J o u r n a l of Chromatographic Science, V o l .

27, A u g u s t

1989

Carbohydrate Analysis by Ion Chromatography

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

* Author to w h o m correspondence should be addressed.

496

sugars, starches, a n d celluloses, but several must be available.

Analyte and matrix determine the separation m o d e . T h e IC
modes considered in this paper are anion exchange a n d ligand
exchange. T h e partitioning of sugars on amine-bonded phases
with an acetonitrile-water mobile phase will be considered out
side the d o m a i n of IC (2).
The second half of the carbohydrate analysis problem is detec
tion. Sugars are not particularly c h r o m o p h o r i c above 210 n m .
T h u s , this most universal detection m e t h o d is not applicable
because of low sensitivity a n d b a c k g r o u n d interference. Fluor
escence detection of course requires the nuisance of pre- or
postcolumn derivatization (3). Refractive index has been the
mainstay of carbohydrate detection methods but does not allow
gradient elution techniques. Conductometric detection is suitable
and quite selective, but also does not allow the use of gradients.
Polarography or optical activity detection has just been com
mercially introduced (4) and is expected to provide sensitive,
selective detection (5). The pulsed amperometric detector ( P A D ) ,
which provides selective a n d sensitive detection and does allow
gradient elution, has been one of the great b r e a k t h r o u g h s in
carbohydrate analysis (6). T h e detection m e t h o d s specifically
considered in this paper are P A D , refractive index, and indirect
conductometric.
This paper contains b o t h a review of the newer IC m e t h o d s
of carbohydrate analysis a n d an account of recent, previously
unpublished, results obtained in the a u t h o r s ' laboratory. W e
will begin with a discussion of the different IC c a r b o h y d r a t e
separation modes, continue with an evaluation of detection prin
ciples, and conclude with a display of the application of I C t o
actual carbohydrate analyses.

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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Journal of Chromatographic Science, V o l . 27, August 1989

glacial acetic acid and glycerol were obtained from J . T . Baker.

Non-Spectro grade acetonitrile was purchased from Burdick and
Jackson. Poly(ethylene glycol), average M W 400, and p-toluenesulfonic acid monohydrate was obtained from Aldrich Chemical
C o m p a n y Inc. S o d i u m nitrate was obtained from Spectrum
Chemical Mfg. C o r p . All carbohydrates and barium hydrox
ide solution were purchased from Sigma Chemical C o m p a n y .
All other chemicals were of reagent grade or better and used
without further purification.
Columns. P R P - X 1 0 0 ( 1 5 0 4.1 m m ) , RCX-20 (250 4.1
mm), and RCX-10 (150 4.1 mm) columns were obtained from
the H a m i l t o n C o m p a n y . P R P - X 1 0 0 is a strong base anion ex
changer with an exchange capacity of 0.2 m e q u i v / g (7). RCX-20
is a hybrid stationary phase and is best described as a continuous
network of 5-m macroreticular poly(styrene divinylbenzene)
with dispersed d o m a i n s of polystyrenesulfonate gel with a
capacity of 2.5 m e q u i v / g . RCX-10 is a strong base anion ex
changer with an exchange capacity of 0.4 m e q u i v / g .
Column packing. T h e packing a n d dispersion solution for
RCX-20 stationary phase was 4:1 acetonitrile-poly(ethylene
glycol) M W 400. C o l u m n s were packed three at a time u p w a r d
at a constant total flow rate of 10 m L / m i n . Final packing
pressure was 4500 psi. T h e packing and dispersion solution for
RCX-10 stationary phase was 9:1 0.56 sodium n i t r a t e glycerol. C o l u m n s were packed three at a time d o w n w a r d at
a constant total flow rate of 22 m L / m i n . Final packing pressure
was 4500 psi.
Counterion conversion procedure. Conversion of the R C X 20 resin from the hydrogen form to either the calcium or
potassium form was d o n e by p u m p i n g 20 m L of 0.5 calcium
chloride or 1.0 potassium hydroxide respectively over the col
u m n . Conversion of the RCX-20 resin between calcium a n d
potassium forms was done by pumping 20 m L of 0.5 calcium
chloride or 0.5 potassium E D T A over the c o l u m n .
Eluent and sample preparation.
Sodium hydroxide eluents
were prepared by s t a n d a r d dilution of a sodium h y d r o x i d e b a r i u m hydroxide concentrate with previously degassed deion
ized water into volumetric glassware. T h e concentrated solu
tion of sodium hydroxide was prepared by weighing out 300
g of sodium hydroxide pellets and adding a n equal weight, 300
g, of degassed deionized water. After the pellets had dissolved,
10 m L of 0.3 barium hydroxide was added to precipitate car
b o n a t e in the form of b a r i u m c a r b o n a t e . T h e concentration of
hydroxide in solution was determined by titration with potas
sium hydrogen p h t h a l a t e . If organic modifiers, such as acetic
acid, were needed in the eluent, they were added to the previous
ly degassed deionized water before the addition of concentrated
sodium hydroxide.

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

Standard carbohydrate sample mixtures were prepared in

deionized water at concentrations listed on the chromatograms.
Beer samples were degassed a n d then diluted with deionized
water as listed on the c h r o m a t o g r a m a n d injected directly o n t o
the c o l u m n . O r a n g e juice samples were diluted with deionized
water, filtered t h r o u g h a 0.2-m A C R O LC13 disposable filter
assembly (Gelman Sciences), then injected. All samples were
stored at 4C until needed.

Results and Discussion

A n i o n - e x c h a n g e separation
One unexpected property of the carbohydrates is that they
are weak acids (8). This is probably why the anion-exchange
separation mode has not been considered for sugar analyses until
recently (9). T h e ionization constants of a few c o m m o n m o n o and disaccharides are listed in Table I (8). The separations were
carried out at mobile phase p H values of 12 or greater with
sodium hydroxide or sodium hydroxide-sodium acetate eluents.
The stationary phase was a trimethylammonium-functionalized
poly(styrene divinylbenzene), that is, a strong base anion ex
changer. A typical separation is shown in Figure 1.
T h e anion-exchange separation of carbohydrates is one of
the most exciting new developments in I C . The method is selec
tive, efficient, a n d acceptably uncomplicated. O n e must avoid
the use of glass solvent reservoirs where etching of silicate into
the high p H mobile phase will occur and alter (shorten) chro
matographic retention. C a r b o n dioxide, which becomes fixed
in the basic mobile phase as carbonate, must be excluded with
an inert gas blanket or soda lime t r a p s . T o obtain very re
producible retention, H a r d y , Townsend, a n d Lee found it
necessary t o flush their column with 200 m M sodium hydrox
ide for 10 min between each 22 m M eluent run (10).
Anion-exchange retention increases with carbohydrate size.
T h u s , sugar alcohols elute first a n d are followed by m o n o s a c
charides, then disaccharides, a n d so o n . As an example see
Figure 1. T h e carbohydrates are expected to accept a single
charge per saccharide unit regardless of degree of oligomerization (8), so elution order must be dependent u p o n hydration
and shielding of the charge and also degree of ionization similar
to the inorganic anion-exchange selectivity order SO
>
B r - > Cl > F - .
C h r o m a t o g r a p h i c selectivity may be affected by a change in
temperature or mobile phase p H . Rocklin a n d P o h l report that
the effect of increasing temperature to decrease retention is
related t o c a r b o h y d r a t e size. T h e magnitude of the effect fol
lows: oligosaccharides > disaccharides > sugar alcohols >
monosaccharides (9). Mobile phase p H affects selectivity by
altering degree of sugar ionization and eluent strength (Figure
2). Prediction of the net o u t c o m e is difficult because these two
effects oppose each other. As mobile phase basicity is increased
t h r o u g h the p H range 12 to 13, the sugars become m o r e ion
ized, interaction with the stationary phase increases, and reten
tion would increase, but when the p H is increased, eluent ion
is necessarily increased and the mass action serves to reduce
retention. For an example, see Figure 2. Notice how sucrose
with the higher pK (Table I) shows a retention m a x i m u m at
higher eluent p H levels t h a n glucose or fructose.
Incorporation of acetate ion in anion chromatographic mobile
phases for carbohydrates serves t o shorten retention but has
very little effect on selectivity. Hydroxide is a very weak eluent
and extremely high concentrations are required to elute the
2

497

Journal of Chromatographic Science, V o l . 27, August 1989

higher oligomers. Addition of a-few-millimolar sodium acetate

reduces the hydroxide eluent requirement by roughly an order
of magnitude.
Anion exchange IC is compatible with m a n y types of detec
tion but it is especially powerful when combined with a m p e r o
metric methods where high sensitivity gradient elution becomes
possible. This will be shown later in the detection a n d applica
tions sections.
L i g a n d e x c h a n g e separation
The ligand exchange partitioning mode is the oldest and most

c o m m o n method for separating carbohydrate mixtures (11,12).

It is the basis for the industrial refinement of high fructose
syrups (13). Ligand exchange separations, for lack of a better
description, are dominated by two analyte-stationary phase in
teractions, steric exclusion and electrostatic attraction between
electronegative sugar oxygen a t o m s and electropositive metal
cations (Figure 3). The stationary phase is a 4 - 8 % cross-linked
polystyrenesulfonate. Deionized water is the usual mobile phase.
Chromatographic selectivity can be changed by adding 10-40%
of acetonitrile to the water mobile phase or by using a different
column that has a different metal counter ion to the polystyrene
sulfonate. T h e calcium form polystyrenesulfonate is the most
popular because it shows excellent glucose-fructose selectivity
(Figure 4). T h e lead form column shows g o o d selectivity for
sugar alcohols, the hydrogen form is used for organic acids,
and the silver form column for oligomeric distributions (14).
T h e reason different columns of different metal forms are us
ed, rather t h a n employing just one column a n d converting, for
example, from the silver form to the calcium form by passing
a concentrated calcium nitrate solution over the stationary
phase, is the strong tendency for the polystyrene gel to swell
or shrink depending u p o n counterion type a n d mobile p h a s e .
Thus conversion of a silver form ligand exchange c o l u m n t o
the calcium form would cause shrinkage of the packed c o l u m n
bed with formation of voids and a catastrophic loss of efficiency
and resolution.
T h e soft pliable n a t u r e of these ligand exchange gels also re
quires that the columns be operated at relatively low flow rates,
0.03 t o 0.05 c m / s , a n d at a n elevated t e m p e r a t u r e to reduce
mobile phase viscosity and pressure requirements.
Heating the columns, typically to 80 t o 90C, also serves t o
speed rates of partitioning between stationary and mobile phase

Figure 1. Separation of a corn syrup sample, 3% w/v in water, by anion

exchange chromatography on a 150- 4.1-mm PRP-X100 column at
1.0 mL/min 7.5 10- potassium hydroxide. Peak assignments
are, 1, glucose; 2, maltose; 3, maltotriose; 4, maltotetrose; 5, maltopentose; 6, maltohexose.
2

498

Figure 2. Effect of pH on anion exchange retention, k', using an RCX-10

column, 150 4.1 mm. All mobile phases contained 5 1 0
sodium acetate.
3

Journal of Chromatographic Science, V o l . 27, August 1989

and thereby improves c o l u m n efficiency. Heating also speeds

the m u t a r o t a t i o n of sugar analytes a n d results in a selectivity
averaging between the a n d a n o m e r s and elution of a single
sharp peak. A t a m b i e n t or subambient c h r o m a t o g r a p h i c con
ditions many sugars are partially resolved into their two anomers
because m u t a r o t a t i o n kinetics are much slower than ligand ex
change kinetics (15). This peak splitting is usually undesirable
and the column is heated t o avoid it.
Despite a few shortcomings of the ligand exchange technique,
that is, the requirement for c o l u m n heating and the need for
different metal form columns t o achieve different selectivities,
it remains the most popular analysis method for carbohydrates
today. Mobile phase requirements are simplejust add water
and complete oligosaccharide profiles are obtained isocratically
in a few minutes because the size exclusion elution order, from
largest to smallest, is imposed. The separations d o not provide
as much information a b o u t distribution of the higher oligomers
as gradient anion-exchange c h r o m a t o g r a p h y does, which re
verses the elution order (Figure 1), but this information is seldom
required. T h e analyst or food technologist needs t o k n o w the
a m o u n t of sweeter m o n o - and disaccharides relative to the t o t a l .
Recent efforts in the a u t h o r s ' laboratory have focused o n
development of an improved stationary phase for ligand ex
change c a r b o h y d r a t e I C . W e have been successful in the past
in producing m a c r o p o r o u s poly(styrene divinylbenzene) sta-

15

Figure 4. Separation of some sugar standards by ligand exchange

chromatography on a 305- 7.8-mm 8% gel polystyrenesulfonate,
calcium form, at 88C and 0.9 mL/min water. Peak assignments are,
1, maltotriose; 2, maltose; 3, glucose; 4, fructose; 5, sorbitol.

Figure 3. Ligand exchange interaction of sugar analyte with

polystyrenesulfonate counterion, calcium.

Figure 5. Sulfonation of polystyrene to form the polystyrenesulfonate

stationary phase.

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Journal of Chromatographic Science, V o l . 27, August 1989

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

Figure 6. Schematic representation of the RCX-20 "hybrid" phase.

sulfonation (17) of the polymeric reversed phase P R P - 1 (Figure

5). The efficacy of this macroporous polystyrenesulfonate quick
ly became apparent u p o n conversion from the hydrogen form
to the calcium form a n d packing it into a column. T h e material
showed n o selectivity between m o n o - , di-, a n d higher oligosac
charides. T h e molecular sieving character of the moderately
cross-linked gels was absent in the m a c r o p o r o u s polystyrene,
and it failed as a carbohydrate IC phase.
The next attempt at a high-performance ligand exchange
phase was successful. T h e requisite properties of the m a c r o
p o r o u s and gel polystyrenes were combined t o form a hybrid
phase, RCX-20 (18). This hybrid phase may be t h o u g h t of as
a continuous network of rigid m a c r o p o r o u s polystyrene where
the pores have been filled with d o m a i n s of polystyrene gel
(Figure 6). The macroporous skeleton gives the stationary phase
rigidity and allows conversion from one metal form to a n o t h e r
while the gel type domains produce the desired chromatographic
selectivity. A typical c h r o m a t o g r a m is shown in Figure 7. Selec
tivity of the column in the potassium, calcium, a n d lead forms
toward a variety of sugar alcohols, m o n o - , di-, a n d trisaccharides is listed in Table II. T h e availability of the stationary
phase to perform well after conversion from one metal form
to another is shown in Figure 8. A m o r e detailed exploration
of this and other IC stationary phases follows in the applica
tions section.

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

Figure 7. Ligand exchange chromatography of, 1, sucrose, 2, glucose,

and, 3, fructose on a 250- 4.1-mm RCX-20 column, potassium form,
at 0.5 mL/min water, room temperature. Detection was by differential
refractive index.

500

maltotriose
raffinose

* 250 4.1 m m , H O mobile phase at room temperature. W h e r e two values are

given, anomers were being resolved.
2

Journal of Chromatographic Science, V o l . 27, August 1989

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).

Figure 9. Anion-exchange separation of some glycoprotein sugar stand

ards on a Dionex 4.0- 250-mm CarboPac PA1 at 1 mL/min 1.5
1- sodium hydroxide. Detection was by pulsed amperometry, gold
electrode after postcolumn addition of 0.3 sodium hydroxide at 1.0
mL/min. Peak assignments are, 1, fucose; 2, galactosamine; 3, gluco
samine; 4, galactose; 5, glucose; 6, mannose. Chromatogram courtesy
of Dionex Corp.
2

Figure 8. Separation of a corn syrup sample on the 250- 4.1-mm

RCX-20 column in the potassium form at, A, 60C and, B, ambient, then
after conversion to the calcium form at, C, 60C and, D, ambient. The
flow rate in all cases was 0.5 mL/min water, detection by refractive index.

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

Figure 10. Gradient anion-exchange separation of glucose oligomers

on a Dionex CarboPac PA1 column from 0 to 100% at 1 mL/min,
where A is 0.1 sodium hydroxide plus 0.6 sodium acetate with
pulsed amperometric detection. The numbers above each peak corre
spond to the degree of polymerization (DP) of the oligomer.
Chromatogram courtesy of Dionex Corp.

501

Journal of Chromatographic Science, V o l . 27, August 1989

Other separation modes for carbohydrate analysis include

reversed-phase (21) and normal-phase partitioning (2). These
two modes are considered outside the scope of the ion chro
matographic technique.
Detection
The number of carbohydrate IC detection methods is roughly
inversely p r o p o r t i o n a l t o the success of any of them for selec
tive, sensitive detection, with the exception of o n e p u l s e d
amperometric detection (PAD). P A D is sensitive (Table III) and
sufficiently selective to allow salt gradient anion c h r o m a t o g
raphy without excessive base line drift (22). The P A D technique,
first reported by Hughes and Johnson (6) and further developed
by J o h n s o n and co-workers (23), involves a three-potentialpulse sequence to maintain electrode performance. Analytical
signal is measured at oxidizing potentional E after some time
interval t . T h e gold or platinum electrode is then switched t o
a higher oxidizing potential, E to oxidatively desorb (clean)

analyte from the electrode surface. A final switch t o a reduc

ing potential, E removes noble metal surface oxide. T h e cy
cle is then repeated at a rate of 1 H z . T h e P A D m e t h o d is
an ideal match for the high p H anion exchange m o d e of car
bohydrate I C . The only weakness of the m e t h o d is the univer
sality of oxidative detection, so that mobile phase modifiers such
as methanol or p r o p a n o l cannot be used.
Of the other detectors listed in Table III, a few deserve special
3,

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

Figure 13. Lead form ligand exchange chromatography of apple juice

on an Interaction Chemicals CHO 682 column, 300 7.8 mm, at 85C
and 0.4 mL/min water with refractive index detection. Chromatogram
courtesy of Interaction Chemicals, Inc.

Journal of Chromatographic Science, V o l . 27, August 1989

mention: refractive index, optical activity, a n d conductivity. T h e

refractive index detector h a s been a n d remains t h e most com
m o n . T h e bulk property detection m e t h o d does n o t allow gra
dient c h r o m a t o g r a p h y , b u t t h e m e t h o d is universal a n d , with
m o d e r n instrumentation, quite sensitive. T h e optical activity
detector seems ideally suited t o carbohydrate IC. It is both selec
tive a n d sensitive (5,24). Gradient elution is possible and a com
mercial instrument h a s just become available. Conductomeric
detection has n o t previously been used for carbohydrate I C ,
but will be d e m o n s t r a t e d here in association with t h e anionexchange separation m o d e . A s expected it is a reliable a n d ac
ceptably sensitive m e t h o d .
Applications
Carbohydrate I C is most frequently applied to food or bever
age samples. In these cases, t h e sugars a r e present at relatively
high levels a n d t h e sample is n o t limited. O n e notable excep
tion is t h e analysis of t h e c a r b o h y d r a t e portion of glycopro
teins (25). These trace analyses are especially well suited t o t h e
anion exchange-pulsed a m p e r o m e t r i c system (Figure 9). A n
other unique application of this I C system is the gradient elu
tion of oligosaccharides (Figure 10). The column selectivity com
bined with high detector sensitivity allows separation of glucose
oligomers from D P (degree of polymerization) 1 to 4 3 .
Anion-exchange separations of carbohydrates can also be
monitored by conductometric detection (Figure 11). T h e peaks
in this detection m o d e are actually negative because the equiva
lent conductance of analyte sugar anion is less than that of eluent
hydroxide i o n .
C a r b o h y d r a t e analyses of malt beverages a r e easily accom
plished with ligand exchange c h r o m a t o g r a p h y a n d refractive
index detection (Figure 12). T h e hybrid phase RCX-20 was us
ed t o rapidly screen for the unfermented sugars in stout. A dif
ferent ligand exchange selectivity is obtained when the lead form
of the polystyrenesulfonate gel is used (Figures 13, 14). Again,
refractive index detection showed adequate sensitivity for these
food samples.
A variety of separation a n d detection principles are available
to t h e I C practitioner for t h e analysis of carbohydrates. Re
cent advances in t h e field allow sensitive, selective determina
tions of this class of biopolymers, t h e carbohydrates.

Figure 14. Lead form ligand exchange chromatography of strawberry

yogurt on a 300- 7.8-mm Bio-Rad HPX-87P column at 80C and ca.
1 mL/min water. Peak assignments are, 1, sucrose; 2, lactose; 3,
glucose; 4, galactose; 5, fructose. Chromatogram courtesy of Bio-Rad
Laboratories.

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Manuscript received May 22, 1989.
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