Carbohydrates of Switch Grass (Panicum virgatum)

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PURDTJE UNIVERSITY

THIS IS TO CERTTFT THAT THE THESIS PREPARED UN DER MY SUPERVISIO N

BY_______ Edward John Deszyck ENTnxED

CARBOHYDRATES OF SWITCH GRASS (PANICUM VIRGATUM)

CO M PUES WITH THE UNIVERSITY REGULATIONS ON GRADUATION TH ESES

AND IS APPROVED BY ME A S FULFILLING THIS PART O F THE REQUIREMENTS

FOR THE DEGREE OF

Doctor of Philosophy

P r o f e s s o r iN C h a r g e o f T h e s i s

H ea d o f S ch oo l or D epa r tm en t

19

TO THE LIBRARIAN:----THIS THESIS IS NOT TO B E REGARDED A S CONFIDENTIAL.

PBO KEBSO B n r

GHAI». SCaOOXi

Foasi e— 3 . 4 9 —u

t

O BA&O S

CARBOHYDRATES OP SWITCH GRASS (PANICUM VIRGATUM)

A Thesis Submitted to the Faculty of

Purdue University

by

Edward John Deszyck

In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy February, 1950

ProQuest Number: 27712188

All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is d e p e n d e n t upon the quality of the copy subm itted. In the unlikely e v e n t that the a u thor did not send a c o m p le te m anuscript and there are missing pages, these will be noted. Also, if m aterial had to be rem oved, a n o te will ind ica te the deletion.

uest ProQuest 27712188 Published by ProQuest LLO (2019). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e M icroform Edition © ProQuest LLO. ProQuest LLO. 789 East Eisenhower Parkway P.Q. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346

ACKNOWLEDGMENT

The author wishes to express his sincere appreciation to Dr. Roy L. Whistler for his guidance and encouragement throughout the course of this work.

TABLE OF OOKTENT3 Page ABSTRACT..............................................

1

INTRODUCTION..........................................

1

REVIEW OF LITERATURE..................................

4 10

EXPERIMENTAL AND RESULTS......... Materials

.....

10

Analytical Methods.................................

10

Chemistry of Grass and its Fractions................

12

Preparation of Grass Fractions ............. Ethanol Subfractionation of Fraction D ........... Acétylation and Purification of Fraction D ...... Preparation of Holocellulose * ...... Extraction of Grass Holocellulose with Potassium Hydroxide Solutions of Various Concentrations.. Subfractionation of Fractions E, F, and G ........ Chemistry of Hémicellulose B.............. .. .....

12 14 14 15 15 17 22

Ethanol Fractionation of Hemicellulose B ......... 22 Intrinsic Viscosities of Hemicellulose BFractions 24 Anhydro-hexuronic Acid in Hemicellulose B Fractions....... 24 Specific Optical Rotation of Hemicellulose B Fractions .... 24 Preparation of Hemicellulose B for Alkali Con­ centrations Versus Anhydro-hexuronic Acid Study. .......... 28 Preparation of Hemicellulose B for Acid Hydrolysis Study............ 28 Polarimetric Study of Acid Hydrolysis of Hemi­ 28 cellulose B ................. Partial Acid Hydrolysis of Hemicellulose B by a Study of Optical Rotation...... 33 Chromotography of the Neutral Acid Hydrolyzate.... 33 lodometric Study of Acid Hydrolysis of Hemi­ cellulose B ...... *................ 35 lodometric Study of Partial Acid Hydrolysis of Hemicellulose..........«..................... 37 Acétylation of Amorphous Xylobiose............... 37 DISCUSSION............................................ Chemistry of Grass and Its Fractions.

.......

41 41

TABLE OF CONTENTS (Continued) Page Nitrogen............................... Crude Fiber ...................... Nitrogen-free Extract............. AliAia-celluloae. ............................... Pentosans............................... Anhydro-hexuronic Acid......... Reducing Sugars................. Invert Sugars................. Total Acid Hydrolyzable Sugars............. Solubles Extracted from Crass and its Preparations. ............. Ethanol Subfractionation of Fraction D. ......... Acetylated Fraction D. .................

41 42 42 42 43 43 44 45 46

The Alkaline Extraction of Holocellulose............

48

The Chromotographic Subfractionation of Fractions E, F, and C .........

49

Chemistry of Hemicellulose B...'..................

50

Ethanol Fractionation of Hemicellulose B........... Specific Optical Rotation of Hemicellulose B Fractions.............. Intrinsic Viscosities of Hemicellulose B Fractions Anhydro-hexuronic Acid in Hemicellulose B .......... Fractions Anhydrohexuronio Acid in Hemicellulose B derived From Various Concentrations of Alkali......... Acid Hydrolysis of Hemicellulose B. ................ Qualitative Study of Hemicellulose B Acid Hydrolysis.......... Polarimetric Study of Acid Hydrolysis of Hemicellulose B. ......... Partial Hydrolysis of Hemicellulose B by a Study of Optical Rotation..................... lodometric Study of Acid Hydrolysis of Hemi­ cellulose B .................................. Amorphous Xylobiose................. «............. Xylobiose Hexaacetate..................

47 47 48

50 5% 51 51 52 53 53 54 55 57 58 58

SUMMARY AND CONCLUSIONS...............................

59

BIBLIOGRAPHY............

61

LISTS OF FIGURES AND TABLES List of Figures Figure

Page

1. Per cent yield of hemicellulose A and B, and residue obtained by alkali extraction of ........... grass holocellulose at 20^).

16

2. Flow sheet for grass fractionation................

21

3. Intrinsic viscosity of hemicellulose B fractions at 250. ......................................

25

4. Per cent of anhydro-hexuronic acid in hemicellulose B fractions.....................

26

5. Specific optical rotation of hemicellulose F fractions..............

27

6. Per cent anhydro-hexuronic acid extracted from holocellulose by various potassium hydroxide concentrations at 20°. ........

29

7* 8.

9*

10.

11.

Sulfuric acid (IN and 0.5N) hydrolysis of hemicellulose Bsolutions at 80°..............

32

Sulfuric acid (0.25N and O.IN) hydrolysis of 1.% hemicellulose B solutions at 80° and reflux temperatures.............

34

Sulfuric acid (O.IN and 0.5N) hydrolysis of hemiQ cellulose B (1.6 and 2.lant is water insoluble, but the pre­ pared hemicellulose B is water soluble.

The filtrate from

hemicellulose B contains hemicellulose C.

This method and

nomenclature had been developed by 0* Dwyer (2). As a rule, grasses contain very little starch.

Instead

they have inulin-like confounds which are called fruotosans. They are composed of anhydro-fructose units and have a negative rotation.

The values of a number of fruotosans from

different sources range

Z] ^

-19^ to -55°.

Fruoto­

sans are readily soluble in cold water and insoluble in ethanol.

They are non-reducing and non-ferment able and can

be hydrolyzed by invertase, but different preparations vary a great deal to invertase hydrolysis. to ptyalin and diastase.

They are resistant

On acid hydrolysis they are very

readily attacked to yield fructose almost entirely.

In some

grasses, fruotosans are as high as 37^ of the dry grass (3)Reducing sugars found in grasses are fructose, glucose, galactose, but there is very little evidence for mannose. Fructose and glucose are predominant in grass.

Their amounts

vary considerably and may be either predominantly glucose or fructose.

Although free pentose (xylose and arabinose)

sugars have not been isolated, they may exist. saccharides, very few have been isolated. sucrose is present in grass extracts. isolated and identified (4). information in this field.

Of the di­

It is assumed that

Gentiobiose has been

In general there is very little As far as is known, no trisac­

charides have been isolated from grass extracts. Very little chemical data are available for switch grass.

Even less carbohydrate data on this grass can be

found in the literature.

The purpose of this investiga­

tion is to provide carbohydrate data, with special reference to hemicellulose B in regards to its chemical properties and its structure.

The grass was fractionated into several

fractions and each fraction was characterized.

Finally,

hemicellulose B was hydrolytically degraded and a disac­ charide (xylobiose) was isolated with the aid of carbon chromotography.

REVIEW OP LITERATURE Very little analytical literature is available on switch grass#

The analyses of 17 sanqples of this grass are reported.

Percentages for the determination were ether extract (2.25^) * crude fiber (33#52^), nitrogen-free extract (51#52^) and crude protein (6.45^).

A single value for pentosans (21.63^)

was also reported (5)# Some workers, in addition to the regular feed analysis, analyzed for carbohydrates in the grasses.

Praps, in his

studies pf digestion coefficients analyzed 10 grasses and straws for their monosaccharides (0.68-4.35/^) » disaccharides (0.07-1#41^), starches (0.05-0.90^) and soluble pentosans (6). Phillips and Smith (7) studied the composition of timo­ thy plant just prior to bloom and found glucose (2.10^), fructose (0.46^), sucrose (3#34^), and fruotosan (0.56^). Pectin was very small and Jalso some starch vdiich was doubtful.

Harper and Phillips found that at the first inter­

node of the timothy stalk fructosan values were 46.545É (8). De Man and De Heus estimated fructose and fruotosan in Lolium oerenne and found free fructose varied from 0.6^1.1^ and the fructosan from 5#0^-9#0^.

Throughout the

growing season both of these carbohydrates were the same on both young and old plants (9) #

The results were lower than

those found by Norman because of difference of preparation of samples (3)# In 1936 Norman began his investigations on the occurrence of fructosan in grass.

Fructose determinations were made in

the aqueous extracts of rye grass (Western Wolths) before and after very mild acid hydrolysis.

He deduced that the

increase in fructose after hydrolysis must be from fruoto­ san.

The fructosan content increased to a maximum value of

37^ and rapidly decreased with maturity {3 ). Norman and co-workers published the results of an in­ vestigation ofthe fructosan content of 7 grass species in Iowa.

In nearly all samples the fruotosan content ranged

between 2 and 4^ of the dry matter; the highest amount found was 7*6^ in Sudan grass at the blooming stage (10). Norman and Richardson found that the second growth of rye grass contained less fructosan than the first growth. The authors also obtained evidence for a water soluble glu­ cose polysaccharide, \diich hydrolyzed less readily than fructosan and was present in small amounts (11). In an investigation on cocksfoot grass (Dactvlis glomerata), Norman found that increase in nitrogen ferti­ lization lowered the fruotosan content.

During this experi­

ment a maximum value for fructosan was 11^ (12). De Man and De Heus worked out a method for the separate determination of aldoses, fructose, sucrose and fruotosan in grass.

The method was based on Q0% aqueous ethanol

extraction of the grass and determination for the individual reducing sugars before and after a mild hydrolysis (30 minutes in 0.05N sulfuric acid).

Fruotosan was determined

by hot water extraction of the ethanol extracted residue, hydrolysis, and determination of fructose (13)#

Ohalllnor and co-workers (4) isolated from Poa triviales a pure, water soluble, laevorotory, non-reducing polysac­ charide.

The fruotosan could be readily hydrolyzed and

fructose was the only product of hydrolysis. both the methylated and acetylated fruotosan.

They prepared The reducing

sugars in the water extract were glucose and fructose and were identified.

These two sugars appeared in equal amounts.

No mannose was detected.

A trace of mucic acid was found

on oxidation with nitric acid, which showed the presence of small amounts of galactose.

They also isolated and identi­

fied gentiobiose but no sucrose. In 1943 MbIlroy isolated a new water soluble polysac­ charide from perennial rye grass.

The polysaccharide con­

tained glucose and galactose residues.

It was suggested

by the author that the polysaccharide occurred as the calcium salt of a sulfuric ester of a galactan with one or more glucose units incorporated in the carbohydrate material (14). Fructose polysaccharides have been known for some time. One of the early fruotosans was isolated from rhizomes of Agronvron renens in I872 by Ludwig and Muller (15)#

Since

then very many fruotosans have been isolated from grasses. Their structure has been investigated but no definite results have been obtained. Colin and de Cugnao found the optical rotation of hydrolysates of two fruotosans and concluded that these materials produce 100^ hexose (I6 ).

Whether or not the

hexose is fructose and glucose or Just fructose has not been

definitely established, but it is the belief that fructose is the only sugar.

Ohallinor and co-workers found fructose

the only sugar of fructosan hydrolysis (4), vAiile Archbold and Barter (17) found 94^ fructose and 6% aldose in barley fructosan. Ohallinor and co-workers (4), through méthylation technique, concluded that the fructosan from Poa trivialis was similar to laevan and contained 10-122 fructo-furanose units linked to carbons 2 and 6.

Haworth and co-workers

(18), through the same technique, concluded that the fructo­ san from barley leaves is similar to laevan and is constituted of 10 fructo-furanose units.

The authors concluded that in

all probability these fruotosans were the same and contain about 10 hexose units per molecule linked in a straight chain. There is very little known about hemicellulose chemistry. The term hemicellulose was first proposed by Schultze in 1892 (19).

Hemicelluloses appear widely in nature, for ex­

ample, in corn cobs, grasses, soft and hard woods. does not contain any hemicellulose.

Ootton

They are laevorotatory

and do not reduce Fehling*s solution. In the preparation of hemicellulose ^ i c h is free from lignin, grasses are first subjected to a delignification process.

There are several such processes, one of vhich has

been used successfully by Wise and co-workers (20).

De­

lignification by this method produced a holocellulose \diich was usually \diite and fluffy in texture.

Holocellulose

8

consisted of nearly all of the water insoluble polysaccharides in their original chemical structure'.

Holocellulose was

extracted with alkali and from the filtrate hemicelluloses A, B, and 0 could be derived (2).

Bennett prepared holo­

cellulose from Kentucky blue grass, oat straw, and timothy in yields amounting to 74, 81, and 75^» respectively (21). Acid hydrolysis of hemicellulose A (xylan) produced mostly D-xylose, sometimes L-arabinose and small amounts of uronic acids, while hydrolysis of hemicellulose B produced D-xylose, hexoses, and uronic acids in large amounts.

Heuser

and Jayme hydrolyzed straw xylan with 3% nitric acid and obtained crystalline xylose in 85^ yield (22).

Hampton and

co-workers obtained a 935? yield of crystalline D-xylose on hydrolysis of esparto grass (23)#

Hydrolysis of esparto

xylan with 0.2^ oxalic acid produced L-arabinose (24). Norman prepared hemicellulose A and B from oat and rye straw.

He found the ratio of A:B was 3:1 for the oat straw

and 2:1 for rye straw.

Uronic acid values were determined

on hemicellulose A (5#2-10.85?) &nd hemicellulose B (28.431*8^), the lower values were found in rye straw.

Pento­

sans for oat straw (A-63^* B-56.5^) were higher than for rye straw (A-49, B-47#8J?) (25)# Hemicellulose of wheat straw contained D-glucuronic acid, L-arabinose, and D-xylose in the ratio 1:0.9:23 (26). Bennett isolated the polyuronide hemicellulose of sheep's fescue and sweet vernal grass.

In the hydrolysis products,

he found uronic acid, L-arabinose and D-xylose in the molar

ratios of,1:0.2:15.7 and 1:2.9s9#5 respectively (27)# Buston prepared hemicellulose from cocksfoot grass and fractionated It into three fractions.

He further subfrac­

tionated two of these fractions by the copper precipitation method.

On hydrolysis he identified galacturonic acid,

xylose, arabinose, and galactose (28). Hemicellulose A is known to be least soluble and first to precipitate because of its low uronic acid content. The higher w ao- the uronic acid content, the more soluble was the polysaccharide because of the presence of larger numbers of polar carboxyl groups.

For this reason, fraction 0

should contain more uronio acid than hemicellulose A or B (29). Esparto grass xylan was used by Haworth and co-workers for the determination of structure by extensive méthylation, hydrolysis and isolation of the methylated products of hydrol­ ysis.

Méthylation studies suggested a branch structure for

esparto grass xylan; however, complete proof of branching was lacking (24).

The esparto grass xylan has a chain length

(D.P.) of approximately 92 units as determined by viscosity measurements (30). The alkali insoluble holocellulose derived from hemi­ cellulose preparation is known to be alpha-cellulose.

Pento­

sans are closely associated to cellulose and for that reason are removed only with difficulty.

Potts and Bridges described

an attempt to effect a systematic and complete fractionation of paper making cellulesio materials derived from esparto

10

grass into fractions soluble in ether and ethanol.

The

authors also prepared calcium pectate, lignin fraction, hemicellulose fractions, chlorolignin and residual cellulose fractions (31). EXPERIMENTAL AND RESULTS Materials The grass was grown on soil receiving no fertilization treatment and it was harvested on August 3* 1947# approxi­ mately three weeks before blooming.

After drying in an

oven at 65^# the grass was stored in drums.

Before analysis,

it was ground in a Wiley mill to pass a 2 mm. sieve. Analytical Methods Anhydro-hexuronic acid was determined by the procedure of Whistler and co-workers (32), pentosans by the procedure of Pervier and Gortner (33)# and corrected for anhydro-hexuronic acid according to Angell and co-workers (34). Fructose, reducing sugars, 0.2N and 0.05N sulfuric acid hydrolyzable sugars were determined by the method of Harding and Downs (35) and modifiW by Van der Plank (36).

Fructo­

san was determined by method of De Man and De Heus (13). îfeterials for the total acid hydrolyzable sugars were pre­ pared, hydrolyzed, and determined according to standard pro­ cedures (37# 38).

Sucrose was determined by invertase hydrol­

ysis of the water extracts.

The aliquots were acidified with

acetic acid to pH 5# 0.5 ml. of invertase (1^) added end allowed to stand at room temperature for 12 hours.

The in­

11

crease in reducing values was considered sucrose (43)* Glarlfication of sugar solutions used for total acid hydrol­ ysis was done by addition of a slight excess of saturated neutral lead acetate solution, and precipitation of excess lead ions with a slight excess of 10^ disodium hydrogen phosphate solution. Other standard methods used were nitrogen (39)# ash (4o), moisture (4l), crude fat and fiber (42), and alphacellulose (44). Determination of the amounts of extractives removed by various solvents was made by Soxhlet extraction. (2 g. ) were extracted for 4 hours.

Samples

The amount of extractives

was determined by evaporating the solvent in a weighed dish, drying the residue in a vacuum oven at 60^ and weighing. Data are listed in Table 1. Table 1.

Per cent of solubles extracted from grass and its

holocellulose with various solvents. Ethanol Soluble 3? Grass

Followed by ethanol^benzene water ______ . %. ..

15*22

0.33

8.34

Holocellulose

1.44

0.07

4.94

Alkali insoluble hole 0ellulose (5#)

0.78

0.10

1.17

Lignin was determined by two different procedures, Ritter and Barbour (45), and Crompton and Maynard (46).

In

12

the latter method the sample was hydrolyzed with pepsin before treatment with 725? sulfuric acid.

To determine

whether an excess of sodium chlorite removed more lignin from the holocellulose, a 255? excess over the amount re­ commended was added, and lignin determined on this holo­ cellulose. Table 2.

Results are shown in Table 2. Per cent of lignin in grass and its holocellulose

by two methods. I—

■ ■ ■

- ■ . I "

I'l

»■ -.

II .I - I - I .

II

M l

I ij

■

I

li ig iin

. —

—

.II

III..

- m —

.

_Per cent lignin Ritter, Crompton, Barbour (45) Maynard (46) Grass

15.13®'

Grass Holocellulose

3*92

Grass Holocellulose (Treatment with extra 255^ sodium chloritej

3*85

Alkali insoluble holocellu­ lose (5%)

1.79

a&

13.34 3*20

-

11*96^ lignin ^dien corrected for crude protein.

Analytical results are reported on a dry weight, ashfree basis. Chemistry of Grass and its Fractions Preparation of Grass Fractions.

Approximately 500 g. of

of air-dried grass were extracted with ethanol for 24 hours The insoluble grass residue was air-dried.

The filtrate

was evaporated at 40° under vacuum to a thick syrup, vôiich

13

was washed twice with iso-octane in a Waring blender, settled and decanted.

Finally thesolubles were washed out with

ether by décantation.

The yield of the dry dark brown

residue (Fraction E) was 4.85?. Approximately 700 g. of the air-dried alcohol insoluble grass residue were extracted in a Waring blendor and allowed to stand at room temperature for 2 hours with 6 1. of water, filtered and washed with an equal volume of water v^ioh was later used to extract another 700 g. of Iherabove grass. Each batch of grass was extracted with a total of 12 1. water.

The residue was used in the preparation of holo­

cellulose.

To the 9 1. of the aqueous extract (pH 4.5) a

saturated solution of neutral lead acetate was slowly added in slight excess, vhich was removed by addition of a 105? solution of dis odium hydrogen phosphate, centrifuged and the precipitate discarded; the centrifugate was neutralized to pH 7> and evaporated under vacuum at 40® to one-twelfth of the original volume.

To this solution, 2 volumes of ethanol

were slowly added with stirring.

The mixture was filtered

and the residue washed with four separate portions of ethanol in a Waring blendor and dried in a vacuum desiccator. dry residue (Ffact ion D) was of a light cream color.

The On the

basis of the original dry grass, the yield was I.15? (4). Approximately 33 S* were prepared. The aqueous ethanol filtrate was evaporated at 40° under vacuum to approximately 0.5 1# # to which was added an equal volume of benzene.

A light brown precipitate (Fraction F)

14

was formed.

It was filtered# washed with benzene and dried.

The yield was 0.9^.

Total amount prepared was 35 6#

The aqueous ethanol-benzene filtrate was further evap­ orated under vacuum at 40° to a dark brown syrup (Fraction G); yield, 2.3^#

Amount prepared was 93 g.

Ethanol Subfraotionation of Fraction D.

Nine liters of the

aqueous grass extract were treated in the same way as in the preparation of Fraction D.

To the concentrated solu­

tion one volume of ethanol was slowly added, allowed to settle overnight, filtered, and the precipitate washed with ethanol in the usual manner (Fraction D-l).

By further additions

of one volume each of ethanol, two more fractions (D-2, D-3) were obtained.

Finally to the aqueous ethanol filtrate

3 more volumes of ethanol were added, and the precipitate collected (D-4).

Results of this sub fractionation are shown

in Table 3. Table 3#

Per cent of Fraction D derived by ethanol sub­

fractionation. Subfraction

Ethanol per cent

Fraction D on basis of original dry grass per cent

Fraction D per cent

D-l

50

0.33

30.0

D-2

67

0.70

63.6

D-3

75

0.07

6.1

D-4

86

trace

————

15

Aqetylatlon and Purification of Fraction D.

Fraction D

was further purified and acetylated In the same manner used by Ohalllnor and co-workers (4). acetate had

The purified fructosan

(o, 0.45 g. in chloroform).

This

fraction contained the fructosan polymer. Preparation of Holocellulose.

The Insoluble grass residue

from the ethanol and water extraction was used for the pre­ paration of grass holocellulose according to the method of TAilstler and co-workers (47) • The air dried product was porous and ^Ite; yield, 76.7^* Extraction of Grass Holocellulose with Potassium Hydroxide Solutions of Various 0oncentrations.

With minor modification

the method used by Whistler and co-workers was followed. Air dried holocellulose (25 g. ) was treated with 250 ml. of 2.5> 5# 10, 15» 17*5^ potassium hydroxide solutions.

Each

suspension was shaken and maintained In an atmosphere of nitrogen at 20® for 20 hours.

The mixture was filtered

and the residue was washed with dilute alkali and finally with water, and air dried.

This residue was the alkali

Insoluble grass holocellulose, yields of which are plotted In Figure 1. to pH 6. fuged.

The clear alkaline filtrate was neutralized

After standing overnight, the mixture was centri­ The precipitate (Fraction A or hemlcellulose A) (2)

was dehydrated by dispersion in ethanol and subsequent filtration.

The dried product was vhlte and finely powdered.

To the slightly opalescent centrifugate, four volumes of ethanol were slowly added with rapid stirring.

The

16

Fig. 1.

Per cent yield of hemlcellulose A, 3, and residue obtained by alkali extraction of grass holocellulose.

lO cr4

4 o ID f -t

lO ♦B iS 05 05

-p O C r4 0) o

k> 0 ID 0*1

to

lO 05

o O ?er cent yield g $ g r-l 0 3 A # hemlcellulose A B SB hemlcellulose B 0 ,Ï Alkali Insoluble holocellulose

17

slightly colored precipitate (Fraction B or hemlcellulose B) (2 ) was filtered and treated as described above*

Yields are

plotted In Figure 1. Evaporation of the filtrate to dryness under vacuum at 4o® produced Fraction C which, vhen dried, contained a high ash content*

Attempts to eliminate the ash from this frac­

tion without loss of carbohydrate materials proved unsuccess­ ful. A partial analysis of grass and Its fractions Is shown In Tables 4 and 5«

The results of the determinations of

sugars In grass and Its holocellulose fractions are found in Table 6. A flow chart of the preparation of the grass fractions Is shown on page 21. SubfraptionatIon of Fractions E, F, and G.

The method used

for subfra011ohatIon was first used by Tlsellus (48) and modified In this laboratory by Whistler and Durso (49)* Ten grams of alr-drled material was dissolved in 200 ml. of water> filtered and passed over a carbon-celite column (260 X 52 mm. )•

It was eluted with sufficient amount of

water (1.5 1# ) until eluate showed no optical rotation. After this It was followed by consecutive elutions with 1.5 1* of 5» 15» 60 and 95^ aqueous ethanol.

Each sub­

fraction was evaporated at 40® under vacuum, dried and weighed.

The residue from the water fractions was redissolved

in 80^ hot methanol and dried as before. Table 7 .

Data are shown in

18

Table 4.

Partial Analysis of Grass and Its Holocellulose

Analysis

Grass per cent

Holocellulose per cent

Asb

5.89

6.121

Nitrogen

0.79

0.29

Orude fiber Ether soluble extract

38.5

45.0 0.19

2.09

Nitrogen-free extract

42.7

42.3

Alpha-cellulese

39.0

51.0

Pentosans In alpha-cellulose

————

13.1

Pentosans

24.3

35.0

Ahhydro-hexuronlc acid Llgnln

3.68

4.58

15.13

3.92

Fructosan

1.60

————

Sucrose

2.73

————

Reducing sugars

0.91

Fructose in reducing sugars

0.81

————

33.52

36.84

Total acid hydrolyzable sugars (as glucose) Hot water extract (acid hydrolyzed) (as glucose)

5.32

Fructose in above

3.52

Ethanol and ether extracts

16.51

0.11

m #

mm

m *

1.44

19

Table 5.

Partial Analysis of Grass Fractions

îfeterlal

Nitrogen

Pentosans

Anhydrohexuronlc acid

% Fraction A

0.64

73.6

5.54

«

B

0.05

61.6

7.63

«

0

0.98

1.0

3.61

«

D

0.08

trace

————

"

E

0.23

0.7

4.36

M

P

1.21

3.4

1.25=

**

G

1.74

0.9

3.61

13.1

2.78

15.7

1.58

Alpha-cellulose (holocellulose) Alkali Insoluble holocellulose (5JÉ)

0.06

20 «s f*

»r4

EH53 Cb

(Q

•iH ai

à 5 ai u

O O

o

r4 CO

a> t>

cû CO

05

1 1 1 1

1 1 1 1

1 ■ 1 1 t

1 1 1 1

1 1 1 1

1 1 1 1

1 1 1 i

1 1 1 1

1 1 1

t

O

CO

I O q)

(â

00 ^