Use of Multidetection for Chromatographic Characterization of

Chapter 12

Use of Multidetection for Chromatographic Characterization of Dextrins and Starch

Downloaded by NORTH CAROLINA STATE UNIV on August 1, 2012 | http://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0458.ch012

A. Heyraud and M. Rinaudo Centre de Recherches sur les Macromolécules Végétales, Centre National de la Recherche Scientifique, B.P. 53 X, 38041 Grenoble cedex, France

Optical rotation and refractometric detectors were combined to characterize a series of dextrins by gel permeation chromatography and high performance l i q u i d chromatography (HPLC). Gel permeation fractionates according to the hydrodynamic volume when adsorption i s avoided, and for this reason separates isomaltodextrins from linear maltodextrins. Specific rotation power [α] is directly obtained and confirms the chemical structure. Elution of cyclodextrins is also tested and discussed. HPLC reverse phase chromatography separates the anomers as shown by optical rotation and allows good resolution in the range of low DP. Light scattering and refractometric detectors were used to analyze starch with different amylose contents. Fractionations were also performed and iodine complexation tested in relation with the molecular structure. Gel permeation chromatography in DMSO was used to determine the molecular weight distribution of different starch samples without any calibration.

Chromatography is one of the best techniques to characterize oligomers and polymers for analytical p u r p o s e . The f r a c t i o n a t i o n i n v o l v e s d i f f e r e n t mechanisms f r o m w h i c h s t e r i c e x c l u s i o n c h r o m a t o g r a p h y (GPC o r SEC) i s t h e most i m p o r t a n t f o r c h a r a c t e r i z a t i o n o f p o l y m e r s . I n SEC t h e elution is c o n t r o l l e d by t h e hydrodynamic

0097-6156/91/0458-0171S06.00/0 © 1991 American Chemical Society

In Biotechnology of Amylodextrin Oligosaccharides; Friedman, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


172

BIOTECHNOLOGY OF AMYLODEXTRIN OUGOSACCHARIDES

volume o f t h e m o l e c u l e s as e x p r e s s e d by t h e p r o d u c t [η]M where [η] is the intrinsic viscosity and M i s the m o l e c u l a r weight. High performance l i q u i d chromatography (HPLC) i n d i r e c t o r r e v e r s e p h a s e w i t h a f r a c t i o n a t i o n b a s e d on s p e c i f i c i n t e r a c t i o n s w i t h t h e s t a t i o n a r y p h a s e i s c e r t a i n l y t h e most i n t e r e s t i n g f o r o l i g o m e r s / e v e n i f sometimes i t becomes n e c e s s a r y t o combine GPC a n d HPLC t e c h n i q u e s t o s o l v e an a n a l y s i s o f a complex m i x t u r e . A r e v i e w on t h e c h r o m a t o g r a p h y o f o l i g o s a c c h a r i d e s h a s b e e n published previously (1). In t h e f o l l o w i n g , t h e c h r o m a t o g r a p h i c s e p a r a t i o n o f s t a r c h a n d o l i g o m e r s r e l a t e d t o t h e o d - » 4) a n d a ( l - » 6) D - g l u c o s e s e r i e s w i l l be p r e s e n t e d a n d d i s c u s s e d .

Separation o f O l i g o s a c c h a r i d e s SEC. P r e v i o u s l y , g e l p e r m e a t i o n c h r o m a t o g r a p h y o f cello and m a l t o d e x t r i n s was i n v e s t i g a t e d (2-4) . I t was shown that Biogel P2 gave good separation based on the h y d r o d y n a m i c volume o f t h e c a r b o h y d r a t e , p r o v i d e d c o l u m n t e m p e r a t u r e was o v e r 6 5 C . E l u t i o n a t 65 *C s u p p r e s s e d t h e l o o s e a d s o r p t i o n w h i c h e x i s t s on t h i s m a t r i x . A p l o t o f [η] M v e r s u s Ve gave a u n i q u e c u r v e f o r t h e two s e r i e s o f d e x t r i n s which corresponds t o a pure s t e r i c exclusion separation process. Assuming a d i f f e r e n c e between the h y d r o d y n a m i c volume o f a b r a n c h e d d e x t r i n and t h a t o f a l i n e a r m a l t o d e x t r i n o f t h e same number o f monomers, a s e p a r a t i o n may be p o s s i b l e , a t l e a s t i n t h e r a n g e o f low degrees of polymerization. Figure 1 contains chromatograms o b t a i n e d f o r a s e r i e s o f m a l t o d e x t r i n s and isomaltodextrins. Partition coefficients (K^) g i v e n in T a b l e I a r e c a l c u l a t e d from t h e i r e l u t i o n volumes (Ve) and p l o t t e d i n F i g u r e 2 . The t o t a l l y i n c l u d e d volume ( V ) was o b t a i n e d f r o m t h e e l u t i o n o f D - g l u c o s e . As u s u a l , a l i n e a r dependency i s obtained for log p l o t t e d as a f u n c t i o n o f t h e d e g r e e o f p o l y m e r i z a t i o n (DP) . u n i v e r s a l c a l i b r a t i o n f o r t h e o l i g o s a c c h a r i d e s was e s t a b l i s h e d ( F i g u r e 3) f o r t h e m a l t o d e x t r i n s b y p l o t t i n g l o g [η] M a g a i n s t Kd (3). In T a b l e I I , t h e p a r t i t i o n c o e f f i c i e n t s are given for the f i r s t oligomers (α 1 -> 6 ; α 1 - » 4) . P a n o s e is c o n s i d e r e d as a model f o r t h e b r a n c h e d t r i s a c c h a r i d e . Differences i n t h e Kd v a l u e s are s i g n i f i c a n t to show the role of the 0C(1 - » 6) linkage on the h y d r o d y n a m i c volume o f t h e c o r r e s p o n d i n g o l i g o m e r s . The use o f o p t i c a l r o t a t i o n ( ΐ . β . [ α ] ρ ) i s also convenient to confirm the chemical s t r u c t u r e of the oligomers. Panose h a s K 3 and [ C C ] D v a l u e i n t e r m e d i a t e between t h o s e o f DP 3 , a ( l - » 6) a n d β ) , a ( l -> 4) c y c l o d e x t r i n s (CD).

a function of oligomers and



12.

HEYRAUD & RINAUDO

Table

I.

α 1 -> 4

α 1 -> β

Characterization of Dextrins and Starch

Characteristics of the(X(l oligomers

- » 4)

a n d ( X ( l - » 6)

DP

Kd

[OC]

1 2 3 4 5 6 7 8 9 10 11

1 0.901 0.800 0.710 0.623 0.540 0.487 0.435 0.382 0.339 0.302

52.7 140.7 153.5 162 164.2 166.3 168.4 179

2 3 4 5 β 7 8 9 10

0.847 0.717 0.610 0.519 0.448 0.381 0.327 0.282 0.245

121.5 133 145 143 145

D

-

-

Column : B i o g e l P o , h = 205 cm, φ = 1.5 cm F l o w r a t e : 30 m l / h ; t e m p e r a t u r e : 6 5 C ; e l u e n t e


: H2O.

175

176

BIOTECHNOLOGY OF AMYLODEXTRIN OLIGOSACCHARIDES


: l^7i IVI

s

Ν

\ Ν Ν

\ \ \

*2 \ •

a

1-6

*

a

1-4

•

CD

*

panose

\\ Kd

I 0.2 Figure SEC.

3.

Universal

I

0.4

» 06

calibration

ι 0-8 curve

obtained


by

12.

HEYRAUD & RINAUDO Table

Characterization of Dextrins and Starch 177

Kd f o r l o w e r D P o l i g o s a c c h a r i d e s during starch hydrolysis

II.


DP

2 (α ι 2 (α ι 3 (α ι Panose 3 (α 1

[CC]

Kd

-> — >

4) 6) 4) l i n e a r 6)

linear

D

140 .7 121 .5

0 .901 0 .855 153.5

0 .800

145 .4

0 .762

->

produced

0 .717

133

The chromatogr am o f α, β, γ c y c l o d e x t r i n s i s shown i n F i g u r e 4. Values o f Kd are given i n Table I I I and compared with t h e values f o r t h e comparable linear oligomers. Table I I I . C h a r a c t e r i s t i c s o f the c y c l i c and corresponding l i n e a r oligomers Oligomers α DP

6 β

7 Y DP 8

DP

Kd

0.628 0.549 0.570 0.487 0.534 0.435

[COD

[η] ml/g

_

2.01 2.55 2.12 2.74 2.09 2.85

166.3

179

168.4

Same c o n d i t i o n s as Table I . From these values, i t i s apparent t h a t t h e hydrodynamic volumes o f the c y c l i c oligomers are lower than the corresponding l i n e a r oligomers. In Table I I I , the values of Kçj, [CX]D and t h e i n t r i n s i c v i s c o s i t y [η] are determined from u n i v e r s a l c a l i b r a t i o n . The values o f [η] are only s l i g h t y dependent on the DP. The r a t i o between the i n t r i n s i c v i s c o s i t y o f the c y c l i c oligomers and t h a t of the l i n e a r forms decreases when DP i n c r e a s e s from 0.788 f o r a , t o 0.773 f o r β and 0.733 f o r γ. Some r e s u l t s e x i s t i n t h e l i t e r a t u r e and as an example, the GPC and HPLC o f the three c y c l o d e x t r i n s were examined (5-7). GPC was then performed on Sephadex G-15 and G-25. In f a c t , due t o the low r e s o l u t i o n obtained i n GPC i t i s sometimes d i f f i c u l t t o determine the p u r i t y o f the c y c l o d e x t r i n s . A chromatogram obtained on B i o g e l P2 f o r the mixture o f t h e l i n e a r and c y c l i c oligomers i s


178



F i g u r e 4. oligomers

Comparison of l i n e a r (M) and c y c l i c s e p a r a t i o n s on a B i o g e l Ρ 2 c o l u m n .


(CD)


12. HEYRAUD & RINAUDO

Characterization ofDextrins and Starch 179

g i v e n i n F i g u r e 4. From t h e s e r e s u l t s , i t seems t h a t DP β l i n e a r and β c y c l o d e x t r i n may n o t be s e p a r a t e d on t h e b a s i s of [η] M ; the of the c y c l o d e x t r i n s are a l s o r e p o r t e d on t h e u n i v e r s a l c a l i b r a t i o n ( F i g u r e 3 ) . From t h e r e s u l t s o b t a i n e d f o r t h e t h r e e s e r i e s , it i s concluded that : - i s o m a l t o d e x t r i n s a r e e l u t e d more r a p i d l y t h a n t h e isomeric maltodextrins (same DP) due t o an increased c o n t r i b u t i o n t o t h e h y d r o d y n a m i c volume f r o m t h e α 1 —» 6 linkage ; the cyclodextrins are eluted later than the l i n e a r o l i g o m e r s ( α 1 - » 4) due t o a r e d u c e d a x i a l r a t i o . Nevertheless, t h e r e s o l u t i o n i n GPC i s o f t e n n o t good enough a n d HPLC g i v e s a b e t t e r s e p a r a t i o n (8). HPLC. Due t o t h e l a c k o f r e s o l u t i o n i n S E C , HPLC was u s e d t o i m p r o v e r e s o l u t i o n . F u r t h e r m o r e a d e t e c t o r b a s e d on o p t i c a l r o t a t i o n was a d a p t e d t o t h e HPLC e q u i p m e n t and connected i n s e r i e s with the d i f f e r e n t i a l r e f r a c t o m e t e r . It is known (9) t h a t molar o p t i c a l r o t a t i o n includes contributions from the chemical structure of the molecules [m], its conformation [A] a n d t h e relative amounts o f anomers a c c o r d i n g t o t h e r e l a t i o n : [φ] = (n-1) + 162 [α] ι _> + 180 [ a ] t a k i n g the c o n t r i b u t i o n determined p r e v i o u s l y . The agreement between the c a l c u l a t e d value (705.60) and the experimental one (732.80) i s s a t i s f a c t o r y . From Figure 6, i t seems that the d i f f e r e n c e between the c o n t r i b u t i o n of a ( l -> 4) linkage compared with t h a t of a ( l -» 6) i s too small, t o separate the c o n t r i b u t i o n s of amylose and amylopectin i n g e l permeation chromatography on the b a s i s of the o p t i c a l r o t a t i o n . The s e p a r a t i o n of oligomers i n HPLC on a C-18 column i s c o n t r o l l e d by t h e i r i n t e r a c t i o n s with the hydrophobic matrix. The s t r u c t u r e o f the oligomer and e s p e c i a l l y i t s s o l u b i l i t y i s based on -OH groups. Due t o the low s o l u b i l i t y of c y c l o d e x t r i n s , the HPLC was performed i n the presence of c o n t r o l l e d amounts of methanol i n the e l u e n t . E l u t i o n volumes o f c y c l o d e x t r i n s decrease when the content of methanol i n c r e a s e s . The same i s observed with the l i n e a r o l i g o s a c c h a r i d e s (Figure 7 ) . Figure 8 g i v e s the s e p a r a t i o n o f the three c y c l o d e x t r i n s i n 15% MeOH. E l u t i o n volume i n c r e a s e d i n the order γ < α < β. This seems t o i n d i c a t e d i f f e r e n t s o l u b i l i t i e s of these c y c l i c oligomers, e s p e c i a l l y f o r γ c y c l o d e x t r i n . Under these c o n d i t i o n s (C-18 columns ; 15% methanol) the l i n e a r oligomers are e l u t e d i n the v o i d volume f o l l o w e d by the c y c l o d e x t r i n s . O p t i c a l r o t a t i o n should be a l s o u s e f u l t o confirm the i d e n t i t y of these oligomers. The r a t i o s between the two s i g n a l s give the following^numbers : α(0.625), β(0.68), γ(0.803). Thus [α] allows a good means f o r i d e n t i f y i n g c y c l o d e x t r i n s . From these data, i t i s c l e a r t h a t HPLC based on s p e c i f i c i n t e r a c t i o n s allows good separations e s p e c i a l l y when the r o l e of the eluent i s a l s o considered. For heterogeneous oligomers, i t i s n e v e r t h e l e s s u s e f u l t o perform f i r s t SEC f r a c t i o n a t i o n and take each peak f o r HPLC s e p a r a t i o n . 4

6

g l u c o s e


12.

HEYRAUD & RINAUDO

7


?

Characterization of Dextrins and Starch181

t Ο

1

I 10

I 20

I 30

I 40

ι 50

Figure 5. Maltodextrins (M) and isomaltodextrins (IM) s e p a r a t i o n b y HPLC on a C-18 column e l u t e d w i t h H^O. R = ref ractometric and Ρ = polarimetric signals.


182



F i g u r e 6. o f (DP-1)

M o l a r r o t a t i o n a t λ = 589 nm as a f u n c t i o n f o r a ( l - » 4) and 6) oligomers.



HEYRAUD & RINAUDO


ο CO

ο CM



184


I

Ο

Li

5

t(mn) I

1

10

15

F i g u r e 8. S e p a r a t i o n o f c y c l o d e x t r i n s C-18 column i n p r e s e n c e o f 15% MeOH.

I — •

20 by

HPLC


on

a

12.

HEYRAUD & RINAUDO

Characterization of Dextrins and Starch 185


Characterization o f Starch Starch c o n s i s t s of amylopectin (highly branched D-glucose polymer with cc(l - » 6) linkages) and amylose (linear polymer with a ( l -> 4) linkages) and a fraction of i n t e r m e d i a t e m a t e r i a l c o r r e s p o n d i n g t o a m y l o s e w i t h some degree of b r a n c h i n g r e d u c i n g the degree of β - a m y l o l y s i s . The GPC i s w e l l a d a p t e d t o c h a r a c t e r i z e a p o l y m e r b y i t s m o l e c u l a r weight d i s t r i b u t i o n . With s t a r c h the m o l e c u l a r w e i g h t d i s t r i b u t i o n i s s u p e r i m p o s e d upon a h e t e r o g e n e o u s d i s t r i b u t i o n i n c h e m i c a l s t r u c t u r e and e s p e c i a l l y i n t h e degree of b r a n c h i n g . A t l e a s t two p r o b l e m s must be s o l v e d t o i n t e r p r e t GPC c h r o m a t o g r a m s o f s t a r c h : First, s t a r c h must be w e l l dissolved w i t h no adsorption on the support. Therefore, starch was d i s s o l v e d i n DMSO, a good s o l v e n t of the constitutive p o l y m e r s . In bad s o l v e n t s a g g r e g a t i o n o c c u r s and l o o s e a d s o r p t i o n g r e a t l y d i s t u r b t h e d e t e r m i n a t i o n s (14,15) . - S e c o n d l y , a r e l a t i o n between e l u t i o n volume and the molecular weight must be established which is i n d e p e n d e n t o f t h e p o l y m e r m o r p h o l o g y . T h i s was o b t a i n e d with a light scattering detector giving M directly w i t h o u t t h e use o f a c a l i b r a t i o n c u r v e . Yamada and T a k i (16) obtained molecular weight d i s t r i b u t i o n s of d i f f e r e n t starches on S e p h a r o s e 2B i n the presence of 0.04% HCIO4. The iodine index was d e t e r m i n e d on t h e c h r o m a t o g r a m and seemed t o i n d i c a t e a good s e p a r a t i o n between h i g h m o l e c u l a r weight a m y l o p e c t i n and a m y l o s e . No v a l u e s o f m o l e c u l a r w e i g h t were g i v e n . Van Dijk et al. (17) p e r f o r m e d GPC o f amylose d i s s o l v e d i n DMSO on a P o r a s i l c o l u m n (Waters ; s i l i c a gel deactivated). By u s i n g u n i v e r s a l c a l i b r a t i o n after determination of the v i s c o s i t y on e a c h f r a c t i o n , they o b t a i n e d g o o d agreement between Mw a n d Mn v a l u e s o b t a i n e d f r o m GPC a n d d i r e c t m e a s u r e m e n t s . F o l l o w i n g t h e s e w o r k s , DMSO has b e e n a d o p t e d as a g o o d s o l v e n t for defatted starch (14) . GPC e x p e r i m e n t s were p e r f o r m e d on silica g e l s g r a f t e d w i t h d i o l (Merck, L i c h r o s p h e r - d i o l ) a t 6 0 ' C . The e l u e n t was DMSO/MeOH 85/15 (v/v) c o n t a i n i n g 0.5 M ammonium a c e t a t e t o r e d u c e i n t e r a c t i o n (H b o n d s ) . The p r e s e n c e o f Me OH d e c r e a s e d t h e v i s c o s i t y o f t h e solvent. Under these experimental conditions, universal c a l i b r a t i o n was shown t o be v a l i d i n d i c a t i n g t h a t there was no a d s o r p t i o n . The l a r g e r p o r e s o f t h e s e s u p p o r t s a r e even t o o s m a l l t o o b t a i n a good f i l t r a t i o n o f a m y l o p e c t i n w h i c h i s u s u a l l y e x c l u d e d and s e p a r a t e d f r o m a m y l o s e . A l l t h e c h r o m a t o g r a m s o f s t a r c h c o n t a i n i n g a m y l o p e c t i n show a peak w i t h a v e r y l a r g e m o l e c u l a r w e i g h t . T h i s peak would contribute greatly to the overall weight average m o l e c u l a r weight of the sample. Some Table IV.

of

the

values

obtained

are

recalled


in

the


186 Table

IV. C h a r a c t e r i s t i c s of


7 8% amyAmylose losis AVEBE from p o t a t o e s

SNOW f r o m waxy m a i z e

>99% amy lopectin

AMILO f r o m Brasil tapioca

some

starchs

Mw

amylose %

CLI

Mn

162000

278000

95

19.5

14.8x10 6

0

0.5

12xl0

6

6

7.31xl0

13.6xl0

6

27

5.9

C L I = c a p a c i t y o f i o d i n e f i x a t i o n e x p r e s s e d i n grm o f I2 for 100 grm o f s t a r c h , t a k i n g as r e f e r e n c e the first f r a c t i o n o f Avebe c r i s t a l l i z e d w i t h b u t a n o l (ie CLI = 20.5 ; 100% amylose) and waxy m a i z e (CLI = 0 . 5 , 100% a m y l o p e c t i n ) i n s o l u t i o n I2 i n 0.05 M IK a t 2 0 C . e

From t h e s e v a l u e s , i t must be p o i n t e d o u t t h a t t h e fraction of amylose expressed from I2 f i x a t i o n is different (generally larger) from the fraction of material crystallized in presence of butanol. In a d d i t i o n , t h e f r a c t i o n c r y s t a l l i z e d from a s t a r c h sample u s u a l l y has a CLI lower t h a n t h a t o f pure amylose (18). The i m p o r t a n c e o f t h e d i r e c t m o l e c u l a r w e i g h t measurement w i l l be u n d e r s t o o d i f one c o n s i d e r s t h e Mark Houwink relation : Amylose D M S O / 2 5 ° C (19) [η] = 1.25 χ ΙΟ" Μ · A m y l o p e c t i n DMSO/MeOH 85/15 [η] = 2.16 χ ΙΟ"" Μ · 0.5Μ AcNH o r [ η ] = 1.84 χ 10" Μ · 60*C (18) The r e l a t i o n s g i v e n f o r a m y l o p e c t i n d e p e n d on t h e d e g r e e o f h y d r o l y s i s . The f i r s t one i s f o r n a t i v e s t a r c h down t o 6.5x10°. Then one gets a lower dependency with M c o r r e s p o n d i n g t o a more compact s t r u c t u r e w h i l e m o l e c u l a r weight d e c r e a s e s . Following these values, it is clear that even u n i v e r s a l c a l i b r a t i o n , [ η ] M ( V ) , c a n n o t be u s e d d i r e c t l y f o r a n a l y s i s o f a s t a r c h s a m p l e . I n a d d i t i o n , t h e Κ and a v a l u e s a r e n o t w e l l e s t a b l i s h e d i n t h e l i t e r a t u r e due t o difficulties in working with these systems. M u l t i d e t e c t i o n GPC a l l o w i n g t h e measurements on l i n e o f Μ, [ η ] and c o n c e n t r a t i o n s h o u l d be t h e b e s t way t o f o l l o w the c h a r a c t e r i z a t i o n of s t a r c h e s from d i f f e r e n t origins (20) . The use o f a v i s c o m e t e r as d e t e c t o r s h o u l d a l l o w d e t e r m i n i n a t i o n of the f a c t o r g which i s r e l a t e d to the degree of b r a n c h i n g . 3

3

1

4

e


0

8

7

0

6

7

0

3

6

12.

HEYRAUD & RINAUDO


Experimental The p u r e o l i g o s a c c h a r i d e s were o b t a i n e d by p r e p a r a t i v e l i q u i d chromatography from p a r t i a l l y h y d r o l y z e d p o l y m e r s . M i x t u r e o f m a l t o d e x t r i n s i s a c o m m e r c i a l sample o b t a i n e d by partial enzymic and acid hydrolysis of starch (Roquette, France). Mixture of isomaltodextrins is prepared by partial acid hydrolysis of dextran. C y c l o d e x t r i n s a r e commercial samples from A l d r i c h . Gel permeation chromatography was performed on B i o g e l Ρ 2 e l u t e d by p u r e w a t e r at 65 C as described previously (2,3). The HPLC was realized with a Waters solvent d e l i v e r y system. S e p a r a t i o n s o f o l i g o m e r s were o b t a i n e d w i t h a N u c l e o s i l 5 μπι C-18 r e v e r s e p h a s e column from S.F.C.C. (France) (c9) . The e l u e n t was d i s t i l l e d water f i l t e r e d t h r o u g h a 0.45 |im M i l l i p o r e membrane. The s t a r c h was d i s s o l v e d i n DMSO a n d t h e e l u e n t was DMSO/MeOH (85/15 v / v ) i n 0 . 5 M ammonium a c e t a t e . The c o l u m n s e t was d i o l s i l i c a g e l f r o m M e r c k : 2 χ S i 1000 D i o l , 1 χ S i 500 D i o l , 1 χ S i 100 D i o l t h e r m o s t a t e d a t 6 0 * C . The d e t e c t o r was e i t h e r a d i f f e r e n t i a l r e f r a c t o m e t e r f r o m W a t e r s (R 401) o r an IOTA ( J o b i n Y v o n ) . The s e c o n d on l i n e d e t e c t o r was a l i g h t s c a t t e r i n g d e t e c t o r ( C h r o m a t i x CMX 100) o r a s p e c t r o p o l a r i m e t e r ( P e r k i n E l m e r model 241 w o r k i n g a t 365 nm w i t h a f l o w c e l l o f p a t h l e n g t h 10 cm a n d a 30 μ ΐ v o l u m e ) . The v a l u e o f [ a l p a r e e x p r e s s e d f r o m [0t]365 d a t a u s i n g a c o r r e c t i v e f a c t o r . The p a r t i t i o n c o e f f i c i e n t Kd i s e x p r e s s e d as :


e

v

Kd = v

e

"

v

o

glucose

~

v

o

w i t h V t h e v o i d volume and V t h e e l u t i o n v o l u m e . V was o b t a i n e d f r o m t h e e l u t i o n volume o f a T70 d e x t r a n sample (Pharmacia). 0

e

0

Conclusion In this paper the behavior of oligosaccharides corresponding to models for starch hydrolysis or amylolysis products was discussed. The role of the c h e m i c a l s t r u c t u r e on t h e e l u t i o n volume was p o i n t e d out i n r e l a t i o n to the nature of the glycosidic linkage. Combination w i t h o p t i c a l r o t a t i o n improves the q u a l i t y of a n a l y s i s . The b e h a v i o r o f c y c l i c a n d b r a n c h e d o l i g o m e r s was a l s o d i s c u s s e d . In t h e second p a r t , the main r e s u l t s obtained i n the domain of starch characterization were recalled showing t h e n e c e s s i t y t o adopt a m u l t i d e t e c t i o n system f o r the a n a l y s i s of s t a r c h m o l e c u l a r weight d i s t r i b u t i o n . Nevertheless, the major problem remains the p r e p a r a t i o n of a good solution avoiding aggregates and w i t h no a d s o r p t i o n on t h e g e l m a t r i x .


188


Literature cited 1. 2 3. 4.


5. 6

RECEIVED

7 8. 9. 10. II. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Heyraud, Α., ; Rinaudo, M. J. Liquid Chromatogr., suppl. 2, 1981, 4, 175-293. Heyraud, A. ; Rinaudo, M. J. Chromatogr. 1978, 166, 149-58. Heyraud, A. Thesis, Grenoble, France, 1978 and 1981. John, M. ; Trenel, G. ; Dellweg, H. J. Chromatogr. 1969, 42, 476-84. Hokse, H. J. Chromatogr. 1980, 189, 98-100. Koizumi, K. ; Kubota, Y. ; Okada, Y. ; Utamura, T. ; Hizukuri, S. ; Abe, J. J. Chromatogr. 1988, 437, 47-57. Mattsson, P. ; Mäkelä, M. ; Korpela, T. J. Chromatogr. 1988, 447, 398-403. Heyraud, A. ; Rinaudo, M. J. Liquid Chromatogr. 1980, 3, 721-39. Lawson, C.J.; Rees, D.A. Nature 1970, 227, 390-92 Bouffar Roupe, C. Thesis, Grenoble, France, 1989. Noble, O. ; Pérez, S. ; Rochas C. ; Taravel, F. Polymer Bull. 1986, 16, 175-80. Lloyd, D.K. ; Goodall, D.M. ; Scrivener, H. Analyt. Chem. 1989, 61, 1238-43. Heyraud A. ; Salemis P. Carbohydr. Res. 1982, 107, 123-29. Salemis, Ph. ; Rinaudo, M. Polymer Bull. 1984, 11, 397-400. Salemis, Ph. ; Rinaudo, M. Polymer Bull. 1984, 12, 283-85. Yamada, T. ; Taki, M. Die Stärke 1976, 28, 374-77. Van Dijk, J.A.P.P. ; Henkens, W.C.M. ; Smit, J.A. J. Polym. Sci. 1976, 14, 1485-93. Salemis, P. Thesis, Grenoble, 1984. Cowie, J.M.G. Makromol. Chem. 1961, 42, 230-47. Tinland, B. ; Mazet, J. ; Rinaudo, M. Makromol. Chem. Rapid Commun. 1988, 9, 69-73.

November 20, 1990


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