27, A u g u s t
1989
D a n P. L e e * a n d M a r k T . B u n k e r
Hamilton C o m p a n y , R e n o , N e v a d a
Abstract
A variety o f separation a n d detection m e t h o d s m a y b e u s e d
in t h e ion c h r o m a t o g r a p h i c determination of c a r b o h y d r a t e s .
A n i o n e x c h a n g e a n d ligand e x c h a n g e separation m o d e s will
b e e m p h a s i z e d , a s will t h e refractive i n d e x , p u l s e d
a m p e r o m e t r i c , a n d c o n d u c t o m e t r i c detection m e t h o d s .
C o m b i n a t i o n o f separation m o d e a n d detection m e t h o d f o r
the ion c h r o m a t o g r a p h i c a n a l y s e s of c a r b o h y d r a t e m i x t u r e s
will b e d e m o n s t r a t e d with s e v e r a l e x a m p l e applications o f
the t e c h n i q u e .
Introduction
Of the three general classes of b i o p o l y m e r s p r o t e i n s ,
polynucleotides, and c a r b o h y d r a t e s t h e carbohydrate class is
currently the most neglected in terms of the development a n d
application of new analytical techniques toward their study. This
is about to change. Progresses in ion chromatography (IC) have
produced m o r e selective and m o r e sensitive methods for car
bohydrate analysis.
T h e carbohydrates, molecular formula ( C H O ) , pose an
especially difficult chromatographic p r o b l e m . Separation dif
ficulties arise from the myriad of different structures available
to and found for the formula ( C H O ) (1). As an example of
the diversity yet similarity of these c o m p o u n d s , the six-carbon
monosaccharides, C H O , are represented by two main classes
of carbohydrates, the aldohexoses and the ketohexoses, which
differ only in the location of the carbonyl carbon at C or C ,
respectively. Glucose, mannose, and galactose are the three most
c o m m o n sugars in the aldohexose family of eight a n d each of
these sugars has a D and L stereoisomer and an and anomer.
A n appreciation of the chromatographic selectivity required is
obtained when one considers separating the 32 members of this
subgroup of C sugars. Fortunately for the analyst, it would
be rare to find all of these in the same sample, nor is it often
required that the stereoisomers or the anomeric forms be sepa
rated. Nevertheless, one finds that there is no single mode ideally
suited to the separation of this entire class of biopolymers in
cluding m o n o - , di-, and oligosaccharides, sugar alcohols, amine
2
12
496
Experimental
Equipment. The two chromatographic systems used for these
experiments consisted of either a constaMetric I or a C M 4000
high-pressure p u m p (Milton Roy, L D C Division), a 3390A in
tegrator (Hewlett-Packard C o m p a n y ) , a 7010 injection valve
(Rheodyne Incorporated), an ICM conductivity detector (Wescan Instruments Inc.), and a differential refractometer (Knauer).
Materials. Sodium hydroxide pellets for preparing eluents
were purchased from Mallinckrodt Inc. a n d were used with
degassed deionized water (Milli-Q, Millipore Inc.). Analyzed
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T a b l e 1. I o n i z a t i o n C o n s t a n t s , p k , o f
Carbohydrates*
a
Compound
glucose
galactose
mannose
fructose
sucrose
lactose
maltose
* From reference 8.
12.35
12.35
12.08
12.03
12.51
11.98
11.94
497
498
15
499
tionary phases for reversed-phase (16), anion (7), and cationexchange chromatography. These macroporous polystyrenes d o
not show measurable swelling or shrinkage even at extremes in
mobile phase conditions and d o not require column heating for
efficient operation. T h u s the first attempt at producing a highperformance phase for ligand exchange I C involved the direct
T a b l e II. R e t e n t i o n of S u g a r s o n t h e R C X - 2 0
Column (mL)*
Metal form
K+
Sample
Ca
2 +
Pb2 +
Sugar
alcohols
sorbitol
dulcitol
xylitol
mannitol
inositol
adonitol
arabitol
1.64
1.58
1.65
1.57
1.66
1.60
1.64
2.67
2.58
2.59
2.13
1.71
1.80
2.17
4.86
3.98
4.28
2.82
2.74
2.29
3.00
1.61
1.63
2.03
1.85
1.74
1.63, 1.72
1.70
1.66
1.73, 1.88
1.61
1.57
1.49
2.48
1.72
1.82
1.54, 1.63
1.61
1.52, 1.68
1.68, 1.85
1.59
1.75
1.57, 1.69
3.28
1.93
2.18
1.75
1.79
1.64
1.95
1.68
1.51
1.51
1.48
1.55
1.47
1.42
1.43
1.41
1.45
1.41
1.57
1.66
1.55
1.58
1.53
1.47
1.42
1.44
1.39
1.56
1.52
Monosaccharides
sorbose
glucose
ribose
arabinose
fructose
galactose
mannose
xylose
fucose
adamnose
Disaccharides
maltose
melibiose
sucrose
lactose
cellobiose
Trisaccharides
500
maltotriose
raffinose
O t h e r separation m o d e s
T h e t w o c o m m o n IC separation m o d e s for carbohydrates
have been described. A n o t h e r very selective carbohydrate sepa
ration technique involves the anion-exchange separation of the
complexes formed between sugars and borate anion (19). While
this separation m o d e is very selective, it is seldom used because
c h r o m a t o g r a p h i c performance is low. T h e slow and complex
equilibration kinetics between sugar and b o r a t e is t h o u g h t to
be the cause of p o o r c h r o m a t o g r a p h i c efficiency (20).
T a b l e III. C o m m e r c i a l IC D e t e c t i o n M e t h o d s
for C a r b o h y d r a t e s
Lower limit
of detection (ng)
Reference
Notes
5000
900
26
this work
190-210 nm
indirect
method
Refractive index
Optical activity
100
100
this work
23
Pulsed amperometric
1-2
Method
Ultraviolet
Conductivity
selective,
gradient
compatible
sensitive,
gradient
compatible
501
Figure 12. Ligand exchange separation of stout malt beverage and some
standards on a 250- 4.1-mm RCX-20 column at 0.5 mL/min water
and 80C. The column was in the calcium form and peak assignments
are, 1, maltotriose; 2, maltose; 3, glucose; 4, fructose; 5, glycerol; 6,
ethanol.
Figure 11. Anion-exchange chromatography of orange juice on a 1504.1-mm RCX-10 column with indirect conductivity detection. The
mobile phase was 5 1 0 - sodium acetate at 2 mL/min. Peak
assignments are, 1, glucose, 2, fructose, and, 3, sucrose.
3
502
References
1. R.T. Morrison and R.N. Boyd. Organic Chemistry,
and Bacon, Inc., Boston, 1966, pp. 982-1037.
2. P.E. Shaw. Handbook
of Sugar Separations
in Foods by HPLC.
4.
5.
6.
7.
8.
9.
10.
11.
Detection
in Liq
uid Chromatography,
I.S. Krull, Ed. Marcel Dekker, Inc., New
York, 1986, pp. 93-95.
ACS Chiramonitor product bulletin. Applied Chromatography
Systems, Macclesfield, Cheshire, U.K., 1989.
E.S. Yeung, L.E. Steenhock, S.D. Woodruff, and J.C. Kuo. Detector
based on optical activity for high-performance liquid chromat
ographic detection of trace organics. Anal. Chem. 52:1399-1402
(1980).
S. Hughes and D.C. Johnson. High-performance liquid chromat
ographic separation with triple-pulse amperometric detection of
carbohydrates in beverages. J. Agric. Food Chem. 30: 712-14
(1982).
D.P. Lee. A new anion exchange phase for ion chromatography.
J. Chromatogr. Sci. 22: 327-31 (1984).
J.A. Pendleman, Jr. In Carbohydrates in Solution. Advances in
Chemistry Series 117. American Chemical Society, Washington,
DC, 1973, pp. 51-69.
R.D. Rocklin and C.A. Pohl. Determination of carbohydrates by
anion exchange chromatography with pulsed amperometric de
tection. J. Liq. Chromatogr. 6: 1577-90 (1983).
M.R. Hardy, R.R. Townsend, and Y.C. Lee. Monosaccharide
analysis of glycoconjugates by anion exchange chromatography
with pulsed amperometric detection. Anal. Biochem. 170:54-62
(1988).
H.D. Scobell, K.M. Brobst, and E.M. Steele. Automated liquid
chromatographic system for analysis of carbohydrate mixtures.
Cereal
Chem.
12. L.E. Fitt, W. Hassler, and D.E. Just. A rapid and high resolution
method to determine the composition of corn syrups by liquid
chromatography. J. Chromatogr. 187: 381-89 (1980).
13. D.E. Just, CPC International, personal communication.
14. Bio-Rad Price List, Richmond, CA, 2989, pp. 40-46.
15. K. Brunt. Influence of mutarotation catalysts on the liquid chroma
tography of malto-oligosaccharides on a cation-exchange resin.
J. Chromatogr. 267: 347-54 (1983).
16. D.P. Lee. Reversed-phase HPLC from pH 1 to 13. J. Chromatogr.
Sci. 20: 203-208 (1982).
17. D.H. Freeman, S. Goldstein, and G. Schmuckler. Homogeneous
sulfonation of styrene-divinylbenzene copolymers with oleum in
organic solvents. Isr. J. Chem. 7: 741-49 (1969).
18. The process whereby RCX-20 is synthesized is proprietary and
details will not be presented.
19. P. Nordin. Monitoring of carbohydrates with periodate in effluents
from high-pressure liquid chromatography columns. Anal. Bio
chem. 1 3 1 : 491-98 (1983).
20. S. Honda. High-performance liquid chromatography of mono- and
oligosaccharides. Anal. Biochem. 140: 1-47 (1984).
21. E. Rajakyla. Use of reversed-phase chromatography in carbo
hydrate analysis. J. Chromatogr. 353: 1-12 (1986).
22. J.D. Olechno, S.R. Carter, W.T. Edwards, and D.G. Gillen. Devel
opments in the chromatographic determination of carbohydrates.
Am. Biotechnol. Lab.: 38-50 (Sept. 1987).
23. D.C. Johnson and T.Z. Polta. Amperometric detection in liquid
chromatography with pulsed cleaning and reaction of noble metal
electrodes. Chromatogr. Forum: 37-43 (November-December
1986).
24. J.C. Kuo and E.S. Yeung. Determination of carbohydrates in urine
by high-performance liquid chromatography and optical activity
detection. J. Chromatogr. 223: 321-29 (1981).
25. M.R. Hardy and R.R. Townsend. Separation of positional isomers
of oligosaccharides and glycopeptides by high-performance
anion-exchange chromatography with pulsed amperometric
detection. Proc. Natl. Acad. Sci. 85: 3289-93 (1988).
26. H. Binder. Separation of monosaccharides by high-performance
liquid chromatography: Comparison of ultraviolet and refractive
index detection. J. Chromatogr. 189: 414-20 (1980).
Manuscript received May 22, 1989.
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