Partial Characterization of the Xylan Fraction of Corn Cob

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P U R D tJE U N IV E R SIT Y

T H IS I S T O C E R T IF Y T H A T T H E T H E S I S P R E P A R E D U N D E R M Y S U P E R V I S I O N

b y ______

E N T IT L E D

Joseph Bachrach__________



Partial Characterization of the Xylan Fraction of

C o m Cob.

C O M P L IE S W IT H T H E U N IV E R S IT Y R E G U L A T IO N S O N G R A D U A T IO N T H E S E S

A N D IS A P P R O V E D B Y M E A S F U L F IL L IN G T H IS P A R T O F T H E R E Q U IR E M E N T S

FO R THE DEG REE O F

Doctor of Philosophy

P

r o f e s s o r in

OF S

C

h a rg e o f

chool

or

D

T

h e s is

epa rtm en t

T O T H E L I B R A R I A N : ----T H IS T H E S I S I S N O T T O B E R E G A R D E D A S C O N F ID E N T IA L .

PR O FB S SO B VSf OHABOB

GRAD. SCHOOL FORM 9 - 3 - 4 0 - l M

PARTIAL CHARACTERIZATION OF THE XYLAN FRACTION OF CORN COB

A Thesis Submitted to the F a c u lty of Purdue U n i v e r s i t y by J o se p h Bachrach I n P a r t i a l F u l f i l l m e n t of t h e R e q u i r e m e n t s f o r t h e Degree of D o c t o r of P h i l o s o p h y January,

1950

ProQuest Number: 27712210

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

uest ProQuest 27712210 Published by ProQuest LLO (2019). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e M icroform Edition © ProQuest LLO. ProQuest LLO. 789 East Eisenhower Parkway P.Q. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346

VITA The a u t h o r was b o r n on F e b . 9 t h 1918 i n P a p e n b u r g , Germany and a t t e n d e d p u b l i c s c h o o l and l a t e r t h e Gymnasium a t Es c hwe g e , Germany,

He p a s s e d t h e M a t r i c u l a t i o n e x a m i n a ­

t i o n o f t h e U n i v e r s i t y o f London, E n g l a n d i n J u n e o f 1935 and a t t e n d e d Queen Mary C o l l e g e , U n i v e r s i t y o f Lo nd o n, E n g l a n d u n t i l Au g u s t o f 1936 i n t h e f a c u l t y o f a p p l i e d science.

S u b s e q u e n t l y he was empl oyed f o r f o u r y e a r s a s a n

a n a l y s t v; i t h De r by and C o . , M i l l m a r s h L a n e , Br i ms down, Middlesex, England. A rts (with honors) Kingston,

He o b t a i n e d t h e d e g r e e o f B a c h e l o r o f i n c h e m i s t r y from Q u e e n ' s U n i v e r s i t y a t

Canada i n 1944 and h e l d t h e M i l t o n H e r s e y F e l l o w ­

ship for the following y e a r.

I n S e p t e m b e r 1945 he r e c e i v e d

t h e d e g r e e o f M a s t e r o f A r t s i n o r g a n i c c h e m i s t r y from t h e same i n s t i t u t i o n .

From Dec. 1945 u n t i l J u n e 1946 he was

empl oyed a s a r e s e a r c h c h e m i s t w i t h Corn P r o d u c t s R e f i n i n g Company, Ar g o ,

Illinois,

From J u n e of 1946 u n t i l

the present

t i m e he h a s be e n e mpl oye d a s a t h r e e - q u a r t e r t i m e r e s e a r c h a s s i s t a n t wit h tne department of a g r i c u l t u r a l ch em i st r y a t Purdue U n i v e r s i t y ,

ACKNOWLEDGEMENT The a u t h o r w i s h e s t o t h a n k Dr. R . L . Vi / hi s t l er f o r t h e d i r e c t i o n o f t h i s work. Kimmel f o r h e r a s s i s t a n c e

He i s a l s o i n d e b t e d t o Mrs. K. i n some of t h e p r e p a r a t i v e work,

TABLE OF CONTENTS P age ABSTRACT.................................................................................................................

1

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

3

LITERATURE SURVEY.......................................................................................

5

The E a r l y P e r i o d .

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

5

The H e r a i c e l l u l o s e s of Corn Cob.........................................

5

The S t r u c t u r e

7

o f X y l a n .....................

R e c e n t Del i g n i f i c a t i o n M e t h o d s ................... EXPERHvIENTAL AND RESULTS

10

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

12

M a t e r i a l s .....................................................

12

A n a l y t i c a l M e t h o d s ...................

12

P r e p a r a t i o n o f H o l o c e l l u l o s e , ..............

12

I n S m a l l Q u a n t i t i e s .................................

12

I n L a r g e Q u a n t i t i e s ................................................

13

O t h e r P r e p a r a t i v e M e t h o d s . , . , . . , . . , . , , , , , , ..............

14

E x t r a c t i o n of Haw Cob and Cob H o l o c e l l u l o s e w i t h P o t a s s i u m H y d r o x i d e S o l u t i o n s of V a r i o u s Concen­ t r â t i o n s .................................

15

P r e p a r a t i o n of X y l a n . . , , , . . , .................................

17

A t t e m p t s t o F r a c t i o n a t e o r P u r i f y X y l a n ........................

20

Use of Co p per Complex F o r ma t i o n .

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

Use of C o t t o n C h r o m a t o g r a p h y . Identification

20 21

of L - A r a b i n o s e a s C o n s t i t u e n t of Xy l a n 21

A c e t y l a t i o n of X y l a n

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

A t t e m p t s t o A c e t y l a t e X y l a n Vi/itnout t h e Use of N i t r i c A c i d ......................................

22 23

Page By Means

o f B a r n e t t ' s R e a g e n t ...............................................

S3

By Means

of Formamide a s a D i s p e r s i n g A g e n t ...............

23

By Means

of Z i n c C h l o r i d e C a t a l y s i s , , ............................ 24

By Means

of P h o s p h o r i c Acid or S u l f u r i c A c i d . . . . , ,

24

A c e t o l y s i s o f Xy l a n A c e t a t e . . . . . . . . . . . . . . . . . . . . . . . . . .

24

A c e t o l y s i s of X y l a n ..............

25

I s o l a t i o n of A l d e h y d o - D - X y l o s e - H e x a a c e t a t e

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

S y n t h e s i s of A l d e h y d o - D - X y l o s e - H e x a a c e t a t e

25

.......... 26

O t h e r A c e t o l y s i s M e t h o d s , . , , . . . , , , * , , , , . . . ..............

27

The Method Used f o r t h e P r e p a r a t i o n of C e l l o b i o s e ,

27

A t t e m p t e d D e g r a d a t i o n of Xy l a n U s i n g A c e t y l B r o m i d e . .

27

H y d r o l y s i s of Xy l a n i n 42;^ H y d r o c h l o r i c A c i d .................... 28 Other Hydrolys is Methods

........................................................ 28

P r e p a r a t i o n of X y l o b i o s e - H e x a a c e t a t e P a r t i a l H y d r o l y s i s of X y l a n . . .

..........

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

31 31

Ch r o ma t o g r a p h y of N e u t r a l L i q u o r . . . .................................

31

A c é t y l a t i o n of Amorphous X y l o b i o s e

32

H y d r o l y s i s of Xyl ob l o s e

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

32

Preparation

o f Me t h y l

p e n t a a c e t y l x y l o b i o s i d e .......

33

Preparation

o f Met hy l

X y l o b i o s i d e . ..

33

Preparation

of Me t h y l

P e n t arne t hyl xy l o b i o s i

Preparation

of Me t h y l

He x a me t n y 1xy1 o b i o n a t e ..

H y d r o l y s i s of Me t h y l H e x a m e t n y l x y l o b i o n a t e . DISCUSSION................................................................................................ D e l i g n i f i c a t i o n M e t h o d s ........................

d

..........

e 35 36 38 38

. 34

Page E x t r a c t i o n of H e m i c e l l u l o s e From Corn Cobs and From H o l o c e l l u l o s e ................

39

A t t e m p t e d P u r i f i c a t i o n and F r a c t i o n a t i o n of X y l a n , , , ,

42

Met hods of A c e t y l a t i o n .....................................................................

43

A c e t o l y s i s S t u d i e s .............................................

44

H y d r o l y s i s S t u d i e s , ........................

47

C h a r c o a l C h r o m a t o g r a p h y ...............................

47

Xylobiose H e x a a c e t a t e

48

S t r u c t u r e of X

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

y l o b i o s e

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

SUMMARY................................................................................................................................

48 51

BIBLIOGRAPHY........................................................................................................ 53

LIST OF TABLES AND FIGURES

LIST OF TABLES Table

Page

1, A n a l y t i c a l D a t a o f P r e e c e ' s H e m i c e l l u l o s e F r a c ­ t i o n s .......................... 2 , Y i e l d and L i g n i n C o n t e n t of H o l o c e l l u l o s e by V a r i ­ ous D e l i g n i f i c a t i o n P r o c e s s e s ...................................... o.

Amount of C a r b o h y d r a t e s and L i g n i n R e c o v e r e d From Corn Cob a t V a r i o u s C o n c e n t r a t i o n s of A l k a l i . . . .

8 16 41

LIST OF FIGURES Figures

Pa ge

1. Y i e l d of A and B H e m i c e l l u l o s e and R e s i d u e O b t a i n ­ ed by A l k a l i E x t r a c t i o n of Raw Corn C o b , . . . . . . . .

18

2 . Y i e l d o f A and B H e m i c e l l u l o s e and R e s i d u e O b t a i n ­ ed by A l k a l i E x t r a c t i o n of H o l o c e l l u l o s e . , ............. 19 3.

H y d r o l y s i s of X y l a n i n

42/q H y d r o c n l o r i c A c i d ................ 29

4.

H y d r o l y s i s of Xy l a n i n

42% H y d r o c h l o r i c A c i d ................ 30

5.

S t r u c t u r a l F o r m u l a e ............................................................................

49

ABSTRACT A s t u d y of t h e x y l a n f r a c t i o n of c o r n c o bs was u n d e r ­ t a k e n a s p a r t o f a p r o g r a m t o o b t a i n more i n f o r m a t i o n on t h e c a r b o h y d r a t e s of corn cobs.

Al mo s t c o m p l e t e d e l i g n i f i c a t i o n

of corn cobs i s a c c o m p l i s h e d w i t h o u t a p p r e c i a b l e

l o s s of

c a r b o h y d r a t e m a t e r i a l by t r e a t m e n t of c o r n c o b s w i t h sodi um chlorite

in d i l u t e a c e t i c a c i d .

fraction,

called holocellulose,

The r e s u l t i n g c a r b o h y d r a t e is fractio n ated into

e< — c e l l u l o s e and h e m i c e l l u l o s e s by e x t r a c t i o n w i t h a l k a l i . The amount of h e m i c e l l u l o s e s e x t r a c t e d i s d e p e n d e n t on t h e c o n c e n t r a t i o n of a l k a l i used.

N e u t r a l i z a t i o n of t h e a l k a l i

e x t r a c t l e a d s t o t h e p r e c i p i t a t i o n of h e m i c e l l u l o s e - A , xylan f r a c t i o n .

the

A d d i t i o n of a l c o h o l t o the n e u t r a l e x t r a c t

p re c ip ita te s hemicellulose-B,

H e m i c e l l u l o s e - B becomes i n ­

c r e a s i n g l y s o l u b l e i f e x t r a c t e d from raw cobs w i t h a l k a l i s o l u t i o n s of i n c r e a s i n g s t r e n g t h .

However,

it

is completely

s o l u b l e i n ÿC a l k a l i i f e x t r a c t e d from h o l o c e l l u l o s e .

Con­

s e q u e n t l y , t h e B - f r a c t ion i s l a b i l i z e d d u r i n g t h e d e l i g n i f i cation process,

Xy l a n wh i c h c o n t a i n s 94^ o f p e n t o s a n s and

"5% o f u r o n i c a c i d can n o t be f r a c t i o n a t e d by t h e c o p p e r com­ p l e x f o r m a t i o n t e c h n i q u e n o r by a d s o r p t i o n on a c o t t o n col umn. films.

Xylan d i a c e t a t e

can be c a s t i n t o s t r o n g p l i a b l e

A c e t o l y s i s of x y l a n y i e l d s t h e u s u a l m o n o s a c c h a r i d e

a c e t a t e s b u t i n a d d i t i o n t h e a c e t a t e of t h e a l d e h y d r o l for m i s p r o d u c e d and c an be i s o l a t e d by t h e u s e of Ma gn e s ol chromatography.

Thus a l d e h y d e - D - x y l o s e h e x a a c e t a t e

is is o ­

l a t e d and shown t o be i d e n t i c a l v/ i t h a s y n t h e t i c s p e c i m e n . This is the f i r s t

reported

D-xylosehexaacetate,

i s o l a t i o n of c r y s t a l l i n e a l d e h y d e -

P a r t i a l a c i d h y d r o l y s i s a t 0*^0. i n 42^

h y d ro ch lo ric acid leads to the

i s o l a t i o n of a d i s a c c h a r i d e

f o r whi c h t h e name x y l o b i o s e i s p r o p o s e d .

Four c r y s t a l l i n e

d e r i v a t i v e s x y l o b i o s e h e x a a c e t a t e , methyl x y l o b i o s i d e , methyl p e n t a a o e t y l x y l o b i o s i d e and m e t h y l p e n t a m e t h y l x y l o b i o s i d e a r e o b t a i n e d in c r y s t a l l i n e

condition.

This is the f i r s t r e ­

p o r t e d o c c u r r e n c e of c r y s t a l l i n e d e r i v a t i v e s of a p e n t o s e p e n t o s e t y p e of d i s a c c h a r i d e .

It

is suggested th a t xylobiose

c o n s i s t s of two x y l o p y r a n o s e u n i t s l i n k e d by a B g l y c o s i d i c bond i n t h e 1,4^ p o s i t i o n s .

T h i s s t r u c t u r e of x y l o b i o s e i s

a d d i t i o n a l e v i d e n c e f o r t h e s t r u c t u r e of x y l a n p r o p o s e d by other workers.

INTHODÜCTION Corn cobs r e p r e s e n t a c o n t i n u o u s l y r e p l e n i s h e d r e s e r ­ v o i r of c a r b o h y d r a t e raw m a t e r i a l wh i c h m i g h t become of i n ­ cre ased i n d u s t r i a l importance

i f more i n f o r m a t i o n were ob­

t a i n e d re g a rd in g the v a rio u s c o n s t i t u e n t c ar b o h y d rat es . The s e c a r b o h y d r a t e s , wh i c h a r e a l m o s t e n t i r e l y of p o l y s a c ­ charide n a t u r e , c o n s t i t u t e

a b o u t 80^ of t h e cob and i n t h e i r

n a t i v e s t a t e are r e l a t i v e l y fir m ly a s s o c i a t e d with l i g n i n , the o t h e r major c o n s t i t u e n t

of c o r n c o b s .

The r e m o v a l of

l i g n i n i s an i m p o r t a n t s t e p i n t h e p r e p a r a t i o n of t h e c a r b o ­ hydrate f r a c t i o n .

L i g n i n must be removed as c o m p l e t e l y a s

p o s s i b l e w i t h minimum d e g r a d a t i o n or l o s s of p o l y s a c c h a r i d e material.

Th e s e r e q u i r e m e n t s a r e met by r e c e n t l y d e v e l o p e d

o l e l i g n i f i c a t i o n t e c h n i q u e s ( 1 , 2 , 3 ) whi ch l e a v e t h e p o l y s a c c h a ­ rid e s in p r a c t i c a l l y t h e i r o rig in a l s t a t e .

Such h i g h l y d e ­

l i g n i f i e d p u l p s a r e known a s h o l o c e l l u l o s e s ( 1 ) .

Holocellu-

l o s e s c a n be f r a c t i o n a t e d by e x t r a c t i o n w i t h a l k a l i whi c h d i s s o l v e s t h e h e m i c e l l u l o s e s and l e a v e s b e h i n d t h e a l k a l i insoluble '^^-cellulose. hemicelluloses extract.

F u r t h e r s u b d i v i s i o n of c o r n cob

i s e f f e c t e d by n e u t r a l i z a t i o n of t h e a l k a l i

T h i s c a u s e s p r e c i p i t a t i o n of h e m i c e l l u l o s e - A ,

r e l a t i v e l y high molecular weight xylan, Depending, t o a c e r t a i n d e g re e, used,

a

a p o l y m e r of x y l o s e .

on t n e met hod of e x t r a c t i o n

a b o u t 20fo of c o r n cob h o l o c e l l u l o s e c o n s i s t s o f x y l a n .

A f t e r r e mo v a l o f x y l a n ,

h e m i c e l l u l o s e - B i s p r e c i p i t a t e d by

a d d i t i o n of a l c o h o l t o t h e n e u t r a l e x t r a c t .

The herni-

cellulose

f r a c t i o n wh i c h r e m a i n s s o l u b l e a f t e r a d d i t i o n of

a l c o h o l has been c a l l e d h e m i c e l l u l o s e - C .

This s ep a ra tio n

of t h e h e m i c e l l u l o s e s i n t o A, B and G i s an a r b i t r a r y one b u t h a s b e e n u s e d by many w o r k e r s i n t h e f i e l d . The p r e s e n t cellulose-A,

investigation

t h e x y l a n f r a c t i o n of c o r n c o b s .

d e l i g n i f i c a t i o n of c o r n c o b s , cellulose,

i s a f u r t h e r s t u d y of h e mi Met h od s of

a l k a l i e x t r a c t a b i l i t y of h o l o ­

h o m o g e n e i t y , p r e p a r a t i o n of d e r i v a t i v e s ,

aceto-

l y t i c and h y d r o l y t i c d e g r a d a t i o n of x y l a n have b e e n s t u d i e d i n some d e t a i l ,

A c e t o l y s i s of x y l a n h a s l e d t o t h e

isola­

t i o n of a nev/ a l d e h y d r o l s u g a r a c e t a t e and a new d i s a c c h a ­ r i d e - w h i c h we h a v e named x y l o b i o s e - h a s be en i s o l a t e d f r om t h e h y d r o l y s i s p r o d u c t s of x y l a n . The t e r m s x y l a n and h e m i c e l l u l o s e - A , a s u s e d i n t h e literature, slightly.

o f t e n r e f e r t o s u b s t a n c e s ’w hich d i f f e r o n l y I n t h i s work t h e name x y l a n w i l l r e f e r t o p r e ­

p a r a t i o n s t h a t a r e b e l i e v e d t o be r e a s o n a b l y p u r e and h e mi c e l l u l o s e - A w i l l r e f e r to crude p r e p a r a t i o n s .

LITERATURE SURVEY

The E a r l y P e r i o d .

The t e r r a h e m i c e l l u l o s e was i n t r o d u c e d

i n t o chemical l i t e r a t u r e

i n 1892 by S c h u l z e ( 4 ) wno a p p l i e d

i t t o a p r o d u c t t h a t he o b t a i n e d from l e g u m i n o u s s e e d s , wheat b r a n ,

and o t h e r m a t e r i a l s d e r i v e d f r om c e r e a l s by e x ­

t r a c t i o n w i t h a l k a l i and p r e c i p i t a t i o n of t n e e x t r a c t w i t h acid.

This product corresponds to hsmicellulose-A or xylan

u s i n g t h e more r e c e n t t e r m i n o l o g y .

P r e v i o u s t o t h e work of

S c h u l z e , Thomsen ( 5 ) o b t a i n e d an a l k a l i e x t r a c t from b i r c h wood t o wni c h he a p p l i e d t h e name "Holzgumrai" - wood gum. Similar,

v e r y c r u d e x y l a n or n e m i c e l l u l o s e p r e p a r a t i o n s were

made by o t h e r w o r k e r s .

Beech wood s a w d u s t ( 6 ) , wh e a t s t r a w

( 7 ) , wheat and r y e b r a n ( 8 ) ,

c o r n cobs ( 9 ) ,

sunflower husks

( 1 0 ) and o t h e r a n n u a l p l a n t s were i n v e s t i g a t e d .

In a l l

t n e se p r e p a r a t i o n s t n e h e m i c e l l u l o s e s were c o n t a m i n a t e d w i t h lignin.

Very o f t e n o n l y i n c o m p l e t e r e m o v a l o f t h e hemi ­

c e l l u l o s e s was a c c o m p l i s h e d due t o t h e low c o n c e n t r a t i o n s (2 t o 4ko) of a l k a l i u s e d f o r e x t r a c t i o n .

E x t r a c t i o n s were

c a r r i e d o u t i n t h e p r e s e n c e of a t m o s p h e r i c oxygen and c on ­ s e q u e n t l y o x i d a t i on p r o b a b l y o c c u r r e d d u r i n g t n e e x t r a c t i o n . Therefore,

t o d a y t n e work of t n e e a r l y i n v e s t i g a t o r s

m a i n l y of h i s t o r i c a l

is

significance.

The H e m i c e l l u l o s e s of Corn Cob.

The f i r s t

extensive

inves­

t i g a t i o n of t h e h e m i c e l l u l o s e s of c or n cob v;as c a r r i e d o u t by P r e e c e

(11).

P r i o r t o h i s work J o h n s o n had o b t a i n e d

x y l a n by h y d r o l y s i s o f c o r n cob h e m i c e l l u l o s e s wni ch he had

r e g a r d e d as a s i n g l e s u b s t a n c e ,

namel y p u r e x y l a n .

Preece

f r a c t i o n a t e d t h e h e m i c e l l u l o s e s of c o r n cob bj' a method he had u s e d p r e v i o u s l y f o r t h e f r a c t i o n a t i o n o f t h e n e m i c e l l u ­ l o s e s of whe at b r a n .

Ground c o r n cobs were e x t r a c t e d w i t h

0.5% ammonium o x a l a t e t o remove p e c t i n and s u b s e q u e n t l y t r e a t e d W'ith 1% sodium h y d r o x i d e lignin.

i n oOfo e t h a n o l t o d i s s o l v e

The u s e of a l c o h o l i c sodium h y d r o x i d e s o l u t i o n f o r

t h i s p u r p o s e h a s be en c r i t i c i z e d by Norman ( 1 2 ) on t h e g r o un d t h a t

i t r emoves a l s o some of t h e h e m i c e l l u l o s e s .

This c r i t i c i s m has been answered, N o r r i s ( 1 3 ) who have shov/n t h a t

i n p a r t , by Ange11 and

f o r ma i z e cobs and ho ps no

a p p r e c i a b l e l o s s of h e m i c e l l u l o s e o c c u r r e d .

Following the

r e mo v a l of l i g n i n t h e n e m i c e l l u l o s e s were e x t r a c t e d w i t h sodium h y d r o x i d e s o l u t i o n . yielded a p re c ip ita te ,

A c i d i f i c a t i o n of t h e e x t r a c t

hemicellulose-A.

volume of a c e t o n e t o t n e f i l t r a t e lose-B.

A d d i t i o n o f one

p r e c i p i t a t e d h e m i c e 1l u -

By a d d i n g l a r g e r q u a n t i t i e s of a c e t o n e a f u r t n e r

f r a c t i o n - h e m i c e l l u l o s e - C - was p r e c i p i t a t e d .

Ea c h one of

t h e s e f r a c t i o n s was t r e a t e d w i t h F e h l i n g s o l u t i o n .

Some of

t h e h e m i c e l l u l o s e s f or med c o p p e r c ompl e xes and were p r e c i ­ pitated. 0^.

Thes e were d e s i g n a t e d h e m i c e l l u l o s e s

A d d i t i o n of a c e t o n e t o t h e f i l t r a t e

and

fr om whi c h t n e

c o p p e r compl e x had be e n removed c a u s e d p r e c i p i t a t i o n of hemi c e l l u l o s e s A , B and C , F o u r nemi c e l l u l o s e f r a c t i o n s 2 2 2 were i s o l a t e d by P r e e c e , namel y A , B , C and Cp, Some 1 1 1 2 a n a l y t i c a l d a t a and p h y s i c a l c o n s t a n t s of t h e s e f r a c t i o n s

a r e g i v e n i n T a b l e 1,

S u b s e q u e n t l y Ange 11 and N o r r i s

(13)

r e p o r t e d t h a t p r e c i p i t a t i o n of r e mi c e 1l u 1 os e-A r e a c h e s an optimum a t pH 3 , 7 t o 4 . 1 , copper s u l f a t e cellulose

Thes e a u t h o r s a l s o recommend

and g l y c e r o l f o r t h e p r e c i p i t a t i o n of h e mi ­

i n s t e a d of F e h l i n g s o l u t i o n , ,

Acetate, propionate,

b u t y r a t e and o t h e r e s t e r s of c o r n ­

cob h e m i c e l l u l o s e ha ve b e e n p r e p a r e d by C a r s o n and Mac l a y ( 14 ) and f o u n d t o be o n l y p a r t l y s o l u b l e i n o r g a n i c s o l v e n t s . Thes e a u t h o r s a l s o p r e p a r e d e s t e r s of t h e h e m i c e l l u l o s e s o f l i m a b e a n p o d s and a t t e m p t e d t o f r a c t i o n a t e witn o rganic s o l v e n t s . an a p p r e c i a b l e

However,

these e s te r s

they f a i l e d t o accomplish

change i n t h e x y l a n c o n t e n t o f t h e r e g e n e r ­

ated hem icellulose.

T h i s t o them i n d i c a t e d t h a t t h e n o n ­

p e n t o s a n p a r t i s c h e m i c a l l y combi ned w i t h x y l a n .

Similar

o b s e r v a t i o n s have b e e n made by O' Dwyer ( 1 5 ) and A n d e r s o n and c o w o r k e r s ( 1 6 ) who a t t e m p t e d t o f r a c t i o n a t e c e l l u l o s e s of E n g l i s h oak,

t h e hemi ­

lemon wood, and b l a c k l o c u s t s a p -

wood. The S t r u c t u r e of X y l a n ,

Ho s t o f t h e s t r u c t u r a l work h a s

b e e n done on x y l a n o f e s p a r t o g r a s s by Ha wor t h

and c o w o r k e r s

(17,

et.

18, 19,

2 0 ) , The e a r l i e r work by Komat su

a l . (21)

i s of l i t t l e

v a l u e s i n c e a c c o r d i n g t o Hawo r t h t h e s e a u t h o r s

were d e a l i n g

w i t h d e g r a d e d p r o d u c t s and f a i l e d

and c r y s t a l l i z e

t h e i r various fragments.

to

identify

Ha wo r t h accom­

p l i s h e d c o mp l e t e m é t h y l a t i o n o f x y l a n by means of p o t a s s i u m h y d r o x i d e and d i m e t h y l s u l f a t e

i n two o o e r a t i o n s .

Dimethyl-

T a b le 1 . A n a l y t i c a l D a t a o f P r e e c e ''8 H e m i c e l l u l o s e F r a c t i o n s ( 1 1 ) Hemicellulose

% Xyl an

4 94.36

% 8 7 .50

8 2 .7 0

fo Âr a b a n % ü r o n i c Anhydride

80.32

3.76 -96°

D

^2

^1

5.20 -1 0 8 °

7 .4 0 -9 0 °

5.56



xylan is lev o ro tato ry

r

22 ^ ^ — 92 i n c h l o r o f o r m ,

Me t h a -

n o l y s i s l e d t o the i s o l a t i o n of methyl 2 , o - d i m e t h y l - D - x y l o p y r a n o s i d e i n 90^ y i e l d ,

methyl 2 , 3 , 5 - L - a r a b o f u r a n o s i d e in

6>o y i e l d and m e t h y l 2 - m o n o m e t h y l x y l o s i d e P a rtial acetolysis

in

yield.

of d i m e t h y l - x y l a n l e d t o t h e i s o l a t i o n of

an u n i d e n t i f i e d d i s a c c h a r i d e d e r i v a t i v e .

This d is a c c h a r id e

was d e a c e t y l a t e d , o x i d i z e d w i t h br omi ne t o t h e c o r r e s p o n d i n g a l d o b i o n i c a c i d and a f t e r m e t h y l a t i o n was h y d r o l y z e d t o y i e l d 2 ,3 ,4-trimethylxylose xyolonolactone

( 8 0 ^ y i e l d ) and 2 , 3 , 5 - t r i m e t h y l - ^ - û -

(73;^ y i e l d ) .

The s e o b s e r v a t i o n s s u g g e s t a

b r a n c h e d c h a i n s t r u c t u r e w i t h one b r a n c h on t h e a v e r a g e f o r e v e r y 1 8 - 2 0 x y l o p y r a n o s e u n i t s l i n k e d t h r o u g h t h e 1 and 4 p o s i t i o n s and t e r m i n a t e d by an L - a r a b o f u r a n o s e u n i t l i n k e d g l y c o s i d i c a l l y to the xylose chain.

I t was f u r t h e r shown

( 2 0 ) t h a t t h e t e r m i n a l a r a b i n o s e u n i t can be s p l i t h y d r o l y t i c a l l y by 0.2N o x a l i c a c i d w i t h l i t t l e the xylan chain.

off

c l e a v a g e of

M e t h y l a t i o n of t h e a r a b i n o s e - f r e e x y l a n

and s u b s e q u e n t m e t n a n o l y s i s gave t h e mono, d i a nd t r i m e t h y l x y l ose d e r i v a t i v e s i n y i e l d s tively.

This experiment

of t h e x y l a n c h a i n , stitute

i.e.

a branch p o in t.

of 6 - 1 0 , 8 5 - 9 0 and 4fo r e s p e c ­

in d ic a te s th a t tne penultimate u nit the l a s t xylose u n i t ,

d oe s n o t con­

Since th e te rm in a l a rab in o se u n i t s

a r e n o n - r e d u c i n g t h e r e woul d be a v a i l a b l e a t most one p o t e n ­ t i a l r e d u c i n g g r ou p p e r m o l e c u l e n h i c n mi gh t e x p l a i n t h e n o n - r e d u c i n g a c t i o n of x y l a n ,

Sc h mi d t ( 2 2 ) c l a i m e d t h e p r e ­

s e n c e of a c a r b o x y l gr oup b u t t h i s

i s c o n t r a d i c t e d by

10

Ha wo r t h a t l e a s t

for esparto grass.

The h i g h n e g a t i v e r o t a t i o n of x y l a n and t h e marked positive

c hange of r o t a t i o n d u r i n g h y d r o l y s i s t e n d s t o i n ­

d i c a t e B - g l y c o s i d l e bon d s . Periodate

oxidation yields re s u lts

in general agree­

ment w i t h t h e s t r u c t u r e p o s t u l a t e d from m e t h y l a t i o n d a t a

(2 3 ). Whi l e m é t h y l a t i o n s t u d i e s p o i n t t o a b r a n c h e d s t r u c t u r e f i n a l proof is s t i l l cast

lacking.

into strong, p liab le

Since xylan a c e t a t e

can be

f i l m s (24) such b ra nc hes i f t h e y

e x i s t mus t be f a i r l y s h o r t . Relatively l i t t l e of x y l a n .

i s known a b o u t t h e m o l e c u l a r w e i g h t

Ha wor t h ( 2 0 ) from v i s c o s i t y d a t a of d i m e t h y l -

x y l a n c a l c u l a t e d a d e g r e e o f p o l y m e r i z a t i o n ( D . P , ) of 7 5 - 8 0 units. D. P .

Os mot i c p r e s s u r e d e t e r m i n a t i o n s ( 2 5 ) i n d i c a t e d a

of 1 1 0 - 1 5 0 ,

Recent D e l i g n i f i c a t i o n Methods.

A c o mp l e t e r e v i e w on r e c e n t

d e l i g n i f i c a t i o n me t h od s i s f o un d i n a p a p e r by Wise, Murphy and D ' A d d i e c o ( 3 ) ,

T h r e e p r o c e d u r e s have been d e v e l o p e d f o r

t h e q u a n t i t a t i v e d e t e r m i n a t i o n of c a r b o h y d r a t e s fr om p l a n t m aterials.

The o l d e s t

i s t h a t of Sc h mi d t

c h l o r i n e d i o x i d e i n p y r i d i n e and w a t e r . time f o r q u a n t i t a t i v e d e l i g n i f i c a t i o n ,

( 2 6 ) wn i c h u s e s I t r e q u i r e d a long

R i t t e r and K u r t h ( 1 )

u s e d r e p e a t e d a l t e r n a t e t r e a t m e n t of e x t r a c t i v e - f r e e wood w i t h c h l o r i n e and a s o l u t i o n of p y r i d i n e

in e t a a n o l ,

f i n a l l y by t r e a t m e n t w i t h a c o l d c a l c i u m h y p o c h l o r i t e

followed solu­

11

t i o n , u n t i l t h e r e s i d u e was w h i t e and f r e e fr om l i g n i n . R i t t e r and K u r t h named t h e l i g n i n - f r e e "holocellulose”.

carbohydrate f r a c tio n

Van Bechum and R i t t e r ( 2 7 ) d e v i s e d a luodi-

f i e d p r o c e d u r e wh i c h s h o r t e n e d t h e t ime o f i s o l a t i o n . s o l v e n t c h o s e n t o remove c h l o r o l i g n i n was a e t h a n o l a m i ne s o l u t i o n ,

The

alcoholic

and a l t e r n a t e c h l o r i n a t i o n s and e x ­

t r a c t i o n s were made a t 7 5^0 ,

The l i g n i n c o n t e n t of h o l o ­

c e l l u l o s e p r e p a r e d by t n e s e me t hods was i n some c a s e s as low a s 2% and s ummat i ve a n a l y s i s t o t a l e d 9 9 , 6 t o 1 0 0 . 4 ^ . ever,

How­

i t was f o u n d t h a t t h e h o l o c e l l u l o s e r e t a i n e d 0 , 4 6 ^ of

n i t r o g e n a nd s i n c e t h e o r i g i n a l woods c o n t a i n e d o n l y 0,081o of n i t r o g e n i t

f o l l o w s t h a t t h e n i t r o g e n r e t e n t i o n of t h e

h o l o c e l l u l o s e ' i s o v e r and above t h e n i t r o g e n o f t h e wood itself.

Thomas ( 2 8 ) h a s shown t h a t i n t h e c a s e o f a s p e n

h o l o c e l l u l o s e 1% of t h e p e n t o s a n s were l o s t

in the d e l i g n i -

fioation process. The most r e c e n t t e c h n i q u e f o r t h e p r e p a r a t i o n o f h o l o ­ cellulose

i s t h a t of Jayme ( 2 9 ) who u s e d 30 p a r t s o f sodium

c h l o r i t e and 6 p a r t s of a c e t i c a c i d a t 6 0 ° C f o r 12 h o u r s . S u c c e s s i v e t r e a t m e n t s f o r 12, 8 and 8 h o u r s gave a w h i t e p r o d u c t t h a t r e t a i n e d 2 , 8 t o 3 . 5 ^ of l i g n i n .

Complete r e ­

moval o f l i g n i n l e d t o l o s s of c a r b o h y d r a t e m a t e r i a l , a nd c o w o r k e r s ( 3 ) s i m p l i f i e d J a y m e ' s t e c h n i q u e .

vilse

They f ound

t h a t c h l o r i t i n g s c o u l d be c o m p l e t e d w i t h i n 4 h o u r s and f r e ­ q u e n t l y p r e l i m i n a r y e x t r a c t i o n o f wood was u n n e c e s s a r y . T h i s was c o n f i r m e d by L o v e l l ( 3 0 ) ,

12

EXPERIMENTAL AND RESULTS M aterials.

C o m m e r c i a l l y gr ou n d cobs s u p p l i e d by Turabl'

Brite,

Inc., Monticello,

I n d . were u s e d i n t h i s

investiga­

tion,

The cobs had be e n gr ou nd t o p a s s a 20- mes h s c r e e n and

t h e p i t h and gl ume s h a d been removed f o r t h e most p a r t by a ir separation. A n a l y t i c a l Methods.

U r o n i c a c i d s were d e t e r m i n e d by t h e

p r o c e d u r e of W h i s t l e r ,

M a r t i n and H a r r i s

(31),

a c e t y l con­

t e n t o f p o l y s a c c h a r i d e s by t n e p r o c e d u r e of Fr ee ma n and P e t e r s o n ( 3 2 ) b a s e d on t h e met hod of F r e u d e n b e r g a nd H a r d e r (33),

a c e t y l c o n t e n t o f mono- and d i s a c c h a r i d e s by t h e

me t hod of Kunz and Hudson ( 3 4 ) , me t h o x y l by t h e me t hod d e ­ s c r i b e d by N i e d e r l and N i e d e r l ( 3 5 ) and l i g n i n by a s l i g h t m o d i f i c a t i o n of t h e me t hod of R i t t e r and B a r b o u r ( 3 6 ) . cellulose

(37),

n i t r o g e n ( 3 8 ) and a s h were d e t e r m i n e d by t h e

u s u a l s t a n d a r d methods.

M o i s t u r e c o n t e n t was d e t e r m i n e d by

d r y i n g 0 . 5 g . s a m p l e s f o r f o u r h o u r s a t 100°C, oven.

-

i n a vacuum

A l l a n a l y t i c a l v a l u e s a r e r e p o r t e d on a d r y w e i g h t ,

ash-free basis. P r e p a r a t i o n of H o l o c e l l u l o s e A.

In S ma l l Q u a n t i t i e s .

g r o u n d t o 20- me s h s i z e , f l a s k and 680 ml ,

A p p r o x i m a t e l y 30 g. of c o b s ,

were p l a c e d i n a 1 1, r o u n d - b o t t o m

o f w a t e r were a d d e d .

The m i x t u r e was me­

c h a n i c a l l y s t i r r e d and h e a t e d t o 74 t 1 ° C . , a t whi c h t e m p e r a ­ ture

i t was m a i n t a i n e d by a t h e r m o s t a t i c a l l y c o n t r o l l e d

water bath.

G l a c i a l a c e t i c a c i d ( 2 . 5 m l . ) was added,

followed

13

by t h e g r a d u a l a d d i t i o n o f sodi um c h l o r i t e b r o u g h t t h e pH of t n e m i x t u r e t o 4 . 5 .

(7.5 g . ).

This

A f t e r 15 m i n . , a c e t i c

a c i d and sodium c h l o r i t e were a g a i n a d d e d i n t h e same q u a n ­ tities

and t h i s a d d i t i o n r e p e a t e d a t 15 mi n.

4 a d d i t i o n s had be e n made,

intervals u n til

A g l a s s tube pl a c e d in the neck

o f t h e f l a s k p e r m i t t e d i n t r o d u c t i o n of a c o n s t a n t

s t r e a m of

c a r b o n d i o x i d e t o d i s p l a c e c h l o r i n e d i o x i d e from t h e f l a s k a s s o o n a s i t was l i b e r a t e d .

T h i s p r e c a u t i o n was t a k e n t o

p r e v e n t t h e c h l o r i n e d i o x i d e c o n c e n t r â t i o n i n t h e a i r above t h e l i q u i d from r e a c h i n g t h a t

of an e x p l o s i v e m i x t u r e .

t h e c a r b o n d i o x i d e f l u s h i n g was o m i t t e d ,

When

the cnlorine d i ­

o x i d e f or med i n t h e f l a s k woul d a t t i m e s i g n i t e s p o n t a n e o u s ­ ly,

The e n t i r e o p e r a t i o n was c a r r i e d out i n a w e l l - v e n t i l a t e d

hood b e c a u s e of t h e h i g h t o x i c i t y of t h e c h l o r i n e d i o x i d e . D u r i n g t h e e a r l y s t a g e s o f t h e r e a c t i o n t h e m i x t u r e foamed c o n s i d e r a b l y , b u t t h e f oa mi ng was c o n t r o l l e d by a d d i t i o n of 1 o r 2 d r o p s of o c t y l a l c o h o l .

A f t e r one h o u r , t h e m i x t u r e

was r a p i d l y c o o l e d t o a b o u t 20°C. and f i l t e r e d t h r o u g h a f i l t e r c l o t h , washed w i t h w a t e r u n t i l f r e e o f a c i d , n a l l y washed w i t h a c e t o n e . and a l m o s t w h i t e .

and f i ­

The a i r - d r i e d p r o d u c t v;as p o r o u s

The y i e l d was 61.5;^.

I t c o n t a i n e d 0,4%

of l i g n i n and 56.6% of ' ^ - c e l l u l o s e . B.

In L a r g e r Q u a n t i t i e s ,

paration,

700 g.

In a t y p i c a l l a r g e s c a l e p r e ­

o f g r o u n d cob c o n t a i n i n g 10% m o i s t u r e was

a d d e d t o w a t e r i n a 9 1. P y r e x serum b o t t l e . level

The w a t e r

i n t h e b o t t l e was a d j u s t e d t o w i t h i n a b o u t 5 cm. of

14

th e neck.

The m i x t u r e was s t i r r e d v i g o r o u s l y w i t h a m o t o r -

d r i v e n Hershberg type s t i r r e r . ml ,

Fo u r p o r t i o n s ,

e a c h o f 55

o f g l a c i a l a c e t i c a c i d and 160 g, of sodi um c h l o r i t e ,

were a d de d s u c c e s s i v e l y . chlorite

Such l a r g e p o r t i o n s o f sodium

c a n n o t be a d d e d i n s t a n t a n e o u s l y b e c a u s e of t h e e x ­

c e s s i v e foaming in d uc ed .

Therefore,

eac h 160 g. p o r t i o n

was s l o w l y p o u r e d i n o v e r a p e r i o d o f s e v e r a l m i n u t e s ,

and

t h e t o t a l r e a c t i o n p e r i o d f o r t h e p r e p a r a t i o n was e x t e n d e d to 1,5 h rs .

I n a l l o t h e r r e s p e c t s t h e p r e p a r a t i o n was

i d e n t i c a l w i t h the s mal l s c a l e p r e p a r a t i o n d e s c r i b e d above. C. O t h e r P r e p a r a t i v e Me t h o d s . 1 . The e f f e c t s odi um c h l o r i t e .

of pH on t h e d e l i g n i f i c a t i o n a c t i o n of The e x p e r i m e n t a l p r o c e d u r e was a s above

except t h a t a t o t a l

of 20 g. o f sodium c h l o r i t e was u s e d f o r

30 g .

o f c o b.

The pH was a d j u s t e d by means o f p h o s p h o r i c

acid,

sodi um h y d r o g e n p h o s p h a t e , and d i s o d i u m h y d r o g e n p h o s -

phate, 2 . The d e l i g n i f i c a t i o n a c t i o n of c h l o r i n e d i o x i d e g a s . C h l o r i n e d i o x i d e was p r e p a r e d by g r a d u a l a d d i t i o n o f 50% s u l f u r i c a c i d t o a c o l d s o l u t i o n of 20 g. o f sodi um c h l o r i t e i n 20 ml , o f HgO,

The c h l o r i n e d i o x i d e was d i r e c t l y p a s s e d

i n t o a s u s p e n s i o n of c o r n c o b s i n w a t e r m a i n t a i n e d a t 6 0 ° C . I n one c a s e t n i s r e a c t i o n was c a r r i e d out i n c o l o r e d f l a s k s i n o r d e r t o a v o i d d e c o m p o s i t i o n o f c n l o r i n e d i o x i d e by l i g h t . 3 . The e f f e c t of r e p l a c i n g p a r t o f t h e c h l o r i n e d i o x i d e g a s by c h l o r i n e .

C h l o r i n e d i o x i d e and c n l o r i n e were b u b b l e d

15

s i m u l t a n e o u s l y a t a p p ro x im a te ly equal r a t e s through a su spen­ s i o n of c o r n cobs i n w a t e r . The r e s u l t s

of the various

d e l i g n i f i c a t i o n methods are

s u mma r i z e d i n t a b l e 2, E x t r a c t i o n of Raw Cob and Cob Hol o c e l l u l o s e w i t h P o t a s s i u m Hydroxide S o l u t i o n s

of V a r i o u s C o n c e n t r a t i o n s

To d e t e r m i n e t h e a l k a l i s o l u b i l i t y cob h o l o c e l l u l o s e

of raw cob and of

t n e f o l l o w i n g e x p e r i m e n t s were p e r f o r m e d ;

a p p r o x i m a t e l y 30 g. s a m p l e s o f cob or cob h o l o c e l l u l o s e

c on ­

t a i n i n g a b o u t lO/o m o i s t u r e were t r e a t e d w i t h 300 ml . of 1, 5, 10,

o r 17% p o t a s s i u m h y d r o x i d e s o l u t i o n s .

Each s u s p e n ­

s i o n was s t i r r e d g e n t l y and m a i n t a i n e d i n an a t m o s p h e r e of n i t r o g e n a t 25 ± l ^ C . f o r 24 h r s .

The e x t r a c t s from h o l o ­

c e l l u l o s e were l i g h t y e l l o w i n c o l o r , w h i l e t h o s e from raw cob were q u i t e d a r k . The m i x t u r e was f i l t e r e d on a f i l t e r

c l o t h i n each

c a s e , and t h e r e s i d u e v. a s h e d w i t n d i l u t e a l k a l i and t h e n W'ith w a t e r , u n t i l no t r a c e s of c o l o r r e m a i n e d .

The s l i g n t l y

a l k a l i n e r e s i d u e was t r e a t e d w i t h a s m a l l amount of 10% a c e t i c a c i d , w a s h e d u n t i l f r e e of a c i d , and a i r d r i e d . The c l e a r a l k a l i n e

filtra te

was a c i d i f i e d w i t h 50%

a c e t i c a c i d t o a p p r o x i m a t e l y pH 5.

A f t e r a l l o w i n g t n e mi x­

t u r e t o s t a n d f o r s e v e r a l n o u r s , i t was c e n t r i f u g e d i n a supercen trifuge

(40,000 r . p . m , ) .

The p r e c i p i t a t e ,

called

t h e A - f r a c t i o n a f t e r t n e n o m e n c l a t u r e of O' Dwyer ( 3 9 ) , was d e h y d r a t e d by d i s p e r s i n g

i t i n 500 ml. of 95y e t n a n o l i n a

16

T a b l e 2, Y i e l d and L i g n i n C o n t e n t of H o l o c e l l u l o s e by Various D e l i g n i f i c a t i o n Processes Method of D e l i g n i f i c a t i o n

^ Yield

fo L i g n i n

NaClOg, pH 4 . 5

86.4

5.8

NaClOg, pH 2

85.1

5.9

86.3

5.6

93.2

15.3

87.8

6.2

CIO. g a s , pH 2 , c o l o r e d r e a c t i o n , 60°C.

82.8

4.2

CIO. g a s , pH 2, 60°C. (three treatments)

56.1

4.4

CIO. and C l . m i x t u r e pH 5 - 6 ^

63.7

5.1

NaCl O- , pH 4 . 5 ,

colored reaction

NaClO , pH 8 CIO

g a s , pH 2,

60°C.

17

Wa r i n g B l e n d o r a nd f i l t e r i n g .

Care was t a k e n t h a t no a i r

was drawn t h r o u g h t h e p r e c i p i t a t e d u r i n g t h e f i l t r a t i o n . i n s u r e t h e r e mo v a l of w a t e r , peated o times.

To

t h e e t h a n o l t r e a t m e n t was r e ­

The p r e c i p i t a t e was f i n a l l y washed w i t h

e t h e r t o remove most o f t h e e t h a n o l ,

and i m m e d i a t e l y p l a c e d

i n a vacuum d e s i c c a t o r o v e r c a l c i u m c h l o r i d e ,

A white,

finely-pov/dered product r e s u l t e d . The . s l i g h t l y o p a l e s c e n t c e n t r i f u g a t e was p o u r e d i n t o 3 times

i t s volume of 95^ e t h a n o l w i t h r a p i d s t i r r i n g .

s l i g h t l y yellow p r e c i p i t a t e ,

The

c a l l e d t h e B - f r u c t i o n ( 3 9 ) , was

f i l t e r e d and t r e a t e d a s d e s c r i b e d a b o v e . The e x t e n t t o whi ch h e m i c e l l u l o s e s a r e removed fr om raw cob and cob h o l o c e l l u l o s e by e x t r a c t i o n w i t h s odi um h y d r o x ­ ide s o l u t i o n s of d i f f e r e n t

s t r e n g t h s a r e shown i n F i g s ,

1

and 2 , P r e p a r a t i o n of X y l a n .

A p p r o x i m a t e l y 400 g. of a i r d r i e d

h o l o c e l l u l o s e was p l a c e d i n a 4 1. r o u n d - b o t t o m f l a s k and e x tra c te d overnight with 3,5

1. of 10^ p o t a s s i u m h y d r o x i d e .

The m i x t u r e was s t i r r e d and m a i n t a i n e d a t room t e m p e r a t u r e i n an a t m o s p h e r e of n i t r o g e n . te re d through a f i l t e r w i t h 300 ml.

The y e l l o w e x t r a c t was f i l ­

c l o t h and t h e r e s i d u e washed t w i c e

of 5y p o t a s s i u m h y d r o x i d e .

n e u t r a l i z e d w i t h 10/b a c e t i c a c i d .

The f i l t r a t e

Sufficient

i c e was added

b e f o r e or d u r i n g t h e n e u t r a l i z a t i o n t o p r e v e n t a r i s e temperature.

The f i n a l pH was 4 , 5 ,

was

The x y l a n ,

i.e.

f r a c t i o n , was t h e n i s o l a t e d and d r i e d a s p r e v i o u s l y

in t h e A-

18

•H •H iH +3 CO ü (d 1—1 P. < «M >sCQ tJ

e

a ü •H o