The polysaccharide from Ulva lactuca

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"She ^l#aaoolmyl&0 frcœs tave iaetuea” Abatreot

o£ Tlieeis subiaitteâ for the Sktgrm of Doctor of fhiXoQophy itt the iMirersity of loMon by

Doreea mrgaret Barây Hoyel ibllcwey ttoXlege,

The polyaaocharid® has oonteat of

hmu pnrltSjed to m ash

In the aeid form,

A oertala quantity

of protein mterial resisted attes^ts at res»raX, fhe purified material ha a been analysed for sulphate sad oroalc sold content, equivalent weight end periodate uptake. The carbohydrate has been subjected to baryta hydrolysle in an

to remove the sulphate groups,

end the product purified and anRlyaed as for the original polysaeoliaride •

The sulphate i@ resistant

to alkaline hydrolysis, The carbohydrate has been degrtviad to mono* and oligo*saccharides, by treatment with methanolio hydrogen chloride, and the reauiting material partially fractionated by solvent extraction,

These fractions have bean

analysed for sulphate, aronio acid and/or pentose, and

roetftyX

pmtom ooateat.

ïha altpogea which la

|sr©®aat doc a aot appaaz* to bo due to the preaaaoo

o£

m

maiao augar,

îh® fractioa®

hme all b e m esasained by the

laathod of » « # r ohroiaatography, which csoafirmed the praseaoe of rharaaos© and whidh establisbad th© proseac© of xyloae*

fho letter attgar has beea ooofiriaed as its

dibeaaylidia© dlriethyl «oetal, has

The preaeat» of glucooa

aim beea iadioatod by ohrometogrephio «vldeaco

o M fojwaeatetiott teste, «ad- the preparatloa of solid derivatives haa bean attoropted,

æ

behaviour of

igalaotose sulpha tea ia paper ohr«KHeto©E*«phy has been lavestigated.

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» «

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l a p t » ; a ;

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'.’tvr?:' ■■.ticv ■

The author wlehee to exprees her alneere thenks to Profoosor Omqrii Wllliaao, Dr. M.M.T Georg and Dr. J.».S. Br*din«,for their adrioe and encouragement throu^out this research.

/3r\Uf

cosîstms. I.

IntroductloB

...................

InTffstlgatlons already carried out on the polyeacoharide from Ulva Xaetuoa IX.

9,

General Account of Work carried out on Ulya polysaooharide Purification

...............

.

..

1

Hemoral of protein

•

9

9 9

Baryta hydrolyeia

9 9

9 9

10

Periodate oxidation

••

••

12

kethanolyaia

9

.•

4r#

.

.

14.

9

Hlaon end Morgan heaotlon ..

..

16,

Preparation of a Sehiff’a base of an affllno sugar

...............

Chromatography...........

gO. gi.

Dibensylldlne s-xyloae dlmethylaoetal

24.

Glucoe&zone

..

gd,

..

24.

..

26.

Bitric acid oxidation Fomentation

..

..........

Méthylation of sethanolysis

fraction

£4 .

Bische colourimetrlc method for detection of glucuronic acid

..

£7 .

Ehrlich reaction for galaeturonie acid £8 . Uronio cold, furfural and methylfurfural estimations ..........

£9.

XIX.

Msausslon section

IV.

Kxperiraental section.............. « Purification

..

..

..

..

..

..

1.

1.

Analysis of purified material

..

0.

Removal of protein

..

..

..

14.

Baryta hydrolysis

..

..

..

16.

Periodate oxidation

..

.*

..

19.

..

£S.

Wetbanolysis Furfural and methy1-furfural estimations

..

..

..

Elson and Morgan reaction ..

.. ..

29. 04.

Preparation of a Sohiff's hase of an amino sugar ..

..

..

..

07.

Mhensylidins i-xylose dimethylacetal 09. Olttcosasone

..

Nitric acid oxidation

..

.,

..

40.

..

..

40.

Méthylation of metbanolysls fraction 46. Fermentation

*•

BO.

..

52.

Msche oolourimetrio method for deteetion of glucuronic acid

Ehrlioh reaction for galaeturonie aeid56. Paper Chroamtography Appendix Bihliogrephy Graphs Diagrams

.,

..

58.

I

1.

IKTROaUCÎIOB The first slgsl polysaeoherlde was Isolated hy Stanford (1) from a meaher of the yhaeophyceae. in 1882, and the material was given the name alginle aeld heoause of its observed sold properties and its origin*

Prior to this algae had themselves been

hydrolysed with aeid and sugar products obtained. Since this date polysaooharide material has been isolated from many speoies of Phaeopfayeeae (brown algae) end Rhodopbyeeae (red algae).

Any attempt to

generalise about Algal Polysaccharide structures can only be in very wide terme, for, in many of the examples studied, knowledge of etructure is far from complete. It is evident, however, that they are generally acidic in nature, due to the presence of either uronio aeid or sulphuric acid eater groups, or both.

The

polyeacoharide, alginio acid, has been identified by Beleon and Créteher (R){2) and Bird and Haas (4) as a poly-D-oannuronic aeid and Hirst et al. (5) have shown the presence of fil’ A

linkages.

The first poJ^saooharlde material, in which it was established that ethereal sulphate groups were present, vms that extracted by water from the red alga Chondrus crispuB.

For a number of years the h i # ash content

I

£.

of m n y elgal polyaoooharides bad been ooomented upon end the presenoe of a large proportion of sulphate In this ash had been obeerred.

It was not, however,

until 19P.1 that Haas (6 ) noted that, althou^ the poXyeaeoharlde, from Chondrus erlspua. gave very strong réactions for lonto ealeian and sulphate vdien it had been ashed, the natural %terlal gave reactions for oaleiun only.

In addition, the large quantity of ash

m s not removed by dialysis.

Haas and Russell-;ells (?)

obtained slmilttr results on the pelysaooharldes extracted from other red and brown algae. They pat forward the theory that the sulphate was imrt of the organic polysaooharide material and that it was attached by à monosulphate ester linkage to a sugar residue. M.C.SOg.OH

where R = sugar residue.

Ignition of the salts of an ester of this type would result In the retention of half of the total sulphate In the ash, but vAen ashed In the presence of excess sodium carbonate, all the sulphate would be retained. S R.O.SOg.OKa ------ > HagSOg + 30g( •+■COg + % 0 ) E R.O.SOg.OSa + SagCOîS

>EHR2S0 4 (+ COg+HgO)

Quantitative estimations of sulphate were carried under these conditions, on the algal polysaccharides

I

0.

and résulté wore oMt lned yàiieb oonflnsed the above postulate (7). More reoent work, on the Chendrua orlanua polyeacoharide, by Perolral et

(8 ), usine

acétylation and méthylation teohnlquee, has shown galaotoee to be the main constituent, and S’îô-dlmetbyl- and R-methyl-galaetopyranose were isolated, after hydrolysis of the methylated product. A small Quantity of glucose has also been Isolated and Young and «lee (9) hare Isolated di-lsopropylldeneg-keto-n-gluoonle add, but 20-80f of the polysaooharide still remains unaccounted for. The sulphate groups were shown to be exceptionally resistant to alkaline hydrolysis, and

of the

sulphate still remained attached to the organic part of the molecule after hydrolysis with K. SaOH at lOO^C for 78 hours.

From a study of the hydrolysis of

simple sugar sulphate esters, it has been observed that hydrolysis occurs readily, with simultaneous anhydroring formation, when there is « free hydroxyl group suitably situated for the formation of a five membered hydro-furanol ring (see li) or a three membered ethylene oxide ring (see vii) by a trans substitution mechanism.

For example with methyl-/)-D-galactopyranoside

6 -sulphate (i) Percival et a^. (10 ) have shown that

I 4. WrytR treatment readily hydrolyeee the eulphate group to form Bethyl-2:6-6nhydro-/i-D-gRl»etopyr»noBlde (11), Gfijj.O.SOgH ' ' 0\

H

\ owe

H

H

H

CHg

Ba(OH)s OH

''H

^

H

OH

(1 ) Similar behaviour has been Aemonetrated (11) with furanoee sugars, and 1 :E-lsopropylldene-gluoofur&noee4 -sulphate (111 ) was readily hydrolysed by alkali to

give 1 :R-lsopropylldene-2 td-enhydroglucofuranose (Iv) and liE-lsopropylldene-gluoofuranose (r). -O3 8 .O.CH2 CH

HO.CHg HO.CH

HO.CM H\ OH H (IT)

0

CMeg

(V)

Ethylene oxide rlnge have also been obtained (IS); for example by treating barium %-methy1-1 :S-1sopro-

I 5. pyîldene-glucofaî«»o8«-6-8ulphate (t1) with soâinn methoxiae, 0 -m@tbyl-l:8-laoppopyliaene-5 ;6 -anhydrogluoofuranoae (t 11) Is ohtained, » M the sulphate group of barium 1 16-auhydro-/3-i»-galaotopyrAUQse»2sulphate (rlil) may be hydrolysed to give l;6 .g;gdlanhydro-p-D-talopyraneee (Ix).

îhe hydrolysis in

the last ease has been aocompanied by maiden inversion at the carbon atom, which carried the sulphate group. This Is oharacteristlc of the hydrolysis of both toluene-p-suphonyl and methanesulphonyl esters. “OgS.O.HgC HO.GH

liMB

----c \

J

V

K

h\

oh

H (Till)

(ix)

Heady alkaline hydrolysis of sulphate groups may therefore be oonaidered to occur ïdienever there is an adjacent, free, trans-hydroxyl group present, or when the sulphate group is on Ce of a hexose, with a free hydroxyl group on C5 or a free ois^ydroxyl group on Gg .

I 6. în the ORse of the Chendrua erispus polyaeoeharide, méthylation work showed positions 2 and 6 to he free. If it is Resumed that the sulphate groups are linked directly to the galaotoee residues, it may he eoneluded that the galaetose units are pyranooe and linked through positions 1 and 3, with a sulphate group on C4 , for if the sulphate wore in any other position, with any other sugar ring form or linkage, the sulphate would he expected to he readily hydrolysed.

oupport for this structure was oht&lned from the fact that the polysaooharide was unattacked hy the periodate ion, W&ich would oxidise any

giyool

groupings if they were present. It is of interest to note that another red alga, Gl/cartina etellata. which shows close morphological resenhlance to Chondrus erispus. contains oarhohydrate material very similar in structure to that from Chondrus orlspus (18)(Id). From other red algae, polysaooharldes of simpler type have heen isolated.

A galaotan sulphate.

I 7. oontalnlng équivalent proportions of galactose and sulphate, was Isolated hy ilassid from Irldeae 9^4*% (1Î5)»

It oeeurs as the sodium salt, and

could he converted to the acid ester, hy removal of the sodium hy eleotrodialysis.

The sulphate groups were

readily removed hy either aeid or alkaline hydrolysis without evidence of anhydro ring fonmtion. Barry and Dillon (16) isolated another galaotan sulphate from the red alga Dilsea edulis.

The

sulphate content of this polysaooharide was much lower than that of those previously desorihed, and corresponded approximately to one sulphate group in five galactose residues.

Only one residue in five was attacked hy

periodic acid, and the sulphate groups were found to he resistant to alkaline hydrolysis.

It was suggested,

therefore, that the galactose residues are linked throu^ positions 1 and 0 , with every fifth residue bearing a sulphate group on C*. In 1912 Kyiin (17) isolated polysaooharide material from species of the brown algae Lamtnaria and Fucgs, to which he gave the name fuooidin, and from which he was able to obtain phenyl -i_-fuoosazone after hydrolysis of the polysaooharide «

In the brown algae the main

carbohydrate skeleton appears to be founded on fuoose.

I 8. whereas In the red algae the ohlef oonetituent ia galaotoee.

Fuooidin oooura quite generally In brown

algae, together with alginio aoid. In 1931 Bird and Haae (18) reoogniaed fuooidin as a oarbohydrate ethereal sulphate, whloh faet was oonfirsed independently by I>unde. Keen and %y (19). More reoent work by Peroiwal et

(RO) on hijiily

purified fuooidin. haa led to the following oonplete analysis of the material. uronio aeid. 3.3f; and itylose l.Gf.

Sulphate. 3 8 , notais, 8 .

fuoose. 5 6 . galaotoee, 4.If; 3-methyl- and R:3-dimethyl- u-fuoose

were isolated from hydrolysed, methylated fuooidin. and a branohed ohain strueture in tdiioh *-l:R-fuoose residues predominate, is suggested for the polysaooharide. The sulphate group, whloh is stable to alkali, is plaeed on C4 .

:i

I or am

9.

GAHHTBs

l^mACCHAKIBff^RpM u m

ouy

oh

LACTUCa.

Ulyn 3rf>otue& Is the first member of the ChloroT;hyeeee («rreen slgse) for which work on the nature of the polysaccharide material has been published.

Ihe early work on the polysaccharide

was carried out by K.M.T. Plant and E.O. Johnson (Rl), A d d polysaccharide material was isolated, as Its sodium salt, by extraction of the fronds with sodium carbonate, and precipitation In alcohol after neutrellsatlon and concentration (22).

A fractional

precipitation of the carbohydrate was attempted at different pK values but the products Isolated did not seem to show marked differences In properties.

The

product had a high ash content of the order of 40f, much of Ydilch was calcium sulphate, and It was established that sulphate ester groups were present. J.W.K. grading (PH) has lowered the ash content of the acid polysaccharide, to 5-65®. by dialysis against ecetle add. K.a.T. Plant and B.D. Johnson identified L-rhamnoee. In the methylated acid hydrolysis products, as the crystalline 2 :0 ;d-trlmetl&yl-i— rhamnose -anlllde end -phenylhydrazone.

Prom the hydrolysed methylated

polysaccharide J.W.K. grading obtained evidence for the

I

10.

preBonee of 2:0:4-trlaethyl rbamnoae anâ another lese methylated rhannose, whloh wotild Buggeet that at least part of the rhaanoae ia present as an end group. Evldenoe for partially methylated hexose, anhydro struetures and an ester, hut no other simple end group, was obtained.

2:%;4*@-tetMimethyl-5»galaot08e-anlllde

was Isolated hy W.V.T. Plant (PdO fro» methylated hydrolysis produets and J.W.R. Bradlng obtained small Q.uantlties of muelo aoid, together with a potassium salt resemhllng potassium hydrogen saeeharate, hy nltrio sold oxidation of the sold polysaecharide. The presenoe of uronio sold was suggested hy the carbon dioxide evolution obtained, by K.M.T. Plant end P.j). Johnson and J.Vt.B, Brading, on distilling the polysaooharide with hydroohloric sold.

44 gm. of

carbon dioxide were yielded hy approximately 1000 gm. of carbohydrate.

Estimations of furfural and methyl-

furfural, after distilling the oarhohydrate with hydroohloric acid, indloated the rhaanose content of the polysaooharide to he 3^5—40^ (R8 }, about half of which wee removed, on autohydrolysis, as small molecular weight material, possibly in a hiuronlo acid.

II 1. a C.C^BHT o f

OAHRISB OOT ON

•JLVA yOLYS/.CCiLi.RIM piArifloation. frolitalmry Inveatigatloma by other workere In this laboratory had ladioated that the orude oarhohydrate eodlua emit, me extracted with sodiua oarbonate and precipitated into alcohol, had a h i ^ ash oontont of about 80?4.

Analysie of the eah gave erldenee for a

large percentage of oaloius and eulphnte together with entiller quantitlee of iron and phosphate. be lowered to a value of

Thia could

for the aoid carbohydrate

by dialysis against C.OSK eoetio acid and distilled mter.

Further improvement in ash ooatent by this

method was not praotioable.

The ehief impurities at

this stage were omloinm and iron* &

In order to obtain accurate analytical figures on the carbohydrate it was essential to obtain nmterial free from ash save that inherent in its structure.

The

first object of the present investigation was, therefore, to devise ft satisfactory method of purification. Following the example of Millis and Reed (F*,) it was attempted to precipitate the calcium as oxalate, •*«

adjusting the pH of tho isrolutlon if thi« woro neoeoR&ry# This proTod unsucoossful owing to the preolpltation of a oomparatively

amount of carbohydrate with

II 2. the ORle1mm oxelate. The oalcium and Iron were successfully romorea tsy the use of e cationic exchange resin, "Zeokarh 215", in the a d d form. S’il, rather than In the sodium salt form, H'Ha.

Kxoheng® of metal Ions for hydrogen (1 )

v.as found to he much more efficient than the exchange of metal Ions for sodium (1 1 ) (I)

2K*H + Ca* ^ Hg’Ca -+ SU*

(II)

2K’Sa

C& ^

Rg'Ca -f

where K* = resin anion. Ihe oarhohydrate was obtained as the free aoid. ,.nalysls:-

C, 40.4^; H, G.Mf ; K, 2.90» ; 3. 4.58?; GMe, 2.22# ;

Ash, 0.8?»; I’otal 30^, 15.9? ; Kquiw. wt., 385 g.; Uronio, 1 COOH per 913 g.; IO4* , 1 mole per 985 g.; Red. fig., 1 red. gp. per 3080 g.,

, -84.2®.

All elemental analysée

are by Aeller & Strauss. The eculvalent weight and ash déterminations were carried out on the free a d d material, but all the other estimations were made on the sodium salt for® of the carbohydrate end figures calculated on the basis of ash free a d d form polysaccharide. The nature of the nitrogen oontaining moiety was IhTcstlgated.

The material gave positive, thou^

weak, Biuret and Aanthoprotelo reactions for protein. Assuming the average nitrogen content of a protein to be 15» , a nitrogen content of approximately 3?, in

ïT. a. the earbohydrete, would Indioate the preseiioe of about of protein.

However, stereh admixed with oe

little aa O.Sf of protein glvee definite Biuret end lanthoprotelo reeetlonB, eo that the poealhlllty of aome other nitrogen oontaining body, poeslbly an amino au#ir, had to be ooneidered. Amino Bugare are deteoted by means of the Klson and Morgan Reaction with £>dim«thyXaainoben3;aldehyde. using eonditiona for this reaetien as given by Slson and Morgan (E8 ), oarbohydrat# material which had been hydrolysed with concentrated hydrochloric acid appeared to give a positive colour test for amino sugar. However, a Sohiff 's base derivative of an amino sugar, with/30K-Maphthftldehyde, eonld not be obtained (see page jj 37 )•

The Klaon and Morgan Reaction was

investigatod more carefully (see page II 15) and after modification of the conditions of reaction, evidence WAS Obtained mieh indicated that amino sugars were not present. The possibility that the nitrogen was present in pigment material ims considered unlikely, since such a relatively low molecular wei; K, l.OSf ; S, 5.11^; Total SO4 , 1 7 . Ash. S.74!#; Ash of Ke Salt, 19.Of; Squlv. wt. 427 g.

The material removed in the chloroform

layer gave the following analysis:- C, 29.6f; H, 6.09#; », 6.82#; S, 1.78f; Ash, 11,8?.

Il s. ïhla prooess hafl obviously proeured »n improvomont In the purity of the m&terlal, but since the presence of »Ey protein materinl m s bound to render Ineccurate the enelytlcal figures obtained, more exhaustive attempts to remove nitrogen were made. During the couz>ae of qualitative investigation of the nitrogen content of various carbohydrate «amples, it was observed that dlalysed carbohydrate vihieh had been precipitated In alcohol at an acid pH, always appeared to contain more nitrogen than that whloh had had a similar purification but which had been precipitated In alcohol at a pH of 7 or above.

This difference was

significant after allowing for differing ash contents. In view of this the following course for nitrogen removal was tried.

Material which had been dlalysed

against acetic acid and distilled water was precipitated three times in alcohol and then redlssolved as a 1# aqueous solution.

A small portion of material was found

to be insoluble in water end was separated off.

It

did not, however, show a particularly h i ^ nitrogen content. The pH of the solution was lowered to % with hydro­ chloric aeid.

Precipitation of floooulent material

began at pH 5 and was complete at pH %.

This material

(approximately 8^ of the original) was separated and the remaining carbohydrate solution concentrated and dlalysed against distilled water before working up.

II ü. The «mterlal whicîi was laaolhble at pH £ had. m aotnawhat h igher n itro g o a eoniaht thaa the htiXh o£ the oarhohydrate

which w®a wor&od up after dialysis, hut tJi® difference fms hot oufficiently great to justify this os a method of nitrogen remivml, Alcohol, into which carbohydrate had bmen precipitated, was always coloured slightly yellow, 'ilM3 iatoaalty of the colour which remmlmd ta t W alcohol decreased upon repeated rcpreoipitatioa of the carbohydrate,

$%terial ia the alcohol was isolatod sad

was found to tiaro m relatively high nitrogen content, giving; positive Biuret& anthoprotelc reactions for proteins and at the amae time a fairly strong Molieoh last for carbohydraie material,

it appeared therefore that aoim

protein material was being preferentially held in solution by the aqueous alcohol.

However, repeated prcoipitntion

of the cnrbohydrat® in alcohol lowered the nitrogen content only slightly.

it seatsed possible tîiat only

protain material which had bsea somewhat degraded by the ml&allhc extraction procoaa was suffioimtly soluble in the aqueous alcohol to be retained ia aolution. Carbohydrate material, which had been aabjootod to the usual aootie aeid dialysis treatnsaat, was precipitated in alcohol four ti£«ea, at which point the alcohol from the lest precipitation was only faintly coloured,

A iojl

il ?*

aQueous solatlaa

o£ the otarboîïÿdrate wae broaght to a

pH of about 11, uith ûauatie aodo, aïWS h®at# 1 6 , t - r . o ^ ,.

■ )* t j#

4s p ro t e l a

p e rc e n to g o o f

,h e laore-fsae l a @f%aiv:%kat

weight iadioate© that t/iQ usaterijil iwaor^d lo prodoffiififoitl^ m o W lo .

If

3*xylose according to Breddy and Jones (46). Attempts to Confirm the Presence of r^Glucose. The Preparation ofjD-Glu£0£az^one_j, An unsuccessful attempt was made to prepare glucosazone from fraction B of the methanolysis syrup. A large variety of conditions were investigated, but none produced any glucosazone even though this was readily obtained in a pure state from a mixture of galaoturonic acid, glucose, xylose, rhamnose, galactose and a mixture of galactose sulphates.

An osazone was obtained which

appeared to be a mixture of xylosazone and rhamnosazone. Nitric Acid Oxidation of Fraction B# Nitric acid oxidation of this fraction was carried out with a view to oxidising any glucose present to saccharic acid, which could be characterised as its acid potassium salt, and to oxidising any galactose present to insoluble mucic acid.

Althou^ no galactose was detected

chromâtographically in any of the current work, its presence had been established by M.M.T. Plant (24\) and

II 28. J «% «E, Brading (20) obtained email quantitlea of mucio a oid «

It is poasiblo that the galaetose is all present

as sulphate eater and that under the aoid eonditions employed hy U.U.T, Plant the sulphate was hydrolysed, without inversion, to give free galaetose.

However,

J.W.S, Brading has oarried out oxalic hydrolyses of the more recently obtained polysaooharlde. which has been used throu#d)out the ourrent work, and no ohromatograpble evidence for galaetose oould be obtained.

It seems

possible that the starting material now being used differs In some structural details from the earlier material. A nitric acid oxidation should hydrolyse off any sulphate groups and oxidise the galactose to muoio acid. A variety of conditions were investigated but no muoic aoid or potassium hydrogen saecherate oould be obtained (see page IV 40

).

Fermentation of Fraction B by Saeoh^rorayoes oerevlsiaej, Saecheroinyoes cerevisiae will ferment glucose, mannoae and fructose but,provided that it has not previously heen 'trained* in a galactose medium, this letter sugar is not fermented.

2 -keto-gluconlo acid is, in common with

ell dL-ketoacids, decarboxylated by yeast with evolution of carbon dioxide (47).

It was established that the strain

of yeest available for these fermentation experiments

IT 26. ferffiented glucose bat did not ferment gslaetose, xylose, rhsfflnose or galaoturonic acid.

Under standard conditions,

ft sample of fraction B, from whloh glycosidlc groups had heen removed, was fermented with evolution of oarhen dioxide.

This result suggests the presence of either

glucose or a 2 -keto-hexonlo sold or both. M«thjrlatJ,on of_Fractlon B. A sample of fraction B was methylated twice with methyl sulphate and alkali, and finally once with Fordie reagents.

The product was fractionally distilled in

vacuo when four fractions were obtained.

The third

fraction distilled at a temperature and pressure that suggested it would contain any fully methylated hexoees that ml#t be present. Fraction 3:- OMe, 66.5^;

^790; n^®® 1.4418.

Chromatographic analysis of fraction 3, after hydrolysis of the glycosidio groups, gave evidence for two components.

One of these ran exactly parallel to on

authentic sample of tetramethyl D-glucose.

The other

slower moving component appeared to be either tetramethyl j)-galectose or 0;4-dlmethyl L-rhemnose.

The methoxyl

content would suggest that it is the latter substance.

II 27. The Mftturg of the Or/mnlo Aeld Ccmponent. The MeoheJJolouriactrlc^Method for Detection of Il«eur£nîc_ft£iî. Clsohe (48) h&8 deaorihed a eolonrlmetric method whereby glucuronic acid may be dlatlnguished from either galaeturonle or mannuronlc aeids.

When glucuronic aoid,

mennoae and thioglycollie acid reacted together in a sulphuric acid medium, he obtained a coloured product ithioh showed a characteristic absorption curve.

By

spectrophotometric enalyeis of the reaction solutions Dische m s able to detect glucuronic aoid in the presence of other sugar substances. The reaction was investigated for mixtures containing glucuronic acid, and for galaoturonic aoid, and for fmotion B of the methanolysis syrup.

It was not found

possible to obtain consistent results even using a standard solution of galaoturonic acid with carefully controlled conditions (see page IV 52 }. The nature of the coloured product, obtained in this reaction, la unknown and, since it is carried out in a 68ç^ sulphuric aoid medium, there must be degradation of the

sugar substances to e mixture of products.

Slight

variation in the proportions of these degradation products could have a marked effect upon the absorption spectrum of the solution.

II 28.

Ehrlleh (49) deaeritees the formation of a brlek-red preeipitftte «Den a solution of galaoturomie aoid or its salts is heated with a solution of basic lead acetate. A yellow preeipltate is obtained with glueuronie and mannuronie aeids. The reaction was carried out on a sample of fraction B, from which the glyeoaldic groups bad been removed.

The

precipitate which was obtained was yellow, from which it was concluded that the organic aoid was not galaoturonic acid.

II 29. Uronie Aoia. Furfural ana Methyl Furfural Eatlmationa. Vihen su^ra and related ooapotmds are boiled with hydrochloric aoid, furfural or substituted furfurals are obtained. Pentoses, hexuronlc acids and 2-keto-hexonlo aeids are converted to furfural with simultaneous liberation of oarbon dioxide from the latter two classes.

It is

probable that the two aeids are first decarboxylated to a pentose with evolution of oarbon dioxide, and that this pentose Is subsequently converted to furfural (i) HC flCl

HCl

(I) C00H.(CHCHÎ4 .CHO ---» CifeOH(CHOHteCHO-^ GO2

» HC

CH II II C.CHO

CHgOK.(CHOH)g.CO.COOH Kethylpentoses yield methyl-furfural (1 1 ) and hexoses give very small quantities ofw-hydroxy-methyl-furfural (ill) HCl

(II)

G%.(CH0H)4.CH0 -- >

HC-- CH

II CHgC

II C.CHO

Y

(ill)

HCl CHoOH.(CHOH)a .OHO >

HC-- CH || ||

V

HOCHp.C

C.CHO

II ao. The erolution of earhon dioxide In (1) le pmctlcally quantitative and uremia and S-ketohexonlo aeids may he determined hy «nestimation of this earhon dioxide.

The

uronie or £«keto-hexonlo acid contents of the purified natural oarhohydrate and haryta hydrolysed material were determined hy this means, following the conditions suggested hy Dioksom £t al. (60). The furfural and suhstltuted furfurals may he distilled off from the mixture of hydrochlorio aoid and sugar suhstanoes. The yield Is not quantitative and with w-hydroxy-methylfurfural It la particularly low.

In addition the yields are

very varlahle unless great precautions are taken to maintain ahsolutely standard conditions.

Marshall and

Norris (61) have shown that the chief factor which causes unreliable estimations, partloularly In the ease of methyl» pentoses. Is variation in the concentration of the hydrooblorle acid during the course of the distillation.

Oxidation

of the aldehydes under the conditions of distillation is also a minor factor which affects yields.

They report

conditions which, when carefully observed, enable reliable estimations of pentose and methylpentoae to be made. Furfural and methyl-furfural are estimated in the distillate by precipitation as their pblorogluelde or thlobarbiturate complexes.

The former Is preferred by these authors.

Methyl-furfural may be estimated In the presence of furfural

II 21. ainee the methyl-farfural phloro^laelâe precipitate la aoluhle in ethanol, vAiile the furfural phlorogluoide precipitate la only very allghtly aoluhle.

For accurate

determinations In a mixture of sugars Marshall and Horrls point out that it is necessary to compare the masses of precipitates obtained with those from a irnown mixture of the same sugare, since the presenee of other sugars affects the yields of furfural and methyl-furfural. If hexoses are present email quantities of w-bydroxymethyl-furfural phlorogluolde are precipitated, but provided thet hexoses do net form the major portion of the mixture, their Influence may be neglected. In the current work the yields of furfural and methylfurfural from fractions A and 0% were determined following the conditions recorded by Marshall and Morris (51). Comparison of the yields of phlorogluelde precipitates with those from erablnose end rhamnose enabled a rough estimation of pentoses and uronie acids and of methylpentoses to be made.

Ill 1. DISCUSSION SEGÏIQlf From a eonsideratlon of this and earlier «ork on the Ulya polyaaeoharide it is possible to draw some eonolusions aboat the general etraetore of the material. It ie first neoesaary to eonaider to what extent the material which has been studied is homogeneous. Sarly extractions were made in two stages.

A water

soluble fraction was first remoTed, and then a fraction soluble in dilute sodium carbonate (%.D. Johnson (28) ), Each fraction contained sulphate aoid ester groups and one mole of carbon dioxide was liberated from the same mass of each fraction when distilled with hydrochloric aoid.

It was therefore assumed that no real fraotionatioA

was aehlcTed by this two stage extraction process, and all subsequent extractions were made with sodium carbonate. A fraotional preoipitation of the sodium carbonate extract was attempted at an aoid pH.

Two fractions were obtained,

but again there was no marked difference in their properties end it seemed probable that the slight differences observed were due simply to different proportions of the aoid and sodium salt forms of the polysaeoharide. The present work has shown that the polysaccharide is oontasinated with a nitrogen containing body, at least part of which ie protein.

This material seems to be

attached very firmly to the carbohydrate since much

Ill 2 . of It l8 rosistant to all attempts at removal and some is oven found in earbohjrdrate fractions which have been methylated In a strongly alkaline medium, la addition to this nitrogen containing Impurity It eeema possible that the polysaccharide material Itself is not homogeneous.

About 9* of the material la

precipitated from a If aqueous solution vd»en the pH Is lowered to 2 *

Iwo-thlrds of a lOf aqueous solution

of the polysaccharide may be precipitated In 90f alcohol at pH 5 but the remaining third Is only precipitated at pH 9,

The material precipitated In alcohol at pH 8

has a hifdi nitrogen content and a total sulphate content of 10f.

That precipitated at pH 9 has a lower nitrogen

content end a total sulphate content of 18f.

(Both values

have been corrected for the presence of ash).

The higher

sulphate content may, of course, simply be due to a lower protein content. The analytical figures on the purified polysaccharide indicate that there Is one acid group In every 2*2 anhydro hsxose residues.

One residue In three contains a sulphate

ester group and one In 8—8 residues contains a group from which one mole of carbon dioxide Is liberated when the polysaccharide la distilled with hydrochloric acid.

This

latter unit may be a uronlo or 2 -keto-hexonlo add.

Six

sugar residues consume one mole of sodium periodate and

Ill 3. one mole of fornle aeld la liberated from 27 reaiduea. One reducing group ie present, on an average, in 3491 g. i.e. 21 reaiduee.

The low periodate eonaunption indioates

that the linksgea between units moat be auoh aa to leave very few adjacent hydroxyl groupa free. The corresponding figures for the material ediioh has been hydrolysed with baryta are, one acid group In 3 residues, one sulphate group in 4^residues and one uronis or 2-keto»hexonie acid in 4-6 residues.

One mole of

periodate is consumed by 3 sugar residues and one mole of formic acid is libsMted from 11 residues. one reduoing group in 3400 g. i.e. SI residues.

There is The

decrease in sulphate content from 17.9* to 13.89& corresponds to the removal of one sulphate group from approximately 14 residues.

This should in fact cause the eguivalent

weight to rise from 427 g. (for the material purified to H, l.Of) to approximately 930 g. instead of 464 g. The deorease in the mass of the carbohydrate which yields one mole of carton dioxide, on hydrochloric acid treatment, from 913 g. (on material with », 3.^) to 770 g., is of the same order as that calculated from the known decrease in sulphate and nitrogen contents (790 g.).

It is assumed

that the 3# drop in nitrogen content is due to removal of protein of nitrogen content 19-20:^.

The decrease from

989 g. to 621 g. in the mass of carbohydrate which

Ill 4. eonsofflvs os* mole of perlodete ia In very elo*« agreement wits that anticipated If each sulphate group that has been removed had two adjacent, free hydroxyl groups; for example, a terminal hexo- or pento-pyranoae 2 -sulphate linked throng Ci{ (xlx) and (xx) respectively) or a hexoss 2 -sulphate linked through Ci and C$ (xxl). — — OHC CHOH

■OHC

— — OHC CHOH

CHOH

CHOSOgH

CHOSOaH

I CHOSOgH CHOH

I

CH----------

I

0 CKOH G H g -----

CH----------

0

I

CHjgOH

(xix)

I

CHOH

CHgO---------

(xx)

(xxl)

Unless the esterlfled hydroxyl group and the two adjacent ones were all ois. It would be expected that ready removal of sulphate would occur, with simultaneous ethylene oxide ring formation.

Ihle la only possible

in allose, telose and rlbose and it seems most Improbable that these sugars ere present.

A posnible explanation Is

that an adjacent trans hydrojqrl group is initially involved In linkage with another sugar residue and that this linkage Is broken at some point in the hydrolysis, after the sulphate has been removed.

However, einoe the

reducing figure Is little changed by the baryta hydrolysis. It seems that degradation of this type has not occurred to

in 5. finy appreciable extent.

Slnee the baryta hyArolysea

material vbloh has been analyaed ferma only IZf- of the original mterial. It la possible that. If the starting material contained more than one polysaeeharlde, some fractionation hes taken place, and the results after hydrolysis will bear little relation to those on the original polysaccharide. Chromatographic analysis of material, idiieh has been degraded by methanolysls, has confirmed the presence of rhamnose and a uronlo or keto-hexonlo acid In UIt s polysaccharide and Indicated the presence of xylose and glucose.

I'he former has been Identified conclusively

as Its dlbensylldlne dimethyl aeetal.

The evidence for

the presence of glucose has been less conclusive. Chromatographic analysis gave quite definite evidence for the presence of glucose In the methanolysls fractions. This tms confirmed by chromatographic evidence for the presence of tstramethyl gluooae In a hydrolysed sample of methylated fraction B;

the tstramethyl glucose oould not

be obtained in a crystalline state. of fraction

0

The specific rotation

of the méthylation syrup. In tdileh two

components were shown to be present. Is high In a positive A direction, [-galaotoseanllide has been isolated, from an oxalic acid hydrolysed sample of the polysaccharide, by h.V.T. Plant (£4i) and the mucle acid mentioned above could be derived from galactose.

There seem to be two possible

explanations for the absence of galactose in the methanolysls. Galactose may be present in the original polysaoeharidc as a sulphate ester.

If this were on Cg with a free

hydroxyl group on Cg (possibly freed during the methanolysis), the aqueous baryta hydrolysis of the methanolysis syrup would cause removal of the sulphate group with 0:6-anbydroring formation.

Consequently Z :6-anhydro-3-galactose

end not p-galactose would be present in the fraotions. Under the sold oondltions employed by H.H.Î. Plant such a

Ill 8. sulphate group would in all probability be hydrolysed directly to leave r-galaotose.

Ho evidenoe for the

preaenee of a 8:6-anhydro sugar, oould be obtained from the Sellwanoff reaotion and only very slight evidence from ohromatographlo analysla (see Page IV 70).

From

some methylated fraotions J.W.S, Brading (2B) obtained positive Sohlff reactions which suggested the prosense of an anhydro-sugar.

If the sulphate group were on

Cg or Cg with an adjeoent free trans-hydroxyl group. It is conceivable that, under the alkaline hydrolysis conditions employed, the sulphate would be removed with initial ethylene oxide ring formation and that this would subsequently be hydrolysed with preferential fissure of one of the orygen links to give r-ldose in both oases., In fractions Ci and Cg one of the unidentified oonstituents, which travels sli^tly more slowly than galaetose, might be D-ldoee for no Ry or Rq value for this could be found quoted In the literature. The other possibility is that the galactose is all removed during methanolysis as part of a dlsaccharide fragment, probably a bluronie acid, for It is known that the linkage between a uronio sold and a sugar is particularly stable.

J.W.S. Brading (2B) had evidenoe for the preeenoe

of a methylated biuronlo acid ester in methylated fractions. However, in spite of these explanations for the

i n 9., abs«ne« of falaotoao In the present work,

Brading

has reoently failed to detest, any galaetose ohroaatograph1sally after oxalle asid hydrolysis of the asid polysaooharide under oondltions similar to those employed by M.M.T. Plant. It is possible, therefore, that the polysaecharids new being studied, differs in some struetural details from that whioh eus first Investigated.

The ülva from

whloh the first polysaooharide material was extraeted was gathered from different parts of the sountry and eertainly the fronds differed in appcaranoe.

This view

is supported by the fact that there was no evidenee for the preaenee of xylose in the first material, while this is quite definitely present in the material whioh has been used throufdiout the present work. By oonsideration of the yields of furfural and methyl-furfuMil from fraotions A and 0% of the methanolysis syrup and by oonsideration of the relative sises of spots of the different oonstituents obtained in the ehromatographie analysis of fraotions A, B, Ci, and Cg, it seems that rhamnose, gluooae end xylose are present in the original polysaooharide to the extent of 29f, 7.B,* and fi.Sjf’ respeotively.

The analysis of the purified polysaooharide

shows that there is 44.1)* of anhydro hexose sulphate and 1 9 . of anhydro uronlo aeid.

If one struotural unit

of the polysaooharide is provisionally oonsidered to

Ill 10. ooat&in twelve sugar residues as follows:4 hexoee sulphate, S uronio aold, 4 rhamnose, 1 gluoose, end 1 xylose, the peroentages of eeoh in the polysseoharlde, expressed in terms of their snhydro forms, are:» hexose sulphate, 44#;

uronio aeid, 16#;

glueose, 7#; xylose, 6#.

rhamnose, 27#;

These proportions agree very

well with those determined experimentally.

The

proportion of aeid groups is seen to be one in every two anhydro sugar units, whioh agrees with e mean figure between the equivalent weights before and after dialysis, that is to say 078 g.

IV 1.

SXyBRIMEaTAL aROl'ION. Purification of Carbohydrete. jJemoTal of__C&loium by Précipitation a« Oxalete. The cr rbohydrr.te wes pertlslly purified by lie lye la oeelnst 0.002 K iJCl end distilled water for e total of seven days.

The solution vsa concentrated In vacuo

at 40Oc, precipitated into 90ÿ alcohol and worked up by trituration with alcohol and ether.

The product had

ft solvent content of &f and an ash content of 11$. 2.5 #r. were dissolved In 400 ml. water and 10 ml. of B saturated solution of sodium oxalate solution were added. The pM of the solution was lowered to S with hydrochloric aold, when a small white precipitate was obtained. was centrifuged off and washed.

This

The solid gave a red

calcium flame, but elso charred considerably when Ignited, owing to the precipitation of organic matter with the calcium oxalate at the aold pH.

______

All material used for the exhaustive purlflcfltlon ^ which follows, had been partially purified by dlnlysls agftlnat 0.02 « acetic acid followed by distilled vmter for a total of eight days. Removal of Cations by Getionic Kxohange Hesln. The cationic exchange resin "Xeokarb 21B", in fairly coarse granular form, was used for this purification.

IV 2. ïhe resin oolumn w«s oonstruoted as Indicated in Fig. I.

A «as B syphon arrangement for introduction of

the impure carhohydrate solution, B and C two reservoirs to receive the effluent solution, with a syphon arrangement for transference of solutions from B to C. The column used was one inch in diameter and three feet in length end was filled with resIn to a depth of sixteen inches, on a base of one Inch of glass wool and one inch of purified sand. The oolumn was evacuated by means of a water pump and water introduced in vacuo to cover the resin. Introduction of water in vacuo eliminated the possibility of a film of air surrounding the reeln particles end preventing effective contact between solution end resin. The resin was used in the acid form, prepared by passing through it twice its bed volume of 2 S hydrochloric acid at a rate of approximately 500 ml. an hour, équivalent weight figures for the resin indicated this to be an adequate excess of acid.

Kxoeas chloride and

hydrogen ions were removed by passage of distilled water through the column until the washings were acid free. 2 g. organic

of partially purified carbohydrate

dissolved in two litres of distilled water, were passed through the acid rosin column at a rate of 500 ml. per hour.

A suction pressure of about 20 cm. of mercury

IV 2. was applied to effect thia.

400 ml, of the effluent

solution were retained for egulralent weight, ash and solrhate determinations. After passing through the resin any Inorganlo chloride, or sulphate would he present as hydrochloric and sulphuric BClds respectively.

Their presence would

Introduce a smeller error into equivalent weight end sulphate determinations, eo the remainder was dlalyaed against distilled water for 108 hours with three changes of water per day.

A further 400 ml. were retained for

equivalent weight, ash, sulphate and specific rotation determinations, and the reminder neutralised with an equivalent volume of standard caustic soda, concentrated on e water hath, precipitated In 90f alcohol and worked up to a powder hy trituration with alcohol and finally with ether.

This earhohydrate sodium salt was used for

uronlo acid and sulphate determinations. Analysis of Purlfle^g^gysaocharlde.

Unless stated otherwise, all figures are calculated on the hasls of ash free aold polyeaecharlde. The sodium salt form has. In every analysis, hsen dried to constant mass over PgOg in vaouo. Complété Analysist« G, 40.4?^; H, 5.04#; H, 2.90f; 3, 4.58#; OMe, 2.28#; Ash, 0.6#; Ash of Ha salt, 19.4#; Total 304, 15.9#; Kqulv. wt., 386 g.; Uronlo, 1 COOK per 913 g.; lOl" , 1 mole per 985 g.; 8ed. Fig., 1 red. gp. per 3080 g.; -» 8 4 .2 ^ «

a n e a 5 u .r « < l

on

N e t “Sexlt.

IV 4. ABü vinalyaea. 0.06 - 0.08 g. of orgfinle carbohydrate were ashed to constant weight in a platinum oruolble and the ash Inreetlgeted qualitatively and roughly quantitatively by means of spot tests* Srot Tests used in Analysis. Chloride. One drop of extract was placed in a small tube and to this one drop of ËS HBOg, followed by one drop of silver nitrate, were added.

Presence of chloride

caused the production of a white cloud and this was compared with that obtained from a standard chloride solution*

The test is sensitive to approximately

1 X 10-6 g, ml,-1 .

Ferric. One drop of extract was placed on a watch glass and 0

one drop of ammonium thioeyanete added to this.

Presence

of ferric ions caused the production of a red colouration wbloh could be compared with a standard.

The teat la

sensitive to approximately E x I0"®g. ml*"^. CftlciUB. One drop of extract was placed on a watch glass, two drops of saturated ammonium ferrocyanlde added and finally one drop of alcohol to increase the sensitivity of the test.

Cloudiness appearing within one minute indicated

IV 5. the presence of calcium ions and the cloud was compared with that from a standard.

The test is sensitive to

about 1 X 1 0 g. ml.*^. Sulphate. One drop of extract and of 2N HCl were placed in a small tube, and one drop of barium chloride added. Sulphate, if present, gave a white cloud which was compared with that from a standard.

The test is sensitivA

to about 5 X 10~^g. ml."l. Magnesium. One drop of extract and one of 2N HCl were placed in a small tube, one drop of a O.Zfo solution of magneson reagent added, and the whole made strongly alkaline with caustic soda.

In the presence of magnesium a sky-blue

precipitate was obtained which might be compared with a standard and with a blank test since the dye itself is purple when in alkaline solution.

The test is sensitive

to 1 X 10-6g. ml.-l. (a) Acid Polysaccharide Passed through Resin but not Bialysedi This material contained approximately If of ash.

The

only inorganic ion which could be detected, by spot tests on extracts of the ash, was the ferric ion.

Any other

ions which are present must constitute less than 0.06f of the acid carbohydrate.

IT 6, (t)

Aoia folyaaeohariae after Dlalysla r M Resin Treatment. Dialysis apparently increasea the ash oontent to

#

of the aold polysaooharide.

of

the

Examination of extroots

ash,by thespottests, indioatod the

presence

of IO9'

oaloium, 0«6firon, and 41^ sulphate, as peroentages

of

ash.

the

(0 ) Purified Sodium Salt. The ash of the sodium salt was approxlnetely 20* of the seIt.

Calelum, iron, sodium, sulphate end a trace of

ohlorldo were found to be present.

Calelum to the extent

of approximately 1.5*, iron 0.4*, sulphate 60* and chloride O . l f of the ash. The Inerease In ash content on dialysis of the acid polysaccharide Is commented upon on page IV 9

.

Purified Carbohydrate. »Ash of fAsh of îüAsh of Acid before Aeid after fie Salt. Dialysis. Dialysis. Batch I (page jv 9 "

II

"

"

TV

”

" V

)

«

0.25 0.61

SO.2 20.6

C.94

4.48

18 .S

2.78

19.3

Baryta Hyd. (page IV 16 )

1.44

15.6

CHClg treated ((ii)page II s)

S.74

19.0

»

VI

"

IV 7. üulpiiRte gstlmatlons. â.^.Pfaatfl In A 8h when Carbohydrate sshed slone. Approximately 0.1 g, of carbohydrate was ashed alone In a platinum oruolble.

The oarbohydrate was charred

and the carbon burnt away at a low temperature In order to lessen the possibility of the carbon oauslng reduction of sulphate to sulphide.

The ash v/&a treated with

ammoniacal hydrogen peroxide in order to reoxldlze» to sulphate, any sulphide present.

A gravimétrie

determination, of the sulphate, present in an aqueous extract of the ash,was carried out. (b) Sulphate in Ash when Carbohydrate ashed with SapCQg,. 0.1 g. of oarbohydrate was ashed with 0.0& g. Â.H. sodium carbonate and treated as in (a). Na Salt ashed ha Salt alone ashed alone 304 ? SO4

Purified Oarbohydrate

Batch I (page iv 9

)

15.4

Batch II

"

lE.B

16.2

Patch IV

"

12.3

20.4

Batch VI

"

12.8

15.9

Baryta jSyd. II (page CHClg treated

IV 16)

(ii) page II 8

9.37 IS.9

IS.a 17.5

IV 8. ggttlvalsnt weight Détermination», A volume of acid carbohydrate solution such as to contain approximately 0.1 g. aah free carbohydrate was used.

This was titrated directly against 0.1 K KaOH

using phenol phthelein as indicator and with nitrogen bubbled through ; a sharp end point was obtained with this precaution.

The mass of oarbohydrate sodium salt

present In the solution was determined by concentrating the titrated solution to dryness and drying it off completely over PgCg in vacuo. Values of Equivalent Weight. All figures are calculated on the basis of dry ash free sold form of the polysaccharide. Specimen Caloulation of Bouivalent sseight. Batch V before Dialysis. 100 ml. of carbohydrate solution = (1) 2.54 ml. (11) 2.55 ml. 0.0954 8 »aOH, Vrss of Nr salt in 100 ml. = 0.0912 g. Mass of acid carbohydrate

ash = 0.0053 g.

(Galoulsted from the WeOH required for equivalence). Ash content of aold form = 0.944 Mass of ash free acid oarbohydrate In 100 ml.= 0.0850 g. i.e. 0.0860 g. of acid carbohydrate

e

2.55 ml. 0.0954 H Nb OH

The equivalent weight = 0.0860 x 1000 X 0.0954 - 349 g.

g.

IV 9. Batch

Before hlalyala After Malrsla

T

248

II

227

III

302

804

IV

318

fill

V

249

430

VI

334

386

Baryta hydrolysed (page IV 16 )

-

454

CHCls Purified

-

427

(ii) page II 8)

The equivalent weif^t figures recorded are for purified samples of separate batches of extracted carbohydrate, and the Tarlatione from one batch to another are probably explained by some slight variation in the conditions of alkaline extraction from the point of view of destruction of uronlo acid and hydrolysis of sulphate groupings. It will be ebsenred that the increase in «ah content of the aold polysaooharide after dialysis, recorded on page

IV 6

, is accompanied by an appreciable increase in

the equivalent weight.

Dialysis effects removal of the

inorganic acids, formed from their salts during passage through "Zeokarb 218".

This will cause an increase in the

equivalent wei#dit, but the final value should be approximately constant for all batches.

The high equivalent weight of

batches III and IV and the high total sulphate content

IV 10. of batch IV are aecouutad for by the fact that the •Cellophane’ ueed In the dlalyale, although rinaed with water, was found to contain appreciable quantities of sodlua, sulphate and chloride Iona.

The 'Cellophane* used in the

later purifications was exhaustively washed before use. The distilled water available throughout contained 0.0004 g. of inorganic-salt per 100 ml.

Sodium, calcium,

sulphate and chloride Ions were present to an extent such R9 to Cftuae an lacreaae of 0.5^ in the ash content of the a d d polysaccharide.

The increase in ash content above

this 0.6»i may be accounted for by the replacement of some hydrogen ions, in the acid carbohydrate, by metal cations. Ihe presence of metal cations would cause an equivalent amount of sulphate to be retained in the ash.

An exchange

of this type could be due to the existence of a honnan Equilibrium or to the possession, by the carbohydrate, of Ion exchange properties similar to those observed with alglnic acid.

"

Uronlo Acid Determinations. The apparatus was constructed aa in Figure II. The reaction flask R was of 500 ml. capacity. Approximately 0.6 g. of carbohydrate vmro introduced into S and warmed with a few millilitres of 2K hydrochloric acid to liberate carbon dioxide from any carbonates present. of 12.1!^ HCl were added and the flask replaced in the

150 ml.

IV 11. apparatus.

Carbon dioxide free air. obtained by passage

through soda lime towers C and D, was drawn through the apparatus, for twenty minutes, to remove carbon dioxide. The rate of flow was Indicated by bubbler A end adjusted by screw clip X.

The absorption tower I contained small glass

beads and after removal of carbon dioxide from the apparatus approximately 20 ml. of 0.2N baryte, were ran Into the tower from the burette, which was filled from stock bottle K.

Réaction flask a was then heated to 125-1400 in an

oil bath, the bubble rate being Increased somewhat during these processes to prevent carbon dioxide or liquid being Buoked back from S Into D.

when the solution was boiling

steadily the bubble rate was adjusted to about three per second, and heating continued for five hours since the material was polysaccharide and not a simple uronlo aold. 0 was an aniline trap to absorb any furfural and most of the hydrogen chloride escaping from S.

The silver nitrate

trap H removed the last traces of hydrogen chloride from the gas passing into the absorption tower. After five hours heating the apparatus was allowed to cool, the baryta tower washed Into the Buchner flask with carbon-dioxide-free distilled water and the excess baryta back titrated with H/10 hydrochloric aold using phenol phthaleln as indicator with nitrogen bubbling throufgi. A blank run was carried out before any actual estimation of uronlo was made in order to detect leaks

IV 12. and o'btaln the small correction to be applied due to absorption of carbon dioxide during washing down of the baryta and titration. Snecimen Calcalation of Uronio Acid Content. Batch I. Carbon dioxide liberated = 18.42 ml. 0.0967 H HCl. Mess of dry He. salt of Batch I = 0.8964 g. Ness of acid form + ash = 0.5604 g. (calculated from equlv. wt. data) Ash = 0.

f/R88 of eah free aeid form - 0.8F91 g. I.e. 12.42 ml. 0.0967 H HOI = COg from 0.5891 g. acid polysaooharide. One uronio aold group is present in 0.5891 x 2000 g. X TÉViz= 929 g. of acid polysaccharide. Carbohydrate Batch I

Mass of Carbohydrate Erolring 44 g. COg 929 g.

II

(a)

882 g.

II

(b)

868 g.

Baryta hydrolysed.

770 g.

ihe above figures are all expressed as mass of dry form of polysaooharide corrected for ash. Xatermlnstion of the Reducing Figure of the T'olyaaecharide. Method according to Auerbach and Bodlfinder (52). Approximately 0.5 g. of the dry sodium salt form of the rolysacoharlde were dlsaolved in 26 ml. water.

20 ml.

IV la, 0.1 tl Iodine were ad,1@d and then a mixture of 50 ml. each of O.a V. sodium carhonate and 0.2 « sodium tlcarbonate. Tho solution was left In the dark for 2 hoars and then eoldlfled with 12 ml.

sulphuric aold and the exceae

Iodine titrated with 0.1 K sodium thlosulphate.

A

blank estimation was carried out with 25 ml. water. R. CttO ^ Ig ^ aSeOix

>K.COOBa + 2KaI + BBgO.

Speolinen Calculation of Reducing Figure. Batch II. fJ&BB of dry sodium salt of polyseocharlde = 0.4825 g.

!'ass of ash free aold polysaccharide - 0.4510 g. (calculated from equlv. wt. data). 0.4510 g. polysaccharide = 2.46 ml. 0.0961 S

HagBgGg

I reducing group = 2 equivalents of iodine Kass of aold polysaccharide containing 1 reducing sro\xp =

&I9, 2.46 X 0.0961

0 4

- 2820 g. Batoh

g. aold polysaccharide contaInlnf: 1 reducing group.

II

2820

VI

2080

Baryta Hydrolysed

2400

A definite smell of Iodoform was detected In each of the oxidised carbohydrate solutions.

This was not

detected In either glucose or rhamnose estimations.

IV 14. Removal of Protein. Method of Seva,g (26). 10 g. polysaccharide sodium salt (7 g. organic material) were dissolved in 200 ml. water.

225 ml.

chloroform and 22 ml. amyl alcohol were added.

The

mixture was shaken for 6 hours and then centrifuged for 1 hour.

The upper water layer was separated from a

white gelatinous lower layer.

Carbohydrate was

extracted from the lower layer by shaking it for 6 hours with 200 ml. water.

The mixture was centrifuged for

1 hour and the upper aqueous layer syphoned off.

This

and all subsequent extracts were kept separate from the main bulk of the aqueous solution. The main solution was treated in the above manner for a total of five times, at whioh stage there was no longer a gelatinous region at the interface between the chloroform and water layers.

The aqueous solution was

concentrated by freeze-drying in vacuo and precipitated into alcohol.

Mass = 2 g.

This product was twice redissolved and precipitated in alcohol»

Mass = 2 g.

The aqueous oarbohydrate extracts from the chloroform layer were worked up in similar manner but without reprecipitation.

Mass = 2.5 g.

The chloroform layer was concentrated in vacuo at 5000.

Mass - 2 g.

IV 16. AnaljBlB of giylflad aelâ yolysaoeharldas-

C, 26.

H, 5.8£f 5 B, 1.08f ; S, 5.11#; Aeh, 2.7#; Aeh of »a Sait, 19.0^; Eq,ulT. wt., 4E7 g.; Total SO4 , 17.5f of aolld from ehloroferm:-

C, 29.6^; H, 6.09^;

N, 6.82f; S, 1.78f; Âsh, 11.#. Ail throe fraotions gave positive Xanthoproteic and Bluret reactions, that from the chloroform giving the strongest.

IV 16. Baryta

liY d r o ly a la .

Carttohydrate sodium salt with £9^ ash oontent was used In these hydrolyses. Ihe preliminary hydrolyses ware earrled out on 14 g. of dry sodium salt form of the polysaooharide (10 g. aotual orgnnle material).

Whls was dissolved In

100 ml. water and heated for IS hours at 30*0. with 100 ml. 2S baryta.

The mixture was neutralised to

litmus with S» sulphuric aold at room temperature. The barium sulphate sludge was centrifuged off end extracted three times with water.

The supematent

solution was eonoentrated by evaporation on a water bath at 8Q0C. and preolpltsted into aloohol.

A 30ÿ» yield of

organic matter was obtained. A strong Kolleeh reaction was given by the barium

sulphate sludge, which indicated the presence of carbohydrate even after further extraction with water.

The most

eatisfeotory method for recovery of this material was found a to be extraction at 60*0. and at pK 8. The yield was further improved by carrying out the hydrolyses at 60*0. for 30 hours, and the following procedure was finally adopted. 48 g. sodium salt (30 g. actual organic material) were dissolved in 300 ml. water and heated for SO hours at 60*0. with 300 ml. £8 baryta.

neutralised to litmus

IV 17. with SS sulpburie acid at 60®C., with stirrlog, kept at 60®C. for two houra and the barium sulphate eludge oentrlfuflred off.

The pH of the sludge was raised to

8 with @odium hydroxide and o&rbohydrete sodium salt was

extracted exhaustively from the sludge at 60®C. under these eoadltions. The extracts were concentrated at 40-45^ In vacuo and then dlalyeed against 0.08 H acetic acid for two days and distilled water for two days.

The aqueous solution

was diluted to 750 ml. and passed through the acid form of the resin column and finally dialysed against water for six days.

The material was analysed)for the natural

polysaccharide and worked up by neutralisation, eonoentration, precipitation in alcohol, and trituration in the usual manner. Yield of purified Ke salt = 5 g. Solvent content, 4. Ash, 15.6f. Yield of actual organic polysaccharide = 4 g. Overall percentage of polysaccharide = 10»^. AT'proxinately 10 g. of organic matter passed through the 'Cellophane* during the first dialysis.

This was

obtained as 80 g. of very dark syrup, containing 50# ash, vdien the dialysis water was concentrated.

It had a

high nitrogen content and contained amino acids (see page

IV 72 ).

The colour of the syrup masked any

purple colour in the Molisoh test.

IV 16. Henee total yield of orfmnie matter (polyeaooharide }#

degraded material) —

14 g. r 60f,

Analyeis of baryta hydrolysed material:»

C, 40.8f;

H, 5,49f ; N, 0.6gf,; S, Z.84?; OHe, 2.9Bf; Total SO4 , 13.8f; Ash, 1.4#; Ash of Ha salt, 13.56*; SqaiT. wt., 454; Uronio 1 coon per 770 g.; TO4 ", 1 mole per 521 3 .;

- -

Red. fig., 1 red. gp. per 3400 g. ,

«

A eeoond hateh of hydrolysed material showed an equivalent weijdit of 550 g. Material which bad been hydrolysed at 90*0. for IE hours had a total sulphate content of 7.7# but was obtained in only 30# yield.

lüB f ,

,

a s

v"'T

: y

‘ t,:-

V:. /';S«

i :w

lîïVTi

-

f

*

.-ir5

v.r ,

It

%.

IV 19. Perlodata Oxidation. Approximately 1 g. of polysaeeharide sodlua salt was dried for three boors In raouo at 50®C., to remove alcohol and ether, and then dried to constant mass over in vaouo, in order to remove the last traces of moisture.

The dried material was transferred to a

flask and dissolved in 100 ml. water.

Ihe solution was

cooled to 0 %. in a refrigerator end 10 ml. 0.2 U sodium periodate were then pipetted in. a temperature of 2>5oc.

îhe flask was kept at

10 ml. samples were removed, and

analysed for periodate content, at intervals from the time of addition of the sodium periodate until SO hours, îhe oxidation was carried out at a low temperature in order to reduce the tendency for the soluble sodium periodate to oxidise the earbohydmte beyond the theoretical limit for A-glycol groupings.

îhese conditions were

found to be satisfactory for the oxidation of starch (see Graph I). The withdrawals for analysis, of the Diva polysaccharide were at first made from the one flask at the required intervals, but titration figures were found to be erratic, due to the separation, with time, of a solid, which liberated iodine from potassium iodide in acid solution (see Graph XI).

Investigation suggested that it was the

relatively insoluble sodium iodate, formed on reduction of sodium periodate.

It was found most satisfactory to

IV 20. pipette the 10 ml. samples after one hour and maintain them at

in separate flasks.

At this time the

gel-like carbohydrate solution was uniform and the inorganic salts were still in solution. It was found that the reaction gradually slowed down to an apparently constant, slow rate after about twelve hours (see Graphs III and IV).

It had been

anticipated that the reaction would come to completion to give a constant thiosulphate titre, but it is possible that the slow periodate uptake after twelve hours is due to the presence of protein in the carbohydrate.

A

graphical or mathematical back extrapolation of this straight portion of the periodate-consumption/time curve then gives the periodate consumption by the carbohydrate. m m

It is, therefore, analyses after twelve hours which are important and which fix the position and slope of the curve for extrapolation. The final procedure adopted was as follows.

1 g.

of dry carbohydrate sodium salt was dissolved in 100 ml. water, cooled to 2C. and 10 ml. 0.2 M sodium periodate added.

After one hour 10 ml. samples were pipetted into

separate flasks and these maintained at 3-fîoc.

They were

analysed in turn, at approximately two hourly intervals, for periodate content, during the period 12-36 hours from the initial time of addition of the periodate.

Periodate

IV 21. ««« estimated by addition of Z ml. BS hydrooblorie aoid and 10 ml. 10)t potassium iodide to the 10 ml. samples, and then titrating the liberated iodine with O.IN sodium thiosulphate. Caleulation of Results. Ihe figures for the periodate ooneumption of 1 g. of the ash free aoid polysaocharide, at the stated times, are glTsn in tables with the appropriate graphs: Using the method of Least Squares, the intercept, on the porlodate-oonsumption axis, of the beat straight line throng the points, vdiieb have time values between IS and 06 hours, has been determined.

This figure gives

the periodate ooneumption of 1 g« of the ash free aeid polysaoeharlde, from which the mass of carbohydrate comsumlng one mole of sodium periodate nay be calculated. Batoh —

Graph Intercept on 104 “ — — axis by Method of Least Squares.

Mass of ^terial consuming 1 mole R8 IO4 . :

stnroh

I

12.09

165

II (a)

III

2.12

940

(b)

IV

£.22

901

(0 )

V

2.11

947

(d)

VI

2.18

917

VII

2.00

986

2.64

521

VI (a)

Baryta Hyd.VIII

IV 2 2 . Ittliatlon of Foralo Aeld liberate! in Oxidation Proagaa. îhe ffletbod adopted was a modification of that used by Meyer (58). 10 ml. samples of the above oxidation mixture were

removed after 50 hours oxidation.

1 ml. ethylene glyoel

wee added to destroy exoess periodate, and, after standing one hour In the dark at room temperature, the solution was titrated against 0 .01 » baryta, using phenol red ae Indicator. A blank estimation was carried out In like manner using CJ. g. carbohydrate, 1 ml. 0.2 M aodlum periodate and 1 ml# ethylene glycol.

The ethylene glycol was added

Immediately to prevent oxidation of the carbohydrate by sodium periodate. Saturai Carbohydrate. Formic acid equivalent to 1.990 ml. 0.0100» baryta was liberated frcm 0.0857 g. of the ash free acid polysaccharide. .‘.1 mole of formic aeld Is liberated fro® 4810 g#

Baryta Hydrolvaed Carbohydrate# Foralo acid equivalent to 2.151 ml. 0.01118N baryta vas liberated from 0.0426 g. of the ash free aeld polysaccharide. . M mole of foralo acid Is liberated from 1775 g. A gravimetric estimation of the formic aeld wae

IV 20. attempted, aeeordlmp to the method reported hy Whiatler {5 4 ). After 60 hours oxidation a 10 ml. sample was extracted with ether for three days in a llould-llguld extractor.

A

slight excess of sodium hydroxide was added to the extract which was then concentrated to about 5 ml.

It was

diluted to SO ml. with water and neutralised v/lth N hydrochloric aoid,

1

ml. excess acid and

acetate were then added.

0

g. sodium

the solution was filtered and

20 ml. 5f mercuric chloride solution were added.

The

solution was heated on a steam bath for several hours but the Quentlty of merourous chloride which was obtained was too small to be weighed. Investigation of the Oxidised Carbohydrate Solution for the Presence of Ammonia. An amino sugar In which there Is an adjacent, free hydroxyl group, Is oxidised by the periodate Ion with liberation of ammonia.

H — n — ÎIHg

H— C = » H >

u— C— OH

I

H -C = 0 -------- »

H-C=0

I

-t- HHg

H-C = C

I

An aqueous solution of the natural carbohydrate and a sample of the oxidised solution were made Just alkaline with caustic soda.

The solutions were boiled and the

aqueous distillate which was obtained was Investigated for the presence of ammonium Ions.

The addition of

IV 24. Nessler’s solution, to a portion of the distillate, produced a slight yellow colouration with each carbohydrate sample.

This indicated that a very small

quantity of ammonia was liberated from the natural carbohydrate.

A slightly larger quantity was obtained

from the oxidised material but the difference was too small to suggest oxidation of an oc-hydroxy amino grouping by the periodate ion.

The presence of ammonia was confirmed

by the p-nitrodiazobenzene test, and by the phenol-sodium hypochlorite test (for details see Appendix).

IV 25. Methanolysls. The polysaccharide material had heen dialysed against acetic acid, neutralised, concentrated, and precipitated in alcohol containing hydrochloric acid equivalent to twice the amount of caustic soda added for neutralisation. 56 g. of the dry product were triturated with anhydrous methyl alcohol and then suspended in 560 ml. anhydrous methyl alcohol.

Dry hydrogen chloride was

passed in to give a 3^ solution, and the mixture refluxed for 24 hours.

A further 20 g. hydrogen chloride were

passed in at this stage to increase the solution to 5^ strength, and to replace hydrogen chloride lost during refluxing.

A further 10 g. was passed in, after a total

of 48 hours refluxing, to maintain strength, and refluxing continued for a total of 72 hours. A large pr/oportion of the hydrogen chloride was bubbled off with a dry air stream at 4Qoc.

The material

remaining insoluble in methyl alcohol was centrifuged off, washed, and worked up by trituration with ether. Yield r

6

g.

The supernatent liquid and washings were neutralised to Congo Red with dry freshly prepared silver carbonate, filtered, hydrogen sulphide passed to precipitate silver ions

88

silver sulphide, filtered, and concentrated to

a syrup at 40® in vacuo.

The silver carbonate-silver

IV 26. chloride residues were suspended in methyl alcohol and hydrogen sulphide passed in order to convert any insoluble organic silver salts to the acids.

The methyl alcohol

extracts, thus obtained, were added to the syrup obtained above, and concentrated. Yield = 50.4 g. syrup. OMe, 15f.;

f

= +

20.

Baryta Hydrolysis of Methanolysis Syrup. 50 g. of methanolysis syrup were hydrolysed with 500 ml. K baryta for

00

hours at ôO^C. with nitrogen bubbled

through to prevent extensive oxidative degradation. Preliminary investigations had indicated that after 00 hours hydrolysis, precipitation of barium sulphate from hydrolysis of carbohydrate sulphate groups, was virtually complete. hot water.

The solid was filtered off and extracted with The supernatent liquid and washings were

neutralised to phenol phthalein with carbon dioxide, filtered, the precipitate extracted with hot water until free from carbohydrate material and the filtrate and extracts concentrated to a stiff syrup at 400/15 mm. Yield of syrup =40.7 g.

SG4 ,

0

.296,

Fractionation of Baryta Hydrolysed Syrup by Solvent Extraction The syrup obtained above was extracted exhaustively with acetone which removed part of the monosaccharide glycosides and left behind disaccharide fragments and

IV 27. sulphate and uronio derlvatlvoe. Fraotlon A.

Yield of syrup

1,8 g.

Ihe residue was exhaustirely extracted with ethanol oontaining 1# of ether in order to remove material other than barium salts.

Ihe addition of Ift by volume of

ether was to decrease the solubility of barium organlo salts present, since these have a small solubility in ethanol. Fraotlon B.

Yield of syrup

22.9 g.

Ihe residue was dissolved in water and filtered off from insoluble baritus inorganic salts. Fraction C .

Yield of solid

15.2 g.

Qualitative Investigation of Fractions. Fraction A. (1) negative barium flame test. (2) negligible organic sulphate. (0) Negative naphthoresonoinol test for uronie acids. (4) Brown colouration but no indication of red in Seliwanoff reaction. (5) Nitrogen absent. Fraction B. (1) Positive barium flame test. (2) Organic sulphate present.

(a)

Positive naphthoresorcinol test for uronio acids.

(4) Negative 3ellwanoff reaction. (5) Nitrogen present.

IV 89.(a)

(1) i*oeitive bmrimi flmme t«8t, ( t i tilgh or^ionlc aiHphate oomteat,

{3J Strong ttaphtiioraisoroiaol t&st for oroaic aoid. (4) tefintioe Seliwtwioff raaetioa, (Si Mitrogen proseat, i?or datalla of ««phtliorosorotooi and ;&%ll»eaoff tests aeo Appaadix, It was obyious that aepnratloa ofelaiaed was poor ead llKitod to the esoliisloa of baritua aroaatae mad barios orgaalc sulphates

£rm /rmtioa A,

itoKSver, further

cross extraction of fractions had little effect oa their ooateat, .-QterificsUoa of BarAaia..,oelts_ in..ijTeetl^Ntt c. Ib.S g, of faction C mere rsfluaed with 800 ml. 8)1 mtheaolio hydrogen chloride for 9 hoars.

Trader

these conditloas the oruaic salts would be converted to ffwthyl eater but tl»e barium orgaaio sulphate would probably remain as the barium salt.

The solution was

nautrallsed to Congo Red with dry freshly prepared silver carbonate, filtered, hydrogen sulphide passed to remove silver ions and concentrated at 40*48®/IB mm,„ îiÿârogca sulphide was passed throu{0 the silver carbonate, silver chloride residue, in methanolic suspension, to redissoive, as acids, any silver organic salts present.

IV £8. eaâ th* imtheaallo oxtreot t«ld©d tu th@ cmia ayrup* ïielâ üJt ayrup = 16,5 g, SU© eaterlfied syrup

wrs

eshaastiveiy «xtracieâ with

Boetoii® to ramuT® lættiyl eater giycosides of urouio aclâa ahd leave hehlnâ tîv® baritua organic aalphate mitai, Y ield o f ayrtîp = 7 ,6 g, Yield of laeolttble k» tori il = 7,9 g, 'î îhe iaaolubie material «ma re-oeterifled by refluxing with 80 rai, of l,8ji Kiethaaolio hydrogen ohloriaë for 9 houra «nâ working up ne above. Yield üf re. aaterifi W syrup = 6,1 g, 11ni« eyrup wma extracted with acetone m à the extract added to the first acetone extract and concentrated at 4i'j-46®/16 mm. y ra o tio a

îotal mma;: o f acetone soluble eyrup - 9 .1 g .

îhe insoluble residue was exhaustively extracted with ethanoi to separate all organic Esatter frMS an appreciable quantity of berim Inorganic salts,

Bxtraction was a alow

process but, tha soivont fractionations after baryta hydrolysis of the mettenolysis syrup, showed that solubility of the berlwa sugar sulphate glycosides in ettenol was suffioeiat to mate this separation possible. fraction %

H%»s of ethanol soluble syrup =2,3 g.

IV m ,

Fv^mots. 4 -’

aolvent Ash ïotai

r..;dijct

: tartia^i

î ' s t h a n o i y e i s

h a r y t »

1

tfstorial

n y d ,

^ r u c t i o n

”

.gti

"

(oif

d#

Ü.2

m

m

A

u . l

«#

1.5

m.

3

‘■ > 5

m

0 . B

**

C l

1.0

V . 9

1. # 8

i'4l

1 , 4

f i g u r & o

© r e

4»

f o6]'^ 3>

T

I 5 . Ê

•

1 3 * 7

t s i c u r r o o i e d

=-goo"

15,0

m

#*

u y r a p

Hotetioa

15.0

7 , g

4»

y r a p

i#e

[=

o”

f o r

i.nilmBtloùa.

«'urfurc,l aaü

. 'o tô T O in e tlo a a o f th e afitsual o f f u r f a r ? î l omî C A jt'n /if a r î u r a i . o b ia ia s d oa d i s t i l l B tio ïi v * iih 5Jij?iiroGhloi’i c n o re o o r r is d o u t o a f r u o t i c t t e

«aâ Cj_ a in c « I t mx» hoped

a

t b e t rhttfîuose # e e coaeeatatutw d l a ia

o c id

a sud u ro u io a c i4

%* £ h e

f m t h o d

t ' a r a h f t l l

« a d

o o n t a i u i s d

svae

u m é

î S ô r p i a

o a i ^

©

K a d i f i c a t i o a

( 51).

glass

j o i a t s ,

'fh#

o f

s p p a r f i t a a ,

Is

s h o w s

l a

t h a t

o f

# i e h

C i g a r e

III.

Su ml. of lë*lû,i hÿâi’«ü.hlaric ©oiû satureWa with soûittti'i chloride w«re rua into the flash which already cuiitaiaed o capsule Midlag approximately syrup under iuvcatiguti o u , b t t b b l a d

i s

s'i

to

b a

j u s t

a

g, of th«

stream of nitrogen was

b e l o w

t h e

i o v e l

of

t b s

IV m , in the fi&eEc*

'*&«

waa lasaersed la an oil

bath to 9ll£htiy above the level of It# llqaid oantenta, aoA the bath teaperatare ralaeâ to, m S raelatalned at, 170-196*0#

The flash wa« immeraed

m

farther in order

that the aides of the flask should not beoome oaked with soAiuffl ohloride «hen distillation was in progress# the flask oontents boiled, m

then

ml. of IB#16;^ iijrdroohlorio

aoid Saturated w i # sodiua ehlorlde were added slowly through the fhsnel#

After SO minutes a further 60 ml#

portion was added, fallowed by two 60 ml# portions of 16.16^ hyhroohlorio aeid at SO minuta intervale# distillation was oontinued for a farther half hour making a total of 1,6 hours, by whieh time distillation of furfural and methyl-:feurfttral was found to be oomplote# rhloroeluoinol solution was prepared as follows# 11 g# «malar phlorogluetnol were dissolved la 800 ml# boiling 18,16^ hydroehlorio aoid, end the solution poured into 1200 ml# 10.16^ hydroahlorlo aoid.

solution

wae ti%en left to stand for one week and filtered before use# 10 ml. of the phlorogluoinol solution were added to the distillate and ^ e volume auule up to 180 ml# with 16#16^ hydroehlorio aeld.

After standing 24 hours the

preeipitate was filtered #irouf^ a ara&e 4 sintered oruolble, wa^ e d with 60 al# of water and dried to

IV 31. oonataiit mass in an air oven at 105*^. The preoipitate oonalated of furAiral and methylforfnral phlorogluoides*

The methyl-fiirfural

phlorogluoida was eatraoted with three 0 ml. portions of 96^ ethanol at SO^G*

The oruolble was placed in a

smaller beaker and heated In a hath at about 70^0* for this purpose.

The oruolble was again dried at 105®0.

The residue ooasisted of furfural phlorogluolde, honoe the mass of methyl-furfural phlurogluoide was obtained* An approximate estimation of the initial pentose or uronio aeid. and methyl pentose oontent. could be made by referonee to the yields of phlorogluoi&ea from standard arsbinose and rhamnose solutions.

The

estimation cannot be of very great aoouraoy, however, sinee the effect of the other constituents of A and on the yield of phloroglucide precipitates is unknown. All figures are based on 0.1 g. of starting material. Arabinoae. Total Phlorogluoide Preoipitate

Part Insoluble in Ethanol

Part Soluble in Ethanol. Ê*

I* 0.0780

0.0722

0.0068

0.0809

0.0726

0.0083

0.0786

0.0726

0.0069

Thus 0*1 g. arabliioee yielded a mean value of 0.0791 g.

:i G

I? as,

of farforol phXero^^ laaia* proaipitato, of wbtoh 0 *ôQ6 e g, was eolutols la 96;| e^anol.

Mtiaatoaas, Total Phlorogluoiâe I’reolpltato £•

Part Xnsolttblo la Sthaaol

Part Soluble la BthaiMl I*

0»OB87

0*0005

0*0884

0.0682

0 *0004

0.0678

0.0592

0.0003

0*0569

Thua 0,1 8 * rhamwae hydrate yielded a mean value of

.

0*0887 g. of raethyl-furfural phlorogluoide preoipitate* of whieh 0.0008 8 * was Insoluble In 96^ ethanol* Fraotlon A. Total ?hlorogluolde Preoipitate iE"

Part Insoluble in Bthanol g.

Part Soluble in Btlianol Ê♦

0,0692

0.0H9

3.0443

0.0627

0.0107

0,0420

0.0541

0.0170

9,0371

A mean value was oaloulated from the first two sets of figures, sines these were in fairly 5-eod agreement with eaoh other. Thus 0.1 g* fraotlon A yielded 0.0844 g# of »?

phlorogluolde preoipitate of whieU 0.0118 §. was liieeluble and 0.0481 g. was soluble in 99^ ethanol. Oomparlaen with the results obtained for arablnoee

IV as.

l&diORtes that an Insolultla residue of O.Olia g, is left by 0.0122 g. total preoipitate.

Xhis eorresponds to am

initial pentose content of 0.0166 g. or to 0.0171 g. of pentose glycoside, that is, 17^ of fraction A.

0.0009 g.

of the soluble part of the precipitate ess furfural pblorogluoide.

By comparison with the results for

rhamnose hydrate, the remaining 0.0422 g. of soluble preoipitate is found to oorrespond to an initial methyl pentose glyooslde content of 0.0702 g. that is, 70# of fraction A. Fraction Ci Total Phlorogluolde Preoipitate g.

Part Insoluble in Bthanol g.

Pert Soluble in Bthanol g.

0.0175

0.00S2

0.0091

0.0117

0.0064

0.0065

0.0106

0.0055

0.0071

The methyl pentose and pentose and/or uronie aoid contents of fraction C,were oaloulated from the first set of resvdts since it is mere likely that the yield of precipitate m a lew than that it m a high.

îiethyl pentose

glycoside was oaloulated to form 14# of fraction Ci. If the furfural were all derived from a uronie aeld, the uronio ester glycoside was oaloulated to form 17# of fraction Ci.

If the furfural were all derived from a

pentose, the pentose glycoside was oaloulated to fora 15# of fraction Ci*

IT 04. îto« résulta Quotaa era obvloualy of little quantitatire 7«lu«. but are of great qualitative interest.

The presenee

of methyl-furfural in the distillate from fraotlon A and of both furfural and «ethyl-furfural from that of fraction Cl was antioipated.

The presence of a large amount of

furfural in fraction A was entirely unexpected.

Since

uronie acid seemed quite definitely absent from this fraction (negative naphthoresorcinol test) it seemed probable that seme as yet undetected pentose was present.

Blmftl,ffmd.Mernan Ce im^ Keaotion. It WAS found necessary, as suggested by Palmer. Smyth and Meyer (00). to purify the reagents used in the Elson and Morgan test.

Aldehydes were removed from the alcohol

by the method of binkler (56). and the eeetyl acetone was purified via its copper salt by the method of Claieen (56). 260 ml. of ethanol were refluxed with 0.28 g. silver oxide end 0.26 g. sodium hydroxide for 0 hours and then distilled. 80 ml. of acetyl aeetone were treated with 276 ml. of a hot saturated solution of copper acetate containing 26 g. of the salt.

The precipitate was filtered after it had

stood for 1 hour, washed, and while damp introduced into a mixture of 40 ml. of water end 100 ml, of ether.

160 ml.

of diluted sulphuric aeld were added (60 ml. sulphuric aeid and 100 ml. water) and the mixture shaken until dissolved.

IV

Z5,

The ether layer was separated and the water layer

re-extraeted three times with ether.

Ihe eomhined extracts

were dried over anhydrous sodium sulphate and the aeetyl acetone distilled after removal of ether. Ihe p-dlmethylaminohenzaldehyde used in the reaction was purified by dissolving/in eonoeetrated hydrochloric aoid, diluting, and fractionally precipitating the aldehyde with saturated sodium aoetate solution.

The

first fractions were yellow coloured, and only the later white fractions were retained. Acetyl Acetone Reagent. O.S ml. acetyl acetone dissolved in 10 ml. 0.6 1 sodium carbonate solution.

Ihe reagent was freshly

prepared each time before use. p-Mmethylaminobensaldehyde Reagent. 0.2 g. of the purified aldehyde were dissolved in 7.5 ml. of purified ethanol, and 7.5 ml. of concentrated hydroehloric aeld. It was fotnd most convenient to carry out the reaction on 1 mg. of glucosamine hydrochloride and 0.1 g. of the Ulva ayrupa under investigation.

These samples were

dissolved in 1 ml. of water, 0.5 ml. of the acetyl acetone reagent added and the test tubes, fitted with small air condensers, heated in a boiling water bath for 15 minutes. The solutions were cooled and 2.6 ml, of purified ethanol

IV 36. anâ 0.5 ni. of the ^•’diiBethylanlno'benealâetayde reegent were added and the Tolame made up to 6 ml. with ethanol.

A

hlank was run simultaneously. Under these conditions a charaeteristie cherry red colouration was obtained immediately when the p-dimethylafflinohenealdehyde reagent urns added to a solution which contained glucosamine.

The amino acids glycine, aleraine,

tyrosine, tryptophane, leucine and glutamic aeld gave no such colouration. Fraction A gave a yellow solution similar to that in the blank test.

Fractions B, Ci and Og all gave similar

yellow solutions initially, but after standing for 15 minutes, a pink colouration appeared which gradually increased over a period of 1 hour. in fraction B.

This was most intense

The pink was more orange then that

obtained with glucosamine, but was such as to agree with that obtained by superimposing the charaeteristie cherry red on the fairly strong yellow colour of the sugar syrup solutions.

An exactly similar colour reaction was

obtained when fraction B was treated with p-dimethylaminobenzaldehyde in the absence of aeetyl acetone.

The

same pink colouration was obtained with a sample of the original polysscoharidc which bad been hydrolysed with hydrochloric acid and neutralised.

Aeetylated amino

sugars are known to give this colour reaction without an

IV av. aeetyl eeetone eoàdenaatlon» but the eolpor la usually mttoh nore purplet end no aeetyl groupe were detected in the tflTft earbohydrate by Weller and Strauae.

8»Hrdroxy~l>iyif|»^thi^dehyae Dérivât Ire of The preparation vas carried out on the degraded fraotlon of the baryta hydrolysed polysaoeharlde. vas first treated as follovs.

This

The ash content of 50V

vas reduced to 30V by extraetlon with 6ÔV ethanol. In idileb much of the Inorganic material was Insoluble. 4 g« Of this syrup were heated on a belling water bath with BO ml, of oeneentrated hydroehloric aeld for 1.8 hours.

From this solution It was hoped to Isolate a

IV as. oryetalllme anlno 8%ig&r hydrochloride, and f M n this the Sohlff’a Base eould be prepared*

0*4 g, of

ehareoal and £0 ml, of ««ter were then added and the mixture maintained at 60«C. for 0,8 hour. Solid was removed by filtration and the solution oeneentrated at 60^0. in Taeuo. all Inorganic»

The oryetale which appeared were

The syrup wae oeneentrated to dryness

and used for the Sehiffe Ease preparation aeeending to the method of folles and Morgan adtm®ar,

îhls was attached to a B Ed

boUlng tube containing 6 ml. of water,

The belling

tube was placed In an oil bath at llt®c, for fifteen

IV 74. miButea dmrlng which time water refluxed down the spiral condenser and glase rod and on to the paper ■trip, from which the anger was oompletely extraoted. Three suoh stripe each oontainlng four sugar spots were extracted, three strips of plain paper over whloh the solvent had passed were extracted to serve ae blanks, using r> ml. of water in every ease.

Kaeh 5 ml. extract

was concentrated to dryness and redissolved in 1 ml. of water.

These solutions were heated in a boiling

water bath with two drops of coneentrated hydrochloric acid for half an hour, and the acidity of each solution reduced with 0.04 g. sodium carbonate.

One drop of

£ a barium chloride was added and the quantity of barium sulphate precipitated from the extract of each of the spots A, B, C, 1), IS and F compared with that from the blanks, in an attempt to identify the sulphate containing body.

The blank contained a fairly heavy

preoipitatc of barium sulphate and it was not possible to detect any marked increase in quantity from azy of the strips containing sugars.

Attempts to lower the sulphate

content of the paper by washing it thoroughly with water before running the chromatogram did not prove very successful.

A 1. Aprmpix (1) yaphtiior8»orcl«t»l Teat, A few milligram# of the aubstanoa to be tested are dissolved 1» 4-5 drops of 5o7.HCl«»d 0.5 ml. of 1;^ alooholle naphthoresorolnol is added.

the solution is

heated in a boiling water bath for Z minutes, cooled and ether added.

If a uronie aold Is present the ether

layer Is a eh&racterlstic mauve colour.

the coloured

products formed from other sugars are all Insoluble in ether. (£) Sellwanoff test. L few milligrams of the aubstanee to be tested ere

dissolved in 4-5 drops of water and S drops of concentrated hydrochloric acid added.

the solution is heated in a

boiling water bath for 0.6 minute and S ml. of 'tf alcoholic resorolnol are added.

A bright red colour develops

quickly with ketoses and anhydro sugars but not with other sugars. (S) Kollsoh lest. A few milligrams of the substance to be tested are dissolved in 4-5 drops of water and 1 drop of 80# alcoholic oL -n&phthol added.

0.5 ml. of concentrated sulphuric acid

is poured down the side of the tube.

A deep violet ring

forms if carbohydrate material is present.

A 2. (4)

tipa^&Mobenzene îest fo>> ,A«iaonl>. 1 g. of 7 >Bltroanlllse is added to 20 ml. of vater

plue 2 ml. ooBoentrated hydrooblorlo aold.

The mixture

le warmed to dleeolve the solid and then diluted with 160 ml. of water.

The solution Is oooled and 20 ml. of

2-5^ sodium nitrite added.

After vigorous shaking the

solution la filtered and used Immediately, since solid separates out on standing.

A drop of the solution to

he tested (neutral or ell^tly add) Is placed on a watch glass, a drop

of the reagent added and then a small

partiole of ealelum

oxide.A red zone forms round

the

oalclum oxide If ammonium Ions are present In solution. It Is claimed that 0.67 y. may he detected In a concentration of Is76,000. (6) Phenol-sodlum hypochlorite Test for Ammonia. 6 ml. of the solution to he tested are placed In a test tube and 1 ml. of 4# aqueous phenol and 1 ml. of sodium hypochlorite

added.

The solution Is heated in a

boiling water hath for 1.5 minutes.

It Is claimed that

a distinct blue or blue-green colouration Is obtained with as little as 0.0001 mg. of the ammonium Ion.

BIBLlOGRAPgy

(X)

3#C#C# St&Bfordl,

Chen. Hews,

(£)

%.L.B«l90D, X.a.Creteher,

J.Amer.Chen.Soe., 51,1914 (1929),

(Z)

i.X.NsIson, L.a.Cratohar,

J.Amer.Chem.8oe., 52.2180 (1920)

(4)

G.U.Blrd» P.Uaas.

Bioohea.J.,

(6)

S.L.Hirst, J.K.R.Jones, %.0.Jones,

47, £54 (1883)