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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
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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
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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
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
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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
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
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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
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
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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
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
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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
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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
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
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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
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
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8/19/2019 Determination of Carbohydrates by Anion Exchange Chromatography With Pulsed Amperometric Detection
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
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
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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
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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
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
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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
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
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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
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
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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
of
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,
4 ,
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F i t t ,
L . E . ;
Hassler,
W . ;
J u s t ,
D.E.
A Rapid and High Resolu-
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2
Chromatogr . 1980,
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81
L a d i sh , M . R . ; Heubner , A . L . ; Tsao, G.T. High Speed Liquid Chro-
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u s i n g
Water
a s E l u e n t ,
J
Chromatogr . , 1978, 147 8 5
3 . Yang,
R.T.;
Mi l l ig a n , L .P .; Ma th i so n ,
G.W.,
Improved Sugar
Se pa r at io n by High Performance Liqu id Chromatography Using
Porous
Mic ro p a r t i c l e C a rb o h y d ra t e C o lu mn s .
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Chromatogr. ,
1981 , 209 316
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
-
8/19/2019 Determination of Carbohydrates by Anion Exchange Chromatography With Pulsed Amperometric Detection
16/16
1590 ROCKLIN
AND POHL
Hendrix , D . L . ; Lee, R.E.; Ba us t, J . G . ; James, H . , S e p a r a t i o n
of Carbohydra tes and Po lyo l s by a Radially Compressed High
Performance L iqu id Chromatographic S i l i c a Column Modif ied
wi t h Te t r a e t h y l e n e p e n t a mi n e ,
2
Chromatogr. ,
1981,
210, 45
b
S i n n e r ,
J . ;
P u l s ,
J .
2 Chromatogr. , 1978, 156, 197, Non-
Co rr os iv e Dye Reagent f o r D e t e c t i o n o f R ed uc in g S u g a rs i n
Bo ra te Complex Ion-Exchange Chromatography
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.
@.
1977, 53 , 247
5.
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
o f Metal Hydroxides and Oxides.
Advances Chemistry, S e r i e s 117, American Chemical
S o c i e t y , 1 9 7 3, p. 5 1
C a r b o h y d r a t e s S o l u t i o n ,
6 .
Neuberger ,
A . ;
Wilson,
B.M.,
The Separa t ion of Gl y c o s i d e s
on
a
S t r o n g l y Basic Ion-Exchange Res in : An I n t e r p r e t a t i o n
i n Terms of Acid i ty , Carbohyd . E.
9 7 1 , 17
9
7 . Brun t , K . , Compari son Be tween th e Pe r fo rmances o f an E lec t r o -
chemica l De tec to r F low Cel l i n a P o t e n t i o m e t r i c a n d a n
Amperometric M easur ing System Using Glu cose
as
a Test Sample,
Ana lys t , 1982 ,
107
261
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712
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