Determination of Carbohydrates by Anion Exchange Chromatography With Pulsed Amperometric Detection

8/19/2019 Determination of Carbohydrates by Anion Exchange Chromatography With Pulsed Amperometric Detection

1/16

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Determination of

Carbohydrates by Anion

Exchange Chromatography

with Pulsed Amperometric

DetectionRoy D. Rocklin

a & Christopher A. Pohl

a

a Dionex Corp., 1228 Titan Way, Sunnyvale, CA,

94086, USA

Version of record first published: 05 Dec 2006.

To cite this article: Roy D. Rocklin & Christopher A. Pohl (1983): Determination

of Carbohydrates by Anion Exchange Chromatography with Pulsed Amperometric

Detection, Journal of Liquid Chromatography, 6:9, 1577-1590

To link to this article: http://dx.doi.org/10.1080/01483918308064876

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D o w n l o a d e d

b y [ N o r t h C a r o l i n a S t a t e U

n i v e r s i t y ] a t 0 7 : 0 9 1 8 F e b r u a r y 2 0 1 3


3/16

JOURNAL

OF

LIQUID

CHROMATOGRAPHY, 6 ( 9 ) , 1577-1590

(1983)

D E T E R M I N A T I O N OF CARBOHYDRATES BY A N I O N EX C H A N G E

CH RO M A T O G RA P H Y W I T H PULSED AMPEROMETRIC DETECTION

t

Roy D . R o c kl in a n d C h r i s t o p h e r A . P o h l

D i o n e x C o r p . , 1228 T i t a n W a y ,

S u n n y v a l e ,

CA

9 4 0 8 6 USA

ABSTRACT

C a r b o h y d r a t e s s u c h as s u g a r a l c o h o l s , m o n o s a c c h a r i d e s ,

d i s a c c h a r i d e s , a n d o t h e r o l i g o s a c c h a r i d e s are s e p a r a t e d a s a n i o n s

b y i o n ex c h a n g e c h r o m a t o g r ap h y w i t h a s o di um h y d r o x i d e e l u e n t . Re-

t e n t i o n t i m e a n d s e l e c t i v i t y a r e c o n t r o l l e d by v a r y i n g e l u e n t s t r e n g t h

a n d co lu m n t e m p e r a t u r e .

The

c a r b o h y d r a t e s

are

d e t e c t e d b y

o x i d a t i o n a t a g o l d e l e c t r o d e . A r e p e a t i n g s e q u e n c e o f t h r e e

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

t h e

e l e c t r o d e s u r f a c e o f

o x i d a t i o n p r o d u c t s a n d o t h e r i n t e r f e r i n g s p e c i e s . D e t e c t i o n

l i m i t s a re a s low

as

30 p pb f o r s u g a r a l c o h o l s an d m o n o s a c c h a r i d e s ,

a n d a b o u t 100 p p b f o r o l i g o s a c c h a r i d e s . O t h e r s p e c i e s c o n t a i n i n g

CHOH g r o u p s c a n a l s o b e d e t e c t e d , s u c h a s a l c o h o l s a n d g l y c o l s .

I N T R O D U C T I O N

T h e d e t e r m i n a t i o n o f c a r b o h y d r a t e s by ch r o m a to g r ap h y h a s b e en

h am pe re d by tw o f a c t o r s : t h e l a c k o f a s u i t a b l e h i g h p e r fo r m a n c e

s e p a r a t i o n m e tho d a n d t h e i n a b i l i t y t o d e t e c t t h e c a r b o h y d r a t e s a t

l o w

l e v e l s .

A

n um ber o f s e p a r a t i o n m e th o d s h a v e b e en u s e d .

C a r b o h y d r a t e s c a n he d e t e r m i n e d b y gas c h r o m a t o g r a p h y , h o w e v e r ,

t h e i r n o n - v o l a t i l e n a t u r e r e q u i r e s

a

time c o ns u m in g d e r i v i t i z a t i o n

p r o c e d u r e (1). The m os t common metho d i s t h e u s e o f h i g h c a p a c i t y

s t r o n g l y a c i d i c c a t i o n ex c h a n g e c o l u m ns i n t h e m e t a l fo rm u s i n g

water

a s t h e e l u e n t

( 2 ) .

C a l c i u m

i s

t h e m os t common metal, a l t h o u g h

s i l v e r a n d

l ead

h a ve a l s o b ee n u s e d .

Copyright 1983 by Marcel

Dekker,

Inc.

1 5 7 7

01

48-39 9/83/0609-1577 3.50/0

D o w n l o a d e d




4/16

1578

ROCKLIN AND PO€

These c o l u m ns s u f f e r

from

s e v e r a l d r a w b a c k s . F i r s t , t h e

organic a c i d s

p r e s e n t i n m any s a m p l e s e l u t e metals f r o m t h e

c a t i o n e xc ha n g e r e s i n . T h i s r e s u l t s i n r e s i n b e d s h r i n k a g e a nd

r e q u i r e s f r e q u e n t r e g e n e r a t i o n of t h e co lu m n b a c k t o t h e o r i g i n a l

metal form. S e c o n d , t h e s e c o l u m ns e x h i b i t p o o r s e l e c t i v i t y f o r

h i g h e r o l i g o s a c c h a r i d e s . Low c r o s s l i n k r e s i n i s r e q u i r e d f o r

r e s o l u t i o n of e v e n t h e lo w e r o l i g o s a c c h a r i d e s s u c h as

DP2

t h r o u g h

DP4.

The r e s i n e x h i b i t s h i g h c o m p r e s s i b i l i t y , m a k in g i t u n s u i t a b l e

f o r h i g h f lo w

r a t e s

a nd r a p i d a n a l y s i s . T h i r d , h i g h c o lu m n

t e m p e r a t u r e s

8 5 O C )

are n e e d e d

f o r

o p ti m um c ol um n p e r f o r m a n c e .

T he se h i g h t e m p e r a t u r e s p r e s e n t a s e v e r e t e s t of t h e p e r f o r m a n c e

o f t h e d e t e c t o r , p a r t i c u l a r l y

f o r

a r e f r a c t i v e i n d e x d e t e c t o r .

F i n a l l y , s i n c e o n l y water o r

water

o r g a n i c s o l v e n t m i x t u r e s

c a n b e u s e d as a n e l u e n t , t h e m e th od s of r e t e n t i o n a nd s e l e c t i v i t y

c o n t r o l

l e f t

o p e n

t o t h e

c h r o m a t o g r a p h e r

a re

s e v e r e l y l i m i t e d .

A

s e c o n d

major

m e t h o d

of

c a r b o h y d r a t e a n a l y s i s i n v o l v e s t h e

u s e of m i c r o - p a r t i c u l a t e s i l i c a w i t h a n am i n o bo nd ed p h a s e

( 3 ) .

While t h i s s y s t e m s u c c e s s f u l l y s e p a r a t e s mono a n d d i s a c c h a r i d e s ,

t h e h i g h e r o l i g o s a c c h a r i d e s

(DP3-DP10)

are e l u t e d a s b r o a d p e a k s

a nd r e q u i r e e x t e n d e d a n a l y s i s t imes . F u r t h e r m o r e , a m in o s t a t i o n a r y

p h a s e s are s u b j e c t t o r e a c t i o n w i t h a l d e h y d e s o r k e t o n e s i n

s a m p l e s ( f r e q u e n t l y p r e s e n t i n f o o d s a n d b e v e r a g e s ) t o

form

a

S c h i f f s b a s e . B e c au se of t h i s , a m in o b on de d p h a s e s t e n d t o

e x h i b i t r e d u c e d l i f e t i m e s w i t h c e r t a i n s a m p l e s .

A

t h i r d m eth od

i s

t h e s e p a r a t i o n

of

s u g a r s v i a t h e i r b o r a t e

c o m p l e x e s

(4).

W h i l e t h e s e l e c t i v i t y of t h e s e s y s t e m s a r e s u p e r i o r

t o t h e a b o v e tw o m e t h o d s , t h e k i n e t i c s o f t h e co m p le x f o r m a t i o n

p r o c e s s

is

slow, t h u s r e q u i r i n g

low

f l o w

ra tes

a n d l o n g a n a l y s i s

times.

W r e p o r t h e r e a n a l t e r n a t i v e t o t h e a b o v e m e t h o d ; t h e

d i r e c t s e p a r a t i o n o f c a r b o h y d r a t e s b y a n i o n e x c h a n g e . B e c au se

c a r b o h y d r a t e s h a v e pK v a l u e s r a n g i n g f r o m 1 2 t o 1 4 ( 5 ) , r e t e n t i o n

is p o s s i b l e o n a s t r o n g l y b a s i c h y d r o x i d e f o r m a n i o n e x c h a n g e co lu m n

w i t h h i g h l y a l k a l i n e e l u e n t s . S u c h a s y s t e m

i s

p r e v i o u s l y

D o w n l o a d e d




5/16

DETERMINATION

OF

CARBOHYDRATES

1579

u n r e p o r t e d , a l t h o u gh r e t e n t i o r : o f m e t h y l g l yc o s i d e s o n a s t r o n g l y

b a s i c h y d r ox i d e f or m a n i o n e x c ha n g e c ol um n h a s b ee n r e p o r t e d

( 6 ) .

Due

t o

t h e a b se n c e o f a s t r o n g l y a b s o r b i n g UV chromophore ,

c a r b o h y d r a t e s

a r e

u s u a l l y d e t e c t e d by

a

r e f r a c t i v e i n de x d e t e c t o r .

R.I. d e t e c t o r s h av e low s e n s i t i v i t y and a r e d i f f i c u l t t o u s e . Low

U V d e t e c t i o n ( < 20 0 nm) h a s b ee n em pl oy ed , b u t t h e n ee d f o r u l t r a

c l e a n e l u e n t s a nd i n t e r f e r e n c e p ro bl em s h av e l i m i t e d i t s u t i l i t y .

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

d e t e c t o r which s u p p l i e s a t r i p l e p o t e n t i a l se q u e n c e t o a g o l d

w or ki ng e l e c t r o d e . Both p o t e n t i o m e t r i c and s i n g l e p o t e n t i a l

D . C . )

a mp e ro me t r i c

de t e c t o r s

h av e be en u s ed t o d e t e c t c a r b o h y d r a t e s ( 7 ) .

P o t e n t i o m e t r i c d e t e c t o r s s u f f e r f rom s l o w r e s p o n s e t imes .

S i ng l e p o t e n t i a l o x id a t i o n o f s u g a r s ca u se s t h e e l e c t r o d e s u r f a c e

t o become c o n t a m i na t e d by t h e p r o d u c t s o f t h e r e a c t i o n , t h u s

r e n d e r i n g t h e d e t e c t o r u n u s a b le . T r i p l e p u l s e a mp er om et ry of

s u g a r s

was

f i r s t r e p o r t e d by H ughes an d J oh tl so n u s i n g

a

p l a t i n u m

e l e c t r o d e

( 8 ) .

A l th ou gh t h e d e t e c t i o n me thod

i s

s u c c e s s f u l ,

t h e p o t e n t i a l a t w hi ch t h e o x i d a t i o n c u r r e n t

i s

measured

(E1,-0 .40V) i s s u f f i c i e n t l y n e g a t i v e t o c au se t h e r e d u c t i on o f

oxygen. Gold

was

i n v e s t i g a t e d

as

a n a l t e r n a t i v e workitlg e l e c t r o d e

mat.eria1.

EXPERIMENTAL

A l l

c h ro ma to g ra p h y was performed on a Dionex System 2011

( P / N

35295 ) Io n Ch roma to g ra p h. The s a mp le lo o p s i z e was 50 p

and t h e e l u e n t f l ow r a t e 1 . 0 mLImin. The d e t e c t o r was a b re a d b o a rd

v e r s i o n o f a Dionex IonChromTM/Pulsed Amperometr ic Det ec to r (P/N

35 227 ) c o n s i s t i n g of a n a mp e ro me t r i c f l o w- th ro u g h c e l l and a

p o t e n t i o s t a t . T h e c e l l

i s a

t h i n l a y e r d e s i gn w i t h a g o l d w o r k i n g

e l e c t r o d e , a s i l v e r / s i l v e r c h l o r i d e r e f e r e n c e e l e c t r o d e , a nd a

g l a s s y c a r bo n c o u n t er e l e c tr o d e . The p o t e n t i o s t a t a p p l i e s

a

s e r i e s

o f u p t o t h r e e p o t e n t i a l s ( E l , E2, E 3 ) i n a r e p e a t i n g w a v e f o r m .

The p u l s e d u r a t i o n s ( t l , t 2 ,

t 3 ) a r e

s e l e c t a b l e by t h e u s e r .

D o w n l o a d e d




6/16

1580

ROCKLI N

AND POHL

The cel l cur r ent i s measur ed onl y dur i ng El

f o r 16. 67

ms ( t o cancel

60

hz l i ne noi se) . A 40 ms del ay f ol l owi ng t he pot ent i al st ep

al l ows char gi ng cur r ent t o decay.

The ani on exchange col umn used was a pr ot ot ype HPI C- AS6

col umn ( PI N 35391). The i on exchange r esi n used i n t he col umn

consi st ed of

10

m cr on subst r at e coat ed wi t h a monol ayer of ani on

exchange l at ex si m l ar t o t hat used i n ot her HPI C ani on I on Chr oma-

t ogr aphy col umns.

The el uent consi st ed of NaOH i n dei oni zed wat er (0. 15 N . 0. 2 M

CH CO Na was added as a pusher t o el ut e ol i gosacchar i des. El uent s

wer e pr epared f r om car bonat e f r ee 50 NaOH sol ut i ons and pr ot ect ed

f r om car bon di oxi de cont am nat i on wi t h Ascar i t e t r aps. Sugar sam

pl es wer e pur chased f r om Pf anst i ehl Laborat or i es ( Waukegan, I L )

and Si gma Chem cal s C o . , St . Loui s , MO. The mal t ose ol i gomer s

wer e a gener ous gi f t

of

t he A. E. St al ey Company.

3 2

RESULTS

DI SCUSSI ON

Cycl i c Vol t ammet r y

of

Gl ucose

The choi ce of pot ent i al s and pul se dur at i ons f or chr omat o-

gr aphi c det ect i on i s most easi l y det er m ned f r om vol t ammet r i c

i nf or mat i on. Accor di ngl y, t he cycl i c vol t ammet r y of s ugar s on

gol d was studi ed and i s i l l ust r at ed i n Fi gur e

1

by gl ucose. The

dashed l i ne i s a backgr ound scan

of

t he

0.1

N NaOH suppor t i ng

el ectr ol yt e. I t shows i r r ever s i bl e oxi dat i on of gol d begi nni ng

at appr oxi mat el y 0. 25

V .

The r educt i on of t he sur f ace oxi de back

t o gol d i s shown as a cat hodi c peak at appr oxi mat el y 0. 1 V

W t h gl ucose added t o t he sol ut i on, oxi dat i on begi ns at

0. 5 V

wn t he posi t i ve goi ng scan. The cur r ent r i ses s l i ght l y

and r emai ns unchanged unt i l i t r i ses at - 0. 15 V t owar ds a peak

at 0. 26 V. On t he r ever se scan, t he cur r ent act ual l y r ever ses

f r om cat hodi c t o anodi c at t he onset of t he gol d oxi de r educt i on.

Thi s i mpl i es t hat gl ucose oxi dat i on whi ch occur s at t he bar e

gol d sur f ace i s i nhi bi t ed upon t he f or mat i on of gol d oxi de. As

soon

as

t h e

r educt i on of gol d oxi de back t o gol d begi ns,

D o w n l o a d e d


n i v e r s i t y ] a t 0 7 : 0 9 1 8 F e b

r u a r y 2 0 1 3


7/16

DETERMIN TION O C RBOHYDR TES

1581

I I

-.5 0 .5

E

vs

Ag/AgCI

Fi gur e 1. Cycl i c vol t ammet r y of 0. 92 m M

( 166

ppm gl ucose on a

gol d worki ng el ect r ode. Dashed l i ne i s 0. 10 2 NaOH suppor t i ng

el ec t r ol yt e. 0. 20 Vl sec sweep r at e.

oxi dat i on of gl ucose t hat had di f f used t owar d t he el ect r ode whi l e

t he sur f ace was cover ed wi t h oxi de t akes pl ace.

Cycl i c vol t ammet r y of t he sugar al cohol s xyl i t ol and sor bi t ol

and t he di sacchar i de sucr ose wer e al so st udi ed. They wer e si m l ar

t o gl ucose, except t he hei ght of t he anodi c peak at 0 V on t he

negat i ve goi ng scan was l arger wi t h t he sugar al cohol s and smal l er

wi t h sucr ose.

Choi ce of Appl i ed Pot ent i al s

Si ngl e pot ent i al (D. C. ) amper omet r y wi t h E set to 0.1 V

aPP

was at t empt ed. 0.1

V

was chosen because at t hi s pot ent i al , t her e

shoul d be a l ar ge f r act i on of unoxi di zed gol d on t he el ect r ode

sur f ace. Al t hough det ect i on was possi bl e, the response decr eased

r api dl y as pr oduct s f r om t he oxi dat i on r eact i on deact i vat ed t he sur f ace.

A

l ar ge i mpr ovement i n r epr oduci bi l i t y was obt ai ned by pul si ng the

appl i ed pot ent i al f r om -0 .8 V t o 0. 2 V and measur i ng t he oxi dat i on

current at 0.2

V

The negat i ve pot ent i al apparent l y ser ves as a

D o w n l o a d e d

b y [ N o r t h C a r o l i n a S t a t e U n i v e r s i t y ] a t 0 7 : 0 9 1 8 F e b

r u a r y 2 0 1 3


8/16

1582

ROCKLIN

AND POHL

c l e a n i ng s t e p , e n s u r i ng t h e r e d u c t i o n o f g o ld o x i d e back t o g ol d .

Also,

t h e m ag ni tu de o f t h e s u g a r o x i d a t i o n c u r r e n t f ro m a v o l t a g e

p u l s e

i s

grea t e r t h a n t h a t c a u se d by t h e h yd rod y na mic s

of

f l o w

when only a s i n g l e p o t e n t i a l i s u s e d. The u s e o f b o t h n e g a t i v e

and p o s i t i v e c l e a n i n g p o t e n t i a l s was f oun d t o p r o vi d e t h e

most

r e p ro d u c ib l e c h ro ma to g ra ms , as well a s minimum in t e r f e r e nc e f rom

o t h e r

s p e c i e s i n t h e s a mp l es . The f o l l o w i n g p o t e n t i a l s and

t imes

( sh own i n F i g u r e 2) have been chosen a s optimum f o r c a r b o h y d r a t e

d e t e r m i n a t i o n : E l :

0. 20

V , 60 ms;

E2:

0.60 V ,

60

m s ; E3: -0 .80 V ,

240

m s .

The s i g n a l - t o - n o i s e r a t i o i s a f f e c t e d t h e

most

by t h e c h o i c e

of

t h e d e t e c t i o n p o t e n t i a l , El . T h e p e a k h e ig h t s f o r t h r e e s u g a r s

a nd th e b ac kg ro un d c u r r e n t

a re

shown i n F i gure 3 . The

l a rge

i n -

crease i n b ac kg ro un d c u r r e n t f ro m 0. 2 Vto 0.4 V i s accompanied by

a 4X i n c r e a s e i n n o i s e . The optimum value

f o r E l

i s 0 . 2 V . Due

t o t h e s i m i l a r i t y of t h e e l e c t r oc h e m i s t ry

of

t h e d i f f e r e n t s u g a r s ,

t h i s v a lu e may b e o pt imum f o r mo s t , i f n o t a l l , s u g a r s .

Small

( 0 . 1

V f

c h a n g e s i n

E 2

and E 3 ,

as

w e l l

a s

t h e i r d ur a-

t i o n s , h a v e o n l y a minor

e f f e c t

o n r e s p o n s e .

Chromatography

One of t h e main a d v a n t a g e s of t h e a n i o n e x c ha n g e c hr om at o-

g r a p h y o f c a r b o h y d r a t e s as compared t o metal

form

c a t i o n e x c h a n g e

i s t h e a b i l i t y t o i n f l u e n c e r e t e n t i o n time a nd r e t e n t i o n o r d e r .

The p ri m a r y o p e r a t i n g v a r i a b l e s

are

e l u e n t s t r e n g t h a n d c o l u m n

t e m p e r a t u r e . The s e p a r a t i o n o f a m i x t u r e

o f

s u g a r a l c o h o l s

a n d s a c c h a r i d e s i s shown i n F ig u re 4 . The a d d i t i o n o f a c e t a t e

i on r e s u l t s

i n

r e d u c t i o n i n t h e K'

of a l l

s o l u t e s . T hi s e n a b l es

t h e e l u t i o n o f t h e o l i g o s a c c ha r i d e s DP2 t h r o u g h

10,

shown i n

F ig u re

5.

I n g e n e r a l , t h e ob s er v ed a n i o n ex cha nge a f f i n i t y f o l l ow s

t h e t r e n d : s u ga r a l c o h o l s

<

mo n o sa c c h a r id e s . d i s a c c h a r i d e s

<

o l i g o s a c c h a r i d e s , a l t h o u g h i n some cases o v e r l a p i s o b se rv e d

i n t h e e l u t i o n o r d e r of m i x t u r e s o f d i s a c c h a r i d e s a nd o l i g o -

s a c c h a r i d e s . F o r a homologous s e r i e s o f c a r bo h yd r at e s , r e t e n t i o n

D o w n l o a d e d


n i v e r s i t y ] a t 0 7 : 0 9 1 8 F e b

r u a r y 2 0 1 3


9/16

DETERMINATION OF CARBOHYDRATES

1583

0.4

m

-

Cleaning

E3

Cleaning

t t

60

120 180 240

300 60 120

F i g u r e 2. T r i p l e p o t e n t i a l program a p p l i e d

t o

t h e g o l d w o r k i n g

e l e c t r o d e f o r t h e d e t e c t i o n of c a r b o h yd r a t e s . O x i d a t i on c u r r e n t

i s

sampled from 40 t o 5 6 . 7

m s

a f t e r t h e b e g i n n i n g o f

E l .

I

100

ol

60

i HA)

40-

20-

0-

I

-0.2 0.2

0.4

E l V

vs

Ag/AgCI)

F i g u r e 3.

100 ppm g l u c o s e ( s q u a r e s ) , a n d

316

ppm s u c r o s e ( t r i a n g l e s ) .

B a c k g ro u n d c u r r e n t a s a f u n c t i o n o f

E l

i s a l s o shown.

p as a f u n c t i o n o f E l f o r 1 00 ppm x y l i t o l ( d o t s ) ,

D o w n l o a d e d


n i v e r s i t y ] a t 0 7 : 0 9 1 8 F e b

r u a r y 2 0 1 3


10/16

ROCKLIN AND POHL

23

I

0 5

10

15

t m i n )

F i g u r e 4 . S e p a r a t i o n o f s u g a r a l c o h o l s a nd s a c c h a r i d e s . L i s te d

i n o r d er , t he y a r e : 25 ppm x y l i t o l ( 1 ) ; 50 ppm s o r b i t o l 2 ) ,

rhamnose ( 3 ) , a r a b i n o s e

( 4 ) ,

g l u c o s e

( 5 ) ,

f r u c t o s e

( 6 1 ,

l a c t o s e

( 7 ) ;

100 ppm s u c r o s e 8 ) , r a f f i n o s e ( 9 1 , s t a c h y o s e (10) ; 150

ppm maltose

(11).

0.15

N a O H e l u e n t

a t

36OC.

0 5 10

t mln)

F i g u r e

5 .

S e p a r a t i o n o f o l i g o m e r s o f m a l t o s e , DP 2 ( m a l t o s e )

t h r o u g h DP

1 0

( m a l t o d e c o s e ) .

0 . 2

M N a O H , 0.2 C H co Na e l u e n t a t 34OC-

-

3 2

D o w n l o a d e d


n i v e r s i t y ] a t 0 7 : 0 9 1 8 F e b

r u a r y 2 0 1 3


11/16

DETERMINATION OF CARBOHYDRATES

1585

1

0 o.;o

o ho

0.50

o bo 0.5.0

o h

0.bo

NaOH Concentration M)

Figur e

6 .

Capac i ty fac tor ve r sus

NaOH

concentra t ion. Column

temper a tu r e : 3 4 O C ; sample concen t r a t ions :

100

ppm each.

i n c r e a s e s a s th e deg r ee o f po lymer iza t ion

(DP) increases.

Se lec -

t i v i t y c han ge s a r e o f t e n o b se rv ed a s the hydroxide ion concen-

t r a t i o n i s v a r i e d . T y p i ca l s e l e c t i v i t y c ha ng es o b se r ve d for

o l i g o s a c c h a r i d e s

are

i l l u s t r a t e d i n F igu re 6 .

sac cha r id e s shown, optimum s e l e c t i v i t y i s observed

a t

0.150

-

N a O H .

sac cha r id e s and suga r a lc oho ls . W hi le i n some cases t h e s e

s e l e c t i v i t y changes may

be

u se d t o a d v an t a g e, i n g e n e r a l h y-

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

a

u s e f u l method f o r c o n t r o l l i n g

s e l e c t i v i t y . O ft en , t h e d e si r e d s e l e c t i v i t y f o r

a

p a r t i c u l a r

mix tu r e o f c a r bohydr a te s i s only observed a t

K ’

va lues which

a re u n a c c e p t a b l y l a r g e o r smal l .

For

t h e o l i g o -

Simila r chromatographic behavior i s obse rved f o r mono-

The hydr ox ide i on c once n t r a t ion ha s

a

s i g n i f i c a n t e f f e c t

on

t h e

o bs er ve d e f f i c i e n c y f o r

a

p a r t i c u l a r s o l u t e .

It i s

g e n e r a l l y o bs er ve d t h a t column e f f i c i e n c i e s i n c r e a s e as t h e

D o w n l o a d e d b y [ N o r t h C a r o l i n a S t a t e U n i v e r s i t y ] a t 0 7 : 0 9 1 8 F e b

r u a r y 2 0 1 3


12/16

1586

ROCKLIN ND

POHL

h y d ro x i d e i o n c o n c e n t r a t i o n i s i n c r e a s e d .

F o r

g l u c o s e , e f f i -

c i e n c y n e a r l y d o u b l e s when h y d r o x i d e i o n c o n c e n t r a t i o n

i s

i n c r e a s e d from 0.10

N a O H t o

1.0

2

N a O H .

A s ec o n d v a r i a b l e u s e f u l f o r c o n t r o l o f s e l e c t i v i t y a n d

r e t e n t i o n

i s

c o lu mn t e m p e r a t u r e . R e t e n t i o n d e c r e a s e s

f o r

a l l s o l u t e s as t h e t e m p e r a t u r e i n c r e a s e s . The d e g r e e t o w h i c h

t e m p e r a t u r e

a l t e r s

r e t e n t i o n

i s

l o o s e l y r e l a t e d t o t h e c a r b o -

h y d r a t e s i z e ; t h e o b s e r v e d m a g n i t u d e

o f

t h e t e m p e r a t u r e e f f e c t

b e i n g : o l i g o s a c c h a r i d e s > d i s a c c h a r i d e s > s u g a r a l c o h o l s > mono-

s a c c h a r i d e s . T h i s c a n b e u s e d t o a d v a n t a g e f o r a d j u s t i n g

t h e

e l u t i o n o r d e r fo r a g i v e n m i x t u r e o f c a r b o h y d r a t e s . The

e f f e c t

o f t e m p e r a t u r e o n

t h e

s e l e c t i v i t y o b s e r v e d f o r som e

c o m m o n c a r b o h y d r a t e s i s s ho wn i n F i g u r e 7 . N o t e t h a t a n

e l u t i o n

o rd e r

r e v e r s a l

i s

o b s e r v e d f o r m a l t o s e

( a

d i s a c c h a r i d e )

a nd s t a c h y o s e ( a t e t r a s a c c h a r i d e ) as

t h e

t e m p e r a t u r e

i s

i n c r e a s e d

T h r e e f a c t o r s s e r v e t o l i m i t t h e e f f e c t i v e u p p e r r a n g e

o f t e m p e r a t u r e i n t h e r e t e n t i o n of s u g a r s on h y d r o x i d e f o r m

a n i o n e x c h a n g e c o l u m n s :

(1) T a i l i n g a n d s e c o n d a r y p e a k s are o b s e r v e d f o r s om e c a r b o -

h y d r a t e s ( p a r t i c u l a r l y g l u c o s e an d m an no se ) when t h e

t e m p e r a t u r e of t h e c o l u m n e x c e e d s 4 5 O C . T h i s

i s

p r o -

b a b l y c a u s e d by r e a r r a n g e m e n t a b o u t t h e an o m e r i c c a r b o n

a t o m ,

t h e

L o b r y de B ruyn-V an E k e n s t e i n t r a n s f o r m a t i o n .

2 )

E f f i c i e n c i - e s t e n d

t o

d e c r e a s e w hen t e m p e r a t u r e s e x c e e d

4 5 O C .

( 3 ) A n i o n e x c h a n g e

mater ia l s

i n t h e h y d r o x i d e form a re s u b -

j e c t t o H o ffm an d e g r a d a t i o n a t e l e v a t e d t e m p e r a t u r e s .

B e ca u s e o f t h e a b o v e , c ol um n s a r e n o r m a l l y o p e r a t e d a t

t e m p e r a t u r e s b e t w e e n 20°C a n d 45OC.

A t h i r d

v a r i a b l e

f o r

c o n t r o l

of

s e l e c t i v i t y a nd r e t e n t i o n

i s

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

The

a d d i t i o n o f o t h e r a n i o n s

i s

s o m e t i m e s

d e s i r a b l e

d ue t o

t he f a c t

t h a t

h i g h l e v e l s of N a O H 0 0 . 2 M c a n r e s u l t i n

i n c r e a s e d d e t e c t o r n o i s e as well

as

p ea k a s s y m e t r y f o r t h e s u g a r

D o w n l o a d e d




13/16

DETERMINATION

OF

CARBOHYDRATES

1587

12

10

8

6

4

2

K’

a

1

*\

Maltose

‘ ‘ q q a c h ose

:

Raffinose

Lactose

.-...\..

3

40

50

T “C)

F i gur e 7 . Capaci t y f act or ver sus t emper at ur e. El uent : 0. 15

NaOH; sampl e concent r at i ons: 100 ppm each.

al cohol s . Acet at e, car bonat e, ni t r at e, and sul f at e have been eva-

l uat ed as e l uent addi t i ves .

I n

our wor k, acet at e was f ound t o be

t he pr ef er r ed el uent addi t i ve due t o t he f ac t t hat i t s af f i ni t y

f or t he ani on exchange r es i n i s s i m l ar t o t hat o f hydr oxi de. The

ot her ani ons wer e f ound t o be compat i bl e wi t h t he det ect or but

t hei r hi gh af f i ni t y f or

t he ani on exchange r esi n r esul t ed i n a

subst ant i al r educt i on of t he col umn l oadi ng capac i t y.

Because of t he el ut i on power of car bonat e, i t

i s

i mpor t ant

t hat car bonat e be excl uded f r om t he el uent syst em Thi s i s

par t i c ul ar l y t r ue

f o r

el uent syst ems used f or t he anal ysi s of

sugar al cohol s and monosacchar i des. Pr oper pr ecaut i onar y measur es

i ncl ude t he use of an Ascar i t e t r ap on t he el uent bot t l e and

ASMT

Cl ass

I dei oni zed wat er when maki ng up el uent s.

Sens i t i vi t y, Li near i t y and Repr oduc i bi l i t y

The det ect i on l i m t s f or t he car bohydr at es shown i n Fi gur e

4

r ange f r o m

3

ppb f o r t he sugar al cohol s and monosacchar i des t o

100 ppb f or t he ol i gosacchar i des . Pl ot s of t he l og of peak

hei ght t he l og of concent r at i on ar e l i near up t o sever al

hundr ed t o

1, 000

ppm wher e over l oadi ng of t he capaci t y of t he

D o w n l o a d e d


n i v e r s i t y ] a t 0 7 : 0 9 1 8 F e b

r u a r y 2 0 1 3


14/16

1588

ROCKLIN AND POHL

,

0

5 I

15 20

t m i d

F i gur e 8. Ext r act f r om f l avor ed pot at o chi ps. Lactose con-

cent r at i on i n t he ext r ac t i s 70 ppm

col umn occur s. Al t hough t he l ogf l og pl ot s ar e l i near , t he

s l opes f o r sever a l sugar s ar e: xyl i t ol ,

1. 07;

gl ucose,

1. 03;

sucr ose, 1. 16. The near l i near i t y of i vs C pl ot s , r at her t han

l / i vs 1 / C as on pl at i num 8 ) i mpl i es t hat adsor pt i on of car bo-

hydr at e mol ecul es dur i ng t 3 ( E3 =

-0. 80 V

i s not par t of t he

r eact i on mechani sm

P -

P -

Repr oduci bi l i t y of peak hei ght s f o r mul t i pl e i nj ec t i ons

of

a s i ngl e sampl e i s gener al l y bet t er t han

1

Peak hei ght s

have a t endency t o i ncr ease wi t h t i me, begi nni ng wi t h t he t i me

at whi ch t he worki ng el ect r ode was cl eaned. Thi s i ncr ease has

been obser ved t o be appr oxi mat el y

5

t o

15

a f t e r 8 hour s of

oper at i on. Erro r s i n concent r at i on measur ement s can be m ni m zed

by t he f r equent use of st andar ds.

CONCLUSION

The ani on exchange separat i on of car bohydr at es wi t h al kal i ne

el uent syst ems pr ovi des a power f ul new t ool i n t he anal ysi s of

car bohydr at es. The combi nat i on of t hi s chr omat ogr aphi c syst em

D o w n l o a d e d




15/16

DETERMINATION

OF

CARBOHYDRATES

1589

w i th t r i p l e p o t e n t i a l d e t e c t i o n p ro v i d es

a

h i gh l y s e l e c t i v e a nd

s e n s i t i v e method p a r t i c u l a r l y well s u i t e d t o complex samples .

For e xa mp le , t h e d e t e r m i na t i o n o f l a c t o s e i n f l a v o r e d p o t a t o

c h i p s , shown i n F i g u r e 8 , was e a s i l y a c c o mp l i s he d e v e n t ho ug h

t h e s o l u t i o n t o b e an al yz ed c o nt a i ne d a h i g h c o n c e n t r a t i o n of

p o t e n t i a l l y i n t e r f e r i n g

s a l t s .

The t r i p l e p o t e n t i a l d e t e c t i o n method c a n a l s o b e u s ed t o

d et e rm i ne o t h e r s p e c i e s c o n t a i n i o g t h e a l c o h o l f u n c t i o n a l g r ou p .

U si ng d i f f e r e n t ch ro ma to gr ap hi c, c o n d i t i o n s , s i m p l e a l c o h o l s

a nd g l y c o l s h a ve be en d e t e c t e d .

NOTE;:

T h e a n a l y t i c a l m e t h o d s

d e sc r i be d i n t h i s p a pe r are t h e s u b j e c t of p e n d i n g p a t e n t s .

ACKNOWLEDGMENTS

The a u t h o r s w i s h t o t h a n k Dr. Dennis

C .

Johnson of Iowa

S t a t e U n i v e r s i t y a nd Dr.

William

E. Rich f o r t h e i r h e l p a nd

a d v ic e , a n d Ka re n P o h l f o r p er f or m in g t h e i n i t i a l e xpe ri me n-

t a t i o n .

REFERENCES

1.

Knapp, D . R . Handbook

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A n a l y t ic a l D e r i v i t i z a t i o n R e a c t io n s ,

W i le y ,

N . Y .

1979 , p . 539

2 .

S c o b e l l , H . D . ; B r o b s t , K . M . ; S t e e l e ,

E.M. Cereal

Chem., 1977,

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Hassler,

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3 . Yang,

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D o w n l o a d e d


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S i n n e r ,

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Verhaa r , L . A . ; Di r k x ,

J . M . H . ,

The Analy s is o f Mixture s

Conta in ing G lucos e , F ru ct os e , and Mannose , Carbohyd.

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Rendleman,

J . A . ,

I o n i z a t i o n of C a r bo h yd r at e s i n t h e P r e s e nc e

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Neuberger ,

A . ;

Wilson,

B.M.,

The Separa t ion of Gl y c o s i d e s

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9 7 1 , 17

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7 . Brun t , K . , Compari son Be tween th e Pe r fo rmances o f an E lec t r o -

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Amperometric M easur ing System Using Glu cose

as

a Test Sample,

Ana lys t , 1982 ,

107

261

8. Hughes,

S . ;

Johnson , D . C . , Amperometric Det ec t ion o f Simple

Carbohydra tes a t P l at in um E l e c t r o d e s i n A l k a l i n e S o l u t i o n s

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Hughes,

S.

; Johnson , D.

C . ,

High Performance Liquid Chromato-

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Agric.

Food Chem., 1982 ,

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712

D o w n l o a d e d


n i v e r s i t y ] a t 0 7 : 0 9 1 8 F e b

r u a r y 2 0 1 3

Determination of Carbohydrates by Anion Exchange Chromatography With Pulsed Amperometric Detection

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