The composition of cholla gum

Item Type text; Thesis-Reproduction (electronic)

Authors Klaas, Rosalind Amelia, 1905-

Publisher The University of Arizona.

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THE COUPOSITIOIT OF OHOLM GUM

By

R osalind A. Klaas

Submitted in p a r t i a l fu lf i l lm e n t o f the

requirem ents fo r th e degree of

M aster o f Science

in th e College o f L ib e ra l A rts and Sciences of the

U n iv e rsity o f Arizona

1 9 2 7

/3dLvyd . XL

ACKH OWLKBCrEIilSIIT

The w rite r w ishes to express h er s in ce re

g ra titu d e fo r th e adv ice , a ss is ta n c e and

encouragement of Dr. L ila Sands, under whose

d ire c tio n t h i s in v e s tig a tio n was c a rr ie d on.

63071

I&3LB OP 001EEII2S

m m

Intro& tto tion .................................................. 1

General P ro p e r tie s o f Cholla G um .... . . . . . . . . 2

Methods o f A n a ly s is ............................................. 5

A nalysis o f th e Gum................................... .............. 7

H ydrolysis o f th e G u m . . . . . . . . . . . .................... 9

Is o la t io n o f 1 -A ra b in o se ....................................... 17

In v e s tig a tio n o f Mother l iq u o r from P ir s tCrop A rabinose........................................ 18

H ydrolysis of Calcium S a lts "A” ........................ 21

Is o la t io n o f d -G alao tose ......................... 24

H ydrolysis of Calcium S a lts ”Brt. . ..................... 8S

I s o la t io n o f 1-Ehamnose............................................ 28

Id e n ti f ic a t io n of d-G alacturonio A c id .. . . . . . 29

D iscussion of a T ypical H ydro lysis. . . . . . . . . . 32

S u m m a r y . . . . . . . . . . . . . ............................................ 34

ZEE COMBOSIfflCfil OP GHOLLA GUM

ETZHODUGZIQS

Gums a re complex subotanoee found w idely d i s ­

tr ib u te d in n a tu re . They a re not polyaaocharides a s i t was

onoe supposed, but th ey a re r a th e r substances o f a g lucosida l

n a tu re , which upon h y d ro ly s is y ie ld sugars and ac ids of a more

or lo s s com plicated s t ru c tu r e . Since th e gams them selves

occur a s potassium , calcium , or magnesium s a l t s , i t would

appear th a t the m olecule of a gum i s composed o f a number of

sugar re s id u es grouped around an a c id nucleus in such a way as

to leave th e a c id group f r e e . Of S ullivan* ha# concluded th a t

gum a ra b le i s a d iarab inan - to tra g a la c ta n - a ra b le mold,

which i s combined w ith oaleium , potassium , and magnesium.

Other workers have shown th a t cherry gum2 i s composed of e ra -

b inose , g a la c to se , and g a lac tu ro n ic a c id ; th a t gum trag ao an th

y ie ld s upon hydro lysis a rab in o se , sy lo so , fucose , g a lac to se ,

g lucose, and g a lac tu ro n ic a c id ; and th a t mosquito gran4 con­

s i s t s o f a rab in o se , g a lac to se , and glucuronic as id e P lm f

gum, a p r ic o t6 gum, and chestnu t? gum, have a ls o been analyzed .

ChollB gam occurs a s an exudation o f th e white

oho lla cac tu s , Opuntia fo lffid a . which i s w idely d is tr ib u te d

throughout sou thern A rizona, Hew Mexico, and northw estern

Mexico, i t appears most freq u en tly on old o r d iseased p la n ts ,

in round, w arty lumps which vary g re a tly in s iz e . Average-

s iz e lumps weigh from 85 to 50 grams, the la rg e r ones weigh

from 100 to 150 grams, while a few weigh over 200 grams. When

th e gum f i r s t appears on the p la n t i t i s p a le yellow in c o lo r ,

and i t i s s o f t and p l a s t i c . As i t d r ie s , i t tu rn s to a dark

rod-brown c o lo r , and becomes heard and s u f f ic ie n t ly b r i t t l e

th a t i t may bo ground to a f in e powder.

3?ho gum has a f a in t but c h a ra c te r is t ic odor and

p r a c t ic a l ly no t a s t e . The d e n s ity v a r ie s from 1 .34 fo r the

s o f t stage to 1.58 fo r the old s ta g e . The dry gum e x h ib its

marked im b ib ite ry powers. When mixed w ith 6 to 10 tim es i t s

weight o f w ater, i t sw ells in to a th ic k g e la tin o u s mass. I t

i s p a r t i a l l y so lub le in w ater. When the f in e ly powdered gum

i s mixed w ith 50, 100, o r 150 tim es i t s weight of w ater and

allow ed to stand 40 hours, 20 p e r cen t o f i t goes in to so lu­

t io n in each ca se . When th e gum i s warmed w ith 3 tim es i t s

weight o f 95 p e r cent e th y l a lcoho l a t 60°0. fo r 7 hours, 1

p e r cent o f i t d is s o lv e s . The m a te ria l obtained by evaporat­

in g th e a lco h o l e x tr a c t to dryness i s brown in c o lo r , has a

s tro n g odor and i s s l ig h t ly so luble in w ater, the aqueous

so lu tio n "being decided ly a c id to litm u s ,

p o rtio n o f t h i s m a te r ia l, le av in g a red re sin o u s

i s so lub le in sodium hydroxide. The e th e r-so lu b le

i s w hite, waxy, and in so lu b le in a lk a l i .n-f-tio-r.

p ro p e r tie s o f c h o lla gam:

(a) She w ater and ammonia so lu tio n s o f th e gam ro ta te

p o la rize d l ig h t s l ig h t ly le v o . ’

(1) She water so lu tio n i s f a in t ly ac id to litm us and to

methyl re d .

(o) Both 95 p er oent a lcoho l and s o lid ammonium sulphate

p re c ip ita te some o f the gum from i t s vmtor so lu tio n , whereas

n e ith e r f e r r i c ch lo rid e nor le a d a c e ta te p r e c ip i ta te the gum.

(d) S o lu tions o f the gum do no t reduce i ’e h lin g ’s so lu tio n .

(e) I f th in sec tio n s o f th e s o f t gum a re t r e a te d w ith

iod ine and examined under the m icroscope, th e presence o f a

few s ta rc h g ra in s i s d e te c te d .

22BSHODS OP A17A1Y3I8

In the a n a ly s is o f the gum and o f th e various

h y d ro ly tic p roduc ts , the pentosans were determ ined according

to the d ire c tio n s given by the A ssoc ia tion o f O ff ic ia l A gri­

c u l tu ra l C hem ists.9 However, d i s t i l l a t i o n was continued u n t i l

the d i s t i l l a t e gave but a very f a in t yellow c o lo r when tre a te d

w ith a few drops o f the ph lo rog luo ino l re a g e n t, U sually fo u r­

te e n o r s ix te e n 5 0 -e .c . p o r tio n s were c o lle c te d . A sm all c i r c le

o f high, q u a l i ty f i l t e r paper was used, in th e Goooh beneath th e

a sb esto s m at, as a fa r th e r p ro te c tio n ag a in s t lo s s o f p re c ip i­

t a t e and asbestos f ib e r s during th e subsequent e x tra c tio n w ith

a lc o h o l.

Methyl pentosans were determ ined according to the

fo lle n a - B l le t t - Haywood method,10 The d iffe re n c e in weight

between the ph lorogluoide p r e c ip i ta te f i r s t ob tained and the

fu r fu ra l ph lo rog lueide rem aining a f t e r e x tra c tio n was considered

as re p re sen tin g the weight o f m eth y lfu rfu ra l ph lorog lueide

o r ig in a l ly p re se n t. This was c a lc u la te d a s rhamnose-hydrate

according to E l l e t t ' s t a b l e .11 Then, inasmuch as ac id s o f th e

glucuronic ac id group y ie ld fu r f u ra l when t r e a te d w ith IS p e r

cen t hydroch lo ric a c id , i t was necessary to deduct the amount

o f fu r fu ra l ph lo rog lueide corresponding to any such ac id s from

the w el# it o f p re c ip i ta te rem aining a f t e r e x tra c tio n .

Lefevre12 has shown th a t th re e p a r ts o f g lucuronic

ac id lactone correspond to one p a r t o f fu r fu ra l p h lo ro g lu e id e ,

and ra n d er Hear assumes th a t the same re la tio n s h ip ho lds fo r

ga lao tu ron ic a c id . A fte r making the c o rre c tio n fo r th e a c id , the

remainder o f th e p r e c ip i ta te was c a lc u la te d a s arabinoae on th e

b a s is o f K rober1 s ta b le . l s The weight o f arab inose thus ob tained

i s d o u b tless a l i t t l e h igh in many oases, fo r g a lac to se , w ith

12 per oent hydrochloric a c id , y ie ld s oxym ethy lfo rfu ral, which

g ives a p r e c ip i ta te w ith p k lo ro g lu c in o l. The e r ro r v a ried

according to the amount of the hexose p re se n t in th e sample.

but a s i t was not g rea t in any case, no c o rre c tio n was made*

The estim atio n of the a c id o f the g lucuronic •

acid group was c a rr ie d out according to the method o f l e f e v r e .14

The weight o f carbon dioxide l ib e ra te d was m u ltip lie d by fou r

to ob ta in the amount o f a c id lac to n e p re se n t.

G alactose was determined by a method which was

developed by combining p a r ts o f van dor Hear1 s p ro c e d u re ^ w ith

th e o f f ic ia l m ethod.16 A sample la rg e enough to y ie ld approx i­

m ately 0.1 gm. o f muoic ac id was taken and enough sucrose added

to make a t o t a l o f 1 gram. 60 c .c . o f n i t r i c acid f s p .g r . 1.15)

were added and th e s o lu tio n evaporated a t 96°0 to a weight o f

25 gm. A fte r coo ling , 0 .5 gm. pure muoic so ld was added. A fter.

24 hours, 10 c .c . o f w ater were added. A f te r ano ther 24 hours

the muoic ac id was f i l t e r e d o ff , washed w ith 10 c .c . o f water

and d isso lv ed in SO c .c . o f ammonium carbonate so lu tio n (con­

s i s t in g o f 1 p a r t by weight ammonium carbonate, 19 p a r ts w ater,

and 1 p a r t s tro n g ammonium hydroxide) by h ea tin g on the w ater

ba th a t 80°C. The re sid u e was f i l t e r e d o f f , washed w ith h o t

w ater, and the f i l t r a t e evaporated to dryness a t 80°0. The

ammonium mueate was decomposed by the a d d itio n o f 5 c .c . o f

n i t r i c ac id (sp . g r . 1 .1 5 ) . The muoic ac id was f i l t e r e d in to

a weighed Gooch, washed fo u r tim es w ith 5 c .c . s a tu ra te d muoic

acid so lu tio n and then w ith 5 c .c . o f w ate r, and d r ie d in the

oven a t 85° - 90°C.

The mucio ac id added was prepared from la c to s e ,

r e c r y s ta l l i s e d , and d ried to constant w eight. Although i t was

pure (m.p* 215°0 .), i t was found th a t i f 0 .5 gn. o f i t were

added to 25 gm. o f the n i t r i c a o i t obtained "by tlie evapora tion

o f 60 c .c . o f n i t r i c aoid (sp . g r . 1 .15) and then taken through

th e re g u la r procedure, only 94-96 p e r oont was recovered . Hence,

95 por oont o f the weight o f nucip ac id added was su b trac ted

from tro i^h t o f the to t a l p r e c ip i t a te . The amount o f g a lac to se

corresponding to, tho nuclc ac id from the sample was found by

re fe ren ce to a ta b le . In van dor H aar.17

,5ho per cent o f g a lac to se thus c a lc u la te d was

high bsoause galao tu ro n ic ac id , the .aldehyde ac id o f the gum,

y ie ld s music so ld upon ox idation w ith n i t r i c a c id . . The exact

re la tio n s h ip between tho weight o f ac id p resen t and th e weight

o f mucic acM obtained has n o t been worked o u t, but. E h rlich

obtained frcm te tra -g a le o tn ro n io ao id a y ie ld o f fO. per cen t or

more o f pure mucic acid.*-8 G alactose g ives about 75 per cent

o f i t s weight o f mucic ac id , so van der Haar concludes th a t one

can roughly determine the ac id ju s t as in th e case o f g a lac to se ,

u sing tho same ta b le . Hence, to determine th e p e r cent of

ga lac to se a c tu a lly p resen t in the sample, the p e r cen t o f a c id

(based on the carbon-d ioxide determ ination) was su b trac te d from

the t o t a l g a lac to so .

Ash was determ ined by tak ing a 0 ,5 gm. sample,

ig n i t in g a t a low tem perature u n t i l thoroughly charred , and then

h ea tin g a t redness fo r S o r 4 hours, or u n t i l th e weight was

constan t •

M oisture was determined hy drying th e sample to

constant weight a t 95-98°C.

H itrogon was doterminad lay th e E ja ld ah l method.

AH&LY3IS OF 2HB GU1I

A complete a n a ly s is o f th e gnm gave th e r e s u l ts

reeorded in fa b le Ho. 1 .

fABhB HO. 1 . - - C0MB0SE?I01T OF GHOIdA GUM

. ~ ' :C onstituen t : P er cent found

i

::

M oisture (o ld gum) *::

6 .9

Ash..................... 7 .8

A cid................... ..%

I

%

10.7

P en to se* * * . . . ........... 49 .6

G a l a c t o s e . . . . . . . . . 7 .8

Bhamnose........... ..%

i5 .1

n i t ro g e n . . . . . . . . . . JQ i±T o ta l ........... ..

%

:88 .3

* M oisture in th e fre sh gum v a r ie s from 20-40 p e r

*’C alcu lated a s a rab inose .

Anderson and Sands19 analyzed tho ash obtained

by burning th e gum a t a low tem p era tu re . The r e s u l t s are g iven

in Table Ho. 2 .

2ABLE BO* 2 . —0(LEP0SI3?I01I OF ASH

tConst i t mmt : For cen t found

S i O g # # * * # # # # * # # # » 1 3 . 5 9

1t T n l m z n e d c a r b o n . / ; 3 . 7 5

%

C 0 2 a » * * Z 2 7 . 5 7

- - - - - : : . .

8 0 ^ * * * * # # * * * # * # # * %

C l * # * # # * * * # » * * * * # - * %'

2 . 9 7

4 * 6 7

. . . . . . - #

# * * # # # # * * # * * * ! " • > 9 4

:

C a O e « # # * a # # # * # ' # * # # ; #

3 6 . 7 1

a

M g O . . » • * •

$

8 . 7

FegOg 4 AlgOg*• .« : .40. ; / ' ' t

Httg 04 . • • • • • • • • • * • #40"

f o t s l •••* ••* • 89*70________ ;________ ;_________! ________________

fhe gum was t r e a te d w ith 100 tim es i t s weight

o f water and allow ed to stand 40 hours. She l iq u id p o rtio n

was separated from th e in so lu b le m a te r ia l by f i l t r a t i o n and

c e n tr ifu g in g , evaporated to d ryness, and ana ly sed . The in s o l­

uble m a te r ia l , which rep resen ted 80 p e r cent o f th e gum, was

d r ie d in th e oren a t 95°C. and analyzed . The r e s u l t s a re given

in Table Bo. 3 . The p e r cen t o f each c o n s titu e n t in th e o r i ­

g in a l gum, c a lc u la te d on th e dry b a s is , i s g iven fo r com parison.

I1BLB HO. S .—COMPOSITIQII OF THE HSOKJBLB AHD THE VIA TER-

SO im m ZHAOTIOHS OF OHOHLA GUM, IH PEE CEHT■/:'L

A ... :- M o n s ti tu e n tA ' ............ ...............

C hoila gum:i

;W ater-soluble

f r a c t io n1 Inso lub le1 f r a c t io n

•' A s h . . . ............... 8 .4:

9 .1 $ 5 .2

A cid................. .. 11.5i: 14.1 s 8 .6

P en tose........... . 53.8t: 42 .2 : 62.5

R h am n o se ...... 5 .5i: 4 .5 t 1 . 1

G a la c to s e . . . . . - M i# ! 15*1T o t a l . . . , 87 .0

e

i:

78 .0 ; 92.4

' ;■,!<

4' ;

HYDROLYSIS OP THE GUM

Tho f in e ly powdered gum was thoroughly mixed with

10 tim es i t s weight o f 2 per cen t su lphu ric ac id so lu tio n and

heated in a w ater h a th a t 60°0. fo r 27-29 hours. A fte r th i s

trea tm en t 10 p e r cent o f th e gam remained in so lu b le . This r e s i ­

due was f i l t e r e d o f f and washed fou r tim es w ith hot w a te r . The

f i l t r a t e was n e u tra lis e d w ith calcium ca rbonate , sm all amounts

of b u ty l a lcoho l being used to prevent excessive foaming. A fte r

th e removal of th e calcium su lphate p r e c ip i ta te , th e f i l t r a t e ,

which was ligh t-brow n in c o lo r , m s deco lo rised by b o il in g w ith

I e r r i t . The r e s u l t in g so lu tio n , which was almost c o lo r le s s , was

concen tra ted in la rg e evaporating d ish es over a low flam e.

Excessive foaming of th e so lu tio n prevented the use o f d ie t I l l a ­

t io n under reduced p ressu re* Calcium carbonate was kept present

during th e evapora tion . At a low volume, th e calcium su lphate

and th e excess eaioium carbonate were f i l t e r e d o f f , and th e

syrup was fu r th e r evaporated u n t i l i t was 44-46 p e r cen t s o lid s ,

a s determ ined by th e Abbe re frac to m e te r , , !

The syrup was then poured in to 3 : tim es i t s v o l­

ume o f 95 per cen t e th y l a lc o h o l, slowly and w ith v igorous

s t i r r i n g . 'A l ig h t-y e llo w , curdy p re c ip i ta te sep a ra ted , f h is

p r e c ip i ta te w a s 'f i l te r e d o f f and washed by t r i t u r a t i n g w ith

warm a lco h o l. A f te r a second f i l t r a t i o n , i t was d isso lved in

hot water and the t o t a l volume ad ju sted to 50-54 p e r cent s o l id s .

One and o ne-ha lf to two volumes o f a lcohol were added to the

syrup, slow ly and w ith s t i r r i n g . A p r e c ip i ta te aga in separa ted

in a s o f t crumbly mass. I t was f i l t e r e d o f f and e x tra c ted sev­

e r a l tim es by warming w ith alcohol on th e w ater b a th . I t was

then in such a co n d itio n th a t i t could e a s i ly be ground to a f in e

powder. A f te r powdering, i t was ag a in washed w ith hot a lco h o l,

f i l t e r e d and pu t on a porous p la te to d ry . A ll the a lco h o lic

f i l t r a t e s were combined and allow ed to stand over n ig h t.

When th e o r ig in a l so lu tio n was being n e u tra lis e d ,

i t was necessary to add more calcium carbonate th an was req u ired

to re a c t w ith th e su lphu ric ac id p re s e n t . The excess carbonate

re ac ted w ith th e ac id complex of th e gum (from which p a r t of

th e sugar had ju s t been separa ted by h y d ro ly s is) to form so lub le

calcium s a l t s , which were l a t e r p re c ip ita te d ' by th e trea tm en t

w ith a lc o h o l. Theso s a l t s w i l l be re fe r re d to a s calcium s a l t s

- 11-

aiaso lved in th e a leo h o l; any nhich remained adhering to th e

p re c ip i ta te m o removed "oy th e second p re c ip i ta t io n and snbss-

quont ex trac tio n s* Owing t e th e s l ig h t s o lu b il i ty , o f th e c a l ­

cium s a l t s in the a lco h o l, th e y were never com pletely separated

from th e sugar. Some remained in th e a lcoho lic e x tra c t where

th ey doubtless in te r f e re d w ith th e c r y s ta l l i s a t io n o f th e sugar,

Before th e above p la n o f h y d ro ly s is was decided

upon, a number of d if fe re n t experiment a were conducted in v&ioh

th e gum was hydrolysed a t d if fe re n t tem pera tu res and in the p re s ­

ence o f vary ing amounts o f ac id s . Anderson and Sands,30 from

th e i r work on mosquito gum, had found th a t 1 -arab inose wxs hy­

drolyzed o f f most r e a d i ly from th e complex gum m olecule. I f th e

pentose in o h o lla gmn wore likew ise loosely a tta c h e d , p a r t i a l

h y d ro ly sis would fu rn ish a means o f separa ting i t from th e o th er

c o n s ti tu e n ts , fo perm it such a se p a ra tio n th e co n d itio n s fo r

h y d ro ly sis would have to bo such th a t :

(a) The pentose would be s p l i t o ff a s com pletely a s p o s s ib le .

(b) The galootoeo and m ethyl pentose would remain u nd istu rbed

(o) The aldehyde a c id would no t be destroyed*

(d) Decomposition in general would be minimized.

In th e attem pt to a so e rta in udiat th e necessary

cond itions m ig h tb e . the ac id co n cen tra tio n m s kept constan t

( a po r cent su lp h u ric ac id so lu tio n was used in each c a se ) , and

th e tem perature was v a r ie d . The p ro g ress o f th e h y d ro ly s is was

follow ed by removing 1 -c .c . samples a t in te rv a ls and te s t in g fo r

reducing sugar accord ing to th e method o f Munson and W alker.31

5ho p e r cent o f reducing sugar was c a lc u la te d as g lucose. She

r e s u l t s a re shown in Table Ho. 4 and in P i gore I .

SABLE HO. 4 . —IPflOT OF SEEBEBATOEE OH KATE OF HYDBOLYSIS IH4 BEE OEHT AO ID SOLOTIQH

Ear cen t Glucosea tu ro l/2 ~ h r; 5 h r . ; 6 'h r . ; 9 h r . ; i 2 h r .-IB h r . • 18 h r .vv . : ’ - : :______ X X : : .

: l ■ : : ’ : . ■•: .80*». • t . .35 : 2 .72 : 3.77 : 4.60 s 5.45 : i 5.58

X. '• .X . » .»9 0 . .* i .25 : 2.67 : 4 .5 2 X 5,78 I X ' '

' : . 2 ' ' " ' ! : /■ ■' -■2.1 0 * . . . : .75 : 5.15 f 5 .45 $ 5.92 X x 5.98 X

■ i ” ■' I : 1 : :80(1): * n x %m i 5 .77 X 4.52 X X ■ X

. . - i ■x : : ■; ■ ' f ■ ■90(2): .75 : 4.07 : 4 .97 : 5 W f : i

: • : : - : : : : :

f l ) and (3) ware Iccpt in th e b o i l in g w ater b a th fo r th e f i r s t

h a lf-h o u r o f th e p e r io d , and th e n continued a t th e tem perature

in d ica ted fo r the rem ainder o f th e tim e.

From th e above r e s u l t s i t was evident t h a t :

■(e) A tem perature o f 100°C. du ring th e f i r s t h a lf-h o u r p ro ­

duced no im portant d iffe re n c e s in th e t o t a l amount o f h y d ro ly s is .

(b) The ra te o f h y d ro ly s is was f a r more ra p id a t 100°C. th an

a t e 0 °0 ., fo r in 6 hours th e hy d ro ly sis was a s f a r advanced as

th a t a t 80°C. was in 12 hours.

(c) In th e case o f th e experim ents conducted a t 90°C. and

100°C. th e amount of h y d ro ly s is reached a maximum w ith in 9 hours.

Continued h ea tin g d id not in crease the p e r cent o f reducing sugar.

(d) The above maximum was not reached a t 80°0. even a f te r

18 hours o f h y d ro ly s is , bu t a p o in t was reached in 12 hours

which appeared to be th e maximum a t th i s p a r t ic u la r tem pera tu re .

oT/me in

3hours

rigu re I.H A T E OF HYDR0LY5IS

ofCHOLLA 6UM IN 4 •/> A ZO

/5 /d 24

oreaao of roducing sugar o f only .0.15 p e r cent*

E v iden tly , th en , a tem perature o f 90°C. o r 100°C.I '

was causing th e hy d ro ly sis o f something th a t a tem perature o f

@0*0. l e f t und is tu rb ed . To determine what t h i s substance was,

th e s a l t s were is o la te d in th e case of a h y d ro ly sis a t 80*0.

and a lso in th e case o f a h y d ro ly s is a t 100*0. The composi­

t io n o f th ese s a l t s i s g iven in Table Ho. 5 .

TABLE 110. 5 . — COHPOSmOir OF C/u-OIOH SALTS "An FEOli HYDROLYSIS

AT 80*0. MB 100*0,*

C onstituen t: :% P er cent- in s a l t s : P er cen t in s a l t s: from h y d ro ly s is a t : from

^ioOOO?318 ^

P e n to se ? .. . i,s 24.9 v;! 21.7

18.7

16.9

29.5

Rharanoso • . . . . 20.0

. »s8

Ash......... .. 10 .4' 8

' |

G a la c to se * ...:

" ■ I •------ :____ _

• As th e ac id in th e se s a l t s was no t determ ined, the percen tages of ga lac tose and pen tose re p re sen t th e amounts of th o se sugars a c tu a l ly p re sen t p lu s th e amounts in d ica ted by the.m usic ac id and fu r f u ra l derived from th e ac id in th e re sp e c tiv e an a ly se s .

The s a l t s from th e gam hydrolyzed a t 100*0.

showed a much h ig h er ash con ten t th an those from tho h y d ro ly s is

a t 80*0., th u s in d ic a tin g a much sm aller m olecule. The p e r­

centages o f pentose and rhamnose d if fe re d bu t s l ig h t ly , hut th e

percentage o f g a lac to se was 7*7 per cent lower in th e s a l t s o f

14-

hlgh ash ooHtont. The conclusion was th a t ga lac to se was hy­

drolysed o f f in more app reciab le q u a n ti t ie s a t 100°C. than a t

»0°Q. The tem perature of 80°C. th u s appeared to "be more favo r­

ab le fo r th e sep a ra tio n o f th e pentose from th e g a la c to se .

The e f fe c t o f varying degrees o f ac id concen­

t r a t io n a t 80°G. was next s tu d ie d . When 4 p e r cent ac id so lu ­

t io n was used , th e so lu tio n became very dark and the s a l t s wore

h igh ly co lored even though the so lu tio n was t r e a te d w ith U o rr it .

I t seemed probable th a t 4 p e r cent ac id was causing decomposi­

t io n th a t a lower ac id co n cen tra tio n might re ta rd or even p re ­

v e n t. Hence, some of the gum was hydrolyzed in 4 p e r c e n t, 2

per ce n t, and in 1 p e r cent su lp h u ric ac id so lu tio n . The hy­

d ro ly s is was followed as befo re and th e reducing sugar c a lc u la te d

as g lucose . R esu lts a re given in Table Ho. 6 and in Figure I I .

TABLE HO. 6 .—EFFECT OF ACID COHCEHTBATIOII OH RATE OF

AT 80°C.

iOLYSIS

Tima, hours$ P er <sent glucose

•4 per cen t a o id •2 p er cent a c id ‘1 p e r cent aoid

S . . . . . . . . : 2.72: z

i6 . . . ......... f 3.77 i !9 . . . . . . . : 4 .60 i 5.75 : 2.55

1 2 . . . . . . . . 5.45 i 4 .00 : 2.571 8 . . . . . . . . : 5.58 I : —1 9 . . . . . . . . : $ 2.872 0 . . . . . . . . * % ** ! 4.40 :8 9 . . . . . . . . ! 4.67 : 5.755 6 ............ . 1 : 4.2745............... 4 .25

-15 -

Hhe so lu tio n s in the case o f th e 3 p e r cen t and

the 1 p e r coat ac id co ncen tra tions were much l ig h te r in co lo r

than th e so lu tio n in th e case o f th e 4 p e r cent a c id . Shey were

not tre a te d w ith H o rr it , hut th e s a l t s were a s lig h t-c o lo re d

as any th a t had been obtained a f t e r th e d eco lo riz in g of a 4 p e r

cent ac id so lu tio n . Here was one in d ic a tio n o f le s s decompo­

s i t io n . Another in d ic a tio n m s fu rn ish ed by the fa c t th a t la rg e r

q u a n ti t ie s o f bo th a rab inose and s a l t s were is o la te d in the cases

where th e ac id co n cen tra tio n was low (Table Ho. 7 ) .

TABLE HO. 7 .—AH0UH2S OP ARABI1I0SB AHD SALTS ISOLATED HtCEI

HYDROLYSES QAH3IED OUT IH DIPPERBUT ACID CQHCEUTBATIOHS .

(Expressed in grams p e r 100 grams of gum hydrolyzed)

:$:

4 p e r cen t a c id

:i:

2 p e r cen t a c id

: 1 p e r cen t $ ac idl _ . _ . . .: : :

S a l ts i s o la t e d . ......... : 20.5 : 84.5 ■ $ 32.88 5• ■ i : 2

Arabinoao i s o l a t e d . . : 88.8 : 33.0 8 31.5: i f: : :

The s a l t s is o la te d had th e com position in d ic a te d

in Table Ho. 8 .

TiSLB HO. 8 .—COMPOSE?IGH OI1 CALCIUM SALTS "A" PROM HYHROLYSRS

AT BIPPSREU2 ACH) COHCBHTRAHOHS

Conet itxient P er cent found|4 p e r cent a c id 2 p e r cen t ac id *1 p e r cent ac id

P e n to s e . . . . 8 .0 4 .5 8 .3

Rhamnose.. . , z 25.0 27.9 21.6

A c id .......... .. ,: 29.7 28.6 25.8

G tilaotoao.. 9 .6 7 .3 8 15.7

A s h . . . . . . . . .: g

U *5 12*0 IL titT o ta l.

t83.8 80.3

8

:82.8

Prom the foregoing da ta i t may he soen th a t the

h ighest y ie ld of oalcima s a l t s mas ob tained in the caso o f th e

hyd ro ly sis in 1 p er cen t ao id , and i t was th e se s a l t s which

had th e h ig h est p e r cent o f g a lac to se p re se n t. However, th e

time repa ired fo r th e hyd ro ly sis (56-40 hours) made th e use o f

th i s o o aeen tra tio n im p rac tica b le . Prom a l l p o in ts o f view, th e n ,

2 p e r cent seomod th e most favo rab le co n cen tra tio n to u se .

Hence, i t was decided to hydrolyse th e gum in 2 p e r cen t a c id

in the manner a lready d esc rib ed .

-17

IS01A2I03 OP 1-A3&BIFOSS

She a lcoho l © stree t ohtainecl ’oy tho p ro o ip itc -

t io n and washing o f calcium s a l t s "An vras f i l t e r e a ana then

concen tra ted on th e b o il in g w ater b a th under rednoed procsure

to 84-86 p er cen t so lid s* Methyl a lco h o l was added to make th e

syrup th in and f lu id . A 5 0 -c .o . p o r tio n m s s u f f ic ie n t fo r th e

syrup obtained from 250 gm. o f gum. E thy l a lcoho l m s not used

a t t h i s p o in t, fo r i t had a tendency t o p r e c ip i ta te s a l t s . The

syrup was then seeded w ith pure arab inose and pu t in to tho r e ­

f r ig e r a to r . C ry s ta l l is a t io n began in an hour or so , and w ith in

12 hours th e whole mass was th ic k w ith c ry s ta ls . I t was allowed

to stand fo r a week, w ith occasional s t i r r i n g , in o rder to allow

c r y s ta l l iz a t io n to become a s complete a s p o s s ib le . A m ixture

o f 1 p a r t g la c ia l a c e t ic ac id and 55 p a r ts 95 p e r cen t e th y l a lc o ­

ho l m s added to make a th in p a s te , and th e c r y s ta ls were th en

f i l t e r e d o f f on a Buchner funne l, removed from th e f i l t e r , mixed

w ith th e a c e tic a c id - a lcoho l so lu tio n , and again f i l t e r e d .

They were washed once more and then put on a porous p la te to dry.

The c r y s ta l l in e product was yellow ish in co lo r and o ften some­

what gummy, owing to th e incom plete removal o f th e m other syrup.

The crude sugar was p u r i f ie d by h e a tin g i t in

th e b o il in g w ater b a th w ith 1-|* tim es i t ' s weight o f g la c ia l

a c e t ic a c id , f i l t e r i n g o f f th e c r y s ta ls , washing them w ith 95

p e r cen t e th y l a lco h o l, and re o ry o ta ll iz in g them from a m ixture

o f t h e i r weight of w ater and 4 tim es t h e i r weight o f a lc o h o l.22

v

One c r y s ta l l iz a t io n gave a white product w ith a m elting po in t

of 149-152% . Tko sp e c if ic ro ta t io n o f t h i s m a te ria l in 4 p e r

cent so lu tio n a t 20% . was 4-105°. The ro ta t io n of 1 -arah inose

as given in th e l i t e r a tu r e i s 4104-105°. 23 This f a c t , to g e th e r

w ith the f a c t th a t th e syrup c r y s ta l l i s e d when seeded w ith pure

arah inoso , id e n t i f ie d th e su g ar.a s l-a ra b in o se .

mBSTIQATIOIT OJ? THE 1I0TE3R LIQUOR FROH FIRST CROP

ARABLOOSE

The y ie ld of th e f i r s t crop o f arab inose v a ried

from 28.5 - 56.4 p e r cen t o f the weight o f th e gum hydrolysed,

th e average fo r n ine hydro lyses b e in g 27.5 p er c e n t . A second

crop o f c ry s ta ls was ob tained by co n cen tra tin g th e m other l iq u o r

from th e f i r s t crop , seeding w ith a rab in o se , and a llow ing to

stand fo r sev e ra l weeks. However, t h i s second crop was always

so small th a t i t was sep ara ted from th e syrup w ith d i f f i c u l ty .

In th e attem pt to increase th e y ie ld , some of th e mother liq u o r

was poured in to a la rg e volume o f §5 p e r cent e th y l a lco h o l,

slow ly and w ith s t i r r i n g . A lig h t-o o lo ro d flo o o u len t p re c ip i­

t a t e sep a ra ted .* The a lc o h o lic s o lu tio n was decanted , evaporated

to sm all volume and seeded w ith arab inose. C ry s ta ls formed, but

not in appreciab le q u e n tl t ie * . Another p o r tio n of th e syrup

was poured in to a m ixture o f 400 c .c . o f 95 p e r cent a lcoho l

♦The exact n a tu re o f t h i s p r e c ip i ta te was no t determ ined.

-1 9 -

and 100 o .o . ab so lu te e th e r . A bronnish gum m s p re c ip i ta te d .

The c le a r s o lu tio n was decan ted , concen tra ted , and seeded w ith

arah inose. Again c ry s ta ls formed, hut not to any g re a t e x te n t.

Since e f f o r t s to secure a second crop o f a rah in ­

ose were f r u i t l e s s , some o f th e mother syrup from the f i r s t crop

was evaporated t o dryness and analyzed. The r e s u l t s a re given

in Table Ho. 9 . The dry syrup c o n s ti tu te d about S3 per cen t

o f th e weight o f th e o r ig in a l gum.

TABLE HO. 9 •—COMPOSITIOU OF MOTHER LIQUOR FROM FIRST CROt

ARAB III OS IS

C onstituen ts::

P e r cent found :Grams p e r 328 •Esams o f syrup

A sh.. . . . . . . . .:

4 .7 *: 15 .3

* 17. *5A c i d . . . . . . . . .:

5 .3

7.7

3.7

G alactose. . . . ! 25.3

: 12.1

: U M

: 183.2:

Rhamnose. . . . .:

P entose. . . . . .' 8

34.4

T o ta l . . .$

55.8:

The a n a ly s is showed th a t only email amounts o f

g a lac to se and rhamnose were hydrolyzed o f f a t 80°0. in 2 p e r

cent ac id s o lu tio n . Consequently, th e co n d itio n s chosen were

very favorab le to tho sep a ra tio n o f the arah inose from those

Although th e s a a ly s is InaicatecL th a t pentose was

p resen t In la rg e cLuantltles ( to the ex ten t o f 54.4 p e r cen t of

th e t o t a l sy ru p ), seeding w ith arabinose re su lte d In the separa-: ' ' ' - 't io n o f only a sm all crop of c r y s ta ls . Beferenoo has a lre ad y

been made to t h i s f a c t . B ertran d ’s t e s t fo r xy lose24 was nega­

t iv e , but th e re were such la rg e q u a n ti t ie s o f o th e r substances

p resen t to in te r f e r e th a t t h i s fa c t d id not prove i t s absence.

On th e co n tra ry , i&en th e syrup was seeded w ith pure xy lose ,

c ry s ta ls formed slow ly . This was a s trong in d ic a tio n o f th e

presence o f xy lose , but no t enough o f th e m a te r ia l sep a ra ted

to perm it a complete id e n t i f i c a t io n . Seeding th e syrup w ith

mannoso and dex trose re su lte d in ho c r y s ta l l i s a t io n .: ' ' .................... ....................................'

When th e syrup was tr e a te d w ith Sellw anoff* s r e ­

ag e n t,25 an orange-red c o lo r formed, follow ed by th e sep a ra tio n

o f a dark-brown p r e c ip i ta te , # iio h was so lu b le in e th y l a lc o h o l.

When the a c id so lu tio n was made a lk a lin e w ith sodium carbonate

and shaken w ith amyl a lcoho l a g reen ish flu o rescen ce26 developed.

The syrup a lso gave a yellow p re c ip i ta te w ith th io b a rb itu r ic

a c id .27 Both o f these t e s t s in d ic a te d th e presence o f a k e to -

hexose. However, the syrup d id not ferment w ith compressed yeast#

thus showing th e absenoo o f fru c to se , a s w ell as o f glucose and

mannose. The id e n t i ty of th e ketose remains to bo e s ta b lis h e d .

•The y eas t a v a ila b le did n e t ferment pure g a la c to se .

was unaccounted fo r . As a fu r th e r a ttem pt to d iscover what the

unknown m a te r ia l n ig h t b e , a le v u lin io a c id determ ination was

c a rr ie d o u t .28 Seventeen grams of the syrup y ie ld ed only 0 .6 gm.

of le v u lin io a c id , whereas 4-5 grams would have re su lte d i f h a l f

o f the sample had been hexos®. l‘he galac to se p re se n t doub tless

gave th e sm all amount th a t was ob ta ined , fhe r e s u l ts o f th i s

t e s t were in accord w ith those o f th e ferm en tation t e s t , bo th

in d ic a tin g the absence of la rg e q u a n t i t ie s o f hezosee o th e r than

g a la c to se . 2he q u a l i ta t iv e t e s t fo r sugar a lco h o ls29 was nega-

t iv e . ' ■ ■ ;• Some o f the syrup was d isso lv ed in 2 p e r cent s u l­

phuric ac id so lu tio n and heated in th e au toclave a t a tem pera­

tu re o f 120°C. fo r 8 h o u rs . Shore was an alm ost n e g lig ib le

increase in the amount of reducing su g ar. Jh irth er h y d ro ly tic

treatm ent was th e re fo re of no p a r t ic u la r v a lu e .

HYDROLYSIS OP CALOIUE SALTS ”An

The calcium s a l t s "A" ob tained by p r e c ip i ta t io n

w ith e th y l a lco h o l c o n s ti tu te d about 28 p e r cen t of th e weight

o f th e gum used in th e h y d ro ly s is . They were an amorphous

powdery p roduc t, ligh t-b row n in c o lo r , and very soluble in w ater.

•The r e s u l t s o f t h e i r a n a ly s is are given in Table Ho. 10.

fABLl 10. 1 0 .—COHPOSIHOIi OP CALCIUM SALTS ttA”

C onstituen t :Per cen t found $ grams of s a l t s

A s h . . . . . . . . . . * 2 ' ' 9 .4 • 2 ■ 25.9 '

: A c i d . . . . . . . . . . t ' S3.9 ■ - t 97.0

G a la c to se ... . . 2 6 .1 2 14.5

P e n t o s e . . . . . . . 2 5 .5 s 15.7

Bhamnoso. . . . . . ? 27.9 ’ ' t

- . ! ' , # * # #t 81.8 : 235.9

, Inasmuch as h y d ro ly s is o f the o r ig in a l gum a t

100oC. haa g re a tly aeoreasea th e ga lao to se con ten t o f the s a l t s ,

i t was decided to fa r th e r hydrolyse a t 100°C. th e s a l t s from a

hyd ro ly sis a t 80°0. They wore d isso lv ed in 10 tim es t h e i r

weight o f 1 p e r oont su lp h u ric ac id so lu tio n to which enough

sulphuric ac id was added to p r e c ip i ta te th e calcium . They were

then hydrolyzed in the l o l l i n g w ater ba th fo r 6 h o u rs . The

choice o f acid co n cen tra tio n and tim e o f hy d ro ly sis was made

a f te r hydrolyzing some o f th e s a l t s in 4 p e r c e n t, 2 p e r c e n t,

and 1 por cen t ac id s o lu t io n . R esu lts are shown in Table Ho. 11

and Pigure I I I .

TABLE 10* 1 1 .—BATE OF HYDROLYSIS OF CALCIGU SALTS "A" AT 100°C.

Baa roachoa a mazlEain In each ease . Since the decom position vms

lo ss in th e 1 p e r cent so lu tio n th a n in th e so lu tio n s of h ig h er

acid co n cen tra tio n , i t was decided to hydrolyze in tho 1 p e r cent

acid fo r a p erio d of 6 hours.

A fte r th e com pletion o f th e h y d ro ly s is , tho ac id

was n e u tra liz e d w ith calcium carbonate, th e so lu tio n f i l t e r e d ,

d e c o lo r iz e ! , and evaporated as in the case o f th o gran i t s e l f .

The syrup was taken to 55 p e r cent so lid s and th en poured in to

3 volumes o f 95 p e r cen t e th y l a lc o h o l. A l ig h t-c o lo re d g ran u la r

p r e c ip i ta te o f calcium s a l t s nBn sep a ra ted . Those were washed

w ith a lco h o l, d isso lv e d in enough water to make th e so lu tio n

65-65 per cent s o lid s , end re p re c ip i ta te d hy pouring two volumes

p f a lco h o l in to th e sy rup . They were th en ex trac ted severa l t in e s

w ith hot a lco h o l, powdered in a m o rta r, and d r ie d .

Pcr-c e

nr Glue 05 e

3.0

*°7- L 2/ //77e m hours 3 3

R A T E

CALC/UM

6 7

F igure _ZZT.o r HYDROLYSIS

o fS A L T S "A " A T 100° C.

8 9 /O / / /2

ISOLAHOH OF d-GALACTOSE

The a lc o h o lic e x tra c t from calcium s a l t s nB"

was allowed to stand over n ig h t . I t was th en f i l t e r e d and con­

ce n tra ted as much as p o ss ib le under dim inished p re ssu re . The

syrup was thoroughly mixed w ith an equal volume of g la c ia l

a c e t ic ac id and seeded w ith g a la c to se . C ry sta ls formed almost

im m ediately. A fte r a llow ing sev era l days fo r complete c r y s t a l l i ­

za tio n , th e c r y s ta ls were f i l t e r e d o f f and washed sev e ra l tim es

w ith a m ixture o f 1 p a r t of g la c ia l ao e tio ac id and 5 p a r ts 95

p e r cent e th y l a lc o h o l. The y ie ld of sugar was 6 .2 p e r cen t o f

the weight o f s a l t s hydrolysed, or 1 .7 p e r cent o f the o r ig in a l

o h o lla gum.

The sugar was p u r i f ie d sev era l tim es by r e c r y s ta l l

i s a t io n from a m ixture of i t s weight o f w ater and 4 tim es i t s

weight of 95 p e r cen t a lco h o l. I t gave mucic a c id upon oxida­

t io n w ith n i t r i c a c id , i t had a m olting p o in t o f 163° - 165°C.,

and in 4 p e r cent so lu tio n th e s p e c if ic ro ta t io n was 4 7 9 .7 ° .

These fa c ts Id e n tif ie d the sugar a s d -g a la c to se .

The mother syrup , which c o n s ti tu te d 2,7 p e r cen t

o f th e weight o f th e o r ig in a l gum, was evaporated to dryness

and analyzed. He s u i t s a re g iven in Table Ho. 12. I t i s evident

th a t th e amount o f methyl pentose th a t hydrolysed o f f w ith the

g a lac to se was sm a ll.

SABLE HO. 1 2 .--COHPOSISIOH' OE IIOSHBE STOOP TOOK

v SHE QALACSOSE

C onstituen t::Per cent found

' :

: gt ^: 53

ams p e r 87 rams o f syrup

: : ■A s h .. . . . . . . . 5 .2 : . .

A c i d . . . . . . . .2

8 .9:: 2 .4

I - - :G a la c to se .. . * 11.5 5 .1

■* : -P e n to s e . . . . . * 29.4 ; 7 .9

2 - :Bhnm noae.... 1 5 i l :

i ■ s ' -T o ta l . . 70.9 : 19.1

:: : '

HYDBO LYSIS Of CALC IOH SALTS "B" HI AOSOCLAVE

She calcium s a l t s nBn were a l ig h t - h r om pro ­

duct which reduced fe h lin g 1 s so lu tio n very slow ly in the co ld .

They c o n s ti tu te d 70 p e r cent o f th e weight of calcium s a l t s "A"

and 20 p e r cent o f th e weight o f gum used* Their, com position

i s shown in Table Ho. IS . They had l i t t l e i f any g a lac to se

attached, and only a tra c e of pen tose .

lBBLE HOw 13 •—COMPOS II11 Oil OF CALCIUII SALES «»*

C onstituen t !£*

**

**

*

**

**

**

I

$. ; :A s k . . . . . . . . . . 9 .5 ■■ i

: $A c i d . . . . . . . . . 2

• • . . . , .. . . 2 . ' ' . 2

G a la c to se .. . . 0 I

Groms per 200 grams of s a l t s

19 .0

81.6

6"

Total

J12

2.7

28.5

# * # # f2

81.5

222I1I

5 .4

163.0

The problem now wee to separate th e methyl pen­

to se w ithout a too g re a t d e s tru c tio n o f th e aldehyde a c id .

Some of the s a l t s were b o iled under a re f lu x

condenser in 1 p e r cent and in 0*5 p er cent su lp h u ric acid so lu ­

t io n . In bo th oases th e re was an increase in th e p e r cent of

reducing sugar, bu t th e increase was so slow th a t i t was no t

p ra c tic a b le to hydrolyse a t such a low tem perature.

Some of the s a l t s were then hydrolysed in th e

au toclave a t 120°G. in 2 p e r ce n t, 1 per cen t, and 0 .2 p e r cent

su lp h u ric ac id so lu tio n . Eke increase in p e r cent glucose i s

shown in Table lio . 14 and F igure IV.

ZABLE 110. 1 4 .—EATS OS HYBBOLYSIS OF GALCIUK SALTS

"B” AT 1S0°C.

Tim©1 P er cent glucose

» In ho u rs;— :— :-------- --------;---------------- -— —r - — ----- :--------- -*2 per cen t acicl*! p e r cent ac id ^0 .2 p e r cen t ac id: • ■ : I

Q # # * * * e * 2 1 .1 2 2 : *8T■ ‘ • .: ’ “ . i ' . :

4se # # * # # #. # 2 .9 0 ' t 2 .3 2 . : 1 .8 7$ i "

8»**»*e*I 3 .7 0 : 3 .2 5 2 2 .4 0: 2

l&*e e . e e : • 3 .7 0 • 3 .1 1• : ...... ' ' .

Inasmuch a s long trea tm en t in th e au toclave was

accompanied by considerab le decom position, i t was decided th a t

hydro lysis in 2 p e r cent ac id fo r a period o f 8 hours was the

bee t mode of p rocedure .

A fte r hyd ro ly sis under those co n d itio n s , the so lu ­

t io n was n e u tra lis e d w ith calcium ca rbonate , f i l t e r e d , d eco lo r­

ized , and evaporated to 50 p e r cent s o l id s . The syrup was poured

in t o th re e volumes o f 95 p e r cen t a lcoho l , and once more a l i g h t -

brown p re c ip i ta te of calcium s a l t s (designated as "C") sep a ra ted .

These s a l t s were washed w ith a lco h o l, d isso lved in w ater, r e -

p re c ip ita te d from a s o lu tio n co n ta in ing 50 p er cent s o l id s , and

e x tra c te d w ith hot a lc o h o l. They were f in a l ly powdered and d ried

5.

4

i

2in H rt / /* - c

o 4 6

o

Figure HZ'RA TE OF HYDROL YSfS

o fCALCIUM SA LTS "B" A T t2 0 ° C .

6 10 12. / 4 .

I30M TI01I OF 1-RH6J2I0SE

$k© alooholt© e x tra c t ±rom s a l t s "0" was f i l t e r e d

and coneeatra ted under reduced p ressu re to a very th ic k syrup.

Siough g la c ia l a c e t ic ac id was added to make a th in syrup. With­

out seeding, t h i s syrup showed no evidence o f c r y s ta l l i z a t io n

a t tho end o f th re e weeks, hut a f t e r adding a few c ry s ta ls o f

rhamnoso, c r y s ta l l i s a t io n began a ln o s t immediately and w ith in an

hour the syrup was th ic k w ith c r y s ta l s . A fte r se v e ra l days the

c r y s ta ls were f i l t e r e d o f f and washed w ith a m ixture o f alcohol

and g la c ia l a c e tic a c id . The y ie ld was 2 ,7 p e r cent o f th e

weight o f th e o r ig in a l gum, or IS . 6 p e r cent o f the m ig h t o f

calcium s a l t s . nBn.

The sugar was p u r i f ie d hy d is so lv in g i t in w ater,

d e c o lo ris in g the so lu tio n w ith H o r r i t , co n cen tra tin g to 68 p e r

cent solid® , and adding o n e-h a lf o f the volume o f g la c ia l a c e tic

a c id . The sugar so ob tained had a m elting p o in t o f 92-94°C., arid

gave a phenylosazone w ith a m eltin g po in t o f 178-180°0. In 4

p e r cent so lu tio n , th e sp e c if ic ro ta t io n a f t e r 5 m inutes was

-5 .1 ° ; in 10 m inutes i t was 0°; and th e f in a l constant value

was + 7 .8°. The sugar was thus id e n tif ie d as 1-rhamnose - hydra te

The mother syrup , which c o n s ti tu te d 2.7 p e r cent

o f th e weight of the o r ig in a l gum, m s evaporated to dryness

and analysed. The r e s u l t s o f the a n a ly s is are given in Table

Ho. 15. Only very sm all amounts o f g a lac to se and pentose wore

present, thus in d ic a tin g th e com pleteness o f th e f i r s t two

h y d ro ly ses. Tho syrup did not ferment with compressed y e a s t .

TABLE ITOe 1 5 .—00HP03ITI01I OF MOTHER LIQUOR FROM

FIRST CROP RHMiUOSS

1 1 riAT 27C onstituen t : P er cen t found : o f syrup

:A sh .................. : 6 .4 1.7

% .

Acid................. s 12.7 :1 3 .4

G a la c to s e . . . . : 7 .6 2 .0

P e n t o s e . . . , . . : 3 .6 1 .02 ' :t

R h am n o se .....: 43*3 : ULzI2 . :

T o t a l . . . : 73.6 : 19.82 :

ZDEIITIHCATICH OF d-GALAOTUROITIC ACID

The calcium s a l t s "C'% is o la te d from the syrup

con ta in ing th e rhamnose, were r a th e r dark-hrorai in c o lo r .

They reduced F eh lin g 1 a so lu tio n almost immediately in th e

co ld . T heir com position i s denoted in Table Ho. 16.

They s t i l l had a sm all amount o f rhamnose a ttach ed , th u s

in d ic a tin g th a t th e h y d ro ly s is in the au toclave had not

been com plete. 86 grams o f th ese s a l t s mere ob tained from

1 kilogram of th e o r ig in a l gum.

SABLE 210. 16 .—GOmOSISIOB OP GALCIUli SALTS "C*

C onstituen t : Per cent found i Grams p e r 86 I grams o f s a l t s

A s h . . . , . ......... .. 17 .0 2 - ■ 14.6

Acid......... ; 56.1 s 31.1

G a l a c t o s e . . . . .$ : 0

P e n to s e . . . . . . • t 2 .9 : 2.5

Bhamnose. . . . . . i 10*5 : ^ 5

T o t a l . . . . : 66.5 : 67.6

V/hen th e s a l t s were t r e a te d w ith bromine w ater

in th e co ld , a white p r e c ip i ta te s e t t le d ou t, which whon d is ­

solved i n . sodium hydroxide and re p re c ip i ta te d w ith hydroch lo ric

a c id , had a m elting p o in t o f 215°. E v iden tly mucic ac id had

been formed. G alactose does not y ie ld mucic ac id when t r e a te d

w ith bromine in th e co ld , whereas d -ga lac tuzon le ac id d o e s .30

O xidation of th e s a l t s w ith n i t r i c ac id ( s p .g r .

1*15) gave a y ie ld o f 20.7 p e r cent th e i r weight o f mucic a c id ,

while ox idation w ith bromine in a sea led tube fo r 6 hours a t

100°C. y ie ld ed 17.7 p e r cent th e i r weight o f th e a c id . Galac­

to s e , vdien heated w ith bromine in a sea led tu b e , g ives a very-

sm all amount o f mucic a c id , whereas d -g a lac tu ro n ic ac id y ie ld s

60-70 p er cent i t s w eigh t.31 The fa c t th a t th e o r ig in a l gam

and th e various s a l t s y ie ld ed carbon d ioxide when b o ile d w ith

12 p e r cen t hydrochloric acid and th a t th e s a l t s mentioned

-31-

abovo gave mucic ao ld when o x id ised w ith bromine in the cold

in d ic a te d th a t d -ga lac tu ron io ac id was p re se n t. $he absence

o f d-g lucuronio ac id was dem onstrated by thd fa c t th a t no p o ta ss ­

ium acid , saocharato was ob tained upon f i l t e r i n g o f f th e nucic

ac id a f t e r o x id a tio n of th e s a l t s w ith n i t r i c .a c id .

Some o f th e s a l t s "Cn were b o iled in 6 p e r cen t

su lphuric ac id fo r 56 h ou rs. The so lu tio n was then n e u tra lis e d ,

f i l t e r e d , evaporated and poured in to a lco h o l. Calcium s a l t s again

p r e c ip i ta te d . They were a brown powder which s l ig h t ly reduced

ffehling*a so lu tio n in the co ld . T heif com position i s in d ic a te d

in Table Ho. 17 . The id e n t i ty o f th e acid p re sen t remains to

be e s ta b lish e d .

TABLE 110. 1 7 .--COMPOSITION 01? CALCIUM

SALTS *B*

C onstituen t ::

P er cent found

:A S ite see # * te 84.8

#Aldehyde a c id . . : 8 .4

%G a l a c t o s e . . . . . . : 0

#B l i a m n o s e . . . . : 0

P e n to s e . . . . . . . . : __0#

T o ta l. . . . . : :

33.2

DISOUSSICEI OF A TYPICM, S r s is

The process o f successive h y d ro ly s is , which has

ju s t been described in d e t a i l , perm itted the sep ara tio n and

is o la t io n o f various o f the c o n s titu e n ts of the gam. When the

o r ig in a l gam was heated in 2 p er cen t ac id fo r 27-29 hours, only

the pentose was hydrolyzed o ff in conspicuous q u a n t i t ie s , A

la rg e p o rtio n o f th i s pentose was is o la te d and id e n t i f ie d as 1 -

a rah inose , but considerab le amounts were re ta in e d in th e m other

syrup. The mother syrup from a kilogram o f gam con ta ined , however

only 25,3 grams of ga lac tose and 12.1 grams o f rhamnose, thus

in d ic a tin g th a t pentose was p r a c t ic a l ly th e only sugar a f fe c te d

by th e f i r s t h y d ro ly s is . Moreover, since th e calcium s a l t s "A"

(weight 286 grams) had only 15.7 grams of pentose a ttached , i t

i s evident th a t th e pentose had been almost com pletely separa ted

from th e gum m olecule. Then, to o , d -g a lac tu ro n ic ac id had not

been destroyed , fo r th e re was as much p re se n t in the s a l t s and

in th e syrup as in the o r ig in a l gam. n e i th e r had any o f th e

©thor c o n s ti tu e n ts been destroyed to any app rec iab le e x te n t, fo r

95.8 p er cen t o f th e sample was found in the dry p ro d u c ts , and

taking in to co n sid era tio n th e 70 grams o f m oisture in the o r ig i ­

nal gam, 102.8 per cen t o f the sample was accounted f o r . I f

th e re wore no decomposition, more than 100 p e r cent should be

present, fo r during the h y d ro ly sis th e pentosan m a te r ia l had

changed in to p en to se .

The h y d ro ly s is o f th e s a l t s "A” a t 100*0. in

1 p e r cen t ac id m s eq u a lly su ccessfu l in th a t p r a c t ic a l ly a l l

th e galac to se jo ined to th e s a l t s was s p l i t o f f , while only

4 .3 grams of rhamnose were separa ted . The pentose th a t s t i l l

adhered from th e f i r s t hy d ro ly sis was a lso removed almost com­

p le te ly , so th a t th e s a l t s nBM were p r a c t ic a l ly fireo from both

g a lac to se and p en to se . However, about 10 grams of ac id were

destroyed during t h i s h y d ro ly s is .

The trea tm en t in th e au toclave m s much more

rig o ro u s , fo r i t re su lte d i n the decom position o f about 45 grams

o f a c id . Tho rhamnose was hydrolyzed o f f to the [extent th a t

only 9 grams were found in the s a l t s "C,\ The high tem perature

was necessary fo r th e se p a ra tio n o f the rhamnose; but u n fo rtu ­

n a te ly th e use o f th a t tem pera tu re was accompanied by a g re a t

lo s s in th e a c id .

When th e calcium s a l t s "C” were decarbozy lated ,

s a l t s o f another acid were ob tained , th u s suggesting th a t th e

s a l t s "O" con tained two ac id s , one o f which was no t destroyed

by th e trea tm en t w ith th e 6 p e r cen t su lphuric a c id . An o u tlin e

o f a ty p ic a l h y d ro ly s is , to g e th e r w ith the amounts of th e v ario u s

substances sep a ra ted , i s shown in Chart I .

CHARS Ie~@B*HSS OF A 2ZPICA& HTStoHSIS

.tJSsOi

SUIEIARY

A nalysis of c h o lla gma showed i t to con tain

pen to se , methyl p en to se , g a lac to se , an& an ac id of th e g lucu r­

onic ac id group. By a p ro cess o f successive h y d ro ly s is , 1 -a ra -

b lo o se , d -g a la c to se , and l-rhamnose were separa ted , is o la te d ,

and id e n t i f ie d , and th e presence o f d -galactuzon io ac id was

eetab liehed . In d ic a tio n s o f o th e r sugars, n o tab ly xy lose , and

o f ano ther ac id were ob tained , but fu r th e r work i s n ecessary fo r

the complete id e n t i f ic a t io n o f those o th e r c o n s ti tu e n ts .

BBratEITCES

1— O 'S u lliv an , J . Chem, S oo ., London, 1884, v o l . 45,41; 1890, v o l. 57,59; 1891, v o l. 59,1029.

2— van dor H sar, A.W., A nleitung sum liaohweis, so r Trennung und Bostimmtmg dor Monosaccharide und Aldehydesauren, Gohruder B orn traoger, B e rlin , 1920, p p .2 -3 .

3— E ef. 2 , pp , 4-5 , 532-340.

4— Anderson and Sands, (a) The p re p a ra tio n o f 1-Arahinose from mosquito gum, Ind. Eng. Chem., v o l. 17, 1257 (1925); (b)5he oomposition o f meaquite gum;, th e I s o la t io n o f d-G alaotose and 1-A rabinose; J . Am. Chem. S oc., v o l. 48, 3172 (1926).

5— Reference 2, pp . 5 -4 .' ■ . ■ ' ;

6— Bef. 2 . pp . 514-322.

7— Hof. 2, pp. 322-332. : /

8— Anderson and Sands, Some p la n t gums of th e Southwestern U .S .:An. J r . Pharm.. v o l . 97, 589 (1925).

9— A.O.A .C., Methods o f a n a ly s is , 1925.

10— (a) Browne, Handbook o f sugar a n a ly s is , John Wiley and Son#,1912. p . 458. :Co) B ef. 2, pp. 67-69.(c) E l io t t and (Pollens. 3 . d e n t. Zuckerind. v o l . 42. 19 (1905%.(d) Haywood: B u ll. 100. O .S.Bur. of Chem.. p . 112 (1907).

11— Hof. 8, p . 82.

12— (a) L efevre, Unterauohungen uber d ie g lucuronsaure , D is s e r ta tio n O o ttin g er, 1907.

13— R ef. 2, p . 77.

14— (a) Hof. 2, pp. 71-76.(b) Lofovro and fo l lo n s , B o r., v o l. 40, 4513 (1907).

15— Hof, 2, pp. 123-130.

16— B ef. 9. p . 121.

17— B ef. 2 , p . 186.

18— E h rlio h , Chem.Z tg . , v o l. 41, 198 (1917).

BEFERBHCES (Conolude6L)

!L9”~Rof • 8*

20— R ef. 4 . . . .

21— Ref * 10 ( a ) .

22— R ef. 4 (1)).

BS— (a) R ef. 2; (b) R ef. 10 ( a ) ; (c) Horro?/, C.A. , E x tra c ts from biochem ical re sea rch methoas, p r e - p r in t , 1925, P a rt n , p . 23.

24—BertranS.. H .O ., Recherches sn r ouelqu.es d e riv e s du X ylose: B u ll. S w . Ohem., (3) , v o l . 5, 554-557 (1891).

BS—Seliw anoff, T . , H otiz uber e ine Pruohtsucker R eaction , B o r,, v o l. 20, 181-182 (1887).

26— Roein, H ., Eine Veraharfung der Seliwaneffeehon R eak tiea ,2 . # y a i o l . GWm.. V@l. 38, 555-556 (1905).

27— P la isan ce , G .P ., IM o b a rb itu r ic ac id a s a q u a l i ta t iv e r e ­agent fo r ke to -bsxoses: J . B io l. Chon., 29, 207-208 (1917).

28— P o llen s , Ann. v o l . 206, 207, 226; v o l. 245, 314; Ber. v o l. 35 1286.

89—Meuxiier, 0<*pt. rend , v o l . 106, 1425, 1732; v o l . 107, 910; v o l . 108, 408.

30—R ef.''1 8 . ' .... . .

31 —Re f . 18. '

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