Reversed-Phase Chromatography of Lignin Derivatives - American

Chapter 13

Reversed-Phase Chromatography of Lignin Derivatives Kaj Forss, Raimo Kokkonen, and Pehr-Erik Sågfors

Downloaded by UNIV OF CINCINNATI on May 31, 2016 | http://pubs.acs.org Publication Date: July 31, 1989 | doi: 10.1021/bk-1989-0397.ch013

The Finnish Pulp and Paper Research Institute, P.O. Box 136, Helsinki, Finland

00101

The paper shows that lignosulfonates and kraft lignin can be fractionated according to their polarities by reversed-phase liquid chromatography. The high molar mass lignin derivatives are fractionated in such a way that those with highest molar mass are eluted last. Lignin-carbohydratecompounds can be separated from virtually carbohydrate-free lignin. In our o p i n i o n , l i g n i n i n w o o d consists of h i g h m o l a r mass g l y c o l i g n i n b o u n d to carbohydrates a n d of a group of low m o l a r mass l i g n i n s collectively referred to as h e m i l i g n i n s . T h e t e r m lignin(s) is used collectively for g l y c o l i g n i n a n d h e m i l i g n i n s . In spruce w o o d , h e m i l i g n i n s representing 15-20% of the t o t a l l i g n i n consist of m o n o m e r i c , d i m e r i c a n d oligomeric molecules. D u r i n g the a c i d bisulfite a n d kraft p u l p i n g processes, the h e m i l i g n i n s a n d g l y c o l i g n i n are rendered soluble; as g l y c o l i g n i n undergoes b o t h d e p o l y m e r i z a t i o n a n d p o l y m e r i z a t i o n d u r i n g the cook, the result is a c o m p l e x m i x t u r e of molecules of different sizes a n d characteristics (1-4). T h e c o m p l e x i t y of this m i x t u r e is i n no way reduced b y the fact t h a t s m a l l fragments peel off the g l y c o l i g n i n d u r i n g the d e l i g n i f i c a t i o n a n d t h a t some of the dissolved lignins are p r o b a b l y b o u n d to c a r b o h y d r a t e s as l i g n i n c a r b o h y d r a t e c o m p o u n d s . It is interesting to note i n the present context t h a t these l i g n i n - c a r b o h y d r a t e compounds are m u c h more p o l a r t h a n the other l i g n i n compounds. A s t u d y of dissolved l i g n i n derivatives first requires t h e i r s e p a r a t i o n f r o m each other. F o r this purpose gel p e r m e a t i o n c h r o m a t o g r a p h y ( G P C ) is w i d e l y used. I n this technique separation of l i g n i n derivatives is based largely o n the size a n d shape of the molecule, n a m e l y its h y d r o d y n a m i c volume. However, i t is possible for c o m p o u n d s w i t h the same m o l e c u l a r size t o have different c h e m i c a l structures. S u c h c o m p o u n d s m a y not be separated 0097-6156/89/0397-0177$06.00A) © 1989 American Chemical Society

Glasser and Sarkanen; Lignin ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

LIGNIN: PROPERTIES AND MATERIALS


178

b y G P C . T h e r e is thus a need i n e x p e r i m e n t a l l i g n i n research for f r a c t i o n a t i o n techniques t h a t separate molecules o n the basis of properties other t h a n size. O n e such f r a c t i o n a t i o n m e t h o d , w h i c h is based o n the p o l a r i t y of the components, is reversed-phase l i q u i d c h r o m a t o g r a p h y ( R P C ) . T h e purpose of t h i s paper is to describe the f r a c t i o n a t i o n of l i g n i n derivatives b y means of t h i s m e t h o d . T h e h y d r o p h o b i c s t a t i o n a r y phase used i n reversed-phase c h r o m a t o g r a p h y is a s i l i c a gel or p o l y m e r i c m a t r i x to w h i c h h y d r o c a r b o n chains have been a t t a c h e d b y s i l y l a t i o n . T h e most c o m m o n l y used are C i s , C g , C e a n d C 2 chains. E l u t i o n i n reversed-phase c h r o m a t o g r a p h y is often c a r r i e d out u s i n g a g r a d i e n t , p r o d u c e d f r o m water a n d some w a t e r - m i s c i b l e organic solvent. T h e solute components are t h u s d i s t r i b u t e d between the s t a t i o n a r y a n d m o b i l e phases m a i n l y o n the basis of their p o l a r i t i e s . I n reversed-phase c h r o m a t o g r a p h y h y d r o p h i l i c compounds elute before h y d r o p h o b i c ones.

Fractionation of Lignosulfonates In order to s t u d y b i r c h lignosulfonates, spent sulfite l i q u o r , f r o m w h i c h monosaccharides h a d been removed by i o n exclusion c h r o m a t o g r a p h y , was f r a c t i o n a t e d o n the basis o f m o l e c u l a r size b y p r e p a r a t i v e G P C ( F i g . 1). It can be seen f r o m the figure t h a t almost h a l f of the b i r c h l i g n o s u l fonates have a m o l a r mass greater t h a n 1000 g / m o l . T h e fractions i n the region 700-1230 m L i n F i g u r e 1 were c o m b i n e d i n order to s t u d y the s t r u c t u r e of the p o l y m e r i c p o r t i o n of the b i r c h l i g n o sulfonates. T h e c o m b i n e d s o l u t i o n was then refractionated b y p r e p a r a t i v e R P C i n t o five fractions ( F i g . 2). F i g u r e 2 shows t h a t the lignosulfonates are f r a c t i o n a t e d i n t o two p o r tions. T h e lignosulfonates eluted i n the retention t i m e range 0-15 m i n u t e s are s t r o n g l y p o l a r , whereas those eluted i n the range 15-40 minutes behave as less p o l a r c o m p o u n d s w i t h p o l a r i t y decreasing w i t h increasing r e t e n t i o n time. It must be n o t e d t h a t lignosulfonates are s t r o n g polyelectrolytes a n d thus p o l a r components. However, p a r t of the h i g h m o l a r mass molecule is n o n - p o l a r i n character, a n d t h i s part of the molecule causes h i g h m o l a r mass lignosulfonates t o elute as n o n - p o l a r c o m p o u n d s . T h e reason w h y lignosulfonates elute i n the r e t e n t i o n t i m e range 3-9 minutes c o u l d be because they are, i n fact, s t r o n g l y p o l a r l i g n i n c a r b o h y d r a t e c o m p o u n d s . T o investigate t h i s p o s s i b i l i t y , fractions I - V were subjected to a c i d h y d r o l y s i s a n d the monosaccharide content a n d c o m p o s i t i o n of the r e s u l t i n g m i x t u r e d e t e r m i n e d b y l i q u i d c h r o m a t o g r a p h y . T h e c a r b o h y d r a t e a n d lignosulfonate contents are s h o w n i n T a b l e I. T a b l e I shows t h a t carbohydrates account for about o n e - t h i r d o f the solids i n f r a c t i o n I. F r a c t i o n s I I I - V c o n t a i n e d considerably less c a r b o h y drates. A f t e r h y d r o l y s i s , f r a c t i o n I contained xylose a n d arabinose i n the r a t i o 10:1. H y d r o l y s i s of fractions I I I - V y i e l d e d very s m a l l a m o u n t s of x y l o s e . T h e other monosaccharides present i n fractions I I I - V were arabinose a n d



13.

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W

179

Reversed-Phase Chromatography

/ { f f { f f / { f f t^f / f f / f f f f /

7 5 0

1

/nnn ° 0 0 . , , , 7000 3000 2000 4 0

0 0

U

Lé

j

la

LA

ι

1250

1500 1750 , RETENTION VOLUME, ml 1000 MOLAR MASS

F i g u r e 1. F r a c t i o n a t i o n o f b i r c h lignosulfonates b y p r e p a r a t i v e gel p e r m e ation chromatography.

RETENTION TIME, min F i g u r e 2. F r a c t i o n a t i o n o f h i g h m o l a r mass b i r c h lignosulfonates (fraction A i n F i g u r e 1) b y p r e p a r a t i v e reversed-phase c h r o m a t o g r a p h y .



180

T a b l e I. C a r b o h y d r a t e a n d Lignosulfonate C o n t e n t s of the H y d r o l y z e d Fractions I - V Fraction Compounds


Monosaccharides, % ( w / w ) Lignosulfonates, % ( w / w )

I 30 70

II

III

IV

V

5 95

2 98

6 94

r h a m n o s e . These results suggest t h a t f r a c t i o n I contains lignosulfonatex y l a n compounds. F i g u r e 2 shows t h a t f r a c t i o n I elutes over a very n a r r o w r e t e n t i o n t i m e range, whereas fractions I I - V are spread over a w i d e range. T o deter m i n e the reason for t h i s , fractions I - V were f r a c t i o n a t e d b y a n a l y t i c a l G P C ( F i g . 3). It can be seen f r o m F i g u r e s 2 a n d 3 t h a t the v i r t u a l l y c a r b o h y d r a t e free fractions I I I - V are eluted b y R P C i n order of increasing m o l a r mass a n d t h a t f r a c t i o n V contains the highest m o l a r mass lignosulfonates. A large p a r t of f r a c t i o n I is eluted b y G P C i n the same region as f r a c t i o n V . T h i s s u p p o r t s the s u p p o s i t i o n t h a t the lignosulfonates of f r a c t i o n I are b o u n d to c a r b o h y d r a t e s . O t h e r w i s e they w o u l d have been eluted b y R P C in fraction V . It s h o u l d be noted t h a t the b r o a d m o l a r mass d i s t r i b u t i o n o f f r a c t i o n I i n F i g u r e 3 reflects the m o l a r mass d i s t r i b u t i o n of the l i g n i n - c a r b o h y d r a t e c o m p o u n d s a n d not t h a t of the l i g n i n p o r t i o n of the l i g n i n - c a r b o h y d r a t e compounds. It was s h o w n t h a t h i g h m o l a r mass lignosulfonate c o m p o u n d s can be f r a c t i o n a t e d by R P C i n t o h y d r o p h i l i c a n d h y d r o p h o b i c c o m p o u n d s . It c a n be seen f r o m F i g u r e s 4 a n d 5 t h a t the b i r c h lignosulfonates w i t h low m o l a r mass (fractions Β a n d C i n F i g u r e 1) were also f r a c t i o n a t e d into h y d r o p h i l i c a n d h y d r o p h o b i c p o r t i o n s w i t h no clearly resolved peaks. O n the other h a n d , fractions D a n d E , w h i c h elute later i n p r e p a r a t i v e G P C ( F i g . 1), show clearly separated peaks i n b o t h the h y d r o p h o b i c a n d h y d r o p h i l i c zones w h e n f r a c t i o n a t e d b y R P C ( F i g s . 6 a n d 7). It can be seen f r o m F i g u r e 3 t h a t the m o l a r mass d i s t r i b u t i o n of the h y d r o p h i l i c c o m p o u n d s (fraction I) is b r o a d a l t h o u g h they are eluted b y R P C i n a n a r r o w zone ( F i g . 2). T h e i r reversed-phase c h r o m a t o g r a p h i c f r a c t i o n a t i o n is t h u s based almost exclusively o n t h e i r p o l a r i t y , m o l e c u l a r size h a v i n g no effect o n the process. T h e results show t h a t R P C w i l l also separate low m o l a r mass l i g n o sulfonates i n t o h y d r o p h i l i c a n d h y d r o p h o b i c fractions as well as i n t o a far greater n u m b e r of i n d i v i d u a l c o m p o n e n t s t h a n o b t a i n e d b y f r a c t i o n a t i o n with G P C .

Fractionation of Kraft Lignin In the same way as w i t h b i r c h lignosulfonates, p r e p a r a t i v e R P C can be used



13.

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CU 10000

5000

3000

181

0.6 0.8 1.0 RELATIVE RETENTION VOLUME 1500 1000 MOLAR MASS

F i g u r e 3. M o l a r mass d i s t r i b u t i o n o f h i g h m o l a r mass b i r c h (fractions I - V i n F i g u r e 2).

lignosulfonates

F i g u r e 4. F r a c t i o n a t i o n o f low m o l a r mass b i r c h lignosulfonates i n F i g u r e 1) b y reversed-phase c h r o m a t o g r a p h y .

(fraction Β


182



MeOH,%

~i 20

1 Γ 30 40 RETENTION TIME

F i g u r e 5. F r a c t i o n a t i o n of low m o l a r mass b i r c h lignosulfonates ( f r a c t i o n C i n F i g u r e 1) b y reversed-phase c h r o m a t o g r a p h y .

MeOH,% *280nm

π 20

1 Γ 30 L0 RETENTION TIME

F i g u r e 6. F r a c t i o n a t i o n of low m o l a r mass b i r c h lignosulfonates (fraction D i n F i g u r e 1) b y reversed-phase c h r o m a t o g r a p h y .



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183

to fractionate kraft l i g n i n i n t o h y d r o p h i l i c a n d less h y d r o p h i l i c c o m p o u n d s ( F i g . 8). T h e figure shows t h a t h y d r o p h i l i c l i g n i n derivatives ( f r a c t i o n I) elute i n the r e t e n t i o n t i m e range 30-80 m i n u t e s , c o m p a r e d w i t h 90-140 m i n u t e s for the h y d r o p h o b i c c o m p o u n d s (fractions I I - I V ) . R e f r a c t i o n a t i o n b y a n a l y t i c a l G P C of fractions I - I V ( F i g . 9) shows t h a t these fractions encompass w i d e m o l a r mass ranges. F r a c t i o n I, w h i c h is less h y d r o p h o b i c t h a n fractions III a n d V ( F i g . 8), contains lower m o l a r mass c o m p o u n d s t h a n f r a c t i o n I I I , w h i c h i n t u r n c o n t a i n s lower m o l a r mass c o m p o u n d s t h a n the most h y d r o p h o b i c f r a c t i o n (fraction I V ) . F r a c t i o n I, w h i c h consists m a i n l y o f low m o l a r mass c o m p o u n d s , also contains a s m a l l a m o u n t of h i g h m o l a r mass l i g n i n derivatives e l u t i n g w i t h relative r e t e n t i o n volumes of 0-0.1. These derivatives are p o l a r a n d some m a y be b o u n d to c a r b o h y d r a t e s , or otherwise they w o u l d have been e l u t e d b y R P C a l o n g w i t h the h y d r o p h o b i c fractions I I - I V . T h e a n a l y t i c a l reversed-phase c h r o m a t o g r a m s i n F i g u r e 10 show t h a t h i g h m o l a r mass h y d r o p h o b i c kraft l i g n i n i n kraft black l i q u o r elutes i n the r e t e n t i o n t i m e range 60-90 m i n u t e s . T h e c o r r e s p o n d i n g lignosulfonates elute sooner i n the r e t e n t i o n t i m e range 30-60 m i n u t e s because of their sulfonate groups a n d consequently their more h i g h l y h y d r o p h i l i c n a t u r e . It can also be seen f r o m F i g u r e 10 t h a t the h y d r o p h i l i c sulfonated h e m i l i g n i n s i n the spent sulfite l i q u o r elute i n the r e t e n t i o n t i m e range 0-15 m i n u t e s . F i g u r e 11 shows t h a t the e l u t i o n of m o n o m e r i c benzene derivatives i n R P C is closely connected w i t h the s t r u c t u r e of their f u n c t i o n a l groups a n d t h u s w i t h their p o l a r properties. T h e figure shows t h a t the s t r o n g l y h y d r o p h i l i c sulfonate is the first of the m o d e l c o m p o u n d s to be e l u t e d . It is also seen t h a t i n R P C m o n o m e r i c acids elute before the c o r r e s p o n d i n g alcohols, w h i c h elute before the aldehydes. G u a i a c y l c o m p o u n d s elute before the c o r r e s p o n d i n g s y r i n g y l c o m p o u n d s , w h i c h i n t u r n elute before the veratryl compounds. Conclusions It has been s h o w n t h a t R P C can be used to f r a c t i o n a t e b o t h l i g n o s u l fonates a n d kraft l i g n i n o n the basis of p o l a r i t y . S t r o n g l y h y d r o p h i l i c l i g n i n c a r b o h y d r a t e c o m p o u n d s can be separated f r o m v i r t u a l l y carbohydrate-free l i g n i n . H i g h m o l a r mass lignosulfonates a n d kraft l i g n i n are f r a c t i o n a t e d o n the basis of m o l a r mass, w i t h the highest m o l a r mass c o m p o u n d s e l u t e d last. P r e p a r a t i v e a n d a n a l y t i c a l reversed-phase c h r o m a t o g r a p h y c o m b i n e d w i t h G P C is a useful t o o l i n e x p e r i m e n t a l l i g n i n research. Experimental Lignosulfonates. Samples of b i r c h a n d spruce w o o d m e a l e x t r a c t e d w i t h e t h a n o l - c y c l o h e x a n e (1:3) were heated f r o m 20°C to 135°C d u r i n g 1 h a n d then cooked for 6 h at 135°C i n 150 m L reactors w i t h s o d i u m bisulfite l i q u o r



184


RETENTION TIME F i g u r e 7. F r a c t i o n a t i o n o f low m o l a r mass b i r c h lignosulfonates ( f r a c t i o n Ε i n F i g u r e 1) b y reversed-phase c h r o m a t o g r a p h y .

RETENTION TIME, min F i g u r e 8. F r a c t i o n a t i o n of p i n e kraft l i g n i n b y p r e p a r a t i v e reversed-phase liquid chromatography.


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185


A280 nm

I 10000

I I 5000 3000

RELATIVE RETENTION VOLUME I I 15001000 MOLAR MASS

F i g u r e 9. M o l a r mass d i s t r i b u t i o n o f pine kraft l i g n i n (fractions I - I V i n Figure 8).

RETENTION TIME, min F i g u r e 10. F r a c t i o n a t i o n o f spruce spent sulfite l i q u o r a n d pine kraft b l a c k liquor.



186


F i g u r e 11. Influence of f u n c t i o n a l groups on retention t i m e .


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c o n t a i n i n g 7% S O 2 a n d 1% N a 0 . T h e spent a n d w a s h i n g l i q u o r s f r o m the p u l p were c o m b i n e d a n d evaporated. Sulfite a n d sulfate ions were p r e c i p i t a t e d f r o m the spent b i r c h l i q u o r ( F i g . 1) w i t h b a r i u m h y d r o x i d e . M o n o s a c c h a r i d e s a n d other low m o l a r mass non-electrolytes a n d weak electrolytes were separated q u a n t i t a t i v e l y f r o m the lignosulfonates b y means of i o n exclusion c h r o m a t o g r a p h y (5). 2

Kraft Lignin. T h e i n d u s t r i a l pine black l i q u o r was d i l u t e d w i t h water (1:10) before a n a l y t i c a l R P C .


RPC

System and

Conditions.

Reversed-phase

columns:

Analytical:

F i g u r e s 4,5,6,7 F i g u r e s 10,11

Preparative:

Figure 2 Figure

8

Spherisorb C 6 , 5 / / m , 140/6.4 m m (Phase S e p a r a t i o n , U K ) Spherisorb O D S 2, 5 / / m , 1 4 0 / 4 m m (Phase S e p a r a t i o n , U K ) L i C h r o p r e p C s , 40-60 μπι, 2 5 0 / 1 0 m m (E. Merck, F R G ) S e p r a l y t e C i , 40 / i m , 3 0 0 / 2 5 m m (Analytichem, U S A ) 8

Solvent delivery s y s t e m : M o d e l L C - 5 0 6 0 ( V a r i a n , U S A ) M o b i l e phase:

F i g u r e s 2,4,5,

Figure Gradient:

F l o w rate:

+ B) A) B)

K O H 1.15 m M / L MeOH H 0 MeOH 2

F i g u r e s 2,4,5, 6,7

T i m e (min) v / v , % (A) v / v , % (B)

96 4

0

10 96 4

30 50 50

F i g u r e s 10,11

T i m e (min) v / v , % (A) v / v , % (B)

0 100 0

60 50 50

90 0 100

Figure

T i m e (min) v / v , % (A) v / v , % (B)

0 100 0

30 100 0

90 50 50

8

F i g u r e s 2,8

2.0 m L / m i n

F i g u r e s 4,5,6,7

1.5 m L / m i n

F i g u r e s 10,11

1.0 m L / m i n

D e t e c t i o n of A280nm · Injection:

6,7,10,11 8

A ) KH2PO4 50 m M / L

S p e c t r o p h o t o m e t r i c Detector M o d e l L C - 7 5 (Perkin-Elmer, U S A )

S y r i n g e L o a d i n g S a m p l e Injector M o d e l 7125 (Rheodyne, U S A ) w i t h a n a l y t i c a l 20 μL loop a n d p r e p a r a t i v e 2 m L loop.


120 0 100


188 GPC

System and

G e l permeation

Conditions.

columns:

Analytical:

F i g u r e s 3,9

Sephadex G - 5 0 , fine, 1500/10 m m ( P h a r m a c i a , Sweden)

Preparative:

Figure 1

Sephadex G - 5 0 , fine, 1400/40 m m ( P h a r m a c i a , Sweden)


Solvent delivery system:

S T A - m u l t i p u r p o s e p e r i s t a l t i c p u m p 13 19 0 0 , (Desaga, F R G )

M o b i l e phase:

F i g u r e s 3,9 Figure 1

H2O 0.5 M N a O H

F l o w rate:

F i g u r e s 3,9 Figure 1

90 m L / h 20 m L / h

D e t e c t i o n o f A280nm : Analytical:

F i g u r e s 3,9

U V - d e t e c t o r U V I C O R D S M o d e l 2138 ( L K B , Sweden)

Preparative:

Figure 1

C o l l e c t e d fractions measured w i t h a spectrophotometer M o d e l P M Q 2 ( C . Zeiss, F R G )

Injection:

Syringe i n j e c t i o n , a n a l y t i c a l volume 0.5 m L a n d preparative volume 100 m L .

Literature C i t e d 1. Forss, K.; Fremer, K . - E . Tappi 1964, 47, 485-93. 2. Forss, K.; Fremer, K . - E . Pap. Puu 1965, 47, 443-54. 3. Forss, K.; Fremer, K . - E . ; Stenlund, B. Pap. Puu 1966, 48, 565-74, 66976. 4. Forss, K.; Fremer, K . - E . Appl. Polym. Symp. 1983, 37, 531-47. 5. Jensen, W.; Fremer, K . - E . ; Forss, K . Tappi 1962, 45, 122-7. RECEIVED March 17,1989


Reversed-Phase Chromatography of Lignin Derivatives - American

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