mixture of its two or more important components [11–13]. For instance, the cold-pressed orange oil is usually treated as a mixture of limonene and linalool, representing the terpenes and the oxygenated fractions, respectively. The solubility of these two components has been studied at different temperature and pressure. The impact of temperature and pressure was studied [14] for limonene at only 44.9 °C and linalool at 44.9 °C and 54.9 °C. As for pressure parameter, a range from 69 to 111 bar was used. In conclusion, the solubility of limonene in supercritical carbon dioxide (SC-CO2) is more than the solubility of linalool under the same conditions. Nevertheless, they set closer under larger pressures. In addition, both systems show a sudden increase in the solubility at pressures up to approximately 80 bar. One year later, a complementary article was published [15] on ternary system CO2– limonene–linalool under the same conditions of temperature and pressure. The study focused on the selectivity data for the system defined as limonene/linalool. The conclu-

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sions were that at a constant temperature, raising the pressure decreases the selectivity (Figure 5.3) because of an increase in the solvating capacity of the SC solvent (effect of higher density of the solvent). On the other hand, at a lower pressure 79–95 bar, raising the temperature causes the selectivity to decrease quickly because of a decrease in the density of the solvent. Finally, increasing the limonene amount fed causes the selectivity to rise. In summary, the selectivity varies for the limonene and linalool because of the variation of solvent density due to their molecular weights and vapor pressures being similar, mainly for the linalool polar nature. Another aspect, partial molar volumes, at infinite dilution of terpenes in SC-CO2 was also studied [16]. The values measured in each ­experimental run were the retention time of the solute, temperature, pressure, and flow rate of the mobile phase. The “unretained” solute retention time was determined from the retention time of ethane. Experimental data obtained for terpenes and the extrapolated values for alcohols in the pressure range of 85–95 bar are reported. As expected, a sharp minimum is obtained at pressures between 80 and 90 bar. Different alcohols show a regular behavior in the sense that with the molecular weight of the alcohol increasing, the absolute value of the partial molar volume increases (it becomes more negative). This behavior is similar to that previously observed for n-paraffins in SC-CO2, but the numerical values are larger and the increment per CH2 group in the homologous series is also larger. Different situation is displayed by terpenes. In this case, the molecular weight of the d ­ ifferent compounds is the same, and it is difficult to clearly understand the effect of different configurations. The terpenes differ mainly by the position of the hydroxyl group. In conclusion, using the SC technique, rapid determinations of the partial molar volumes are possible. Starting from experimental determinations of capacity factors under conditions not so close to the critical ones for the mobile phase, it is possible to predict the minimum value of the partial molar volume of solutes. 7 6

y1/y2

5 4 3 2 1 70

80

90 P (bar)

100

110

Figure 5.3: Selectivity data (y1/y2) for the system limonene (1) + linalool (2) + CO2 (3). About 40 mass% limonene + 60 mass% linalool: (□) 318.2 K. About 60 mass% limonene + 40 mass% linalool: (○) 318.2 K and (∆) 328.2 K splint line. From [15].



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The influence of plant matrix on extraction was studied [17] using α-cellulose to simulate it. Different terpenes and derivatives were used such as limonene, caryophyllene, carvone, eugenol, and santonin. A known quantity of these compounds was deposited on the matrix and extracted by SFE. Moreover, a large range of temperature and pressure was examined from 10 °C to 80 °C and 50–250 bar. Recovery yields were used for each component to determine best parameters for extraction. It appears that under subcritical dense gas conditions (−10 °C and 250 bar), the extraction of the less polar compounds was initially rapid except for santonin, which was slowly extracted with less than 60% for the recovery yield. At a higher temperature but lower pressure (40 °C and 55 bar), the extraction was also rapid for the less polar terpenes carvone and caryophyllene, but with a yield of 55%. The yield was very low for the more polar lactone santonin. The extraction of the test compounds must, therefore, depend on their distribution between the carbon dioxide and sorptive sites on the sample matrix as well as diffusion from the matrix into the SC fluid. To conclude, optimum conditions seem to be 40 °C and 250 bars. Last part of this theoretical overview concerns the use of ethanol as cosolvent and its effect on extraction of terpenes and derivatives [18]. Two binary systems were studied at 156.1  °C/69 bar and 167.2  °C/100 bar: CO2 + limonene + ethanol at 156.1 °C and 69 bar; CO2 + linalool + ethanol and one ternary CO2 + limonene + linalool + ethanol at 156.1 °C/69 bar. The experimental results showed that the mole fractions of limonene and linalool in vapor phase are not affected by the addition of ethanol. Ethanol is not effective as an entrainer on solubility of limonene and linalool in SC-CO2 at the experimental conditions. The reduction rates for mole fraction of limonene in liquid phase by the addition of ethanol are higher than those of linalool. The relative volatilities between limonene and linalool increase by factors of 1.2–1.5 with the addition of ethanol. Ethanol is effective as an entrainer on separation of limonene and linalool in SC-CO2. Some studies [19] described other compounds such as α-pinene and cis-verbenol present in Boswellia sacra tree resin. The authors showed that CO2-expanded ethanol is a high-diffusion extraction phase that can be used in fast and efficient extraction of medium polar compounds from solid complex samples – in this case α-pinene and cis-verbenol.

5.2 Supercritical fluid extraction (SFE): Application The citrus family was the first plants typology studied because of their high level of terpenoids. Grapefruit flavedo (Citrus paradisi Macf.) was extracted by different methods [20] such as hydrodistillation and solvent extraction using pentane, ethanol as well as SC-CO2 at two fluid densities. The SC-CO2 extracts were orange solid oleoresins at 4–5 °C, with an intense smell of fruit, and a total of 55 components was separated, 50 of which were identified. The five peaks that were not identified from the tandem

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mass spectrometry (MS) experiments were probably sesquiterpenes hydrocarbons and oxygenated derivatives such as sinensal. Because of its extreme volatility, CO2 can be separated from solutes without loss of the volatiles. The solvent power of SC-CO2 is strictly related to its density. To obtain an extract containing more volatiles, the fluid has to be used at very low solvent power; otherwise, a “total extract” is achieved. Limonene, the main component in grapefruit essence, decreased from 94.9% in the hydrodistillate to 84.1% in the SC-CO2 extract; this decrease is not relatively influenced by the different behaviors of other monoterpenes hydrocarbons. All the relative concentration of sesquiterpenes revealed a tendency to increase in the extracts obtained by SC-CO2. Like linalol, the other terpene alcohols increased their relative concentration in the solvent extracts. The esters, represented by geranyl acetate, contributed to 0.54% of the total GC peak area in the oleoresin extracted at 250 bar of SC-CO2 and were less abundant in the other extracts and distillates. An increase of solubility in the SC-CO2 was observed for nootkatone, a sesquiterpenes ketone. The solubility of organic compounds in SC-CO2 is increased by raising the fluid density. From the reported data, high-quality grapefruit extracts were obtained by SC-CO2 extraction. Nevertheless, at high CO2 density, a lot of high-boiling compounds were coextracted, indicating that the best conditions to obtain a good grapefruit aroma extract are at low CO2 density. Bergamot flavedo (Citrus bergamia Risso) was extracted by SC-CO2 at three different conditions [21]: 80 bar of pressure at 40 °C of temperature, 90 bar at 50 °C, and 100 bar at 60 °C. The citrus essential oils are contained in particular glands located in the flavedo portion of the fruit. The solvent extraction of citrus oils is influenced by the surface area, so the rupture of the matrix gland walls allows the fluid to reach the oil and dissolve it. Particle size plays an important role in the mass transfer process during SC-CO2 extraction of essential oils: the reduction of the material particle size increases the surface area and a high oil recovery is obtained. Vapor pressure and polarity of molecules have an influence on solubility behavior in dense carbon dioxide. Compounds with low polarity, small molar mass, and large vapor pressure are more soluble. Among the essential oil components, the terpene hydrocarbons are solubilized by SC-CO2 more than the other flavor compounds. Bitter orange (Citrus aurantium L. var amara) peels were studied using SC-CO2 extraction with ethanol as cosolvent [22]. GC-MS analysis showed 43 phytochemicals. The peel extracts contained a wide range of compounds including mainly fatty acid esters, terpenes, coumarins, and their derivatives. Compounds such as osthole (­O-methylated coumarin), squalene (triterpenes), and hexadecane have been also found in this study. A modification on the experimental conditions causes the selective extraction of certain compounds. As described in [23], limonene oxide and α-caryophyllene were obtained under a first set of experimental conditions (pressure of CO2: 210 bar, static time: 70 min, CO2 flow rate: 3.3 kg/h) but not in a second one (pressure of CO2: 130 bar, static time: 30 min, CO2 flow rate: 2.1 kg/h). In contrast, compounds such as sabinene, ocimenol, or α-farnesene were only extracted under the second set of conditions.



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Other families of plants containing terpenoids were also studied in extraction experiments. First example is Juniperius communis L. fruits using different sizes of particles 0.250–0.400  nm and with an optimization of the experimental pressure (80–100 bars) at 40 °C [23]. The constituents of the extracts were ranked into five main groups: monoterpenes (M), sesquiterpenes (S), oxygenated monoterpenes (OM), oxygenated sesquiterpenes (OS), and the fifth group, labeled as the O group, comprises higher molecular mass compounds (such as fats, waxes, and some other unidentified peaks). Extract yield during the first 0.6 h consisted predominately of monoterpenes (47.45%), with the highest levels of α-pinene (21.45% of the total yield), sabinene (11.75%), β-myrcene (4.97%), limonene (2.82%), β-pinene (1.96%), ­terpinolene (1.14%), and γ-terpinene (1.12%). The content of sesquiterpenes was at the level of 35.82% with γ-cadinene (12.12%), β-caryophillene (4.22%), germacrene D (3.85%), and α-humulene (3.09%) obtained in the highest yield. Oxygenated monoterpenes were found in this extract at 14.03%, with the main components being terpinen-4-ol (4.60%), pinocarveole (1.52%), and α-terpineol (1.50%). The reported quantities of oxygenated sesquiterpenes were very low, leading to a total amount of 1.93%. Other compounds such as fats, waxes, and some other unidentified peaks with higher molecular masses were present at 0.26%. After the 0.6 h of extraction, concentrations of the monoterpenes in the extract fractions significantly decrease, while the quantities of other groups increased. At all pressures tested, 99% of monoterpenes from the berries were extracted before the end of the first 0.6 h. Another example compared hydrodistillation and SFE on a leaf of E. camaldulensis Dehn. from Mozambique [24]. A reduced percentage of terpenes was obtained under SFE when compared to the distilled extracts. Terpene alcohols such as terpinen-4-ol and α-terpineol are present in lower percentages in SFE extracts compared to hydrodistilled extract because of SC fluid fractionation of the low polarity of CO2. However, this observation is not applicable to globulol, for which higher extraction occurred in SC-CO2 extracts. The sesquiterpene allo-aromadendrene found in low abundance in adult leaves of the Mozambican E. camaldulensis was recovered in the extracts obtained under SFE and hydrodistillation. Allo-aromadendrene was found in greater amounts in SC-CO2 extracts. High molecular weight compounds including esters, fatty acids, and waxylike compounds are more likely to be extracted by CO2 rather than by hydrodistillation, which is more likely to extract the volatile compounds. Investigations on the SFE of geranium (Pelargonium graveolens) were performed too [25]. The main constituents of geranium oil were citronellol (sweet roselike odor) and geraniol (flowery roselike odor). Compounds were classified as terpenes hydrocarbons, terpenols, citronellol, citronellyl esters, and geraniol and geranyl esters. The authors concluded that terpenes hydrocarbons, geraniol, and geranyl esters were better extracted with CO2, but terpenols, citronellol, and citronellyl esters were less extracted. Time of extraction did not exceed 2 h and a compromise had to be found for the pressure. The last example was extraction from Turkish mint-plant leaves [26]. The study concluded that monoterpenes fraction in extracts was inversely correlated with relative oil yields. Mono-

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terpenes were preferentially extracted by SC-CO2, due to their high vapor pressure, low molar mass, and low polarity. Carvone constitutes bulk of this spearmint oil, followed by limonene, 1,8-cineole, and caryophyllene. It is known that the quality of ­essential-oil products decreases with increasing fraction of the monoterpenes hydrocarbons. Decomposition of these unstable hydrocarbons generates compounds that impair the quality of the essential-oil products. On the other hand, the real flavor of essential oils is due to oxygenated hydrocarbons. Therefore, the fraction of the monoterpene hydrocarbons in the samples collected with long-time (high-yield) extractions decreases as their amount in the mint leaves decreases with time. Due to their higher mass and/or polarity, certain type of compounds such as oxygenated ­monoterpenes and sesquiterpenes hydrocarbons are harder to extract and their concentration in the integral samples increases with time. This suggests a possible feasibility of a twostage SC extraction, that is, a short-time extraction conducted at higher temperature and lower pressure to remove the undesired monoterpene hydrocarbons from the plant structure, followed by a long-time extraction conducted at higher temperature and higher pressure to obtain a spearmint-oil extract possibly free of monoterpene hydrocarbons [27].

5.3 Supercritical fluid chromatography (SFC) In SFC SC-CO2 is used as the nonpolar constituent of the mobile phase and generally mixed with organic solvents, such as alcohols. The lower viscosity and thus a higher diffusion rate of the SFC mobile phase allow higher flow rates relative to high-­ performance liquid chromatography (HPLC), whereas a large variety of stationary phase chemistry is available compared to GC and HPLC. Due to its low polarity and volatility, SC-CO2 is expected to be an ideal mobile phase for the separation of nonpolar and volatile compounds such as terpenoids. This chapter will only focus on packed-column SFC; capillary SFC has more or less completely disappeared from analytical laboratories in the last 10 years. Surprisingly, reports of monoterpinoids analysis by SFC are very scarce in the literature. This can be explained by the high efficiency of GC coupled to flame ionization detector or MS for the detection, identification, and quantification of this family [28]. The separation of spirocyclic C13-norisoprenoid flavor compounds using SFC was reported. A set of 16 commercially available chiral stationary phases largely based on polysaccharides were screened and no acidic modifiers such as trifluoroacetic acid were used [29]. Analysis of sesquiterpene was first achieved by Morin et al. in 1991 [30]. They used either Nucleosil 100 or Spherisorb packed column (250 mm × 4.6 mm × 5 µm) for the separation of standards and natural extracts (ylang-ylang oil) by isocratic mode with 100% of SC-CO2. Detection was achieved by Fourier-transform infrared spectroscopy



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instrument equipped with a special flow cell designed to maintain a 240 bar pressure gradient across the windows. More recently, sesquiterpene lactones from Artemisia umbelliformis were successfully analyzed by packed-column SFC coupled to evaporative light-scattering detector (ELSD) in less than 8 min (Figure 5.4) [31]. Calibration curves were obtained leading to a limit of detection (LOD) down to 0.01  µg/mL for artemisinin. SFC was also coupled to ultraviolet (UV) detection at 254 nm for the qualitative analysis of sesquiterpene lactone standards and of extracts of Cardus benedictus L. and Artemisia umbelliformis Lam using SC-CO2 modified with methanol/water (95/5) eluent combined with a nitrile stationary phase [32]. Elution conditions were optimized to reduce matrix interferences as UV detection at 254 nm has a low specificity. In order to improve the identification of terpenoids in complex samples, SFC coupled with MS is becoming a gold standard. Dost and Davidson reported the quantification of artemisinin in plant extracts using SFC-MS equipped with an atmospheric pressure chemical ionization source. The average absolute retention time was 3.54 min, with a standard deviation of 0.017 min, whereas correlation coefficient was 0.998 and limit of detection 370 pg on the column [33]. Chamomille extracts containing up to 0.6% of sesquiterpene lactones of the germacranolide type, mainly nobilin and 3-epinobilin, were analyzed by SFC-UV-MS with sub-2  μm particle stationary phase and electrospray ionization [34]. In order to differentiate Roman versus German 6.0 Me

O O O O

H Me

H Me

O Artemisinin (1)

H

Me

H COOH

Artemisinic acid (2)

Artemisinic acid

Artemisinin

2.79

Me

(mV )

6.85

3.0

0

Time (min)

5

10

Figure 5.4: SFC-ELSD chromatogram of an extract of Artemisia annua obtained on a Nucleosil 100–5 NH2 column (125 × 4 mm i.d.; Macherey-Nagel, Oensingen, Switzerland). The polar modifier (methanol) gradient employed was initially 1% methanol in carbon dioxide for 3 min and then increased to 10% methanol within 0.2 min and held for 5 min. The initial pressure was 17.18 MPa and the flow rate was 4 mL/min. From [29].

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chamomiles, multivariate statistical analysis approaches were performed, leading to identifying sesquiterpene lactone among other macular classes as key markers. To the best of our knowledge, no structural data acquired on high-resolution mass spectrometer or tandem mass spectrometer has been published on SFC-MS(/MS) analysis of sesquiterpenes yet. Like for monoterpenes, articles reporting experimental data related to SFC analysis of high-order terpenoids are rare in the literature. One important example is the pioneer work of L. Taylor’s group in 1996, describing the SFC-ELSD separation of ginkgolides A, B, C, and J and bilobalide diterpenoids from gingko extract in less than 10 min by using an amino column and isocratic mode of 12% m ­ ethanol in CO2 [35]. The method was recently refined by Xin-Guang et al. for the pharmacokinetic study of three ginkgolides and their six hydrolyzed metabolites after intravenous administration of total ginkgolide extract to rats requiring quantitative data obtained by coupling SFC with a triple quadrupole mass spectrometer [36]. Nothias et al. utilized porous graphitic carbon (hypercarb) for the separation of diterpene esters from Euphorbia semiperfoliata [37]. The chromatographic method was also scaled-up for semipreparative approaches, allowing the isolation of major secondary metabolites and their complete characterization by NMR. Finally, triterpenes, including pentacyclic triterpenes in an apple pomace extract [38] or ergostane triterpenoids in the medicinal mushroom Antrodia camphorata [39], were also efficiently separated by SFC.

5.4 Conclusion and perspectives This chapter clearly demonstrated that SC-CO2 is perfectly suited for the extraction and analysis of terpenoids in complex matrices. Thanks to its low environmental impact, low cost, and to its physical chemical properties, SC-CO2 is expected to become the future gold standard for all scientific and industrial studies in the field of terpenoids. These conclusions can be easily extended to other classes of apolar natural molecules from the prenol categories, including quinones (ubiquinones [40], vitamins E [41], and vitamins K [42]) or polyprenols [43]. Together with recent advances in the development of SFC and SFE instruments, it is only a matter of time until SC-CO2 spreads its legitimate place in analytical sciences.

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Vriet C, Russinova E, Reuzeau C. From squalene to brassinolide: the steroid metabolic and signaling pathways across the plant kingdom. Molecular Plant Pathology, 2013, 6(6), 1738–1757. Goodman DS, Huang HS, Shiratori T. Mechanism of the biosynthesis of vitamin A from beta-carotene. Journal of Biological Chemistry, 1966, 241(9), 1929–1932. Fahy E, Subramaniam S, Murphy RC, Nishijima M, Raetz CR, Shimizu T, Spener F, van Meer G, Wakelam MJ, Dennis EA. Update of the lipid maps comprehensive classification system for lipids. The Journal of Lipid Research, 2009, 50 Suppl, S9–14. Tongnuanchan P, Benjakul S. Essential oils: extraction, bioactivities, and their uses for food preservation. Journal of Food Science, 79(7), July 2014, R1231–R1249. Visht S, Chaturvedi S. Isolation of Natural Products. Current Pharma Research, CPR 2(3), 2012, 584–599. Chemat F, Abert Vian M, Cravotto G. Green extraction of natural products: concept and principles. International Journal of Molecular Science, 2012, 13(7), 8615–8627. Durđević S, Milovanović S, Katarina Šavikin K, Ristić M, Menković N, Pljevljakušić D, Petrović S, Bogdanović A. Improvement of supercritical CO2 and n-hexane extraction of wild growing pomegranate seed oil by microwave pretreatment. Industrial Crops and Products, 104, 2017, 21–27. Hu A, Zhao S, Liang H, Qiu T, Chend G. Ultrasound assisted supercritical fluid extraction of oil and coixenolide from adlay seed. Ultrasonics Sonochemistry, 14(2), 2007, 219–224. Mason TJ, Chemat F, Vinatoru M. The extraction of natural products using ultrasound or microwaves. Current Organic Chemistry, 15(2), 2011, 237–247(11). Mira B, Blasco M, Subirats M, Berna A. Supercritical CO2 extraction of essential oils from orange peel. The Journal of Supercritical Fluids, 1996, 9, 238–243. Kalra H, Chung SY, Chen GJ. Phase equilibrium data for supercritical extraction of lemon flavours and palm oils with carbon dioxide. Fluid Phase Equilibria, 1987, 36, 263–278. Sato M, Goto M, Hirose T. Supercritical fluid extraction on semi batch mode for the removal of terpene in citrus oil. Industrial & Engineering Chemistry Research, 1996, 35, 1906–1911. Berna A, Chafer A, Monton JB. Solubilities of essential oil components of orange in supercritical carbon dioxide. Journal of Chemical and Engineering Data, 2000, 45, 724–727. Chafer A, Berna A, Monton JB, Mulet A. High pressure solubility data of the system limonene + linalool + CO2. Journal of Chemical and Engineering Data, 2001, 46, 1145–1148. Cortesi A, Kikic I. Determination of partial molar volumes at infinite dilution of alcohols and terpenes in supercritical carbon dioxide. The Journal of Supercritical Fluids, 1996, 9, 141–145. Smith RM, Burford MD. Optimization of supercritical fluid extraction of volatile constituents from a model plant matrix. Journal of Chromatography, 1992, 600, 175–181. Iwai Y, Ichimoto M, Takada S, Okuda S, Arai Y. Entrainer effect of ethanol on high-pressure vapor-liquid equilibria for supercritical carbon dioxide + limonene + linalool system. Journal of Chemical and Engineering Data, 2005, 50, 1844–1847. Al-Hamimi S, Abellan Mayoral A, Cunico LP, Turner C. Carbon dioxide expanded ethanol extraction: solubility and extraction kinetics of α‑Pinene and cis-Verbenol. Analytical Chemistry, 2016, 88, 4336−4345. Poiana M, Sicari V, Mincione B. Supercritical Carbon Dioxide (SC-CO2) Extraction of Grapefruit Flavedo. Flavour and Fragrance Journal, 13, 1998, 125–130. Poiana M, Fresa R, Mincione B. Supercritical carbon dioxide extraction of bergamot peels. Extraction kinetics of oil and its components. Flavour and Fragrance Journal, 14, 1999, 358–366.

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[22] Trabelsi D, Aydi A, Wüst Zibetti A, Della Porta G, Scognamiglio M, Cricchio V, Langa E, Abderrabbaa M, Mainar A M. Supercritical extraction from citrus aurantium amara peels using CO2 with ethanol as co-solvent. Journal of Supercritical Fluids, 2016, 117, 33–39. [23] Barjaktarivica B, Sovilj M, Knez Z. Chemical composition of Juniperus communis L. fruits supercritical CO2 Extracts: dependence on pressure and extraction time. Journal of Agricultural and Food Chemistry, 2005, 53, 2630−2636. [24] Da Cruz FJ, Järvenpää EP, Huopalahti R, Sivik B. Comparison of eucalyptus camaldulensis dehn. oils from mozambique as obtained by hydrodistillation and supercritical carbon dioxide extraction. Journal of Agricultural and Food Chemistry, 2001, 49, 2339−2342. [25] Peterson A, Machmudah S, Roy CB, Goto M, Sasaki M, Hirose T. Extraction of essential oil from geranium (Pelargonium graveolens) with supercritical carbon dioxide. Journal of Chemical Technology and Biotechnology, 2006, 81, 167–172. [26] Ozer EO, Platin S, Akman U, Hortacsu O. Supercritical carbon dioxide extraction of spearmint oil from mint-plant leaves. The Canadian Journal of Chemical Engineering, 74, 1996. [27] Platin S, Ozer EO, Akman U, Hortagsu O. Equilibrium distributions of key components of spearmint oil in sub/supercritical carbon dioxide. JAOCS, 71, 1994, 8. [28] Tranchida QP, Bonaccorsi I, Dugo P, Mondello L, Dugo G. Analysis of citrus essential oils: state of the art and future perspectives. A review. Flavour and Fragrance Journal, 2012, 27, 98–123. [29] Schaffrath M, Weidmann V, Maison W. Enantioselective high performance liquid chromatography and supercritical fluid chromatography separation of spirocyclic terpenoid flavor compounds. Journal of Chromatography A, 2014, 1363, 270–277. [30] Morin P, Pichard H, Pichard H, Caude M, Rosset R. Supercritical fluid chromatography of sesquiterpene hydrocarbons on silica packed columns with on-line Fourier transform infrared detection. Journal of Chromatography 64, 1991, 125–137. [31] Kohler M, Haerdi W, Christen P, Veuthey JL. Supercritical fluid extraction and chromatography of artemisinin and artemisinic Acid. An improved method for the analysis of artemisia annua samples. Phytochemical Analysis, 8, 1997, 223–227. [32] Bicchi C, Balbo C, Rubiolo P. Packed column supercritical fluid chromatography of sesquiterpene lactones with different carbon skeletons. Journal of Chromatography A, 1997, 779, 315–320. [33] Dost K, Davidson G. Analysis of artemisinin by a packed-column supercritical fluid chromatography-atmospheric pressure chemical ionisation mass spectrometry technique. Analyst, 2003, 128(8), 1037–1042. [34] Jones MD, Avula B, Wang YH, Lu L, Zhao J, Avonto C, Isaac G, Meeker L, Yu K, Legido-Quigley C, Smith N, Khan IA. Investigating sub-2 μm particle stationary phase supercritical fluid chromatography coupled to mass spectrometry for chemical profiling of chamomile extracts. Analytica Chimica Acta, 2014, 17, 847, 61–72. [35] Strode JTB, Taylor LT, vanBeek TA. Supercritical fluid chromatography of ginkgolides A, B, C and J and bilobalide. Journal of Chromatography A, 738, 1996, 1, 115–122. [36] Xin-Guang L, Lian-Wen Q, Zhi-Ying F. Accurate analysis of ginkgolides and their hydrolyzed metabolites by analytical supercritical fluid chromatography hybrid tandem mass spectrometry. Journal of Chromatography A, 1388, 2015, 251–258. [37] Nothias LF, Boutet-Mercey S, Cachet X, De La Torre E, Laboureur L, Gallard JF, Retailleau P, Brunelle A, Dorrestein PC, Costa J, Bedoya LM, Roussi F, Leyssen P, Alcami J, Paolini J, Litaudon M, Touboul D. Environmentally friendly procedure based on supercritical fluid chromatography and tandem mass spectrometry molecular networking for the discovery of potent antiviral



[38]

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compounds from Euphorbia semiperfoliata. Journal of Natural Products, 2017, 27, 80(10), 2620–2629. Lesellier E, Destandau E, Grigoras C, Fougere L, Elfakir C. Fast separation of triterpenoids by supercritical fluid chromatography/evaporative light scattering detector. Journal of Chromatography A, 2012; 1268, 157–165. Qiao X, An R, Hunag Y, Ji S, Li L, Tzeng YM, Guo DA, Ye M. Separation of 25R/S-ergostane triterpenoids in the medicinal mushroom Antrodia camphorata using analytical supercritical-fluid chromatography. Journal of Chromatography A, 2014, 1358, 252–260. Yang R, Li Y, Liu C, Xu Y, Zhao L, Zhang T. An improvement of separation and response applying post-column compensation and one-step acetone protein precipitation for the determination of coenzyme Q10 in rat plasma by SFC-MS/MS. Journal of Chromatography B, 2016, 15, 1031, 221–226. Méjean M, Brunelle A, Touboul D. Quantification of tocopherols and tocotrienols in soybean oil by supercritical-fluid chromatography coupled to high-resolution mass spectrometry. Analytical and Bioanalytical Chemistry, 2015, 407(17), 5133–5142. Irvan Atsuta Y, Saeki T, Daimon H, Fujie K. Supercritical carbon dioxide extraction of ubiquinones and menaquinones from activated sludge. Journal of Chromatography A, 2006, 1113(1–2), 14–19. Bamba T, Fukasaki W, Kajiyama S, Ute K, Kitayama T, Kobayashi A. High-resolution analysis of polyprenols by supercritical fluid chromatography. Journal of Chromatography, 2001, 911(1), 113–117.

Alexander Marziale

6 Synthesis in supercritical carbon dioxide Abstract: Over the course of the past two decades supercritical carbon dioxide (scCO2) has found numerous applications in both industry and academia. The most prominent being supercritical fluid extraction (SFE) and supercritical fluid chromatography (SFC). Beyond that supercritical fluids in general and scCO2 in particular represent intriguing alternatives as reaction media for chemical synthesis. Supercritical carbon dioxide is abundantly available, inexpensive, non-toxic, non-flammable and offers the perspective of simplified catalyst recovery as well as product isolation. These favorable properties have sparked vivid research activities and ultimately resulted in a number of industrial-scale synthetic processes. Keywords: Physical properties, chemical synthesis, hydrogenation, environmentally friendly, green chemistry, reaction medium , solvating power, chemical reactions, product isolation, solubility, catalyst, hydroformylation, enantioselectivity, solvent, chiral, stereoselective, regioselective, oxidation, metathesis, Pauson-Khand reaction, Friedel-Krafts alkylation, Friedel-Krafts acylation, allylic alkylation, cross-coupling, organic chemistry, Heck reaction, Suzuki reaction, Sonogashira reaction, polymerization, reduction, condensation, radical, halogenation, cycloaddition, Diels-Alder reaction, combustion

6.1 Introduction The development of environmental friendly and efficient reaction procedures has become a major concern for synthetic organic chemists in both academia and industry [1]. These endeavors toward sustainable concepts in chemical synthesis have led to a considerable increase in research activities in the field of green chemistry [2]. In addition to the reduction of chemical waste and the number of synthetic steps, catalyst recovery, and safer as well as more efficient reaction conditions, another critical aspect is the application of environmental friendly solvents such as water, ionic liquids, or supercritical carbon dioxide (scCO2) [1a, 2c, 3]. Organic solvents are often hazardous, especially on large scale, due to their flammability, toxicity, and harmful environmental impact. The use of water in this context might solve some of the problems that are associated with volatile organic solvents but is at the same time hampered by

Alexander Marziale, Novartis Pharma AG, Novartis Institutes for Biomedical Research (NIBR), ­Klybeckstrasse 141, 4057 Basel, Switzerland, [email protected] https://doi.org/10.1515/9783110618983-006

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the creation of large amounts of potentially hazardous aqueous waste. scCO2, on the other hand, offers a number of advantages that make it not only particularly environmentally friendly but also render it an interesting alternative with respect to economic considerations [4]. CO2 is nontoxic, nonflammable, and abundantly available at low cost as a by-product of fermentation, combustion, and ammonia synthesis [4, 5]. The benign character and the generally favorable ecologic footprint of scCO2 are among the main drivers for replacing conventional organic solvents in chemical synthesis. The use of CO2 as a solvent, even in an industrial setting, does not contribute to the greenhouse effect since no additional carbon dioxide is created and released into the atmosphere. The previously mentioned lack of toxicity further warrants application on large scale; these factors have rendered scCO2 the solvent of choice for a variety of diverse industrial processes such as extractions [4], material processing, and polymer modification [6]. One remarkably successful application of scCO2 is the decaffeination of coffee beans, among other CO2-based processes that have been successfully implemented in the food and cosmetics industry, such as the extraction of hops, spices, flavors, and fragrances [7, 8]. On a laboratory preparative scale, scCO2 was successfully used for the extraction of biologically active natural products such as Taxol [9]. Despite the considerable potential of scCO2 in the pharmaceutical industry, supercritical fluid chromatography (SFC) remains to this date the predominant application in this area [10]. With respect to the application of scCO2 as a reaction medium in chemical industry, radical polymerizations [6] and heterogeneous hydrogenations are among the most prominent processes [11]. The manufacture of fluoropolymers is one such example for a commercial utilization of scCO2 [12]. Furthermore, the hydrogenation of isophorone to trimethylcyclohexanone at Thomas Swan & Co Ltd. and an industrial-scale hydrogenation as part of vitamin synthesis at DMS Vitamins deserve mentioning in this context [13, 14].

6.2 Physical properties of scCO2 Besides economic and ecologic factors, there are more striking reasons that make scCO2 an interesting and viable alternative to classic organic solvents. These factors are closely related to the fundamental physical properties of supercritical fluids (SCFs) in general and scCO2 in particular. An SCF is defined as a substance above its critical temperature (Tc) and pressure (Pc), which is reached at the so-called critical point. The critical point represents the highest temperature and pressure at which a substance forms an equilibrium between the gaseous state and the liquid state [15]. This behavior can easily be visualized and understood when referencing a phase diagram (Figure 6.1).



Synthesis in supercritical carbon dioxide 

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10,000 Solid

p [bar abs]

1,000

Transcritical fluid

Liquid 100

Critical point (31 °C /72.8 bar g)

10 1

Triple point (–57 °C / 4.2 bar g) –70

–40

–10

gas

50

20 T [°C]

80

110

Figure 6.1: Phase diagram for CO2.

What differentiates scCO2 from other SCFs is the straightforward accessibility of the supercritical point at 304.1282 K (31.1 °C) and 7.3773 MPa [4]. The critical conditions for other SCFs are listed in Table 6.1. The extreme conditions and in particular the high critical temperatures for these solvents severely limit their application in chemical synthesis. Table 6.1: Critical points for various solvents [16]. Critical temperature (°C)

Critical pressure (MPa)

Critical density (g/mL)

31 374 239

7.4 (73 atm) 22 (218 atm) 8.1 (80 atm)

0.47 0.32 0.27

CO2 Water Methanol

The properties of SCFs vary between those of liquids and gases while maintaining the inherent molecular structure of liquids. Hereby, the density varies by three orders of magnitude from 1 to 10−3 g/cm3. Other density-dependent properties such as viscosity and diffusivity can be influenced by changes in temperature and pressure [17]. An overview of these properties for gases, liquids, and SCFs is given in Table 6.2. Table 6.2: Physical properties for liquids, SCFs, and gases [18].

Density (g/cm ) Viscosity (Pa s) Diffusivity (cm2/s) 3

Liquid

SCF

Gas

1

0.1–0.5 10−4–10−5 10−3

10−3 10−5 10−3

10 10−5 −3

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While the density of a SCF is about two orders of magnitude higher than the density of a gas, it remains significantly lower than the density of a typical liquid. The closely related viscosity of SCFs is about a factor of 10–100 lower compared to a conventional liquid phase and finally the diffusivity is two orders of magnitude higher when comparing SCFs over liquids. These unique properties not only make scCO2 an ideal solvent for chromatography, but also render it an interesting reaction medium for chemical synthesis.

6.2.1 Supercritical carbon dioxide as a reaction medium As described in previous chapters, scCO2 has gained tremendous importance as an attractive solvent for extraction and separation processes over the course of the last few decades [19]. This development can in large parts be attributed to the unique physical properties of SCFs in general and scCO2 in particular. Among the most important features is the easy accessibility of the supercritical point (31.1 °C and 73.8 bar) as well as economic and ecologic factors. However, with respect to its application in chemical synthesis, another unique feature of scCO2 is most decisive and intriguing – the tunability of physical properties such as density, diffusivity, and viscosity through changes in temperature and pressure [15, 20]. Manipulating the density enables control over the solvating power of CO2. When the pressure is increased at a given temperature, the density of scCO2 will increase. While at the critical point the density is 0.47 g/cm3, it can be increased to 0.7 g/cm3 at 120 bar (40 °C). In conclusion, the solubility of scCO2 increases with density at a constant temperature. Increasing the temperature at a given density will increase the pressure in a sealed system. Remarkably, at high temperatures and given pressure, the density of scCO2 is typically lower than expected; for example, at 100 °C and 120 bar, the density is as low as 0.24 g/cm3 [20]. Consequently, at constant pressure, raising the temperature may either increase or decrease solubility. The solvation power of scCO2 as a function of density can be fine-tuned and altered by more than an order of magnitude by adjusting the temperature and pressure [4]. This introduces the possibility of controlling chemical reactions by precipitating products and facilitates purification or catalyst recovery. When working in pure scCO2, product isolation to complete dryness is achieved by simple evaporation. As mentioned earlier, viscosity and diffusivity are pressure and temperature dependent. The decreased viscosity and increased diffusivity of scCO2 compared to liquids enable high diffusion rates. For fast chemical reactions where diffusion processes can be rate limiting, the use of scCO2 as a solvent can lead to significant reaction rate enhancements [15, 21]. In addition, SCFs possess no surface tension due to their gaslike properties, and in combination with the high diffusivity this results in complete miscibility



Synthesis in supercritical carbon dioxide 

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of SCFs with gases and renders them perfect solvents for hydrogenations, hydroformylations, and other reactions involving gases. The rates of many hydrogenation reactions are proportional to the concentration of hydrogen in the liquid phase and diffusion limitations can consequently be rate limiting; this can be circumvented by performing hydrogenations and other reactions involving gases in scCO2 [17, 22]. In conclusion, scCO2 is a nonpolar solvent with a low dielectric constant and a low Hildebrand solubility parameter not unlike saturated organic solvents and consequently also have similar solvent characteristics. Despite the tunability of the solvation properties, only relatively nonpolar compounds possess significant solubility in pure scCO2 [4]. On the other hand, carbon dioxide exhibits a significant quadrupole moment and can interact with polar molecules such as water or ionic liquids and its solvation power is not generally predictable [23]. Common strategies to enhance solubility include increasing density, addition of cosolvents, and modification of the solute [24]. Modifiers such as alcohols can be added to increase polarity, which is a common practice in SFC. Alternatively, one can modify the solute to increase its solubility in scCO2. Hydrocarbon chains, organic fluorocarbons, and silanes exhibit increased solubility in scCO2 through increased solute–solvent Van der Waals interactions [25]. Finally, scCO2 exhibits a number of additional interesting and potentially useful properties. It is chemically inert to most reaction conditions; it is nonflammable, nonprotic, only weakly Lewis acidic or basic, and inert to radical and oxidizing ­conditions [15, 26].

6.3 Chemical transformations in scCO2 6.3.1 Hydrogenation Hydrogenation reactions are among the most successful and best-studied transformations in scCO2. Much of this success can be attributed to the excellent solubility of hydrogen in scCO2, which prevents potential rate-limiting effects caused by diffusion limitations from the gas to the liquid phase [27]. However, insertion of CO2 into the metal–hydride bond of a catalytic species and the subsequent formation of formate complexes could lead to inhibition of the reaction [28].

6.3.1.1 Homogeneous hydrogenation To the best of our knowledge, the first example of a homogenous hydrogenation of organic substrates in scCO2 was the hydrogenation of a cyclopropene by MnH(CO)5. The reaction occurs via a radical mechanism and can be performed both catalyti-

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C6H5

C6H5

+

MnH(CO)5

C6H5

C6H5

C6H5 +

scCO2, 200 atm, 60 °C

C6H5 CHO

Hydrogenation product

Hydroformylation product

Scheme 6.1: Reactions of MnH(CO)5 with 3,3-dimethyl-1,2-diphenylcyclopropene.

cally (in the presence of H2 and CO) and stoichiometrically [29]. Given the nature of the catalyst, the cyclopropene can undergo either hydrogenation or hydroformylation (Scheme 6.1); in this particular case, the outcome of the reaction is driven by the choice of solvent. The selectivity of the reaction is a measure of the strength of the solvent cage and hydroformylation is favored with stronger cages. This behavior is reflected by a very low yield of 8% for the hydrogenation product when the reaction is performed in a micellar solution. On the other hand, yields as high as 66% for the hydrogenated product were reported when alkanes or scCO2 were chosen as the reaction medium (Table 6.3). Table 6.3: Reactions of MnH(CO)5 with 3,3-dimethyl-1,2-diphenylcyclopropene [30] Solvent

Micelle Pentane Hexane None scCO2

Gas (atm)

CO CO CO CO2 (5) CO2 (200)

(M)/(olefin) (mM)

8/2 89/87 3,200/1,100 20/6 20/6

T (°C)

t (h)

50 60 55 60 60

Product yield (%)

15 24 5 4 3.5

Alkane

Aldehyde

8 63 66 66 66

92 37 34 34 34

In 1998 Leitner and coworkers demonstrated that the CO2-soluble Rh catalyst Rh(hfacac)(R2PCH2CCH2PR2) (with R = C6H4-m-(CH2)2(CF2)5CF3) is an active catalyst for the regioselective hydrogenation of isoprene (Scheme 6.2) [31]. +

H2

Rh catalyst

+

scCO2, 80 °C Major

Rh catalyst: F3C

F3C

Ar2 O P Rh O P Ar2

Ar = (CH2)2(CF2)5CF3

Scheme 6.2: Regioselective hydrogenation of isoprene [31].

Minor



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 133

Despite the lowered reaction rate as compared to related catalysts in conventional media, the conjugated diene is selectively hydrogenated with only minor overreduction to the corresponding isopentane. A more recent example for a selective hydrogenation in scCO2 using perfluorinated ligands was presented by Altinel et al. in 2009. Highly selective hydrogenation of styrene to ethylbenzene was observed with low loadings (0.2 mol%) of the cationic rhodium complex and up to 96% conversion were achieved (Scheme 6.3) [32]. +

Rh catalyst

H2

H2, MeOH/scCO2, 70 °C

Rh catalyst: LRh(COD)BArF 4

L=

C8F17 PPh2 PPh2 C8F17

Scheme 6.3: Selective hydrogenation of styrene to ethylbenzene in scCO2.

The solubility of the cationic catalyst species was enhanced through utilization of perfluorinated ligands and addition of methanol to the reaction mixture.

6.3.1.2 Asymmetric hydrogenation In asymmetric hydrogenations of prochiral olefins, the enantioselectivity is often times strongly dependent on the hydrogen concentration and thus offers a handle to manipulate the outcome of the reaction [33]. The high solubility of hydrogen in scCO2 is particularly valuable in this context. Burk and Tumas investigated the asymmetric hydrogenation of α-enamides in scCO2 and other solvents such as methanol and hexane (Scheme 6.4). A cationic rhodium complex in combination with the Et-DuPHOS ligand was used under mild reaction conditions (40 °C, 14 atm partial H2 pressure). The obtained enantioselectivities in scCO2 were good to excellent with ee values as high as 99.7%. For a number of selected substrates, enantiomeric excess in scCO2 was considerably higher as compared to reactions that were performed in conventional media (Table 6.4) [34]. Noyori et al. reported the asymmetric hydrogenation of α,β-unsaturated carboxylic acids such as tiglic acid in scCO2 using a ruthenium-BINAP catalyst (Scheme 6.5). Enantioselectivities in scCO2 and in methanol were comparable with 81% and 82%, respectively (Table 6.5).

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R

COOMe

R

NHCOMe

+

Rh catalyst

H2

H2, scCO2, 40 °C

R

COOMe

R

NHCOMe

[(R,R)-Et-DuPHOS-Rh](BArF4)

Rh catalyst

Et (R,R)-Et-DuPHOS:

Et P

P Et

Et

Scheme 6.4: Asymmetric hydrogenation of α-enamides in scCO2.

Table 6.4: Asymmetric hydrogenation of a-enamides in scCO2. Product

ee [%]

COOMe

scCO2

MeOH

Hexane

96.8

81.8

76.2

84.7

62.6

69.5

NHCOMe

COOMe NHCOMe

COOH

Ru catalyst

COOH

H2, scCO2, 180 atm, 50 °C Ru catalyst

(S)-H8-binap:

Ru(OCOCH3)2[(S)-H8-binap]

PPh2

PPh2

Scheme 6.5: Asymmetric hydrogenation of α,β-unsaturated c­ arboxylic acids in scCO2.

*



 135

Synthesis in supercritical carbon dioxide 

Table 6.5: Asymmetric hydrogenation of α,β-unsaturated carboxylic acids in scCO2. Solvent

Yield (%)

ee (%)

99 23 99 100 100

81 (S) 71 (S) 89 (S) 82 (S) 73 (S)

33 7 5 30 30

scCO2 scCO2 scCO2 (RFOH)a MeOH Hexane a

Product

H2 (atm)

RFOH = CF3(CF2)6CH2OH.

The addition of a fluorinated alcohol (CF3(CF2)6CH2OH) to the reaction mixture in scCO2 increased the conversion to 99% and the enantioselectivity to 89%, respectively. The authors reasoned that the co-solvent might have either increased the solubility of the catalyst in the reaction mixture or enabled a different catalytic pathway [35]. Besides prochiral olefins ketones and imines can also be reduced in an enantioselective manner; the resulting chiral alcohols and imines are of great interest in various industries. A perfluorinated BINAP ligand was used in the asymmetric hydrogenation of β-ketoesters in scCO2 (Scheme 6.6(A) and (B)). Despite the installation of a perfluorinated tail on the BINAP ligand, small amounts of fluorinated co-solvent such as 1,1,1,3,3,3-hexafluoro-2-propanol or trifluorotoluene were necessary to trigger product formation and drive the reaction to full conversion [36]. A preformed Ir-catalyst composed of [Ir(COD)2]BArF4 and a perfluorinated phosphinooxazoline ligand was used in the hydrogenation of imines to chiral amines C6F13

(a)

Ru/L catalyst

O

O

OEt

H2, scCO2, 50 °C

OH

O OEt

*

(b)

Ligand:

PPh2 PPh2

C6F13

(c)

N Ph

Ph

Ir/L catalyst H2, scCO2, 50 °C

HN Ph *

Ph (d)

Catalyst: (C6F13(H2C)2-p-C6H4)2P

O Ir

N

Scheme 6.6: Asymmetric hydrogenation of ketones and imines in scCO2 with the corresponding catalytic systems.

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 Alexander Marziale

(Scheme 6.6(C) and (D)). Notably, a catalyst loading of only 0.09 mol% was sufficient to drive the reaction to full conversion and 80% ee in scCO2.

6.3.1.3 Heterogeneous hydrogenation In heterogeneously catalyzed processes, hydrogen is mixed with a solubilized or liquid substrate and a solid catalyst, resulting in a mass transport resistance between the gaseous and the liquid phase. Such limitations can be avoided by working in SCFs, where a homogeneous reaction mixture is formed. Due to the full miscibility of hydrogen in scCO2, the hydrogen concentration at the catalyst surface can be increased tremendously, resulting in very high reaction rates compared to conventional approaches [37]. An important application of heterogeneously catalyzed hydrogenation processes in the food industry is the saturation of vegetable oils. Hereby, the hydrogenation of fats and oils results in hardening and consequently yields the desired melting profile and textures, in combination with reduced sensitivity to oxidation and prolonged stability. In 1995 Tacke et al. reported the hydrogenation of fats and oils in scCO2, using a supported palladium catalyst. The observed space–time yields were up to six times higher compared to conventional hydrogenation processes. The utilization of scCO2 furthermore resulted in an extended catalyst lifetime and improved selectivity [38]. Hitzler and Poliakoff used a continuous flow reactor to hydrogenate a variety of organic compounds (acetophenone, cyclohexene, and 1,2-(methylendioxy)-4-nitrobenzene) in scCO2 and supercritical propane employing supported palladium catalysts (Scheme 6.7(A)—(C)). For the hydrogenation of acetophenone, the authors demonstrated how conducting chemical reactions in SCFs enables controlling reaction conditions (temperature, pressure, hydrogen concentration, etc.) by operating in a single phase. This approach enabled tuning of the selectivity of the reaction and hence maximizing the yield of a particular hydrogenation product (Scheme 6.7(A)). The hydrogenation of cyclohexene proceeded rapidly and in the absence of external heating, temperatures of up to 300 °C were observed in the catalyst bed, generated by the exothermic nature of the reaction (Scheme 6.7(B)). Finally, the reduction of 1,2-(methylendioxy)-4-­nitrobenzene to the corresponding amine was achieved with quantitative conversion at 90  °C in scCO2 (Scheme 6.7(C)) [38, 39].

6.3.1.4 Continuous hydrogenation An early example for a continuously operated hydrogenation process by Poliakoff et al. was earlier discussed in this chapter [39], as well as the commercial-scale hydrogenation of trimethylcyclohexenone at Thomas Swan & Co Ltd, which was developed in collaboration with the same group (Scheme 6.8) [7, 14].



 137

Synthesis in supercritical carbon dioxide  O

Me

5% Pd APII Deloxan

(a)

HO

Me

H2, scCO2, 90–300 °C

HO

Me

Et

+

Et

+

+

5% Pd APII Deloxan

(b)

H2, scCO2, 40–320 °C

(c)

NO2

O O

1% Pd Deloxan

O

H2, scCO2, 90 °C

O

NH2

Scheme 6.7: Continuous heterogeneous hydrogenation in scCO2. O

O

2% Pd catalyst H2, scCO2, 56–100 °C

Scheme 6.8: Continuous hydrogenation of trimethylcyclohexenone in scCO2.

Another more recent example for a continuous hydrogenation was reported by the Leitner group in 2013. The asymmetric hydrogenation of methyl propionylacetate to the corresponding hydroxy ester was performed in a biphasic system composed of an ionic liquid and scCO2. The catalyst was prepared in situ from [Ru(benzene)Cl2]2 and (S)-BINAP; with a catalyst loading of 0.2 mol%, the reaction proceeded with 77% conversion and the desired alcohol was obtained in 97% optical purity [40]. O

O

OH

0.2 mol% [Ru(benzene)Cl2]2, (S)-BINAP O

O O

[dMEIm][BTA], [Bu4N]Cl, H2, scCO2, 60 °C Me [dMEIm]:

Et

N

N Me

[BTA]:

O F3C

S

N O O

S

O CF3

Scheme 6.9: Continuous asymmetric hydrogenation of trimethylcyclohexenone in scCO2.

The Leitner group further refined the approach of using biphasic reaction mixtures consisting of ionic liquids and scCO2 for continuous hydrogenations and other chemical transformations in flow mode. Hereby, the ionic liquid holds the catalyst and is itself immobilized on a chemical support [41]. These catalyst beds are called supported ionic liquid phases (SILP) and the concept was successfully applied in the

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 Alexander Marziale O

O

O O

Rh-QUINAPHOS catalyst [EMIM][NTf2], H2, scCO2

[Rh(cod)(Sa,Rc)-1-naphthyl-QUINAPHOS)][NTf2]:

O O

*

O

O

N O P P Rh O

Scheme 6.10: Continuous asymmetric hydrogenation of dimethylitaconate in supported ionic liquid phases/scCO2.

enantioselective hydrogenation of dimethylitaconate to dimethyl-2-methylsuccinate (Scheme 6.10). An excellent optical purity of 98% was obtained consistently over 30 h in con­ tinuous flow, which corresponds to a TON of about 70,000. The pure product was obtained from a single reactor with space–time yields of up to 0.7 kg/L/h and productivities of more than 100 kg product per gram of rhodium or 14 kg per gram of ligand [42].

6.3.2 Hydroformylation 6.3.2.1 Homogeneous hydroformylation The hydroformylation reaction is a catalytic transformation of utmost importance in chemical industry; it enables the production of aldehydes from olefins and syngas, a mixture of hydrogen and carbon monoxide [43]. Aldehydes are used in various industries and are key intermediates for the synthesis of alcohols, carboxylic acids, esters, acetals, amines, and as substrates for the aldol reaction [44]. Typically, a terminal olefin is converted with syngas in solution in the presence of a solid transition metal catalyst. This constitutes a major similarity to hydrogenations. Along those lines, the hydroformylation reaction suffers from the same drawbacks – the low solubility of the gaseous reagents in organic solvents and the resulting transport limitations [15, 43]. This renders scCO2 an interesting and valuable alternative for hydroformylation ­processes.



Synthesis in supercritical carbon dioxide 

 139

The first example for a hydroformylation reaction in scCO2 was reported by Rathke et al. in 1991. The conversion of propylene to n-butyraldehyde was catalyzed by 10 mol% of Co2(CO)8 and proceeded in 88% yield at 80 °C [45]. In the late 1990s, both Leitner and Erkey developed a number of fluorinated hydroformylation catalysts in order to increase their solubility in scCO2 and consequently boost their catalytic activity in this medium. The solubility of trans-RhCl(CO) (P(p-CF3C6H4)3)2 and RhH(CO)(P(p-CF3-C6H4)3)3 was two orders of magnitude higher than the corresponding Rh-triphenylphosphine catalysts and exhibited comparable catalytic activity [15, 46]. The Leitner group reported a similar trialkylphosphine ligand with fluorinated side chains in 1997 (Scheme 6.11). The hydroformylation of oct-1-ene in scCO2 resulted in a 95% conversion and 8.5:1 selectivity toward the linear product (Scheme 6.11).

C5H11

0.125 mol% Rh(hfacac)(η4-C8H12), ligand H2, CO, scCO2, 60 °C

C6F13 Ligand:

C5H11

O

+

C5H11 O

C6F13 P

C6F13 Scheme 6.11: Hydroformylation of oct-1-ene in scCO2.

The application of trialkylphosphines as a simple alternative to fluorinated ligands resulted in comparable solubilities in scCO2. The hydroformylation of hex-­ 1-ene in presence of an in situ prepared catalyst made from Rh2(OAc)4 and PEt3 resulted in complete conversion within 2 h at 100 °C [47]. The hydroformylation of more challenging substrates such as disubstituted alkenes in scCO2 was enabled through the application of a bulky phosphite ligand, tris(2,4-di-tert-butylphenyl)phosphite, in combination with Rh(acac)(CO)2. With a catalyst loading of only 0.1 mol%, excellent conversion and selectivity toward hydroformylation at the internal position of prop-1-en-2-ylbenzene was achieved (Scheme 6.12) [48].

6.3.2.2 Asymmetric hydroformylation In asymmetric hydroformylation reactions, the branched aldehyde is the desired product. For the enantioselective hydroformylation of styrene, Rh(CO)2(acac) in com-

140 

 Alexander Marziale

0.1 mol% Rh(acac)(CO)2, ligand

CHO

H2, CO, scCO2

Ligand:

P

O

3 Scheme 6.12: Hydroformylation of prop-1-en-2-ylbenzene in scCO2.

bination with (R,S)-BINAPHOS and fluorinated variations thereof were used to catalyze the reaction (Scheme 6.13). Remarkably, the enantioselectivity was dependent on the density of scCO2, at higher densities (0.75 g/mL) the chiral induction dropped from 66% to 10% ee when using the nonfluorinated ligand system. The authors reasoned that at lower densities no true single phase SCF was present in the reaction mixture and that hydroformylation occurred in the liquid phase. At higher densities however, the conventional (R,S)-BINAPHOS ligand exhibited only low solubility, resulting in vastly reduced enantioselectivities. Through introduction of fluorinated side chains on the ligand, the solubility was improved and the enantioselectivity could be raised significantly from 66% to 94.8% ee [49]. An elegant approach to optically pure cyclic α-amino acids through a consecutive hydrogenation–hydroformylation one-pot synthesis was reported by Robinson et al. in 2005. A catalytic system consisting of an Rh(I) precursor and Et-DuPHOS was used to promote the step-wise synthesis. Under optimized conditions with careful pressure

HRh(CO)2(ligand)

CHO

H2, CO, scCO2

Ligand:

PPh2 O P

O O

C6F13

C6F13 Scheme 6.13: Asymmetric hydroformylation of styrene in scCO2.



 141

Synthesis in supercritical carbon dioxide 

Rh(I)-Et-DuPHOS

O O N H

OMe O

N H

Rh(I)-Et-DuPHOS

OMe

H2, CO, scCO2

O

H2, scCO2 N H

O

O

OMe

N

O

O

3

Selectivity: O

OMe +

N

1

OMe O

Scheme 6.14: Asymmetric one-pot synthesis of cyclic α-amino acids in scCO2.

control of both hydrogen and carbon dioxide, reduction of the terminal alkene was avoided and a yield of 88% and a selectivity toward the desired enamine of 82% were achieved. Consequently, the reactor was vented and set under pressure again with syngas and carbon dioxide. The hydroformylation of the terminal double bond was followed by spontaneous condensation to give the cyclic amino acid in a total yield of 70% and 98% enantiomeric excess (Scheme 6.14) [15, 50].

6.3.3 Photochemical and radical reactions The distinct physical properties of scCO2, that is, the low viscosity and the resulting high diffusion rates, make it an interesting solvent for radical reactions. Even though the first photochemical transformations in scCO2 were reported as early as 1987 [51], to this day examples in the literature remain scarce [15]. Johnston reported a [2 + 2] cycloaddition reaction in scCO2 and scCHF3. The photodimerization of isophorone resulted in three isomeric products; the stereoselectivity is hereby predominantly influenced by solvent reorganization and regioselectivity is controlled by both solvent polarity and solvent reorganization (Scheme 6.15). O

O h󰣍

O

O

O

+

+

SCF O

O

Scheme 6.15: Photodimerization of isophorone in SCFs.

In an effort to replace carbon tetrachloride in free radical halogenation reactions with a more environmental friendly and nontoxic alternative, Tanko et al. performed the radical bromination of toluene and ethyl benzene in scCO2 (Scheme 6.16). The corresponding benzylic bromides were isolated in good to excellent yields. In the conversion of toluene, small amounts (11%) of p-bromotoluene were found as well, originating from the competing electrophilic aromatic substitution [53].

142 

 Alexander Marziale Me

h󰜈, Br2

Br

scCO2, 40 °C

Me + Br

Br Me

h󰜈, Br2

Me

scCO2, 40 °C Scheme 6.16: Free radical halogenation of toluene and ethylbenzene with bromine in scCO2. Br AIBN, (CF3(CF2)5CH2)3SnH scCO2, 90 °C Scheme 6.17: Radical dehalogenation of bromoadamantane in scCO2.

An example for a thermally induced radical reaction in scCO2 was published by Beckman and Curran in 1997. A fluorinated tin hydride reagent was used in combination with AIBN in the reduction of bromoadamantane (Scheme 6.17) [54]. The reaction proceeded in high yield (90%) and is further characterized by clean and facile work-up through partitioning between benzene and perfluorohexane [15].

6.3.4 Cycloaddition reactions The first reports of cycloaddition reactions in scCO2 originate from the late 1980s; these pioneering works studied the effects of pressure and temperature on the selectivity and the speed of well-known Diels–Alder reactions [55]. It was found that for the conversion of cyclopentadiene with n-butyl acrylate, the diastereoselectivity was strongly affected by the density of the supercritical reaction medium, with decreasing selectivity at higher densities (Table 6.6). Table 6.6: Diastereoselectivity of the Diels–Alder ­reaction between cyclopentadiene and n-butyl acrylate. Solvent Toluene scCO2 1.03 g/mL scCO2 1.20 g/mL

Endo/exo ratio 10 24 5



 143

Synthesis in supercritical carbon dioxide 

The reaction was performed at 50 °C in the presence of 6.5 mol% of a Lewis acid catalyst (Sc(OTf)3), which drove the reaction to completion within 15 h (Scheme 6.18) [56]. O OBu

+

Sc(OTf)3

+

scCO2, 50°C

CO2Bn

CO2Bn endo

exo

Scheme 6.18: Sc(OTf)3 catalyzed Diels–Alder reaction in scCO2.

Similar investigations elucidating reaction rates and regioselectivities of Diels–Alder reactions in scCO2 were performed by Ikushima et al. They reported the cycloaddition of methyl acrylate and isoprene in scCO2 and organic solvents [57]. At 40 °C, increasing the pressure of scCO2 resulted in a faster transformation, and reaction rates were increased from 0.022 h−1 at 7.4 MPa to 0.099 h−1 at 19.1 MPa (Scheme 6.19). O

Me +

O OMe

scCO2, 40 °C

OMe Me

Me +

O OMe

Scheme 6.19: Diels–Alder reaction of isoprene and methyl acrylate in scCO2.

Furthermore, the selectivity of the reaction could also be controlled by pressure. When the reaction was performed in conventional media at ambient pressure, the 1,4-product was predominantly formed (98% yield). In a supercritical environment, the product ratio was pressure dependent (Table 6.7). Table 6.7: Pressure dependence of the Diels–Alder reaction in scCO2. Entry 1 2 3

Pressure (MPa)

Major product (yield)

15.69 20.60 7.45

1.4 (75%) 1.4 (86%) 1.3 (61%)

Notably, the 1,4-cycloaddition product is favored at higher pressures, while at the critical pressure point, the selectivity is inversed in favor of the 1,3-addition product [58]. Attempts to develop a stereoselective variant of the Diels–Alder reaction in scCO2 have been explored as well. The diastereoselectivity of the cycloaddition between

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 Alexander Marziale

Evan’s chiral dienophile and cyclopentadiene could be considerably improved upon performing the reaction in scCO2 (Scheme 6.20 and Table 6.8) [59]. O

O N

O

+

O

10 mol% Yb(ClO4)3 . 9H2O O

endo Scheme 6.20: The diastereoselective Diels–Alder reaction. Table 6.8: Diastereoselectivity in the Diels–Alder reaction. Conditions CH2Cl2, 0 °C, 2 h scCO2, 40 °C, 100 bar, 0.5 h

Yield (%)

Endo/exo

de (%)a

90 49

75/25 83/17

61 77

Endo.

a

While increased stereoselectivity is commonly associated with low temperatures, the nature of scCO2 requires performing the reaction above the critical temperature. Remarkably, the diastereoselectivity was increased to 77% under supercritical conditions at 40 °C. This phenomenon can be attributed to the unique solvation characteristics of scCO2 [43].

6.3.5 Oxidation reactions Oxidation reactions are among the most important chemical transformations and constitute some of the largest scale industrial processes. For example, the global production of ethylene oxide is estimated at 58 million tons per year [60]. Once again, the excellent miscibility of gases in CO2 under supercritical conditions makes scCO2 a viable choice for aerobic and other oxidation processes, offering potential rate benefits. A first attempt to perform the catalytic oxidation of alkenes in scCO2 was undertaken in 1998 by Haas and Kolis. They used Mo(CO)6 as the catalyst precursor and tert-butyl hydroperoxide as the oxidant in the catalytic oxidation of cyclohexene [61]. Notably, complete conversion of the substrate was achieved with only one equivalent of an aqueous solution of tBuOOH, yielding three oxidation products (Scheme 6.21).



Synthesis in supercritical carbon dioxide 

Mo(CO)6, tBuOOH

+

O

scCO2

OH

 145

OH +

OH

Scheme 6.21: The catalytic oxidation of cyclohexene in scCO2.

Hereby, anti-1,2-cyclohexanediol was formed by hydrolysis of the epoxide and correspondingly cyclohex-2-en-1-ol resulted from water elimination of the former. When an excess of anhydrous alkyl peroxide in decane was used as oxidant, cyclohexene was obtained as the sole reaction product in quantitative yield. The aerobic oxidation of cyclohexene in scCO2 was enabled through fluorinated iron porphyrin complexes (Figure 6.2). The observed selectivity for this transformation was higher under supercritical conditions (34%) than in dichloromethane s­ olution (21%) due to the absence of frequently occurring solvent oxidation. At 80 °C turnovers of up to 580 were observed over the course of 12 h [62]. Recently, the enantioselective epoxidation of styrene in scCO2 was reported. A chiral manganese salen complex bearing a fluorinated binaphtyl moiety was used to catalyze the reaction (Scheme 6.22). The desired oxirane was obtained in 85% yield and 76% ee. The formation of cleavage oxidation products was avoided and only cyclic carbonates were observed as side products, originating from reaction of the epoxide with CO2 [63]. A sharpless-type asymmetric epoxidation of allylic alcohols in liquid CO2 was reported by Tumas and coworkers in 1998 (Scheme 6.23). While the reaction gave F

R F

F

F

F

F

F

R

R

R N

F

N

F

F

Fe F

F

F

N Cl N

R R

R

F

R F

F

F F

F

F

R = H, Br

Figure 6.2: Iron porphyrin complex for the aerobic oxidation of cyclohexene in scCO2.

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 Alexander Marziale

O Mn-salen complex, tBuOOtBu scCO2

O

O *

O

+

C8F17 OMe O Mn OCOMe O N OMe N

Mn-salen complex:

C8F17 Scheme 6.22: The enantioselective epoxidation of styrene catalyzed by a manganese salen complex in scCO2. OH

Liq. CO2 OH Ligand:

OH

3.5 mol% Ti(OiPr)4, ligand O

O

O

O O

OH

Scheme 6.23: The asymmetric epoxidation of allylic alcohols in liquid CO2.

only poor enantioselectivity when performed at room temperature (93% yield, 16% ee), at 0  °C an optical purity of 87% ee at quantitative conversion could be achieved [64].

6.3.6 Other catalytic reactions 6.3.6.1 Olefin metathesis Fürstner and Leitner used standard Grubbs catalysts of the first generation for olefin metathesis in both supercritical and liquid carbon dioxide. Ring-opening metathesis polymerization of norbornene and cyclooctene proceeded in excellent yields under both conditions [65]. In later reports also, second-generation Grubbs catalysts bearing an N-heterocyclic carbene ligand were applied, namely, in ring-closing metathesis (RCM); the high catalytic activity was hereby fully retained in scCO2. Conversion of the toslyated bisallyl amine resulted in an 85% yield of the desired pyrrole (Scheme 6.24(A)).



Synthesis in supercritical carbon dioxide 

Ts N

(a)

2.5 mol% Grubbs II

Ts N

N

scCO2, 40 °C, 24 h

Cl Cl

N Ru PCy3

Ph

Grubbs II

O O

O (b)

 147

1.0 mol% Grubbs I

O

Cl Cl

scCO2, 40 °C

PCy3 Ru PCy3

Ph

Grubbs I Scheme 6.24: RCM in scCO2.

The authors found that the RCM was highly sensitive to density. The conversion of hex-5-en-1-yl undec-10-enoate to the 16-membered ring proceeded with an excellent yield of 90% at densities greater than 0.65 g/mL (Scheme 6.24(B)). At lower densities however, mostly oligomers were formed and the desired macrocyclic product was only found in 10% yield. It was reasoned that a dilution effect caused the density dependence, since at higher densities more solvent molecules are present in the reaction mixture and the resulting more dilute conditions favor the intramolecular reaction and vice versa [15, 66].

6.3.6.2 Pauson–Khand reaction The Pauson–Khand reaction is defined as the co-cyclization of an alkyne with an olefin in the presence of carbon monoxide, resulting in the formation of cyclopentanones. Jeong et al. demonstrated that this reaction can be performed in scCO2. In the intramolecular reaction of an enyne and CO, catalyzed by 2.5 mol% of octacarbonyldicobalt at 69 °C, a yield of 85% was obtained (Scheme 6.25) [67].

EtO2C EtO2C

2.5 mol% Co2(CO)8 scCO2, 69 °C

EtO2C EtO2C

O

Scheme 6.25: The Pauson–Khand reaction in scCO2.

6.3.6.3 Friedel–Crafts alkylation and acylation In 1998 Poliakoff et al. reported a continuous Friedel–Krafts alkylation in scCO2, using a fixed bed heterogeneous polysiloxane supported acid catalyst. In this example, mesitylene was converted with isopropanol as the alkylating agent (Scheme 6.26).

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 Alexander Marziale

Deloxan®

+

scCO2, 200 °C

Scheme 6.26: Continuous Friedel–Crafts alkylation in scCO2.

The reaction proceeded with good selectivity in 46% conversion, the desired mono-alkylated product was hereby formed in 40% yield, and 5% of the double-alkylated compound was found in the reaction mixture [68]. Similar reports on the continuous catalytic Friedel–Crafts acylation in scCO2 were published in 2008 by the Leitner group in Germany. They used oxophilic Lewis acids immobilized in ionic liquids to promote the Friedel–Crafts acylation of anisole with acetic anhydride (Scheme 6.27). O + MeO

O

O O

In(OTf)3 scCO2, IL, 60 °C

+ MeO

O OH

Scheme 6.27: Continuous Friedel–Crafts acylation in scCO2.

A screening of various Lewis acids identified In(OTf)3 as the catalyst of choice for this transformation. At 60 °C a conversion of 66% was achieved, which corresponds to a TON of 177. The biphasic IL/scCO2 conditions furthermore allow for straightforward recycling of the homogeneous, soluble catalyst [69].

6.3.6.4 Enantioselective allylic alkylation Asymmetric allylic alkylation offers a straightforward route to complex chiral molecules and allows for the construction of challenging structural motifs such as all carbon quaternary centers with excellent enantiocontrol [70]. This synthetically important transformation was successfully applied under supercritical conditions in the palladium-catalyzed conversion of diphenylallyl acetate and diethyl malonate (Scheme 6.28). A chiral diamidophosphite ligand bearing a carborane moiety was used to generate chiral induction and good enantioselectivity (81% ee) at moderate conversion (60%) was achieved [71]. The application of a bidentate diamidophosphite ligand resulted in quantitative conversion and the desired product was isolated in high optical purity (90%) [72].



 149

Synthesis in supercritical carbon dioxide 

OAc Ph

Ph

Monodentate ligand:

+

EtO2C

N

P

CO2Et

N Ph

EtO2C

[Pd(allyl)Cl]2, ligand Base, scCO2, 25 °C

Bidentate ligand:

Ph

Ph N

O

P N

CO2Et

O

Ph

H

H

Me

N P O N Ph

Me Scheme 6.28: Asymmetric allylic alkylation in scCO2.

6.3.7 Cross-coupling reactions Transition metal catalyzed cross-coupling reactions are among the most important and powerful synthetic transformations in organic chemistry and have become indispensable tools in various industries [73]. Attempts to apply these reactions in alternative and sustainable reaction media such as water and SCFs have been the focus of many research groups for decades [74].

6.3.7.1 Heck reaction The first successful application of a Heck reaction in scCO2 was reported in 1996 by Reiser and coworkers. They used 0.001 mol% of Pd(OAc)2 as a metal precursor in combination with PPh3 to catalyze the arylation of 2,3-dihydrofurane with iodobenzene. Extremely high turnover numbers (>106) and a quantitative yield could be achieved this way (Scheme 6.29). Furthermore, 2-phenyl-2,3-dihydrofuran was formed as the major product in a 6:1 ratio [75]. I O

+

0.001 mol% Pd(OAc)2, PPh3 scCO2, 60 °C, 24h

O

Ph

+

O

Ph

Scheme 6.29: Pd-catalyzed Heck reaction in scCO2.

In order to improve the solubility of phosphine ligands in scCO2, a fluorinated ligand was introduced and the resulting palladium complex exhibited significantly increased solubility. This novel catalytic system promoted the Heck reaction between iodobenzene and methyl acrylate in high yield (91%) (Scheme 6.30). Notably, the

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 Alexander Marziale

reaction gave superior results in scCO2 as compared to conventional solvents and also Suzuki and Sonogashira reactions were successfully performed under supercritical ­conditions [76]. I

O O

Me

O

0.05 mol% Pd(OAc)2, ligand

+

Ph

scCO2, 60 °C

O

Me

Ph P

Ligand: C6F13

C6F13

Scheme 6.30: Pd-catalyzed Heck reaction in scCO2 employing a fluorinated ligand.

A different approach to enhance the solubility of the catalyst was described by Rayner and coworkers; they used palladium salts with fluorinated counterions such as Pd(OCOCF3)2 and Pd(hfacac)2. The use of fluorinated ligands was rendered obsolete and only low catalyst loading was required to promote the intramolecular Heck reaction in scCO2 (Scheme 6.31).

I

Pd(OCOCF3)2, P(2-furyl)3 +

EtNiPr2

O

O

O

Scheme 6.31: Pd-catalyzed intramolecular Heck reaction in scCO2.

Remarkably, in scCO2 the selectivity toward the desired chromane was higher than in organic solvents and the undesired double-bond isomerization was largely ­suppressed (Table 6.9). Table 6.9: Pd-catalyzed intramolecular Heck reaction in various solvents. Conditions

Neat MeCN Toluene scCO2

Conversion (%)

95 95 85 95

Selectivity (%) Chromane

Chromene

71 24 45 83

29 76 55 17



Synthesis in supercritical carbon dioxide 

 151

6.3.7.2 Suzuki reaction Ley and Holmes developed a novel and effective phosphine-free supported Pd catalyst through microencapsulation that efficiently catalyzes a number of carbon–carbon bond-forming reactions, among them is the Suzuki–Miyaura reaction. A range of aryl halides was coupled with tolylboronic acid in scCO2 in the presence of 0.4 mol% of Pd(OAc)2 encapsulated in polyurea (MC-[Pd]) (Scheme 6.32). X R

B(OH)2

+

Me

0.4 mol% MC-[Pd], Bu4NOAc scCO2, 100 °C

Me

R Scheme 6.32: Pd catalyzed Suzuki–Miyaura reaction in scCO2.

The yields were comparable to those obtained in organic solvents. Notably, both electron-rich and electron-poor aryl halides as well as an aryl chloride were successfully converted under these conditions (Table 6.10) [77]. In a batch-wise synthesis, the heterogeneous catalyst could be recovered by means of simple filtration and recycled up to four times without loss of catalytic activity [78]. Table 6.10: Pd catalyzed Suzuki–Miyaura reaction in scCO2. Entry 1 2 3 4 5

Transformation R=H R = OMe R=F R = NO2 R = NO2

X = Br X = Br X = Br X = Br X = Cl

Yield (%) 99 60 98 78 60

The polyurea-encapsulated Pd(OAc)2 catalyst was commercialized ([PdEnCat]™) and packed into columns. This enabled the creation of a phosphine-free continuous flow Suzuki cross-coupling reaction under mild reaction conditions. The coupling of tolylboronic acid and iodobenzene at 55  °C was chosen as a model reaction. In order to isolate the desired biphenyl in high yields (up to 85%), the reaction mixture had to be passed through the [PdEnCat]-packed column three times [78]. Notably, this catalytic system was also successfully applied in the catalytic hydrogenolysis of epoxides and the palladium catalyzed transfer hydrogenation of aryl ketones to benzyl alcohols [79, 80]. A different approach to the Suzuki–Miyaura reaction in scCO2 was reported in 2011. An oxadiazoline palladium(II) complex was used to catalyze the reaction of 4-bromoanisole with phenylboronic acid in various solvents (Scheme 6.33).

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 Alexander Marziale

Br

B(OH)2

+

0.025 mol% Pd catalyst, K2CO3

MeO MeO Pd catalyst:

Cl2Pd

O N 2

Scheme 6.33: Pd-catalyzed Suzuki–Miyaura reaction in various solvents.

Remarkably, the catalytic activity in scCO2 was much higher than that in conventional organic solvents with a TON of 4,000 and a quantitative yield for the desired coupling product (Table 6.11) [81]. Table 6.11: Pd-catalyzed Suzuki–Miyaura reaction in scCO2. Entry

Conditions

Yield (%)

1 2 3 4

MeOH, reflux Toluene, reflux THF, reflux scCO2, 120 bar, 120 °C

72 67 Traces 100

6.3.8 Polymerization A number of commercial polymerization processes are currently being conducted in scCO2, due to the unique properties of this solvent. Among those characteristics are the adjustability of fluid properties, the environmental sustainability, and the inertness to polymerization conditions. Other advantages of performing polymerization reactions in CO2 are related to facile downstream processing and product isolation [15].

6.3.8.1 Free radical polymerization The enhanced solubility of fluorinated compounds in scCO2 makes this reaction medium an interesting choice for the synthesis of fluoropolymers. DeSimone and coworkers reported the free radical polymerization of fluorinated acrylates in scCO2, using AIBN as an initiator (Scheme 6.34). The polymerization reaction gave the corresponding perfluoropolymer in 65% yield, with a molecular weight of 270,000 g/mol [82].



Synthesis in supercritical carbon dioxide 

O

AIBN C7F15

O

 153

n

scCO2, 207 bar, 59.4 °C, 48 h

O

O C7F15

Scheme 6.34: Free radical chain growth polymerization of fluorinated acrylates in scCO2.

The same group reported a heterogeneous polymerization process, in which the synthesized polymer precipitated from the reaction mixture upon formation. The copolymerization of tetrafluoroethylene and an excess of perfluoro(propyl vinyl ether) in scCO2 was described. A fluorinated peroxy anhydride was used as radical starter and the desired copolymer could be isolated in quantitative yield (Scheme 6.35) [83]. F

F

F

F

+

F

F

F

OC3F7

scCO2, 207 bar, 59.4 °C, 48 h

CF3 Radical starter:

FF

Radical starter

C3F7O

FF FF

n F O(C3F7)

O O

OC3F7

O

O

CF3

Scheme 6.35: Free radical precipitation polymerization in scCO2.

6.3.8.2 Cationic polymerization An example for a cationic precipitation polymerization reaction was published in 1995. The polymerization of isobutyl vinyl ether in scCO2 was initiated by 1-isobutoxyethyl acetate and promoted with catalytic amounts of EtAlCl2 in combination with ethyl acetate (Scheme 6.36). 1. EtAICI2/ethyl acetate, initiator 2. NaOEt

O

scCO2, 345 bar, 30–60 °C Me

Initiator:

OEt n

Me

O O

O H

O

Scheme 6.36: Cationic precipitation polymerization in scCO2.

The latter was added as a weakly Lewis acidic deactivator. Upon formation, the polymer precipitated from solution. When cyclohexane was used as a reaction

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 Alexander Marziale

medium, the reaction remained homogeneous, while the yield remained equivalent to the one obtained in carbon dioxide. Both vinyl ethers and oxetanes were successfully converted through cationic polymerization. The cyclic ethers, however, were polymerized under noncritical conditions at −10 °C using BF3·THF as a catalyst. As before the reaction was performed in both carbon dioxide and an organic solvent (methylene chloride). Yields and molecular weights were similar between polymers synthesized in methylene chloride and carbon dioxide [84].

6.3.8.3 Metal-catalyzed polymerization In 2011 Leitner and Milani reported the copolymerization of CO and 4-tert-butylstyrene as well as CO and styrene in scCO2 and in CO2-expanded liquids. They used novel dicationic and monocationic palladium complexes with 2,2’-bipyridine ligands bearing perfluorinated ponytails to catalyze the reactions (Scheme 6.37).

R

+

[Pd]

CO

O

CO2, Δp

R

R = C6H5, 4-tert-Bu-C6H4

n

[Pd] =[Pd(ligand)2[X]2, [Pd(CH3)(NCCH3)(ligand)][X] Ligand:

Rf

Rf N

N

Scheme 6.37: Palladium-catalyzed polymerization in compressed CO2.

The most active catalysts resulted in productivities of up to 1.5  kg CP/g Pd and 6.1 kg CP/g Pd of predominantly syndiotactic polyketones with a MW of 149,500 and 222,000 g/mol for the monocationic and the dicationic precatalysts, respectively. The authors furthermore investigated the effect of the total pressure on the catalytic performance in the presence of a dicationic catalyst precursor (Table 6.12). Table 6.12: Effect of total pressure on copolymerization of CO and TBS. Entry 1 2 3 4

CO2 (g)

Total P (atm)

Productivity (g CP/g Pd)

MW (g/mol)

11.1 8.5 5.1 1.8

320 200 115 69

260 816 1,008 1,379

96,200 134,000 106,800 167,000



Synthesis in supercritical carbon dioxide 

 155

While under fully homogeneous conditions (11.1 g CO2, total pressure 320 atm) a highly syndiotactic polyketone with a productivity of 260 g CP/g Pd was obtained (entry 1, Table 6.12), decreasing the amount of CO2 to 8.5 g (total pressure 200 atm) resulted in the formation of a biphasic system consisting of a supercritical phase at the top and a liquid phase at the bottom. Hereby, the productivity was increased significantly to 816 g CP/g Pd, while simultaneously enhancing the molecular weight of the synthesized polyketones from 96,200 to 134,000 g/mol (entry 2, Table 6.12). Further lowering the amount of added CO2 to 1.8 g (total pressure 69 atm) resulted in even higher productivity (1,379 g CP/g Pd, 167,000 g/mol) (entry 4, Table 6.12). The authors reasoned that the higher concentration of catalyst and monomers in the liquid phase as compared to the homogeneous supercritical phase may account for the observed increase in productivity [85].

6.3.9 CO2 fixation and utilization While hydrogenation reactions have been discussed in detail earlier in this chapter, it was omitted that the very first hydrogenations in scCO2 used carbon dioxide itself as the substrate. The reduction of CO2 offers a powerful path to bulk chemicals such as methanol or formic acid. These compounds are typically prepared from CO and consequently CO2 hydrogenation could offer a safer and more sustainable approach, especially if the hydrogen can be produced using energy from renewable sources [43]. In 1994 Noyori and coworkers reported the ruthenium-catalyzed hydrogenation of CO2 under supercritical conditions (Scheme 6.38). The reactions were performed in a mixture of hydrogen and carbon dioxide at pressures over 200 bar at 50 °C. A TON of 7,200 could be reached. Notably, the observed reaction rates in scCO2 were typically two orders of magnitude higher than that in organic solvents [86].

Catalyst:

Me3P

Me3P

O

Catalyst, NEt3

CO2 + H2

scCO2, 50 °C

Cl Ru Cl

H

OH

PMe3 PMe3

Scheme 6.38: Ruthenium-catalyzed hydrogenation of CO2 under supercritical conditions.

By addition of methanol or dimethylamine to the reaction mixture, the production of methyl formate and dimethylformamide was enabled via a two-step process. Initially, the carbon dioxide is reduced to formic acid, followed by condensation with the additive (Scheme 6.39). For the synthesis of methyl formate, a turnover number

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of 3,500 was obtained, while the production of dimethylformamide reached a TON of 420,000 [87]. O

CO2 + H2 +

(CH3)2NH (CH3)OH

[Ru(PMe3)4]Cl2]

H

NMe2 O

scCO2, 80–100 °C H

OMe

Scheme 6.39: Ruthenium-catalyzed synthesis of dimethylformamide and methyl formate in scCO2.

Leitner et al. reported a continuous-flow hydrogenation of carbon dioxide to formic acid in scCO2 using a biphasic reaction system. In analogy to previous works from this group the catalyst was immobilized in an ionic liquid (Scheme 6.40). Remarkably, in this process, scCO2 was not only used as a reactant and a solvent, but also as an extractive phase for continuous removal of formic acid from the reactor, thus forcing the reaction equilibrium to adjust toward the reaction product. Isolation of the formic acid was then achieved by simple decompression of CO2. CO2 + H2

IL:

N

+ N

O

[Ru(cod)(methallyl)2], PBu4TPPMS, IL scCO2, 50°C

H

OH

– HCO2

Scheme 6.40: Continuous-flow hydrogenation of carbon dioxide to formic acid.

High catalytic activity with a TOF up to 314 h−1 was observed, exceeding the performance of comparable ruthenium catalysts in conventional solvents [88]. In the same year, the group described a ruthenium-catalyzed homogeneous hydrogenation of CO2 to methanol. The catalyst was formed in situ from [Ru(acac)3] and a tridentate Triphos (1,1,1-tris(diphenylphosphinomethyl)ethane) ligand (Scheme 6.41). CO2 + 3H2

Catalyst:

[Ru(acac)3], Triphos, HNTf2 THF/EtOH, 140 °C

MeOH + H2O

PPh2 Ph2P Ru P Ph2

Scheme 6.41: Hydrogenation of carbon dioxide to methanol.



Synthesis in supercritical carbon dioxide 

 157

In the presence of the organic acid HNTf2 (bis(trifluoromethane)sulfonimide), a TON of 221 could be reached with a hydrogen pressure of 60 bar, demonstrating the feasibility of methanol production from carbon dioxide with a single, homogeneous catalyst system [89]. Shortly thereafter, the Leitner group investigated the direct N-methylation of amines using carbon dioxide and hydrogen. While N-methylation was traditionally achieved through alkylating agents, orthoesters, dimethyl carbonate, or via reductive alkylation, using formaldehyde and formic acid, in this report carbon dioxide was utilized as a C1 building block. Using virtually the same catalytic system as for the reduction of carbon dioxide to methanol, the authors were able to methylate a variety of primary and secondary aromatic amines in good-to-excellent yields (Scheme 6.42). For the methylation of N-methylaniline, a quantitative yield was obtained. This methodology is particularly sustainable with respect to its atom efficiency [90]. H N

2.5 mol% [Ru(triphos)(tmm)], HNTf2

N

CO2/H2, THF, 150 °C

[Ru(triphos)(tmm)]:

PPh2 Ph2P Ru P Ph2

Scheme 6.42: Ru-catalyzed direct N-methylation using CO2 and H2.

Utilization of carbon dioxide as a sustainable source for C1 building blocks has become a research area that has attracted much interest in recent years [91]. Along those lines Hong and coworkers developed a methodology that enables CO2 capturing from combustion and utilization of the latter in organic synthesis. Hereby, the carbon dioxide that is generated from fossil fuel combustion is captured with an alkanolamine solution forming the corresponding carbamates. The carbon dioxide is then released from the capturing solution and applied in synthetic transformations. The authors were able to demonstrate that carbon dioxide originating from combustion could be used for the catalytic carboxylation of alkynes (Scheme 6.43) [92]. A variety of alkynes could be converted this way in good yields. It was furthermore shown that the alkanolamine solution could be recycled up to 55 times without significant reduction in yields. Additionally, this approach was used in the synthesis of cyclic carbonates from carbon dioxide and epoxides. The reaction utilizes catalytic amounts of ZnBr2 in combination with an N-heterocyclic carbene ligand (Scheme 6.44). Despite the air and moisture sensitivity of such a catalytic system, the obtained yields were excellent (R = phenyl: 93%, R = benzyl: 92%) [92].

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R

1. 2.5 mol% Agl, Cs2CO3, DMF, CO2 2. HCl

H

R

25 °C, 16 h

O OH

CO2 125 °C

H N

HO

OH O

NH2

HO 25 °C CO2

Scheme 6.43: CO2 capture and release for alkyne carboxylation. O

2.0 mol% ZnBr2, 2.0 mol% K2CO3, 2.0 mol% NHC

O

O

DMSO, 80 °C, 24 h

R

O

R CO2 125 °C

HO

H N

HO

OH O

NH2

NHC:

N

N + – Cl

25 °C CO2

Scheme 6.44: Synthesis of cyclic carbonates from epoxides and CO2.

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Paola Donato, Danilo Sciarrone, Paola Dugo, Luigi Mondello

7 Applications of supercritical fluid chromatography in the field of edible lipids Abstract: Lipid analysis and profiling is a subject of great interest in different fields of research, including pharmaceutics, cosmetics, clinical, and food analysis. Among the different separation techniques, supercritical fluid chromatography (SFC) has shown a number of positive features, especially in combination with mass spectrometry. In this chapter, an overview of the feasibility of SFC for lipid analysis is given with regard to the specific application field of edible fats and oils. The topic of lipid c­ lassification and nomenclature is first introduced, before discussing some representative ­examples of the application of SFC in the field of lipid analysis in foodstuffs. Keywords: supercritical fluid chromatography, lipids, edible fats, edible oils

7.1 Introduction and contents Lipid analysis and profiling is a subject of great interest in different fields of research, including pharmaceutics, cosmetics, clinical, and, eventually, food analysis [1]. As a fundamental class of biological molecules, the importance of lipids in living organisms is well established. In addition to serving as a major source of energy, lipids play a crucial role as structural components in cell membranes, and are involved in several cellular functions and metabolic processes, every single lipid class having a highly specific function [2–4]. As a consequence, the study of lipids in humans and biological fluids is of great interest both in health and disease, and a number of pathologies such as diabetes [5], obesity [6], atherosclerosis [7], schizophrenia [8], Alzheimer [9], and even cancer [10] may derive from alterations in lipid structure, function, or metabolism. Lipids are used in the pharmaceutical and cosmetic industry for biomarker ­discovery, disease prevention and treatment, as excipients and coadjuvants, transdermal carriers, and skin emolliency agents [11]. In the food industry, the ­characterization of the lipid fraction present in fats and oils is very important, since they will i­ nfluence

Paola Donato, Dipartimento di Scienze Biomediche, Odontoiatriche e delle Immagini Morfologiche e Funzionali, University of Messina, via Consolare Valeria, 98125–Messina, Italy Danilo Sciarrone Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, University of Messina, Polo Annunziata, viale Annunziata, 98168–Messina, Italy Paola Dugo and Luigi Mondello, Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, University of Messina, Polo Annunziata, viale Annunziata, 98168–Messina, Italy Chromaleont S.r.l., via Leonardo Sciascia Coop. Fede, Pal. B, 98168–Messina, Italy; University Campus Bio-Medico of Rome, via Álvaro del Portillo 21, 00128–Rome, Italy, [email protected] https://doi.org/10.1515/9783110618983-007

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the ­nutritional value of a given food and, furthermore, for their functionality and ­emulsifying qualities in food systems. Emulsifiers are very frequently used as food ­additives, as they have a strong influence on the structure and texture of many foods, and also can help to make them more appealing. Emulsifying agents are also used in food processing, and may contribute to maintain quality and freshness of final food formulations [12]. Besides the tasks of food quality and processing, the accurate determination of the food fat content has also legal implications, to assess conformity to standards of identity and nutritional labeling laws, and for the possible recovery of expensive ingredients from waste products [13]. Unlike other molecules such as carbohydrates and proteins, lipids are not characterized by a common chemical structure, and their great structural heterogeneity represents a major challenge for their comprehensive analysis. As a consequence, a variety of different instrumental techniques have been exploited for lipid separation and detection, including capillary gas chromatography (cGC) [14], thin-layer chromatography [15], high-performance and ultrahigh-performance liquid chromatography (HPLC, UHPLC) [16, 17], capillary zone electrophoresis [18], capillary electromigration [19], and supercritical fluid chromatography (SFC) [20], especially in combination with mass spectrometry (MS) [21, 22]. An extensive literature survey by Christie is freely available at the website owned and managed by the American Oil Chemists’ Society (lipidlibrary.aocs.org). Although not equally widespread, SFC may represent a convenient alternative to other conventional chromatographic techniques, commonly employed for lipid analysis, and, not surprisingly, the latter has represented one of its major application area [23]. One major advantage of using SFC for lipid analysis consists in the capability of hyphenation to universal detectors like evaporative light scattering (ELSD) and flame ionization detector (FID), beside MS. Furthermore, analysis by SFC does not require any derivatization step, unlike GC, and is capable of superior resolution in shorter analysis time with respect to HPLC [24]. SFC was very popular at the end of the twentieth century, as scientists were very interested in the unique selectivity of this separation technique, associated with a very low consumption or organic solvent and, thus, reduced costs and environmental impact, but soon declined at the shadow of more established (cGC) or expanding (HPLC) techniques. After almost 30 years, a new generation of robust commercial instruments and specifically designed separation columns have given the technique a new impetus, and new applications are being developed in different fields, including lipid research [25]. In this chapter, an overview of the feasibility of SFC for lipid analysis is given with regard to the specific application field of edible fats and oils. The topic of lipid classification and nomenclature is first introduced to facilitate the readers’ understanding of the sections that follow. The latter section will discuss some SFC applications, as representative examples of the use of this technique for lipid analysis in foodstuffs.



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7.2 Lipids: Classification and structures A very generic definition describes lipids as biological molecules characterized by poor solubility in water and high solubility in nonpolar solvents, such as hydrocarbons and ethers; however, such a description is quite inaccurate, since it does exclude several compounds that are commonly regarded as lipids, but actually do not show such behavior. In this regard, the topic of lipid classification is controversial, as no universal definition exists, unanimously accepted for such a wide range of compounds that greatly differ in their structures, properties, and functions. Not even the definition by Bloor [26], classifying lipids into simple lipoids (fats and waxes), compound lipoids (phospholipoids and glycolipoids), and derived lipoids (fatty acids [FAs], alcohols, and sterols) seems satisfying, since it indeed leaves out compounds such as terpenes and steroidal hormones, commonly regarded as lipids because of their solubility properties. According to Christie and Han [27], the term “lipids” should be restricted to “fatty acids and their derivatives, and substances related biosynthetically or functionally to these compounds”; again, terpenes and steroidal hormones would be left out. Analysts commonly distinguish between simple lipids, which on saponification would yield at most two primary hydrolysis products per mole, and complex lipids, which would produce three or more primary hydrolysis products per mole. According to this classification, triacylglycerols (TAGs), which are esters of glycerol with FAs, would be grouped into simple lipids, also termed as neutral lipids. Glycerophospholipids (PLs), which not only are esters of glycerol with FAs but also contain a polar head group, would be considered as complex or polar lipids. In the field of food science, it is also common to group lipids according to their physical state and rheological properties: those lipids that are liquid at ambient temperature are regarded as oils, while those that are solid at ambient temperature are regarded as fats. The International Union of Pure and Applied Chemists and the International Union of Biochemistry and Molecular Biology have provided a classification scheme and related definitions, based on both structural and biosynthetic properties of lipids, freely available for consultation and accessible online at http:// www.chem.qmul.ac.uk/iupac/. Classification, nomenclature, and structural representation are periodically updated on the LIPID MAPS website at http:// www.lipidmaps.org, by the International Lipid Classification and Nomenclature ­Committee. Several textbooks and articles are available, to which the interested reader is referred, dealing with the topic of lipid nomenclature [28, 29], classification [30, 31], biochemistry, and properties [32–34]. In the following sections, only major lipid classes will be illustrated, in relation to their role as food constituents, on which most applications of SFC technique have focused.

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7.2.1 Free fatty acids (FFAs) An FA is a carboxylic acid with an aliphatic chain, either saturated or unsaturated, which confers hydrophobic properties to the molecule. FAs are synthesized by chain elongation of an acetyl-CoA primer with malonyl- or methylmalonyl-CoA groups, the latter may contain heteroatoms and/or a cyclic functionality. These simple lipids ­represent the major building blocks of complex lipids and are important dietary sources of energy for animals, since they produce large quantities of adenosine triphosphate, upon metabolism. Short- and medium-chain FAs are able to cross the blood–brain barrier and thus can also fuel the cells of the central nervous system, while long-chain FAs cannot. Naturally occurring FAs in plants and animals have an unbranched chain consisting of an even number of carbon atoms (most commonly from 14 to 22), with a carboxyl group at one end and with double bonds of the cis or Z configuration in a specific position. In the systematical nomenclature, FAs are named from the saturated hydrocarbon that contains the same number of carbon atoms, by changing the final “e” to “oic.” Also trivial names and shorthand designation are frequently used. For example, the saturated (linear) FA with 18 carbon atoms, with chemical formula: CH3(CH2)16COOH, is called “octadecaenoic acid,” but in the literature it is also known by the trivial name “stearic acid.” The shorthand designation for this molecule is “18:0,” where 18 is the number of carbon atoms and 0 the number of double bonds. Table 7.1 lists the most common FAs, according to their systematic and trivial names, as well as shorthand designations. Monounsaturated FAs, called “monoenoic fatty acids,” contain one double bond usually in the cis configuration. The position of the double bond in the aliphatic chain is designated by “n-x,” where x notes the number of carbon atoms between the double bond and the carboxyl group. For example, the monounsaturated (linear) FA with 18 carbon atoms, with chemical formula: CH3(CH2)7CH=CH(CH2)7COOH, is called “cis-9-octadecenoic” acid, also found in the literature with the trivial name “oleic acid,” and shorthand designated as “18:1” or “18:1(n-9).” Its chemical structure is illustrated in Figure 7.1(A). Table 7.1: Official nomenclature, trivial names, and shorthand nomenclature of the most common FAs. Systematic name

Trivial name

Shorthand designation

Butanoic acid Hexanoic acid Octanoic acid Decanoic acid Dodecanoic acid Tetradecanoic acid Hexadecanoic acid Octadecanoic acid

Butyric acid Caproic acid Caprylic acid Capric acid Lauric acid Myristic acid Palmitic acid Stearic acid

4:0 6:0 8:0 10:0 12:0 14:0 16:0 18:0



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Table 7.1 (continued) Systematic name

Trivial name

Eicosanoic acid 9-Hexadecenoic acid 9-Octadedecenoic acid Trans-9-octadedecenoic acid 11-Octadecenoic acid 9,12-Octadecadienoic acid 9,12,15-Octadecatrienoic acid 6,9,12-Octadecatrienoic acid 5,8,11,14-Eicosatetraenoic acid 5,8,11,14,17-Eicosapentaenoic acid 4,7,10,13,16,19-Docosahexaenoic acid

Arachidic acid Palmitoleic acid Oleic acid Elaidic acid Vaccenic acid Linoleic acid α-Linoleic acid γ-Linoleic acid Arachidonic acid EPA DHA

Shorthand designation 20:0 16:1(n-7) 18:1(n-9) 18:1(n-9) 18:1(n-7) 18:2(n-6) 18:3(n-3) 18:3(n-6) 20:4(n-6) 20:5(n-3) 22:6(n-3)

O OH

(A) R

O

O

O

R1

O

R

O

R (B)

O

O

O

O O

R2

H

–O

O X

O

(C)

O

H

P

H

H H H

H

(D) HO Figure 7.1: The chemical structures of most common lipid classes: (A) a monounsaturated fatty acid (oleic acid), (B) a generic triacylglycerol, (C) a generic phospholipids, and (D) a sterol lipid (cholesterol).

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Monoenoic FAs with double bonds of the trans configuration are less common in nature; however, they are present in foodstuffs, as a result of industrial processing (hydrogenation) of oils and fats. Moreover, trans isomers are formed in the intestine of ruminants after ingestion of plants, and thus they are present in in meat and diary products. Remarkably, the length of the aliphatic chain, the degree of unsaturation, and the double bond configuration will affect the chemical properties and the physical state of these molecules. In detail, long-chain saturated FAs are less reactive and are solid at room temperature, while monounsaturated FAs are more chemically reactive and easily undergo oxidation. Among the latter, isomers of the cis configuration are prone to melt below room temperature, while trans isomers will have higher melting points. Another important subclass is represented by the polyunsaturated fatty acids (PUFAs), containing two to six methylene-interrupted double bonds in the cis ­configuration; due to the high degree of unsaturation, these compounds are liquid at room temperature, and can very easily undergo autoxidation or deterioration. PUFAs are designated in the same way as the monoenoic FAs, and regarded to as “essential FAs,” since they are required for the optimal growth of animal tissues, but cannot be synthesized by animal enzymes. Examples of this lipid subclass are “cis-9,cis-12-octadecadienoic acid” (18:2(n-6), commonly termed as “linoleic acid”), representative of the n-6 PUFAs, and “cis-9,cis-12,cis-15-octadecatrienoic acid” (18:3(n-3), commonly termed as “linolenic acid”) , representative of the n-3 PUFAs, mainly found in fish and marine organisms.

7.2.2 Triacylglycerols (TAGs) TAGs, sometimes also called triglycerides, belong to the subclass of glycerolipids (GLs), which are mono-, di-, and tri-acyl-substituted esters of glycerol with different FAs. TAGs represent the main constituents of both animal and vegetable fat; in mammalian tissues, they also comprise the bulk of storage fat. GLs are considered as simple lipids, since upon hydrolysis they would yield only glycerol, plus one (monoacylglycerols), two (diacylglycerols), or three (TAGs) mole of FAs per mole of glycerol. The chemical structure of a generic TAG is illustrated in Figure 7.1(B). As for FAs, TAGs can be classified into saturated and unsaturated, where the degree of unsaturation affects the physical state: the former (major constituents of fats) are characterized by higher melting point and tend to be solid at room temperature, while the latter (major constituents of oils) are more likely to be in a liquid state. Saturated fats are generally found in animal food sources like meat and dairy, while unsaturated fats are found in plant food sources like olives and nuts. Most natural fats and oils actually consist of complex mixtures of different TAGs and, as such, they melt over a broad range of temperatures. The three FAs esterifying the glycerol backbone may be equal or different; the FAs chain lengths may vary. However, most natural FAs found in plants and animals are typically composed of only even numbers of carbon atoms. If the first and third FA chains are different, enzymatic biosynthesis of these compounds creates a center



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of asymmetry at carbon-2 of the glycerol backbone, so that different enantiomeric forms may exist. Provided that the molecule is not racemic, and its stereochemistry is known, the official nomenclature system recommends the prefix “sn” to be placed before the name of the compound.

7.2.3 Phospholipids (PLs) PLs, also termed as glycerophospholipids, are also esters of glycerol with FAs, but also contain a polar head group; thus, they are regarded as complex, polar lipids. PLs commonly consist of two hydrophobic FA chains (equal or different) linked at a glycerol backbone (tails), and a hydrophilic phosphate group (head); the latter can be modified with different organic moieties. As a result of their amphiphilic characteristics, PLs are able to form lipid bilayers and constitute major components of cellular membranes, as well as binding sites for intra- and intercellular proteins. Furthermore, PLs play a crucial role in trafficking and signaling in eukaryotic cells, which also act as membrane-derived second messengers, or as precursors of them. These complex lipids can serve as emulsifiers (in water–oil emulsions like margarine), and are used as food additives or dietary supplement (e.g., lecithin from egg yolk). The precursor of most PLs is phosphatidic acid (PA, 1,2-diacyl-sn-glycerol3-phosphate), consisting of a glycerol backbone, with two (saturated and unsaturated) FAs at the sn-1 and sn-2 positions, and a phosphate group bonded at the sn-3 ­position. According to the type of the polar headgroup at the sn-3 position of the ­glycerol backbone, PLs are grouped into different subclasses: phosphatidylethanolamine, phosphatidylcholine (PC), phosphatidylserine, and phosphatidylinositol. The chemical structure of a generic PL is illustrated in Figure 7.1(C). In the official nomenclature, PLs are termed after the common polar moieties or head groups (X), and in turn further differentiated on the basis of the sn-1 and sn-2 FA substituents at the glycerol backbone (e.g., PC(18:1/18:1)). Those PLs containing only one FA moiety in their structures, usually linked at the sn-1 position of the glycerol backbone, are termed by adding the “lyso” prefix to the name of the corresponding GL, for example, lysophosphatidic acid (Lyso-PA), lysophosphatidylserine (Lyso-PS), and so on.

7.2.4 Sterol lipids Sterols, also known as steroid alcohols, are a subgroup of the steroids naturally occurring in plants (phytosterols) and animals (zoosterols). The most known type of sterols in animals is cholesterol, characterized by a tetracyclic ring system, with a double bond and a free hydroxyl group; its chemical structure is illustrated in Figure 7.1(D). ­Cholesterol

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is an important component of membrane lipids, maintains membrane fluidity, and also serves as a precursor to fat-soluble vitamins (vitamin D) and steroid hormones. Most common sterol lipids found in plants are sitosterol, ergosterol, and campesterol. Foods of animal origin, known for high in dietary cholesterol, include egg yolks and shrimp (sterols are also a minor fraction of total milk fat), while vegetable sterols are found in plant oils, nuts, seeds, and legumes.

7.3 SFC: Technology and applications SFC is a separation technique similar to GC and HPLC, in that it uses a chemical stationary phase (column) for selective separation of compounds in a mixture; different from both techniques, SFC uses a highly compressed gas as mobile phase that has a density similar to that of a liquid. A supercritical fluid is a substance that has gaslike compressibility and liquidlike solvating power; such behavior only occurs when the substance is above its critical temperature and pressure [35]. Besides carbon dioxide (CO2), a number of different gases can behave as SFs, including hydrogen, neon, nitrogen, argon, methane, ethane, ammonia, and water. However, CO2 is the most commonly used SF; since it is a nontoxic, nonflammable, and inexpensive solvent, furthermore conditions for supercritical state of CO2 are reached above the critical temperature of 31 °C and critical pressure of 74 bar, which can be easily maintained and are suitable for the analysis of most type of compounds. Moreover, its separation properties can be widely and precisely manipulated by changing the pressure and temperature. As a general trend, increasing pressure at a given temperature will result in increased density and solvent strength, while an increase in temperature at a constant pressure will reduce density and solvating power. Thus, the retention becomes shorter at lower temperature, which is in contrast to normal retention behavior in GC and HPLC [36]. Today, most SFC separations are achieved using a mobile phase consisting of a mixture of CO2  and organic cosolvents, called modifiers, which are added in small amounts (typically, in the 2–40% range). Organic solvents like methanol, ethanol, isopropanol, and acetonitrile will extend the range of SFC-amenable analytes, with respect to the use of pure CO2 as mobile phase, which is highly nonpolar and therefore only allows for analysis of more hydrophobic molecules. Effects resulting from the addition of a modifier are better solubility of more polar compounds, increased elution strength of a mobile phase, and, thus, shorter retention. Elution order will follow the polarity of analytes, and chromatographic peaks will show better peak shape, with less tailing. Moreover, the addition of an organic modifier will result in a shift of the critical temperature and pressure to higher values, depending on nature and amount of the modifier itself. For a typical separation using a mobile phase gradient of methanol into CO2, ending with 20–30% of organic solvent, the critical point will reach 135 °C and 168 bar; since the first parameter would be detrimental for the



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compounds to be separated, the practical consequence is that most of SFC separations are carried out under “subcritical” conditions, rather than “supercritical” [37]. While in the past SFC was commonly performed under a pressure gradient of neat CO2 on capillary columns, currently most SFC separations utilize modified CO2 mobile phases and packed columns. Repeatability and efficiency are ensured in modern instruments, designed to allow precise and reliable control of both pressure and temperature at the column inlet and outlet. Lipid analysis and profiling is a major target of SFC applications, due to the high solubility of these molecules in SFCs, that is, carbon dioxide (CO2). SFC allows nontargeted lipidomics without the need for derivatization prior to analysis, and is amenable to coupling with most widespread detectors such as UV, ELSD, FID, and MS. Capillary SFC (cSFC) analysis of lipids with MS detection is straightforward when atmospheric pressure chemical ionization (APCI) or photoionization are used, while the use of an electrospray interface (ESI) would require a make-up solvent delivered by an additional pump for the optimal transferring of SFC-separated compounds to the MS source and subsequent ionization [38].

7.3.1 Separation of FFAs Determination of the FFAs often represent the first step in lipid analysis, since they not only serve as nutritional compounds in living organisms, but are also useful to assess food quality and properties. The type and amount of FFAs affect both the physical and the chemical properties of edible lipids including oils, fats, and waxes: their viscosity, boiling point, melting point, aroma, and taste, apart from their nutritional value. As a consequence, the analysis of FFAs may serve many purposes, like determining the origin, authenticity, and quality of an oil, and furthermore monitoring the industrial processing and storage. The FFA content is useful to assess the nutritional value of fish oils as related to the content of PUFAs (ω-3 and ω-6 FAs) with beneficial physiological effects, as well as to characterize high-quality pressed oils, such as extra virgin olive oil. FFAs are primary products of hydrolysis of TAGs and, being less stable than the latter, are prone to oxidation and degradation processes, which would cause rancidity of oils and fats. Thus, FFAs are used as a parameter for quality control during production, refining, storage, and cooking of edible oils, as the FFA content is likely to be affected by different conditions of temperature, moisture, light, oxygen, as well as acidic and alkaline solutions [39–41]. FFAs are separated according to the chain length, degree of unsaturation, and cis/ trans configuration of double bonds. SFC separation of FFAs, either in their native form or as fatty acid methyl ester (FAME) derivatives, has been accomplished by SFC using both open and/or packed columns. The use of open tubular columns is advantageous for the high efficiencies, with many stationary phases of different selectivity, achievable at low pressure drop. On the other hand, packed columns are available with any

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 Paola Donato, Danilo Sciarrone, Paola Dugo, Luigi Mondello

separation material employed in HPLC and have higher loadibility, thus allowing for higher sample capacity and reduced analysis time. Packed capillary columns deliver high efficiency at low mobile phase flow rate and, thus, reduced solvent consumption typical of the open tubular columns, and at the same time offer the advantages of packed columns in terms of short separation time and sample load [42]. A meaningful example of the employment of packed capillary SFC for the analysis and quality control of the various process steps in the refining of fish oils is shown in Figure 7.2 (A)–(C) and Figure 7.3 (A) and (B) [43]. In this work, the molecular weight distributions of FFAs and other lipid constituents of natural and processed oils have been analyzed on a fused-silica capillary column (20 m × 0.1 mm i.d. × 0.4 µm df) packed with a 5% phenyl-methylpolysiloxane DB-5 phase. The chromatographic run (160 min) was performed isothermally at 170 °C, using neat CO2 as mobile phase, under a density gradient starting from 0.300 g/mL increased to 0.468 g/mL at a rate of 0.003 g/mL/min, held constant for 30 min, and subsequently increased to 0.920 g/mL at 0.001 g/mL/min. The chromatograms in Figure 7.2(A)–(C) show the analysis of three samples of sand eel oil: crude (A), alkali-refined, bleached, and deodorized (B), alkali-refined, bleached, and interesterified with sodium methoxide (C). The nonpolar DB-5 column afforded separation of the sample components into lipid groups, and further within each group by the molecular weight; little separation was indeed attained, based on the degree of unsaturation or position of the double bond(s). Table 7.2 lists the peak numbers and retention times of the lipid compounds identified in the three samples by FID detection, together with their percentage area and relative deviation. The influence of industrial processing on the oil composition can be seen by a visual comparison of the three chromatograms: from Figure 7.2(A) and (B), it is observed how the FFAs were removed by the refining process, and the amount of cholesterol reduced; on the other hand, the deodorization process did not affect the amounts of all other compounds, as can be seen from the data listed in Table 7.2. In the chromatogram in Figure 7.2(C), it can be seen how the interesterification process led to formation of FAMEs, as expected, and also higher amounts of the heavier TAGs; it was concluded that subsequent fractionation of the TAGs resulted in a concentration of the long-chain FAs, mainly PUFAs. The SFC chromatogram obtained for a salmon crude oil is shown in Figure 7.3(A): the lipid distribution is very similar to that shown in Figure 7.2(A), except for the reduced amounts of FFAs and cholesterol. On the basis of this, the salmon oil could be overall considered of better quality than the sand eel oil. The chromatogram in Figure  7.3(B) was obtained after lipase-catalyzed transesterification of the salmon crude oil with decanoic acid methyl ester: the amount of FFAs is clearly higher than shown in Figure 7.3(A), while almost all TAGs nearly disappeared as a result of the transesterification. Further examination of the results showed high 1,3-specificity of the lipase enzyme, and furthermore demonstrated that the heavier FAs, primarily consisting of PUFAs (docosahexaenoic acid, eicosapentaenoic acid), were preferably positioned in sn-2 at the glycerol skeleton.

4 8 26

11 12

10

28

26 20 24 15 22

13

(C)

30

32

0

34 36

1

39

38

40 41 42 43

34

32

(B)

30 28 26 9 24 3 7 11 16 20 5 10 12

37

36 37 38

0

39

41

40

42

11 12 15

13

43

26 20 24 22

28

30

32

34 36 37

39

38

40 41 42

43

 Applications of supercritical fluid chromatography in the field of edible lipids 

Figure 7.2: Supercritical fluid chromatograms of sand eel oil: crude (A), alkali-refined, bleached, and deodorized (B), and alkali-refined, bleached, and randomly interesterified (C). Marked peaks: (0) solvent, others as in Table 7.2. Reproduced from Springer Nature, with permission.

(A)

0

  173

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 Paola Donato, Danilo Sciarrone, Paola Dugo, Luigi Mondello

0

36

37

34 38 32

39

30

6 10 4 8

40

41

28

13 18

11

20 24 22

42

26

43

(A) 0

C DE F

6

2

4

8

10

A 11

B

G 28

30 32

34

36 37

38 39

40

41 42

(B) Figure 7.3: Supercritical fluid chromatogram of salmon oil: crude (A) and lipase-catalyzed transesterified (B). Marked peaks: (0) solvent, (A) TG30, (B) TG34, (C)TG36, (D) TG38, (E) TG40, (F) TG42, (G) TG44, others as in Table 7.2. TG, triglyceride. Reproduced from Springer Nature, with permission.



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Table 7.2: Compounds, chromatogram peak numbers (No.), retention times (tR), and composition of the three sand eel oils.a No.

tR (min)

FAME14

1

FFA14

Compound

Oil A (area %)

Oil B (area %)

Oil C (area %)

19.11

n.p.b

n.p.

0.24 ± 0.02

2

19.36

0.08 ± 0.01

n.d.c

0.06 ± 0.01

FAME16

3

20.52

n.p.

n.p.

0.14 ± 0.01

FFA16

4

20.94

0.35 ± 0.01

n.d.

0.05 ± 0.02

FAME18

5

21.99

n.p.

n.p.

0.13 ± 0.02

FFA18

6

22.54

0.34 ± 0.02

n.d.

0.05 ± 0.01

FAME20

7

23.65

n.p.

n.p.

0.16 ± 0.02

FFA20

8

24.35

0.38 ± 0.02

n.d.

0.06 ± 0.01

FAME22

9

25.65

n.p.

n.p.

0.21 ± 0.02

FFA22

10

26.47

0.58 ± 0.02

n.d.

0.07± 0.00

SQU

11

30.73

0.03 ± 0.00

0.03 ± 0.01

0.02 ± 0.01

TOC

12

35.64

0.01 ± 0.01

0.01 ± 0.01

n.d.

CHO

13

37.69

0.74 ± 0.01

0.59 ± 0.01

0.02 ± 0.01

DG28

14

38.25

0.01 ± 0.01

0.01 ± 0.01

0.01 ± 0.00

WE32(+ DG29)

15

39.34

0.06 ± 0.00

0.06 ± 0.01

n.d.

DG30

16

41.07

0.03 ± 0.00

0.03 ± 0.01

0.08 ± 0.02

WE34(+ DG31)

17

42.12

0.08 ± 0.01

0.08 ± 0.01

0.04 ± 0.01

DG32

18

43.89

0.10 ± 0.01

0.10 ± 0.01

0.17 ± 0.02

WE36(+ DG33)

19

45.09

0.07 ± 0.01

0.08 ± 0.02

0.03 ± 0.01

DG34

20

46.72

0.17 ± 0.01

0.18 ± 0.02

0.26 ± 0.02

WE38(+ DG35)

21

48.06

0.06 ± 0.00

0.07 ± 0.01

0.03 ± 0.01

DG36

22

49.79

0.20 ± 0.01

0.20 ± 0.02

0.39 ± 0.04

WE40(+ DG37)

23

51.31

0.05 ± 0.00

0.05 ± 0.00

0.02 ± 0.01

DG38+ TG42

24

53.13

0.28 ± 0.01

0.27 ± 0.02

0.48 ± 0.04

WE42(+ DG39)

25

54.33

0.04 ± 0.01

0.02 ± 0.00

0.01 ± 0.01

DG40+ TG44

26

56.13

0.50 ± 0.02

0.50 ± 0.03

0.66 ± 0.04

WE44(+ DG41)

27

57.94

0.02 ± 0.02

0.04 ± 0.02

0.02 ± 0.01

TG46 + DG42

28

59.87

1,63 ± 0.02

1.68 ± 0.08

1.36 ± 0.03

TG47 + DG43

29

61.72

0.23 ± 0.02

0.11 ± 0.02

0.12 ± 0.06

TG48(+ DG44)

30

63.81

4.62 ± 0.01

4.68 ± 0.14

2.75 ± 0.08

CE14+ TG49

31

65.10

0.61 ± 0.05

0.52 ± 0.11

0.66 ± 0.06

TG50

32

67.92

9.10 ± 0.03

9.26 ± 0.05

5.45 ± 0.17

CE16+ TG51

33

69.83

0.95 ± 0.03

0.95 ± 0.08

0.85 ± 0.26 (continued)

176 

 Paola Donato, Danilo Sciarrone, Paola Dugo, Luigi Mondello

Table 7.2 (continued) No.

tR (min)

TG52

34

72.28

14.66 ± 0.09

Compound

Oil A (area %)

Oil B (area %)

Oil C (area %)

15.00 ± 0.04

9.40 ± 0.03

CE18+ TG53

35

74.16

c.w. TG52

c.w. TG52

1.06 ± 0.25

TG54(+ CE20)

36

76.83

16.17 ± 0.17

16.55 ± 0.26

14.00 ± 0.13

TG56(+ CE22)

37

81.43

15.67 ± 0.05

16.08 ± 0.10

16.31 ± 0.21

TG58

38

86.01

13.40 ± 0.07

13.71 ± 0.12

15.89 ± 0.26

TG60

39

90.57

9.31 ± 0.12

9.40 ± 0.09

12.96 ± 0.19

TG62

40

95.28

5.20 ± 0.19

5.39 ± 0.01

8.31 ± 0.20

TG64

41

100.07

2.79 ± 0.12

2.81 ± 0.08

5.00 ± 0.15

TG66

42

105.45

1.10 ± 0.07

1.20 ± 0.07

2.24 ± 0.27

TG68

43

111.47

0.17 ± 0.03

0.14 ± 0.03

0.35 ± 0.11

TG70

44

117.91

0.02 ± 0.01

n.d.

n.d.

d

Note: The subscript numbers refer to the number of acyl carbon atoms of each molecule, except for WEs, where the subscript number refers to the total number of carbon atoms. Reproduced from Springer Nature, with permission. a Abbreviations: FFA, free fatty acids; SQU, squalene; TOC, tocopherol; CHO, cholesterol; DG, diacylglycerol; WE, wax ester; TG, triglyceride; CE, cholesteryl ester. b n.p., Not present. Fatty acid methyl esters (FAMEs) are usually not constituents of nonesterified fish oils. c n.d., Not detected, below detection limit. d c.w.TG52, coelutes with TG52.

While earlier SFC separations were achieved on capillary columns, using FID detection and a density gradient of pure CO2, the subsequent improvements in instrument and stationary phases have pushed CO2-based separation to evolve from SFC to “ultra high-performance supercritical fluid chromatography” (UHPSFC), performed with packed columns and LC-type detectors. The limitations related to using first-­ generation instrumentation (gradient delivery was unreliable, and pressure control not fixed, due to the use of laboratory-made backpressure regulators) were overcome by substantial progress in the hardware, allowing for high precision for modified flow and electronic backpressure regulation, higher pressure limits (1,100 bar), and a remarkable reduction in void volumes. The use of state-of-the-art SFC instrumentation with sub-2 µm particle size columns (consisting of both polar and nonpolar stationary phases) has also afforded considerable improvements in the chromatographic efficiency and sensitivity, and FFAs could be separated in their native forms. Such features are illustrated in a recent work [44] focused on the separation of FFAs and other lipid constituents in fish oils of different brands, without any sample ­derivatization or saponification. A preliminary screening of different stationary phase selectivity was performed, comparing a polar column (ethyl pyridine) with one c­ ontaining an



 Applications of supercritical fluid chromatography in the field of edible lipids 

 177

­embedded polar group and an octadecyl-bonded silica (ODS); the latter provided the best separation and was carried out at 25 °C with the use of methanol as the modifier into compressed CO2, employing ELSD and ESI-MS detection. In this regard, formic acid was added to the modifier to improve the peak shapes by reducing unwanted interactions of the analytes with the residual-free silanol groups of the stationary phase, and furthermore suppress the ionization of the analytes. Figure 7.4 shows the UHPSFC-MS analysis of fish oil obtained on a 150 mm × 3.0 mm i.d., 1.8 mm d.p. ODS column, under a gradient going from 2% to 20% of methanol (with 0.1% formic acid) into neat CO2, at a mobile phase flow rate of 1.0 mL/min. Isopropanol was added as makeup solvent, at 0.2  mL/min, prior to the ESI probe, operated under polarity switching to maximize the sensitivity of detection for FFAs (negative ionization mode) and TAGs (positive ionization mode). While ELSD was employed for fast screening of the sample lipid components, the hyphenation to a triple quadrupole MS detector was undertaken to decrease detection limits and attain more positive identification of the analytes. The elution pattern was much similar to that of RP-LC, where the separation was achieved on the basis of the alkyl chain length and degree of the alkyl chain length and degree of saturation, that is, FFAs with longer chains and saturation were more retained. Detection limits of the developed UHPSFC-MS method were about 0.5 ng/FFA in full scan mode, and therefore similar to those achieved by HRGC-MS analysis of FAMEs; however, the separation obtained by UHPSFC was about three times faster. Moreover, the separation of isobaric FFA species was also obtained, based on the chain position of the double bonds contained therein, as shown in Figure 7.5. Some key applications of SFC for the analysis of FFAs can be found in References [40, 45–50].

7.3.2 Separation of TAGs TAGs represent important dietary sources of essential FAs and fat-soluble vitamins, and are used as a source of energy for the organism. As the main constituents of natural vegetable oils and animal fats, analysis of intact TAGs (without presaponification) is useful to provide a characteristic fingerprint of the food matrix. Determination of the TAG profile in foods serves many purposes, that is, confirm the origin and authenticity of oils, and detect possible adulterations; control the manufacturing process and the storage conditions; and evaluate the type and amount of compounds to be used for the formulation of nutraceuticals or to be used as food additives or nutritional supplements [51]. A huge number of possible combinations of FAs esterified at the sn-1, sn-2, and sn-3 position of the glycerol backbone exist, and they are classified according to the structure of each substituent, in terms of total carbon number (CN), as given by the sum of alkyl chain length of the three FAs; degree of unsaturation (DB), as given by the sum of double bonds contained in the three FAs; and position and configuration

100

100

2.00

%

4.00

3.00

4.00

Positive mode

3.00

3.45 255.23

FFA

3.21 3.45 301.22 327.23

5.00

6.99 868.74

7.00

7.06 868.74 7.26 916.74 7.70 870.75

7.00

7.02 868.74

6.67 6.31 842.73 888.70

6.00

5.78 814.69

6.10 840.71

6.09 840.71

6.00

6.07 840.71

5.16 834.66 5.14 834.66 5.49 4.97 860.68 786.66

5.00

TAGs

9.00

8.98 327.23

10.00

10.17 9.80 333.26 415.36

10.93 642.58

11.00

10.43 333.26

10.27 333.26

10.19 255.23

8.00

9.00

10.00

11.00

8.65 922.78 9.75 8.28 10.24 8.70 9.50 950.82 978.85 896.77 10.74 922.78 924.80 1006.88 8.94 876.80

8.05 896.78

8.00

8.36 7.31 255.23 301.22 7.69 8.32 301.22 255.23

Negative mode

Time 12.00

12.00

Figure 7.4: UHPSFC-MS of fish oil in positive and negative modes (single injection for each mode). Chromatography conditions: 0–0.5 min, 98/2 (A/B), 8 min, 96/4, 9 min, 80/20, 11 min, 80/20, 12 min, 98/2. A = CO2, B = MeOH with 0.1% (w/w) formic acid, flow: 1 mL/min, oven temperature: 25 °C, ABPR 1,500 psi, column: HSS C18 150 × 3.0 mm, 1.8 mm, make-up flow rate: 0.2 mL/min. Reproduced from Elsevier, with permission.

0 2.00

%

178   Paola Donato, Danilo Sciarrone, Paola Dugo, Luigi Mondello

100

100

0 2.00

%

2.20

2.20

2.40

2.40

2.80

2.60

2.80

3.00

3.40

3.20

3.60

3.40

3.60

C18:3(∆9,12,15)

277.22

3.20

277.22

3.00

C18:3(∆6,9,12)

2.60

305.25

C20:3(∆8,11,13)

3.80

3.80

4.00

4.00

4.20

4.20

C20:3(∆11,14,17)

305.25

4.40

4.40

4.60

4.60

4.80

4.80

Time 5.00

5.00

 Applications of supercritical fluid chromatography in the field of edible lipids 

Figure 7.5: Extracted ion chromatogram showing the separation of isobaric free fatty acid species based on the chain position of the double bonds contained therein (peak with retention time of 3.3 min is impurity). Reproduced from Elsevier, with permission.

0 2.00

%

  179

180 

 Paola Donato, Danilo Sciarrone, Paola Dugo, Luigi Mondello

of DBs in acyl chains. As in RP-HPLC, TAGs with higher CN are more h ­ ydrophobic and will be more retained on nonpolar phases; on the other hand, the number of double bonds will confer a more polar character to a molecule and, thus, TAGs will be more retained on polar phases, with the increasing degree of unsaturation. Moreover, regiospecific and/or stereospecific TAG isomers may be differentiated by determining the exact position of each of the three FAs at the glycerol backbone. First applications of SFC for TAG analysis were performed on capillary columns, packed with polar stationary phases (e.g., polyethyleneglycol, cyanopropyl–­phenyl– methylpolysiloxane, phenyl–cyanopropyl–polysiloxane) as well as nonpolar ones (e.g., phenylmethylsiloxane, polymethylsiloxane, octyl-methylpolysiloxane). Separations were achieved using neat CO2 as a mobile phase, under a density/pressure gradient, and usually at temperatures above 130 °C (occasionally, under a temperature program). Some key applications of cSFC for the analysis of TAGs can be found in [43, 45, 46, 51–61]. Nowadays, separations of TAGs are performed by SFC on packed columns (pSFC), based on ODS or silver ion-exchange columns (SIC); both techniques benefit from the use of mild temperatures (below 100  °C), so that any risk of degradation for longchain PUFAs can be avoided. A typical SFC analysis on packed columns is carried out using a gradient of small amounts of an organic modifier (usually methanol, ethanol, isopropanol, or acetonitrile) into compressed CO2, and is therefore performed under subcritical conditions. Universal detection by ELSD is widespread for fast screening of sample TAG profile, while more positive identification on the separated species may be attained by MS, most commonly APCI interfaced and, more recently, using an ESI probe. Detection of absorbance in the ultraviolet (UV) range is not capable of giving useful information, given the absence of chromophore groups with absorption wavelengths over the 210 nm, while universal refractive index detection is hampered whenever gradient elution is adopted [39]. The retention behavior of TAGs on ODS-packed columns and CO2/modifier mobile phase has been extensively studied, and the results were similar to those obtained by RP-HPLC. In fact, the retention of TAGs is related to the “equivalent carbon number”, which is given by CN-f·DB (where f is a double bond coefficient varying with the experimental conditions and the type of FA). Moreover, a prediction model for the retention of TAGs as a function of temperature has been developed, and is shown in Figure  7.6(A) and (B) [62]. For saturated TAGs, a linear relationship was found between log k′ and CN; for a chosen set of saturated TAGs, the plot of log k′ versus CN is also shown at different temperatures in Figure 7.6(A). It is evident that TAG retention (log k′) increases with the increasing CN, and is higher at the lower temperatures. For various series of saturated and unsaturated TAGs, the plot of log k′ versus DB is shown in Figure 7.6(B), at 20 °C. The Ln-L, L-O, and Ln-O series have the same CN; in the Ln-L and L-O series, DB varies by one, and in the Ln-O series by two. In the L-P series, both CN and DB are simultaneously changed. It can be seen how TAG retention (log k′) decreases with the increasing DB, allowing for faster elution of TAGs with the same CN.



1.9

1.4

; 5 °C ; 15 °C ; 25 °C

1.7

SSS

1.1

log kʹ

log kʹ

20 °C

1.2

1.3

PPP

0.9 0.7

40

LLO

PPP

LLP

LLL

0.9

LaLaLa 35

OOO

1.1 1.0

MMM

0.5 0.3

SSS

1.3

1.5

(A)

 181

 Applications of supercritical fluid chromatography in the field of edible lipids 

45 Carbon number

50

0.8

55 (B)

LnLnO LnLnL LnLnLn

0

1

2

3

4

5

6

7

8

9

Number of double bonds

Figure 7.6: (A) Plots of log k′ versus carbon number for saturated series. (B) Plots of log k′ versus number of double bonds for saturated and unsaturated TGs. Symbols: La, laurate (12:0); M, myristate (14:0); P, palmitate (16:0); S, stearate (18:0); O, oleate (18:1); L, linoleate (18:2); Ln, linolenate (18:3). Reproduced from Elsevier, with permission.

The separation mechanism in operating in pSFC on SIC is complementary in nature to pSFC on ODS columns, for structure elucidation of TAGs, in that it is controlled by the degree and distribution of unsaturations. In detail, retention will increase with the higher number of double bonds and sometimes additional separation is observed, within the same DB group, based on CN. An example is illustrated in Figure 7.7 for the separation of TAGs in a soybean oil, employing both UV and APCI-MS detection [63]. A sulfonic silica-based strong cation-exchanger column (25 cm×4.6 mm i.d., 5 µm d.p.) was loaded with silver ions, and operated at a flow rate of 1.0  mL/min, using a mixture of acetonitrile/isopropanol as the modifier. Detection performed by APCI-MS (Figure 7.7(B)) provided more information on the TAG structure with respect to UV at 210 nm (Figure 7.7(A)), but required the addition of a make-up fluid, consisting of methanol, through a T-piece placed at the column outlet before the probe. This avoided peak tailing and distortion arising from precipitation of the analytes, and furthermore assisted ionization of the column effluent. Besides separation into groups of increasing DB, an additional separation was also observed according to the CN of the three FAs linked at the glycerol backbone; TAGs having the same CN and the same DBs eluted according to the DB distribution in each FA, that is, SLL eluted before OOL. Some key applications of pSFC for the analysis of TAGs can be found in [40, 47–49, 62, 64–67] achieved on ODS columns and in [63, 68–70] achieved on SIC.

40

50

30

1=

40

2=

50

3=

(b) *MSD1 TIC, MS File (1 WORK\SOJA2MS.D) APCI, Pos, Scan

30

60

60

4=

5=

70

70

6=

OLnLn

80

80

7=

90

8=

90

min

min

Figure 7.7: SI-pSFC separation of soybean oil with UV at 210 nm (A) and APCI-MS (B). Symbols: P, palmitate (16:0); S, stearate (18:0); O, oleate (18:1); L, linoleate (18:2); Ln, linolenate (18:3). Reproduced from Elsevier, with permission.

0

100,000

200,000

300,000

400,000

500,000

600,000

0

PLP PSL

50

POO SOO

100

PPLn

150

OOO

PLL

200

POP

POS

SOS

POL SLO

LOO

SLL

OLL PLLn OLnO

250

LnLO

(a) DAD1 A, Sig=210,4 Ret=450,80 (1WORK\SOJAOIL.D)

LLLn

mAU

LnLLn

182   Paola Donato, Danilo Sciarrone, Paola Dugo, Luigi Mondello



 Applications of supercritical fluid chromatography in the field of edible lipids 

 183

7.3.3 Separation of other lipids Minor applications of SFC for lipid separation in foodstuffs have regarded the analysis of PLs and sterol esters. Due to their amphiphilic properties, PLs are used in the food industry as emulsifying agents. These compounds were separated using an apolar (silica) column using isocratic conditions and ELSD, with a mobile phase consisting of CO2 with a mixture of methanol/water/triethylamine as modifier, at low temperature (45 °C). Fractionation of the PL content of soya lecithin into classes of different polarity could be achieved, eluted in the following order: PC, PA, phosphatidylinositol, and phosphatidylethanolamine [71]. Both cSFC and pSFC has also been applied, as niche techniques, for the analysis of sterols, sterol esters and waxes in foodstuffs. Fractionation of the sterolic fraction in sunflower oil was obtained on aminopropyl silica-gel column; on this stationary phase, the more hydrophobic TAGs and diacylglycerols were first eluted along with cholesteryl esters, followed by the free sterols [41]. Some studies were also undertaken, to determine the cholesterol content of nutritional fats, oils and other foods; example are the determination of cholesterol in milk fat [72], egg yolk [73], and dietary supplements like fish oil capsules [74].

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 Paola Donato, Danilo Sciarrone, Paola Dugo, Luigi Mondello

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 Applications of supercritical fluid chromatography in the field of edible lipids 

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Index Active pharmaceutical ingredient 26, 27, 93–109 Additive(s) 21–24, 37, 40, 46, 51, 55, 58, 60, 61, 66, 95, 96, 105, 155, 164, 169, 177 Aerosol 4, 39 Alcohols 5, 17, 18, 19, 23, 24, 89, 95, 104, 116, 118, 119, 120, 131, 135, 137, 138, 145, 151, 165, 169 Allylic alkylation 148–149 Amylose 15, 16, 50, 95 Atmospheric pressure chemical ionization 10, 38, 121, 171 Back-pressure regulator 4, 37, 81, 83–84, 86, 90 Basic additive 21, 23, 95 Bile acids 61 Bioanalysis 33–66 Bioanalytical methods 33–37, 41, 42, 51 Biological molecules 163, 165 Biomarker discovery 163 Cannabinoids 53–55 Cannabis 55 Carbon dioxide 2, 10, 26, 90, 115, 117, 118, 121, 127–158, 170, 171 Catalyst 89, 127, 130, 132, 135–137, 138, 139, 143, 144, 146, 147, 148, 150–152, 154, 155, 156, 157 – catalytic system 140, 149, 151, 157 – precatalysts 154 Cellulose 15, 16, 50, 95, 117 Chemical reactions 130, 136 – chemical transformations 131–158, 137 Chemical synthesis 127–130 Chiral 1–27, 37, 42, 47–51, 66, 77, 79, 80, 86, 89, 90, 94, 95–102, 107, 109, 135, 140, 144, 145, 148 Chiral drugs separations 42 Chiral screening 18, 47, 96, 97, 99 Cholesterol 169, 170, 172, 183 Clinical research 34 Collection 4, 10, 16, 34 Combustion 128, 157 Compressibility 3, 4, 14, 38, 170 Condensation 141, 155 Connection tubing 80, 84, 90 https://doi.org/10.1515/9783110618983-008

Cosmetic(s) 113, 114, 128, 163 Cross-coupling 149, 151 Cycloaddition 141, 142, 143 Cyclone 3, 4 Degradation 23, 39, 94, 105, 106, 171, 180 Density 14, 17, 37, 39, 82, 83, 84, 90, 116, 118, 129, 130, 131, 140, 142, 147, 170, 172, 176, 180 Detectors 2–4, 10, 37, 39, 79, 80, 82–84, 87, 102, 120, 121, 164, 171, 176, 177 Diacylglycerols 168, 183 Diastereoselectivity 142–144 Dichloromethane 19, 23, 25, 95, 145 Diels–Alder reactions 142–144 Dietary supplements 169, 183 Diffusivity 17, 129, 130 Digital filters 79, 84–85, 90 Diode array detectors 4, 10 Distribution isotherm 5, 23 Diterpenes 113, 122 Drugs 1, 8, 11, 33, 34, 41–43, 47, 50, 51–55, 77, 93, 94, 96, 99, 100, 106, 108, 109 Electrospray interface 171 Electrospray ionization 38, 121 Emulsifiers 164, 169 Enantiomeric excess 12, 27, 107, 133, 141 Enantiomers 2, 4, 8, 9, 10, 11, 12, 15, 16, 18, 21, 23, 26, 27, 47, 50, 51, 77, 79, 82, 85, 86–87, 89, 90, 95, 99, 100, 107, 133, 141, 169 Enantioselective epoxidation 145–146 Enantioselectivity/Enantioselective 21, 23, 133, 135, 138, 139, 145, 139, 140, 145, 148 – enantiomeric excess 12, 27, 107, 133, 141 – optical purity 137, 138, 140, 146, 148 Environmental friendly/environmentally friendly 127, 141 – environmental sustainability 152 Epoxidation 145, 146 Essential oils 113, 114, 115, 118, 120 Ethyl pyridine 176 Eutomer 12, 23 Evaporative light-scattering detector (ELSD) 10, 37, 121, 122, 164, 171, 177, 180, 183 Extra-column dispersion 80–81 Extra-column volume 80, 82

190 

 Index

F sub/supercritical fluid 90 Fish oil 171, 172, 176, 177, 183 Flame ionization detector 36, 120, 164 Flow injection analysis 87 Fluid density 82, 118 Fluorinated 135, 139, 140, 142, 145, 149, 150, 152, 153 Food fat content 164 Food science 165 Foodstuffs 164, 168, 183 Fourier transform 65, 84, 120 Fraction collections 4 Friedel–Crafts acylation 148 Friedel–Krafts alkylation 147 Gaussian kernel 85 Glycerophospholipids 62, 66, 165, 169 Green chemistry 24, 114, 127 Halogenation 141, 142 – bromination 141 Hazardous solvents 24, 25 Heck reaction 149, 150 Hemiterpenes 113 High throughput analysis 87, 89, 90 High-throughput screening 37, 62, 77, 79 Hydrophilic interaction chromatography (HILIC) 46, 60, 61, 62 Hydroformylation 131, 132, 138–141 Hydrogenation(s) 128, 131–138, 140, 151, 155, 156, 168 Hyphenation 37, 38, 164, 177 Immobilized 6, 15, 16, 19, 23, 137, 148, 156 Lipid 33, 41, 62–66, 113, 163–183 Lipidomic analysis 41, 42, 62, 66 Lipidomics 41, 42, 58, 62, 65, 66, 171 Lipophilic 55, 66 Liquid–liquid extraction 34 Loadability 8, 15 Loading 4, 6, 8, 10, 15, 17, 107, 133, 136, 137, 139, 150 Mass flow 4, 14 Mass spectrometry 10, 34, 40, 41, 96, 118, 164 Matrix effects 34, 40–41, 53, 60, 66 Metabolic processes 163

Metabolites 33, 42, 45, 46, 47, 51, 55, 57, 58, 61, 62, 122 Metabolomics 42, 58–62 Metathesis 146 MISER 87 MISER analysis 87, 89 MISER SFC 87–90 MISER SFC analysis 90 Modifier 2–4, 6, 8, 10, 15, 17–19, 21, 23–26, 35, 37, 38, 39, 46, 51, 55, 58, 60, 66, 95, 102, 120, 131, 170, 177, 180, 181, 183 Monoterpenes 113, 118–120, 122 Narrow bore tubing 82, 83 Natural products 2, 113, 128 Nebulization 10, 38, 39 Organic chemistry 149 Outlet pressure 4, 14, 83 Oxidation 136, 144–146, 168, 171 Parallel SFC 97, 99 Pauson–Khand reaction 147 Peak broadening 5, 8, 23 Peak distortion 5, 8, 21, 35 Pharmakokinetic 45 Phosphatidic acid 169 Physical properties 17, 128–131, 141 – critical points 17, 128, 129, 130, 170 – critical temperature 128, 129, 144, 170 – supercritical point 129, 130 Polymerization 128, 146, 152–155 – copolymerization 153, 154 Polymerization conditions 152 Polysaccharide 2, 6, 15, 18, 19, 23, 50, 95, 96, 120 Polysaccharide amylose 50 Polyunsaturated fatty acids 168 Pressure drop 14, 15, 39, 87, 99, 171 Prochiral 89, 133, 135 Product isolation 130, 152, 156 Productivity 1, 6, 8, 9, 10, 12, 16, 19, 23, 95, 155 Racemization 23, 24 Radical 128, 131, 141, 142, 152, 153 Radical halogenation 141, 142 Radical polymerization 128, 152 Reaction media 149 – conventional media 133, 143

Index 

Reaction medium 128, 130, 132, 142, 152 Reduction 25, 39, 89, 117, 118, 127, 133, 136, 141, 142, 155, 157, 176 Regioselectivities/regioselectivity 141, 143 Resolution 5, 6, 8, 11, 16, 18, 23, 41, 58, 66, 79, 86, 90, 94, 95, 96, 97, 100, 103, 104, 105, 106, 109, 122, 164 Reynolds number 82 RP-LC 177 Salmon crude oil 172 Sample preparation 34–37, 51, 55 Sampling frequency 79, 87 Sand eel oil 172 Saturated fats 168 Selectivities/selectivity 6, 15, 17, 18, 19, 21, 23, 24, 34, 37, 45, 46, 55, 79, 93–95, 99, 100, 103–106, 115, 116, 132, 136, 139, 141, 142, 143, 145, 148, 150, 164, 171, 176 Sesquiterpenes 113, 118, 119, 120, 122 Sesterpenes 113 Solubility 6, 11, 16, 19, 23, 94, 113, 115, 117, 118, 130, 131, 133, 135, 138, 139, 140, 149, 150, 152, 165, 170, 171 Solvating power 39, 130, 170 – solvation properties 131 Solvent(s) 3, 5, 6, 8, 15, 16, 17, 19, 23–26, 34, 35, 37, 38, 39, 40, 46, 51, 55, 62, 66, 79, 93, 94, 95, 97, 100, 102, 105, 113, 114, 116–118, 120, 127–133, 135, 138, 141, 143, 144, 145, 147, 150, 151, 152–156, 164, 165, 170, 171, 172, 177 Sonogashira 150 Stereoselective/stereoselectivity 141, 143, 144 Steroids 45, 66, 77, 113, 169 Sterols 66, 165, 169, 170, 183

 191

Sub-2 μm 13, 34, 62, 66, 79, 86, 121, 176 Sub-2 μm particle 13, 34, 62, 86, 121, 176 Subminute separations 77 Supercritical fluid chromatography (SFC)  1, 33–66, 77–90, 93–109, 120–122, 128, 163–183 Supercritical fluid extraction (SFE) 8, 117–120 Suzuki 150–152 Suzuki reaction 151–152 – Suzuki cross-coupling reaction 151 – Suzuki–Miyaura reaction 151, 152 Synthesis 27, 127–158, 168 Tandem chromatography 99, 104 Teicoplanin 86 Terpenes 113–122, 165 Terpenoids 113, 114, 117, 119, 120, 121, 122 Tetraterpenes 113 Triacylglycerols 165, 168–169 Triple quadrupole MS 177 Triterpenes 113–122 Turbulence 82, 90 2D LC-SFC 107 2D SFC-SFC 106, 107 2-ethylpyridine 37 Ultrafast chiral separations 79, 86, 90 Ultrafast chiral SFC assay 89 Ultrafast chromatography 77 Ultrafast SFC 77, 79–80, 82–87 Viscosity 17, 82, 86, 87, 90, 99, 120, 129, 130, 141, 171 Vitamins 42, 55–58, 66, 113, 122, 128, 170, 177 Xenobiotics 33