Molecular Weight Distribution of Aspen Lignins Estimated by Universal

Chapter 6

Molecular Weight Distribution of Aspen Lignins Estimated by Universal Calibration M . E . Himmel , K. Tatsumoto , K. K. Oh , K. Grohmann , D. K. Johnson , and Helena L i Chum 1

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Downloaded by UNIV OF CINCINNATI on May 31, 2016 | http://pubs.acs.org Publication Date: July 31, 1989 | doi: 10.1021/bk-1989-0397.ch006

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Applied Biological Sciences Section, Biotechnology Research Branch, Solar Fuels Research Division, Solar Energy Research Institute, 1617 Cole Boulevard, Golden, CO 80401 Chemical Conversion Branch, Solar Fuels Research Division, Solar Energy Research Institute, 1617 Cole Boulevard, Golden, CO 80401 2

This study describes the application of differential viscometry as a G P C detector to the problem of determining molecular weight distributions of acetylated hardwood lignins in tetrahydrofuran. Molecular weight distributions of ball-milled, organosolv, alkali-extracted/mild acid hydrolyzed, and alkali-extracted/steam exploded aspen lignins were estimated using universal calibration. Low molecular weight lignin model compounds (synthetic phenyl-tetramers and Igepals™) were found to fit universal calibration. Fractions from preparative G P C , when analyzed by universal calibration, yield molecular weight distributions which add to a similar value to that found for the unfractionated parent sample. Lignins are irregular phenylpropane polymers that represent approximately 20-30% by weight of the available polymeric content of hardwood tree stems (1-3). This material offers, therefore, a valuable resource that must be utilized as fully as possible if the full value of harvested tree crops is to be attained. The understanding of the macromolecular properties of lignins requires a reliable method for estimating the molecular weights (MW or M) and distribution of molecular weights (MWD) in a suitable solvent. Suitable solvents must be defined here as those that minimize interactions of solutesolute (aggregation), solute-solvent, and solute-column packing material. Although important contributions have been made to this field historically through packed-bed and high performance size exclusion chromatography (HPSEC) (4-17), the design of a chromatography system (solvent and stationary phase) that performs optimally has not been reported. Indeed, the more hydrophobic solvents such as dioxane and tetrahydrofuran ( T H F ) , which work well to minimize solute-column and solute-solute interactions, 0097-6156/89/0397-0082$06.00/0 © 1989 American Chemical Society

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


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MW Distribution by Universal Calibration

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p e r f o r m p o o r l y at t h e task o f s o l u b i l i z i n g l i g n i n s over a w i d e range o f M W . However, these solvents work very w e l l w i t h p o l y s t y r e n e - d i v i n y l b e n z e n e (e.g., / / - S t y r a g e l ) c o l u m n p a c k i n g m a t e r i a l s , causing n o p e r c e p t i b l e c o l u m n d e t e r i o r a t i o n (18). A p a r t f r o m these i m p o r t a n t l i m i t a t i o n s presented b y c o n v e n t i o n a l " G P C " m e t h o d o l o g y t o t h e s t u d y of l i g n i n M W D is t h e issue of the c o m p l e x i t y of these p o l y m e r s even i n i d e a l solvents. C o n v e n t i o n a l G P C is effective i n e s t i m a t i n g M W of u n k n o w n p o l y m e r s o f s i m i l a r or i d e n t i c a l c h e m i c a l s t r u c t u r e s t o those used t o c a l i b r a t e c o l u m n s . T h e e l u t i o n of p o l y m e r s of u n k n o w n (or i m p r e c i s e l y k n o w n ) chemistry, degree o f b r a n c h i n g , shape, degree o f s o l v a t i o n , a n d degree o f r e p e a t i n g u n i t s (as i n c h e m i c a l c o p o l y m e r s ) c a n be t r e a t e d o n l y " p h e n o m e n o l o g i c a l l y " w i t h c o n v e n t i o n a l S E C . T h e result o f the size e x c l u s i o n m e t h o d ( i f c h e m i c a l i n t e r a c t i o n s c a n be assumed t o be negligible) is t h e s e p a r a t i o n of solutes o n t h e basis of their respective h y d r o d y n a m i c r a d i i , T h i s i n f o r m a t i o n , alone, is n o t o f great u t i l i t y . Size e x c l u s i o n c h r o m a t o g r a p h y has been g r e a t l y e n r i c h e d recently b y the advent of two c o m m e r c i a l detectors, t h e r e a l - t i m e differential viscometer ( D V ) a n d the low angle laser l i g h t s c a t t e r i n g ( L A L L S ) p h o t o m e t e r . T h e o n l y D V detector c u r r e n t l y available is offered b y V i s c o t e k i n P o r t e r , T X , as the m o d e l 100. T h i s s t u d y reports the first a p p l i c a t i o n of u n i v e r s a l c a l i b r a t i o n v i a H P S E C - D V t o four a c e t y l a t e d h a r d w o o d l i g n i n s o b t a i n e d f r o m aspen (Populus tremuloides) w o o d m e a l b y b a l l m i l l i n g a n d solvent e x t r a c t i o n ; s t e a m e x p l o s i o n followed b y alkaline e x t r a c t i o n ; organosolv p u l p i n g followed b y water e x t r a c t i o n of the associated sugars; a n d d i l u t e s u l f u r i c a c i d h y d r o l y sis followed b y s o d i u m h y d r o x i d e e x t r a c t i o n .

Materials and Methods Chemicals and Standards. A l l chemicals a n d H P S E C eluants used i n this s t u d y were o b t a i n e d f r o m m a j o r c h e m i c a l suppliers ( J . T . B a k e r , F i s h e r Scientific, a n d A l d r i c h ) . T h e T H F used was F i s h e r H P L C grade. T h e M W s t a n d a r d s used t o c a l i b r a t e the three c o l u m n s y s t e m were o b t a i n e d f r o m A m e r i c a n P o l y m e r L a b s , M e n t o r , O h i o [polybutadienes (narrow M W D ) : P B 9 0 0 , P B 1 K , P B 3 K , P B 5 K , P B 2 3 K , P B 4 3 K ; p o l y - a methylstyrenes ( n a r r o w M W D ) : P A M S 6 K , P A M S 2 3 K , P A M S 6 6 K ; p o l y methylmethacrylates (broad M W D ) : P M M A 1 7 K , P M M A 3 5 K , P M M A 7 5 K , P M M A 1 0 0 K ] a n d f r o m P o l y m e r L a b s , E n g l a n d [polystyrenes ( n a r r o w M W D ) : PS1250, PS1700, PS2450, PS3250, PS5050, PS7000, PS9200, PS11600, P S 2 2 K , P S 3 4 K , a n d P S 6 8 K ; a n d polymethylmethacrylates (narrow M W D ) : P M M A 3 0 0 0 , P M M A 1 0 K , P M M A 2 7 K , P M M A 6 0 K , a n d P M M A 1 0 7 K ] . T h r e e s y n t h e t i c p o l y s t y r e n e s t a r - p o l y m e r s f r o m Polysciences, W a r r i n g t o n , P e n n s y l v a n i a , were also used [ M n = 7000, l o t # 55687; M n = 5 9 , 2 0 0 , l o t # 71520; M n = 126,900, l o t # 55690; a n d M n = 116,700, l o t # 55689]. O t h e r M W s t a n d a r d s e x a m i n e d i n c l u d e d four p h e n y l - t e t r a m e r s w h i c h were s u p p l i e d as generous gifts f r o m D r . J . A . H y a t t at E a s t m a n K o dak L a b s . These m o d e l c o m p o u n d s were prepared b y a m o d i f i e d enolate a d d i t i o n m e t h o d (19) a n d i n c l u d e b i p h e n y l t e t r a m e r hexaacetate (C54H6602o>


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M W = 1034), b i p h e n y l t e t r a m e r h e x a o l ( M W = 782), / ? - 0 - 4 t e t r a m e r heptaacetate (C55H66O22, M W = 1078) a n d / ? - 0 - 4 t e t r a m e r h e p t a o l ( M W = 784). T h e I g e p a l ™ ( G A F C o r p . , sold t h r o u g h A l d r i c h ) s t a n d a r d s F . W . = 749 a n d 1982 were also e x a m i n e d . T w o s y n t h e t i c p o l y m e r s p r e p a r e d b y a n i o n - i n i t i a t e d p o l y m e r i z a t i o n s of a q u i n o n e m e t h i d e a c c o r d i n g t o the p r o cedure o f C h u m et al. (20) were treated as i n t e r m e d i a t e M W l i g n i n m o d e l polymers. Lignin Samples. B a l l - m i l l e d ( B M ) aspen l i g n i n was p r e p a r e d f o l l o w i n g the procedure of L u n d q u i s t et al. (21). T h e y i e l d of p u r i f i e d m i l l e d w o o d l i g n i n o b t a i n e d was u s u a l l y a b o u t 1 0 % w / w t h a t of e t h a n o l / b e n z e n e - e x t r a c t e d aspen w o o d . A l k a l i n e - e x t r a c t e d / s t e a m - e x p l o d e d ( A E S E ) aspen l i g n i n samples were p r e p a r e d f r o m s t e a m e x p l o d e d w o o d samples (55 s residence t i m e at 2 4 0 ° C ) o b t a i n e d f r o m Iotech C o r p . E x p l o d e d w o o d p u l p was treated w i t h a series o f c a r b o n t e t r a c h l o r i d e a n d alkaline e x t r a c t i o n s (12). A l k a l i n e - e x t r a c t e d / a c i d h y d r o l y s i s ( A H / N a O H ) l i g n i n samples were p r e p a r e d by s u b j e c t i n g aspen w o o d flour t o a one h o u r cook at 120°C i n 0 . 0 5 N s u l f u r i c a c i d (22), followed b y m i x i n g the clarified s u p e r n a t a n t w i t h 1% w / w N a O H at 25°C w i t h a W a r i n g blender. T h e i n s o l u b l e l i g n i n s were p r e c i p i t a t e d b y a d d i t i o n of a c i d a n d water washes ( 3 2 % y i e l d ) . T h e organosolv ( O S ) l i g n i n was prepared f r o m the l i q u o r o b t a i n e d b y t r e a t i n g aspen w o o d flour w i t h a 7 0 : 3 0 M e O H : w a t e r ( v / v ) e x t r a c t i o n at 165°C for 2.5 hours i n a r o c k i n g autoclave as described i n ref. 23. L i g n i n samples were a c e t y l a t e d f o l l o w i n g a m e t h o d developed b y G i e r e r a n d L i n d e b e r g (24) w h i c h allows q u a n t i t a t i v e recovery of l i g n i n s . L i g n i n samples were stored frozen d u r i n g the course of the s t u d y . T h r o u g h o u t the s t u d y , freshly prepared solutions were i n v e s t i g a t e d . However, no t i m e dependence of M W d a t a was observed i n any of the techniques e m p l o y e d w h e n samples were o c c a s i o n a l l y r e e x a m i n e d after i n i t i a l p r e p a r a t i o n . Chromatography System. T h e H P S E C - D V s y s t e m used i n t h i s s t u d y c o n sisted o f a B e c k m a n M o d e l 1 0 0 A d u a l - p i s t o n H P L C p u m p fitted w i t h extern a l pulse d a m p e n i n g , a B e c k m a n M o d e l 210 i n j e c t i o n valve fitted w i t h a 250 fiL l o o p , an S S I i n j e c t i o n valve filter, a H e w l e t t - P a c k a r d M o d e l 1 0 3 7 A h i g h s e n s i t i v i t y R I detector, a n d a V i s c o t e k M o d e l 1 0 0 L C differential v i s c o m e ter. F o r studies of l i g n i n c o n c e n t r a t i o n effects, a K n a u e r U V detector set at 280 n m was used as w e l l . T h e c h r o m a t o g r a p h y c o l u m n s y s t e m was c o m posed of three 7.8 x 30 m m c o l u m n s ( B e c k m a n ^ - S p h e r o g e l , 10,000, 1,000, a n d 5 0 0 A ) connected i n series i n order of increasing pore size. C a l c u l a t i o n s were p e r f o r m e d u s i n g the V i s c o t e k U n i c a l 2.71 software. A l l injections o n the H P S E C - D V s y s t e m were m a d e b y o v e r f i l l i n g the 2 5 0 / / L l o o p , thereby p r o v i d i n g a true 250 fiL i n j e c t i o n . N a r r o w a n d b r o a d M W s t a n d a r d s were injected o n t o the H P S E C - D V s y s t e m at concentrations near 1 m g / m L a n d 2 m g / m L , respectively. I n i t i a l l y , i n order to o b t a i n a usable differential pressure c h r o m a t o g r a m , the l i g n i n samples were injected at concentrations near 20 m g / m L , w i t h a n i n s t r u m e n t ( A - D amplifier) g a i n s e t t i n g o f 1 (0-1.0 volt F u l l Scale). A s the



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w o r k proceeded, a g a i n s e t t i n g o f 2 (0-0.1 v o l t F S ) a l l o w e d e x a m i n a t i o n of l i g n i n samples at concentrations o f 4 a n d 8 m g / m L . A s a test for l i g n i n a s s o c i a t i o n i n T H F at these concentrations, the organosolv l i g n i n was c h r o m a t o g r a p h e d at i n i t i a l i n j e c t i o n concentrations of 0.5 a n d 1 m g / m L as well. C o n c e n t r a t i o n s o f a l l s t a n d a r d a n d s a m p l e s o l u t i o n s s t u d i e d were precisely d e t e r m i n e d by w e i g h i n g the d r y m a t e r i a l s to the nearest 0.0005 m g u s i n g a S a r t o r i u s U l t r a m i c r o balance M o d e l 4504 M P 8 . L i g n i n samples e x a m i n e d b y H P S E C - D V were m a d e t o c o n c e n t r a t i o n i m m e d i a t e l y before i n j e c t i o n . T h e organosolv l i g n i n samples were s u b j e c t e d t o p r e p a r a t i v e c o l u m n c h r o m a t o g r a p h y i n T H F u s i n g a t w o c o l u m n , μ-Styragel s y s t e m f r o m Y M C , J a p a n (5 c m x 2 0 0 c m , 500 a n d 1000Â). A B e c k m a n M o d e l H O B p u m p , M o d e l 210 i n j e c t i o n valve w i t h a 2 m L l o o p , a n d a M o d e l 153 U V detect o r w i t h s e m i - p r e p a r a t i v e flow cell were used w i t h these c o l u m n s . S a m p l e l o a d i n g s were u s u a l l y 60 m g . F r a c t i o n s were collected w i t h a n Isco F o x y f r a c t i o n collector w i t h p r e p a r a t i v e c a p a b i l i t y a n d stored i n K i m a x screwt o p p e d glass tubes (25 x 150 m m ) w i t h T e f l o n - l i n e d caps. Before c h r o m a t o g r a p h i c a n a l y s i s these fractions were p o o l e d i n t o five m a s t e r f r a c t i o n s a n d c o n c e n t r a t e d b y r o t o - e v a p o r a t i o n at 25°C. V a l u e s f o u n d for the e x t i n c t i o n coefficient of organosolv a n d b a l l m i l l e d l i g n i n b y c o n v e n t i o n a l g r a v i m e t r i c a n a l y s i s , e 70nm (g m L ~ c m " " ) = 15,300 a n d 10,100, respectively, were used to estimate the dried-weight equivalent of the concentrated fractions recovered f r o m p r e p a r a t i v e c h r o m a t o g r a p h y . These c o n c e n t r a t i o n values are c r i t i c a l for m e a n i n g f u l e s t i m a t i o n o f M W u s i n g the U n i c a l software. 2

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Calculation of Results from Differential Viscometry and SEC. T h e f a m i l i a r r e l a t i o n s h i p first expressed b y M a r k (25) a n d H o u w i n k (26) i n the 1940's is c e n t r a l to the concept of u n i v e r s a l c a l i b r a t i o n first suggested by B e n o i t et al. (27). I n t h i s r e l a t i o n s h i p , [η] = K'M

a

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[η] is the i n t r i n s i c viscosity, a n d K' a n d a are k n o w n as the M a r k - H o u w i n k constants a n d are specific t o a p o l y m e r - s o l v e n t - t e m p e r a t u r e s y s t e m . F o r flexible, linear p o l y m e r s , values of α are l i m i t e d t o the range 0.50 t o 0.80. C o n s i d e r i n g the derivations of e q u a t i o n (1), i t can be p r e d i c t e d t h a t a l l molecules h a v i n g the same value o f [η]Μ w o u l d have the same v a l u e of v/», the h y d r o d y n a m i c v o l u m e . A l s o , i f ν Λ is the p a r a m e t e r t h a t u n i q u e l y determines the e l u t i o n v o l u m e , V , these molecules s h o u l d have the same e l u t i o n v o l u m e . T h e arguments presented b y these a u t h o r s d o n o t p r e d i c t t h a t the r e l a t i o n s h i p between these parameters s h o u l d necessarily be l i n e a r . M o s t u n i v e r s a l c a l i b r a t i o n curves s h o w n i n the l i t e r a t u r e t h a t cover 4 to 6 decades o f M show a definite u p w a r d c u r v a t u r e at h i g h values o f M (28). Sources o f error i n t h i s a p p r o a c h arise f r o m b o t h e x p e r i m e n t a l a n d the o r e t i c a l grounds. M o d e r n theories of S E C r e t e n t i o n m e c h a n i s m are based o n the a s s u m p t i o n t h a t the size e x c l u s i o n process u n i q u e l y determines the e l u t i o n v o l u m e , a n d yet the p o s s i b i l i t y of reversible a d s o r p t i o n is difficult to dismiss a n d , where i t occurs, errors i n the i n t e r p r e t a t i o n m a y easily result. A s a w a r n i n g for the a p p l i c a t i o n o f universal c a l i b r a t i o n m e t h o d o l o g y , C a s sassa (29) i n d i c a t e s i n a l a t e r paper t h a t the q u a n t i t y [η]Μ is not a t r u l y e



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u n i v e r s a l e l u t i o n p a r a m e t e r for S E C , b u t t h a t b o t h t h e o r y a n d experience i n d i c a t e t h a t g o o d results can be o b t a i n e d for e l u t i n g species of s i m i l a r t y p e (e.g., r o d - l i k e macromolecules of s i m i l a r cross-sectional d i m e n s i o n i n a r e s t r i c t e d size range or l i n e a r flexible p o l y m e r c h a i n s ) . C a s s a s s a predicts f r o m theory, however, t h a t over r e s t r i c t e d ranges of M, a c o m m o n [η]M dependence between r a n d o m c o i l p o l y m e r s a n d r o d - l i k e s t r u c t u r e s s h o u l d exist. Divergence often increases, however, w h e n considering fit u s i n g d a t a over three orders of m a g n i t u d e i n M (30). A p p l i c a t i o n of u n i v e r s a l c a l i b r a t i o n to u n k n o w n p o l y m e r s u s i n g the V i s c o t e k U n i c a l software, once the c o l u m n s y s t e m has been c a l i b r a t e d w i t h n a r r o w M W D s t a n d a r d s , is q u i t e s t r a i g h t f o r w a r d . A m a s t e r c a l i b r a t i o n file of n a r r o w M W D s t a n d a r d s was developed w h i c h i n c o r p o r a t e s the "peak p a r a m e t e r " values c a l c u l a t e d f r o m one (or averaged f r o m several) w e l l be haved n a r r o w M W D s t a n d a r d . These values correct for c h r o m a t o g r a p h i c m i s m a t c h of the two detectors ( R I a n d differential pressure) used i n the s y s t e m a n d l e a d t o the c a l c u l a t i o n of values t h a t a p p r o x i m a t e a correc t i o n for peak b r o a d e n i n g ( r ) a n d peak t a i l i n g ( σ ) . T h e s e peak parameters represent effects specific t o the c h r o m a t o g r a p h i c s y s t e m used i n each l a b o r a t o r y , a n d were d e t e r m i n e d for t h i s s t u d y to be 0.256 m L , 0.280 a n d 0.256 for σ, τ ( V ) , a n d r ( C ) , respectively. T h e c o n c e n t r a t i o n value for each s a m p l e processed b y t h i s procedure m u s t be k n o w n accurately, as t h i s t e r m enters i n t o c a l c u l a t i o n s of reduced viscosity, measured here d i r e c t l y as specific viscosity, a n d the M W averages. A n a s s u m p t i o n c e n t r a l t o the d a t a processing is t h a t under c h r o m a t o g r a p h i c c o n d i t i o n s s a m p l e d i l u t i o n is sufficient to assume t h a t the reduced v i s c o s i t y a p p r o x i m a t e s the i n t r i n s i c v i s c o s i t y (or l i m i t i n g v i s c o s i t y n u m b e r ) . T h e software c a n be used t o c a l culate the M a r k - H o u w i n k plots ([η] versus M) for each s t a n d a r d p o l y m e r series. A l l p o l y m e r s t a n d a r d s are t h e n used t o c o n s t r u c t a u n i v e r s a l c a l i b r a t i o n p l o t of [η]M versus e l u t i o n v o l u m e . T h e software can also be used t o recalculate the values of M , M , M a n d M * + i for the n a r r o w M W D s t a n d a r d s used to construct the curve ( a p p r o x i m a t e l y ± 1 0 % d e v i a t i o n for M is observed i n these r e c a l c u l a t e d values w h e n c o m p a r e d w i t h the v a l ues entered i n i t i a l l y ) . M o l e c u l a r weight averages are f o u n d for u n k n o w n p o l y m e r s (accepting the l i m i t i n g a s s u m p t i o n s discussed above) i n a s i m i l a r way. A p p l i c a t i o n of U n i c a l software also requires the selection of c h r o m a t o g r a p h i c baselines, thus selecting the specific d a t a t a k e n for further a n a l y s i s . In the studies r e p o r t e d here, we choose to analyze o n l y the p o l y disperse en velope f r o m l i g n i n e l u t i o n , so t h a t the d i s t i n c t c o m p o n e n t w h i c h often elutes near Y (the t o t a l c o l u m n v o l u m e ) was not i n c l u d e d i n the a n a l y s i s . n

w

Z1

w

t

Results and Discussion Universal Calibration. T h e aspen w o o d l i g n i n samples chosen for t h i s s t u d y were prepared b y organosolv, s t e a m e x p l o s i o n , d i l u t e a c i d h y d r o l y s i s , a n d b a l l - m i l l i n g procedures. C a l i b r a t i o n curves were developed for H P S E C - D V u s i n g p o l y m e r s t a n d a r d s i n c l u d i n g n a r r o w M W D polystyrenes, p o l y butadienes, p o l y m e t h y l -


6.

HIMMEL ET A L

MWDistribution

by Universal Calibration

87

m e t h a c r y l a t e s , a n d p o l y - a - m e t h y l s t y r e n e s . A set of b r o a d M W D p o l y m e t h y l m e t h a c r y l a t e s was also e x a m i n e d . A l l five s t a n d a r d curves i n d i c a t e d very g o o d fit to a l i n e a r f u n c t i o n over the range of M W tested. T h e M a r k H o u w i n k parameters for a a n d —K were f o u n d to be 0.73 a n d 4.35, 0.60 a n d 3.76, 0.72 a n d 3.87, 0.69 a n d 4.26, 0.73 a n d 4.55 for the P S , P A M S , P B , P M M A , a n d P M M A - 6 s t a n d a r d s , respectively. A l l M a r k - H o u w i n k p a rameters, a a n d K\ w i t h the exception of α for the p o l y - a - m e t h y l s t y r e n e s , c o m p a r e closely w i t h those p u b l i s h e d b y H a n e y a n d A r m o n a s (31) u s i n g identical conditions and instrumentation. A u n i v e r s a l c a l i b r a t i o n p l o t (log [η]Μ vs. e l u t i o n v o l u m e ) u s i n g these five s t a n d a r d s series was c o n s t r u c t e d ( F i g . 1). Several other s t a n d a r d M W p o l y m e r s a p p r o p r i a t e t o l i g n i n m o d e l studies were also e x a m i n e d . T h e s e i n c l u d e d two Igepals, four p o l y s t y r e n e star p o l y m e r s , a n d four s y n t h e t i c p h e n y l t e t r a m e r s (two b i p h e n y l s a n d two β-Ο-4 l i n k e d t e t r a m e r s ) . O f a l l the s t a n d a r d s e x a m i n e d , o n l y the p o l y s t y r e n e star p o l y m e r p r e p a r a t i o n i n d i c a t e d p a u c i d i s p e r s i t y . H e r e , the e l u t i o n o f the lower M W c o m p o n e n t ( u s u a l l y i n preponderance) was considered i n the c a l c u l a t i o n s . W i t h the e x c e p t i o n o f one h i g h M W s t a r p o l y m e r , a l l these c o m p o u n d s were f o u n d t o fit u n i v e r s a l c a l i b r a t i o n at least as w e l l as the c o m m e r c i a l p o l y m e r s t a n d a r d s ( F i g . 1). Indeed, the fit o f the low M W p h e n y l t e t r a m e r s ( M W « 800-1000) was i m p o r t a n t . T h e c a l i b r a t i o n curve c o n s t r u c t e d for use i n t h i s s t u d y shows l i t t l e or not c u r v a t u r e over the five decade range o f log[?;]M. T h i s o b s e r v a t i o n is consistent w i t h t h a t of other workers ( 2 8 , 3 1 ) , where the lowermost p o r t i o n o f such curves approaches l i n e a r i t y , w h i l e over a w i d e r range o f l o g [η] M some u p w a r d c u r v a t u r e is e v i d e n t . U n i c a l 2.71 software allowed the c a l c u l a t i o n of a u n i v e r s a l c a l i b r a t i o n curve f r o m a b r o a d M W D s t a n d a r d , P M M A 1 7 K - 6 . T h i s curve appears i n F i g u r e 1 as a dashed l i n e . A l t h o u g h some d e v i a t i o n at d a t a extremes is a p p a r e n t , the fit near the l i g n i n e l u t i o n region is n e a r l y i d e n t i c a l to the curve f r o m n a r r o w c a l i b r a t i o n .


1

MWD of Acttylated Lignins. T h e first a t t e m p t s at l i g n i n c h r o m a t o g r a p h y e m p l o y e d 250 / i L injections of samples m a d e to 20 m g / m L . T h i s p r o c e dure p r o d u c e d acceptable differential pressure signals; however, the very h i g h c o n c e n t r a t i o n was undesirable as i t is k n o w n t o i n d u c e solute-solute i n t e r a c t i o n . A f t e r r e s e t t i n g the amplifier g a i n t o 2 ( a s e t t i n g o f 1 be i n g the s t a n d a r d " d e f a u l t " value for the i n s t r u m e n t ) , differential pressure c h r o m a t o g r a m s w i t h l i g n i n s at loadings o f 2 m g (250 μΐι injections f r o m 8 m g / m L stock solutions) were very w e l l behaved ( F i g s . 2-4). I n s p e c t i o n of F i g u r e s 2 a n d 3 reveals t h a t the r e l a t i v e m a g n i t u d e of the differential pressure s i g n a l at higher M W is greater t h a n t h a t f r o m the differential refractive i n d e x detector. T h e four l i g n i n s were also injected o n the c h r o m a t o g r a p h y s y s t e m at a l o a d i n g of 1 m g (250 / i L f r o m a 4 m g / m L stock s o l u t i o n ) . These d u a l c h r o m a t o g r a m s proved t o i n d i c a t e the l i m i t i n g sen s i t i v i t y for the b r o a d l y p o l y disperse l i g n i n samples s t u d i e d here. A l t h o u g h the " s m o o t h i n g " routines i n U n i c a l were capable of r e n d e r i n g these noisy differential c h r o m a t o g r a m s usable, i t was clear t h a t a m o r e d i l u t e injec t i o n s a m p l e w o u l d not be m e a n i n g f u l . A n e x a m p l e o f the best d u a l c h r o m a t o g r a m f r o m a 1 m g l o a d i n g (before s m o o t h i n g ) is s h o w n i n F i g u r e 4 U .



88


F i g u r e 1. M a s t e r u n i v e r s a l c a l i b r a t i o n curve o b t a i n e d w i t h a B e c k m a n μ-Spherogel c o l u m n s y s t e m i n T H F . T h e fit of n a r r o w M W D s t a n d a r d s , p o l y s t y r e n e star p o l y m e r s , a n d a single b r o a d M W D s t a n d a r d ( c a l c u l a t e d w i t h U n i c a l 2.71 software) are s h o w n .


R I M M E L ET AL.

6.


89


DUAL CHROMATOGRAM

25.0 RET

30.0 VOL (ml)

35.0 CONC CHROMATOGRAM ?

MOLECULAR HEIGHT DISTRIBUTION

S

6.00

LOG M

Figure 2 A . Dual chromatograms showing the elution of aspen A E S E lignin from the H P S E C - D V system. A 250 /zL sample was injected from a freshly prepared 8 mg / m L stock solution. Viscotek A / D amplifier gain setting of 2 and R I detector setting o f l x . Calculated molecular weights are also shown.


90


8.00 y

DUAL CHROMATOGRAM


5.00

2.00

V l.oo

25.0

RET VOL

CJ

θ.00

30.0

(ml)

35.0

40.0

CONC CHROMATOGRAM ?

HEIGHT DISTRIBUTION

7.00

5.00

1.00

Figure 2 B . D u a l chromatograms showing the elution of ball-milled lignin from the H P S E C - D V system. Sample loading was the same as in Figure 2 A .


6.

H I M M E L ET AL.


7.00

91

DUAL CHROMATOGRAM


4.00

1.00

.000 15.0

25.0 RET

VOL (ml)

30.0

35.0

40.0

CONC CHROMATOGRAM !


5.00

4.00

3.00

.000 2.00

Figure 3 A . Dual chromatograms showing the elution o f aspen organosolv lignin from the H P S E C - D V system. Sample loading was the same as i n Figure 2 A . Calculated molecular weights are also shown.



92


DUAL CHROMATOGRAM

25.0

RET VOL

30.0

(ml)

35.0

40.0

CONC CHROMATOGRAM ?


Figure 3 B . Dual chromatograms showing the elution of A H / N a O H lignin from the H P S E C - D V system. Sample loading was the same as i n Figure 2 A .



Figure 4 A . D u a l chromatogram showing the elution of A E S E aspen lignin from the H P S E C - D V system with an amplifier gain setting of 2 and an R I setting of l/4x. This experiment represents the minimum loading of such a sample which results i n usable signal.

RET VOL (ml)

DUAL CHROMATOGRAM


OS


Figure 4Β. Concentration chromatogram of three column loadings (1 mg, 0.2 mg, and 0.1 mg) of oganosolv aspen lignin on the H P S E C - D V system. R I detector setting was l x .


ο

1 Ι

S

Β ο

so

H I M M E L ET AL.

6.


95

V a l u e s for s y s t e m " p e a k p a r a m e t e r s " were f o u n d u s i n g a n a r r o w d i s t r i b u t i o n p o l y s t y r e n e s t a n d a r d ( P S 6 8 K ) before c a l c u l a t i n g M W D d a t a for the l i g n i n samples f r o m u n i v e r s a l c a l i b r a t i o n . T o check software a n d i n s t r u m e n t o p e r a t i o n , several n a r r o w M W D p o l y s t y r e n e a n d one b r o a d M W D p o l y m e t h y l m e t h a c r y l a t e s t a n d a r d s were t r e a t e d as u n k n o w n samples a n d s u b j e c t e d to analysis w i t h the u n i v e r s a l c a l i b r a t i o n curve assembled f r o m a l l p o l y m e r s t a n d a r d s files. It was f o u n d t h a t the M W D c o u l d be e s t i m a t e d for the " r e c a l c u l a t e d " p o l y m e r s t a n d a r d s w i t h errors between ± 5 a n d 1 0 % of the o r i g i n a l value i n d i c a t e d b y the s u p p l i e r of the s t a n d a r d (e.g., M for P S 1 1 K and M a n d M for P M M A 1 7 K - 6 ) . w


w

n

T a b l e I illustrates the m o l e c u l a r weight averages f o u n d f r o m u n i versal c a l i b r a t i o n ( n a r r o w s t a n d a r d s ) for four aspen l i g n i n s a n d t w o q u i n o n e m e t h i d e - d e r i v e d p o l y m e r s u s i n g the U n i c a l software. T h e p o l y dispersities were the same w i t h i n the e x p e r i m e n t a l errors for a l l l i g n i n s a m ples. I n c o n t r a s t , the p o l y d i s p e r s i t i e s f o u n d for the q u i n o n e m e t h i d e - d e r i v e d p o l y m e r s b y u n i v e r s a l c a l i b r a t i o n were near 1.1. T h e m o l e c u l a r weight av erages f o u n d for the four a c e t y l a t e d l i g n i n s s t u d i e d b y u n i v e r s a l c a l i b r a t i o n were s u b s t a n t i a l l y larger t h a n those d e t e r m i n e d f r o m p r e v i o u s work u s i n g c o n v e n t i o n a l G P C (e.g., A E S E a n d B M l i g n i n s f r o m refs. 7 a n d 12 h a d a p p r o x i m a t e l y o n e - t h i r d those values f o u n d b y H P S E C - D V i n the present study). T a b l e I. M W D of A c e t y l a t e d A s p e n L i g n i n s a n d M o d e l C o m p o u n d s f r o m Universal Calibration with Narrow Standards 1

M /M„

Samples/loading

M„

M

A E S E / 1 mg A E S E / 2 mg

1100 1900

7300 7100

34500 27000

3300

OS/1 mg OS/2 mg

1300 1000

5200 4400

16000 18000

3200

A H / N a O H / 1 mg A H / N a O H / 2 mg

2200 1300

8100 6600

38000 34000

4600

B M / 1 mg B M / 2 mg

3600 9000

17300 22000

46800 47000

11500

—

4.7 2.4

8700 14700

10300 16700

12400 20000

9900 16000

1.1 1.1

Q M 34/0.25 mg Q M 33/0.25 m g 1

w

M

z

M

u

— — —

w

6.7 3.7 4.0 4.4 3.7 5.0

O b t a i n e d i n T H F at 20°C w i t h R I d e t e c t i o n a n d U n i c a l 2.71 software ( V i s c o t e k , Inc.). F o r h i g h loadings the s y s t e m was set at R I = lx a n d 20 P A F S , g a i n = 2; injections (250 μΐ,) m a d e f r o m 8 m g / m L stock s o l u t i o n s . F o r l o w l o a d i n g s the detector settings were R I = l/4a? a n d 20 P A F S , g a i n = 2; injections (250 |/L) m a d e f r o m 4 m g / m L stock solutions.

T h e issue of c o l u m n l o a d i n g was further investigated b y i n j e c t i n g 200 μL samples f r o m stock organosolv l i g n i n s o l u t i o n s of 5, 1 a n d 0.5 m g / m L .



96

T h e c o m p o s i t e c h r o m a t o g r a m s h o w n i n F i g u r e 4 L clearly indicates t h a t a n a p p a r e n t increase i n higher M W content occurs w h e n c o l u m n l o a d i n g s increase f r o m 0.1 m g to 0.2 m g l i g n i n . T h e 0.1 m g l o a d i n g represents the s e n s i t i v i t y l i m i t , however, of the h i g h s e n s i t i v i t y refractive i n d e x detector used i n t h i s s t u d y . H o w e v e r , the 0.2 a n d 1 m g loadings (where m o s t l i g n i n d a t a were collected i n t h i s s t u d y ) were n e a r l y i d e n t i c a l i n d i s t r i b u t i o n (see F i g u r e 4 L ) . C o m p a r i s o n of the curves i n F i g u r e 4 L w i t h closely e l u t i n g p a i r s o f s t a n d a r d p o l y m e r s i n d i c a t e d t h a t the a p p a r e n t increase i n M induced b y t h i s c o n c e n t r a t i o n effect w o u l d be < 1 0 % .


w

Chromatographic Fractionation of Organosolv Lignin. T h e preparative μS t y r a g e l c o l u m n f r o m Y M C was l o a d e d w i t h 60 m g of organosolv l i g n i n . T h e r e s u l t i n g e l u t i o n profile is s h o w n as a n insert i n F i g u r e 5. T h i s figure shows the relative d i s t r i b u t i o n of the c h r o m a t o g r a p h i c fractions p o o l e d t o generate the five m a s t e r fractions used for f u r t h e r s t u d y . These f r a c t i o n s were chosen so t h a t the relative areas of a l l five zones were n e a r l y i d e n t i c a l . F i g u r e 5 also shows the s u p e r p o s i t i o n of the r e s u l t i n g c h r o m a t o g r a p h i c a n a l y s i s of four of these f r a c t i o n s . F r a c t i o n n u m b e r five was o m i t t e d f r o m t h i s a n a l y s i s because t h i s peak was not i n c l u d e d i n the s t a n d a r d procedure used i n e s t a b l i s h i n g baselines for the four n a t i v e l i g n i n s described earlier. T h e t o t a l weight average m o l e c u l a r weight for the entire d i s t r i b u t i o n (32) was e s t i m a t e d u s i n g the r e l a t i o n s h i p M ot W)t

= EaMi/Eci

(2)

where c is the c o n c e n t r a t i o n (here i n m i l l i g r a m s ) of each master f r a c t i o n , i , a n d M is the e s t i m a t e d M o f each master f r a c t i o n f r o m u n i v e r s a l c a l i b r a t i o n . T h e value of M f o u n d u s i n g these four master fractions was 3800. W h e n c o n s i d e r i n g the M f r o m the c h r o m a t o g r a p h y of the u n f r a c t i o n a t e d organosolv l i g n i n at a 1 m g l o a d i n g was e s t i m a t e d t o be 4400, the value o b t a i n e d f r o m the organosolv fractions is i n g o o d agreement. T h i s e x p e r i m e n t was designed to e x a m i n e the possible bias the b r o a d p o l y d i s p e r s i t i e s o f the l i g n i n samples m a y have o n e s t i m a t i o n of M W D b y u n i v e r s a l c a l i b r a t i o n . T h e s e d a t a i n d i c a t e t h a t n o such c o n t r i b u t i o n exists, since the s u m m a t i o n of the i n d i v i d u a l fractions of n a r r o w ( e r ) d i s p e r s i t y l e a d t o values of M W D s i m i l a r t o those f o u n d u s i n g U n i c a l software for the unfractionated lignin sample. w

W)tot

w

Conclusions A l t h o u g h evidence exists t h a t c o n c e n t r a t i o n effects m a y be i m p o r t a n t w i t h even a c e t y l a t e d l i g n i n s i n T H F , the effect o f i n c r e a s i n g c o l u m n l o a d i n g s f r o m 1 t o 2 m g seems u n l i k e l y as the cause of the v a r i a n c e i n M W s h o w n i n T a b l e I. T h i s observation i l l u s t r a t e s the m o r e general p r o b l e m i n c u r r e n t S E C - b a s e d " a b s o l u t e " M W measurement: t h a t of a l i m i t e d c o n c e n t r a t i o n w i n d o w for a n a l y s i s . T h e l i m i t i n g value for s a m p l e c o n c e n t r a t i o n appears t o be near 1 m g per i n j e c t i o n for H P S E C - D V , w h i c h is c o m p a r a b l e t o the 0.2-1 m g per i n j e c t i o n range usable i n H P S E C - L A L L S (33). F o r studies



W

2.50

3.00 LOG M

3.50

4.00

F i g u r e 5. C o m p o s i t e M W D d i a g r a m s h o w i n g the c a l c u l a t e d d i s t r i b u t i o n s of four of the five master fractions o b t a i n e d f r o m p r e p a r a t i v e c h r o m a t o g r a p h y of organosolv aspen l i g n i n ( f r a c t i o n n u m b e r 4 to 1 f r o m left to r i g h t ) . T h e M values for the m a s t e r f r a c t i o n s f r o m left to right are 1,110, 1,310, 2,420, a n d 8,050, respectively. T h e insert shows the e l u t i o n profile of o r g a n o s o l v aspen l i g n i n f r o m the Y M C p r e p a r a t i v e μ-Styragel c o l u m n (5 x 200 c m ) . T h e 30 f r a c t i o n s collected were p o o l e d i n t o the five m a s t e r f r a c t i o n s s h o w n .

2.00


4.50

LIGNIN: PROPERTIES A N D MATERIALS


98

such as these, a n increase i n s e n s i t i v i t y o f one order o f m a g n i t u d e w o u l d be h i g h l y v a l u e d a n d s h o u l d b e considered a n area o f focus for suppliers o f S E C detection equipment. T h e d e t e r m i n a t i o n t h a t the low M W , acetylated aspen lignins e x a m i n e d a c t u a l l y fit u n i v e r s a l c a l i b r a t i o n , however, must be deferred t o future studies c o m p a r i n g these d a t a t o results f r o m L A L L S a n d s e d i m e n t a t i o n e q u i l i b r i u m analysis ( i f possible). A s a result o f the dependence o f u n i v e r s a l c a l i b r a t i o n o n c o l u m n e l u t i o n b e h a v i o r (i.e., anomalous behavior due t o a d s o r p t i o n o r e x c l u s i o n ) , the c o n t r i b u t i o n o f the p o l y m e r "core" a n d " s h e l l " c o m p o n e n t s ( 3 3 , 3 4 ) t o h y d r o d y n a m i c b e h a v i o r m u s t be f u l l y u n d e r s t o o d i f c o m p e t e n t a n a l y s i s o f b l o c k copolymers a n d b r a n c h e d heteropolymers is t o be m a d e . It is h o p e d t h a t w i t h the advent o f a p p r o p r i a t e M W , c o m p o s i t i o n , a n d b r a n c h e d p o l y mer s t a n d a r d s , the l i m i t s o f fit o f u n i v e r s a l c a l i b r a t i o n t o b i o p o l y m e r s such as l i g n i n can b e j u d g e d . Acknowledgments W e t h a n k D r . J . A . H y a t t for the gift of s y n t h e t i c p h e n y l t e t r a m e r s a n d M a x H a n e y for h e l p f u l discussions. T h i s work was funded b y t h e B i o c h e m i c a l C o n v e r s i o n P r o g r a m at the D O E Biofuels a n d M u n i c i p a l W a s t e T e c h n o l o g y D i v i s i o n t h r o u g h F T P N o . 658. Literature Cited 1. Brauns, F . E . ; Brauns, D. A . The Chemistry of Lignin; Academic Press: New York, 1960. 2. Sarkanen, Κ. V . ; Ludwig, C . H . , Eds. Lignins: Occurrence, Formation, Structure and Reactions; Wiley-Interscience: New York, 1971. 3. Sarkanen, Κ. V . In The Chemistry of Wood; Browning, B. L . , E d . ; R. E . Krieger Publ. Co.: Huntington, 1975. 4. Marchessault, R. H . ; Coulombe, S.; Hanai, T . ; Morikawa, H . ; Robert, D. Can. J. Chem. 1982, 60, 2372. 5. Kolpak, F . J . ; Cietek, D. J . ; Fookes, W.; Call, J . J . J. Appl. Polym. Sci., Polym. Sci. Symp. 1983, 31, 491. 6. Himmel, M . E . ; Oh, Κ. K.; Sopher, D. W.; Chum, H . L . J. Chromatogr. 1983, 267, 249. 7. Faix, Ο.; Lange, W.; Beinhoff, Ο. Holzforschung 1980, 34, 174. 8. Faix, Ο.; Lange, W . ; Salud, E . C . Holzforschung 1981, 35, 3. 9. Lange, W . ; Schweers, W.; Beinhoff, O. Holzforschung 1981, 35, 119. 10. Meier, D.; Faix, O.; Lange, W . Holzforschung 1981, 35, 247. 11. Lange, W.; Faix, O.; Beinhoff, O. Holzforschung 1983, 37, 63. 12. Chum, H . L . ; Johnson, D. K.; Tucker, M . P.; Himmel, M . E . Holz forschung 1987, 41, 97. 13. Pellinen, J . ; Salkinoja-Salonen, M . J. Chromatogr. 1985, 328, 299. 14. Connors, W . J.; Sarkanen, S.; McCarthy, J . L . Holzforschung 1980, 34, 80. 15. Sarkanen, S.; Teller, D. C . ; Hall, J . ; McCarthy, J . L . Macromolecules 1981, 14, 426.



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Molecular Weight Distribution of Aspen Lignins Estimated by Universal

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