C196-E036

High Performance Liquid Chromatography

Analysis of Saccharides
 Contents

1. Introduction .................................................................................................................. 1

2. Methods of Saccharide Analysis ............................................................................ 1

3. HPLC Separation Methods for Saccharides ........................................................ 1

3.1 Size Exclusion Chromatography .............................................................................. 1
3.2 Normal Phase Chromatography ............................................................................... 2
3.3 Ligand Exchange Chromatography .......................................................................... 5
3.4 Anion Exchange Chromatography ........................................................................... 7

4. Methods of Saccharide Detection .......................................................................... 8

4.1 Absorption Detection ............................................................................................... 8
4.2 Differential Refractive Index Detection ................................................................... 8
4.3 Derivatization (Chemical Reaction Detection) ........................................................ 9

5. Conclusion .................................................................................................................... 11
1. Introduction
Saccharides, or sugars, are a group of organic compounds that occur extensively in nature. They
play important roles in the human body, as sources of energy, constituents of our bodies, and as
substances that take part in various physiological functions. The analysis of saccharides is,
therefore, an essential part of physiological studies.
One of the most popular methods of analysis for this application is high performance liquid
chromatography (HPLC).
This paper describes the HPLC analysis of saccharides, concentrating mainly on separation and
detection methods.

2. Methods of Saccharide Analysis

There are a wide variety of saccharides. Their molecular weights range from about 180 for
monosaccharides (e.g. glucose) to hundreds of thousands, or even several million for starch.
Monosaccharides are classified into neutral saccharides (e.g. glucose), amino sugars (e.g.
glucosamine), uronic acids (e.g. glucuronic acid), and sugar alcohols (e.g. sorbitol). In addition, all
saccharides have many isomers.
The HPLC analysis of saccharides, therefore, must be performed using the most suitable methods
of separation and detection for the compound(s), taking the types and concentrations of the sample
components under study into consideration.
Table 1 lists the methods of separation and detection used in the HPLC analysis of saccharides.
This paper describes some typical methods for this application.

Table 1. Separation and detection

Size Exclusion Chromatography

Normal Phase Chromatography
Separation
Ligand Exchange Chromatography
Anion Exchange Chromatography
UV Absorption Detection
Detection Differential Refractive Index Detection
Derivatization (Chemical Reaction Detection)

3. HPLC Separation Methods for Saccharides

3.1. Size Exclusion Chromatography
Size exclusion chromatography is used to separate saccharides on the basis of molecular size.
Dextran and polyacrylamide gels have been conventionally used as the column packing
material for this application. The drawback to these gels, however, is that they cannot be used
under high pressure conditions. Sodium sulfonated polystyrene and glyceryl polymethacrylate
gels, which have been specifically developed for use under high pressure conditions, are now
most popularly utilized for this analysis.

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 Figure 1 shows calibration curves
Sample: polyethylene glycol
for standard polyethylene glycols,
B-

molecular weight (log)

with several size exclusion columns B- 80
80 6
5
with different pore sizes.
B-
Figure 2 shows typical elution 80
4
patterns of standard dextrans with a B-
80
3
size exclusion column; the pore sizes
of the column packing were selected
to be suitable for the sample
molecular weight range. (min)
It must be noted that a sodium Fig. 1. Calibration curves of polyethyleneglycol
sulfonated polystyrene gel column obtained with size exclusion columns with
elutes acidic sugars rather quickly different pore sizes.
due to ion exclusion, and that basic
saccharides elute slowly because they
are retained by ion interaction.

3.2. Normal Phase

Chromatography
Normal phase chromatography is
effective for analysis of saccharides
ranging from monosaccharides to
oligosaccharides. This method even
separates oligosaccharides into
individual constituents, facilitating
identification.
In normal phase chromatography Conditions
of saccharides, anion exchange resins column: Size exclusion column (500mm × 8.0mm i.d.)
of sulfate salt type and cation mobile phase: water
exchange resins of lithium salt type flow rate: 1.0 mL/min
or of ammonium salt type were temperature: 25˚C
detection: refractive index
conventionally used, with ethanol-
Peaks
water mixtures as the mobile phase. 1: dextran MW500K, 2: dextran MW250K, 3: dextran
Unfortunately, an analytical run takes MW150K, 4: dextran MW70K, 5: dextran MW20K,
over an hour. 6: dextran MW10K, 7: ethyleneglycol.
For the HPLC analysis of
Fig. 2. Elution pattern of dextran from size
saccharides, a silica gel stationary
exclusion column.
phase on which aminopropyl groups
are chemically bonded is now used as the column packing, and acetonitrile-water mixture as the
mobile phase. The retention of saccharides by this type of column can be attributed to the
partitioning of saccharides.

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 Figure 3 shows a typical
chromatogram of a saccharide
standard, obtained using a Shim-pack
CLC-NH2 column. Figure 4 shows
the retention times of the saccharides
under the same set of
chromatographic conditions.
Normal phase chromatography
separates disaccharides; this is
difficult in both size exclusion
chromatography and ligand exchange
chromatography (described in
Section 3.3).
In normal phase chromatography, Conditions
column: Shim-pack CLC-NH2 (150mm × 6.0mm i.d.)
the separation and retention times of mobile phase: water / acetonitrile = 7 / 3 (V/V)
saccharides can be controlled by flow rate: 1.0 mL/min
changing the mobile phase concentra- temperature: 25˚C
detection: refractive index
tion ratio of acetonitrile and water. Peaks
Increasing the water concentration 1: glycerol, 2: xylose, 3: arabinose, 4: fructose,
results in faster elution of saccharides 5: glucose, 6: galactose, 7: sucrose, 8: maltose,
9: isomaltose, 10: maltotriose.
and allows components of higher
molecular weights to be eluted in a Fig. 3. Chromatogram of saccharides obtained

reasonable length of time. Figure 5 with Shim-pack CLC-NH2.

Saccharides
1. sample solvent 14. sucrose
2. rhamnose 15. cellobiose
1 2 3 4 5 6 7 8 910111213 14
3. xylose 16. maltose
4. lyxose 17. maltitol
5. fucose 18. inositol
6. arabinose 19. trehalose
7. sorbose 20. isomaltose
8. fructose 21. lactose
9. mannose 22. melibiose
10. glucose 23. maltotriose
11. sorbitol 24. raffinose
1 15161718192021222324 25 26 12. mannitol 25. maltotetraose
13. galactose 26. maltopentaose

Conditions
column: Shim-pack CLC-NH2 (150mm
× 6.0mm i.d.)
mobile phase: water / acetonitrile = 7 / 3 (V/V)
Fig. 4. Retention times of saccharides separated on
flow rate: 1.0 mL/min
Shim-pack CLC-NH2. temperature: 25˚C
detection: refractive index

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shows example chromatograms of
glucose and its oligomers (ranging
from dimer to hexamer). In this
analysis, water concentration in the
mobile phase was 40%, as opposed to
the 30% used for the data in Figure 3.
A further increase of the water
content enabled the separation of
compounds having 10 to 15 glucose
molecules. A decrease in water
content, on the other hand, somewhat
improved the separation of
monomers. However, a water content
Conditions
below 20% sometimes resulted in column: Shim-pack CLC-NH2 (150mm × 6.0mm i.d.)
unsymmetrical peaks, due to low mobile phase: water / acetonitrile = 6 / 4 (V/V)
solubility of saccharides. flow rate: 1.2 mL/min
temperature: 25˚C
For the mobile phase, an acetone-
detection: refractive index
ethyl acetate-water mixture can also Peaks
be used. It may be superior to an 1: glucose, 2: maltose, 3: maltotriose, 4: maltotetraose,
acetonitrile-water mixture, because it 5: maltopentaose, 6: maltohexaose.
is less toxic. Fig. 5. Chromatogram of glucose oligomers
Recently, resin, rather than silica, obtained with Shim-pack CLC-NH2.
columns have become popular for
this type of normal phase analysis. The resin column is more durable, since the packing
material does not hydrolyze easily.
Some studies also refer to the HPLC analysis of saccharides using columns of silica only or
of silica gel on which octadecyl groups are chemically bonded, and acetonitrile-water mobile
phase to which amines have been added.

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 3.3. Ligand Exchange Chromatography
A sodium sulfonated polystyrene gel column
(first mentioned in Section 3.1) cannot separate
dimers or larger saccharides of the same molecular
weight, but it can separate some monosaccharides
of the same molecular weight, such as glucose and
fructose.
Ligand exchange chromatography is another
type of HPLC analysis. By selecting a counter-ion,
such as calcium, sodium, or lead, the
monosaccharides’ selectivity can be changed and
Conditions
their separation improved. column: Shim-pack SCR-101N
Ligand exchange chromatography has a (300mm × 7.9mm i.d.)
noteworthy advantage, in that it uses water alone mobile phase: water
flow rate: 0.6 mL/min
as mobile phase, simplifying the analysis. As a temperature: 60˚C
result, columns with various counter-ions have detection: refractive index
recently become commercially available. Peaks
1: dextran MW10K, 2: sucrose,
In this type of HPLC analysis, saccharides are
3: glucose, 4: fructose, 5: ethanol.
retained mainly by complexing with metallic
Fig. 6. Chromatogram of saccharides
counter-ions. The retention may be related to the
obtained with Shim-pack SCR-101N.
stability of the complexes formed when the
saccharides exchange for the hydration water molecule of the metallic counter-ions. Figures 6,
7, and 8 show the chromatograms of saccharides obtained using sodium, calcium and lead
sulfonated polystyrene gel columns, respectively.

Conditions Conditions
column: Shim-pack SCR-101C column: Shim-pack SCR-101P
(300mm × 7.9mm i.d.) (300mm × 7.9mm i.d.)
mobile phase: water temperature: 80˚C mobile phase: water temperature: 80˚C
flow rate: 1.0 mL/min detection: refractive index flow rate: 0.6 mL/min detection: refractive index
Peaks Peaks
1: dextran MW10K, 2: maltotriose, 3: maltose, 1: sucrose, 2: glucose, 3: xylose, 4: galactose,
4: glucose, 5: fructose, 6: glycerol, 7: ethanol, 5: arabinose, 6: fructose, 7: glycerol, 8: mannitol,
8: sorbitol. 9: ribose, 10: sorbitol.
Fig. 7. Chromatogram of saccharides Fig. 8. Chromatogram of saccharides
obtained with Shim-pack SCR-101C. obtained with Shim-pack SCR-101P.

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 Figure 9 lists the retention times of the saccharides. The calcium type of column separates
monosaccharides better than the sodium type. The lead type gives even better separation. The
calcium and lead types retain sugar alcohols longer and permit separation of glucose and
sorbitol, which is impossible with the sodium type of column packing.
In ligand exchange chromato-graphy, the column is kept at high temperature. Figure 10
shows how the chromatograms change by varying the column temperature. The column in the
example is a Shim-pack SCR-101C (a calcium type).
When the column is kept at room temperature, glucose, which is one of the reducing sugars,
splits into two peaks. This is the separation of the anomers. When the column temperature
increases, the two peaks combine into one sharp peak due to the enhanced speed of
mutarotation. Since hydroxide ions also serve as an effective catalyst for mutarotation, the
addition of a basic compound, such as triethylamine, to the mobile phase can have the same
effect as increasing the column temperature. In this case, however, the column packing must be
regenerated by adding calcium ion.

temperature (25˚C)

Conditions
mobile phase: water
flow rate: 0.8 mL/min
temperature: 60˚C (for SCR-101N), 80˚C
(for SCR-101C and SCR-101P)
detection: refractive index
Saccharides Conditions
1: raffinose, 2: maltotriose, 3: sucrose, 4: maltose, 5: lactose, column: Shim-pack SCR-101C
6: glucose, 7: mannitol, 8: rhamnose, 9: sorbitol, 10: galactose, (300mm × 7.9mm i.d.)
11: mannose, 12: xylose, 13: fructose, 14: inositol, 15: xylitol, mobile phase: water
16: fucose, 17: arabinose, 18: glycerol, 19: ribose, 20: ethanol. flow rate: 0.8 mL/min
detection: refractive index
Fig. 9. Elution times of saccharides separated on
Peaks
Shim-pack SCR series columns.
1: sucrose, 2: glucose, 3: sorbitol.
Fig. 10. Effect of temperature on
separation of saccharides on
Shim-pack SCR-101C.

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3.4. Anion Exchange
Chromatography
The hydroxyl groups of saccha-
rides are weakly acidic; their dissoci-
ation constants are only at about the
10-12 level. In anion exchange
chromatography of saccharides, there-
fore, it is necessary to adjust the pH
value of the mobile phase to greater Fig. 11. Chemical structures of saccharide-boric
than 12, and use an HPLC system acid complex.
which can withstand basic conditions.
However, there is another method
of exchange chromatography. Since
polyhydroxy compounds, such as
saccharides, react with boric acid to
form a negatively charged complex
(as shown in Figure 11), complexes of
saccharides and boric acid can be
created and analyzed by using a boric
acid buffer solution as the mobile
phase
Figure 12 shows a typical
chromatogram of anion exchange
chromatography of saccharides,
obtained with a Shim-pack ISA-07
column. The anion exchange Conditions
chromatography of saccharide/ boric column: Shim-pack ISA-07 (250mm × 4.0mm i.d.)
acid complexes is quite effective for mobile phase: 300mM (potassium)borate buffer <pH8.5>
separating disaccharides and flow rate: 0.6 mL/min
monosaccharides into their individual temperature: 65˚C
constituents. It provides far better detection: post-column derivatization with arginine,
separation of monosaccharides than fluorescence at Ex:320nm, Em:430nm.
Peaks
any other method. Unfortunately, the
1: mannose, 2: fructose, 3: galactose, 4: xylose, 5: glucose.
retention time difference between
components is very large; the Fig. 12. Chromatogram of saccharides obtained
separation of monosaccharides and
with Shim-pack ISA-07.
disaccharides into individual com-
ponents takes too long, and peaks are
very broad. High analytical efficiency is not possible under isocratic conditions. It is now
mostly used in gradient mode, in which the concentration of the boric acid buffer solution and
the pH value of the mobile phase are changed with respect to time. This also requires a
detection method suitable for gradient mode.

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4. Methods of Saccharide
Detection
4.1. Absorption Detection
UV (ultraviolet) absorption
spectrophotometry is the most popular
method of detection in HPLC.
However, this method is not very
effective for the analysis of
saccharides, because saccharides N-acetylneuraminic acid

absorb at low wavelengths (around

190nm), due to the ns transition. In
this wavelength range, the analysis is
often affected by the absorption of the
mobile phase and interfering
substances. As a result, this detector is
used only for the analysis of uronic
and sialic acids, which absorb in the
200 to 210nm wavelength range. Conditions
Figure 13 shows a chromatogram of a column: Shim-pack SCR-101H (300mm × 7.9mm i.d.)
sialic acid obtained in acid hydrolysis mobile phase: 10mM phosphoric acid aqueous solution
flow rate: 1.5 mL/min
of serum.
temperature: 55˚C
detection: absorbance at 205nm
4.2. Differential Refractive Index Fig. 13. Absorption detection for determination of
Detection sialic acid in hydrolyzed human serum.
Differential refractive index
detectors are now widely used for the
HPLC analysis of saccharides. Highly
sensitive and stable refractive index
detectors are now commercially
available, permitting detection of
Conditions
saccharides at concentrations of less
column: Shim-pack SCR-
than 100ng. Figure 14 shows an 101N (300mm ×
example of high-sensitivity HPLC 7.9mm i.d.)
analysis of saccharides, using a mobile phase: water
differential refractive index detector. flow rate: 1.0 mL/min
Though often used for the HPLC temperature: 50˚C
detection: refractive index
analysis of saccharides, differential
Peaks
refractive index detectors do not
1: sucrose, 2: glucose,
always give satisfactory results at the 3: fructose, each 200ng.
trace quantity level, when analyzing
Fig. 14. Refractive index detection for
samples which contain interfering
determination of saccharides.

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 substances, like biological samples. Another drawback to this type of detector is that it is
incompatible with gradient elution. This means that it cannot be used for anion exchange
chromatography by the boric acid buffer solution method (see Section 3.4), effective for the
analysis of multi-component samples.

4.3. Derivatization (Chemical Reaction Detection)

In HPLC, detection sensitivity and selectivity are often enhanced by derivatizing (chemically
treating) the target compounds with suitable reagents, changing them into compounds that
absorb ultraviolet and/or visible light, or that fluoresce under excitation. This method is
especially effective for biochemical applications, where it is necessary to accurately and
precisely detect trace components in complex samples. Many methods of derivatization have
been developed and have come to be widely used.
Derivatization is also useful for the HPLC analysis of saccharides, where both the pre-
column and post-column methods are used.
4.3.1. Pre-Column Derivatization
In pre-column derivatization, since the target compounds are derivatized prior to
introduction into the analytical column, the reagents, reaction time, reaction temperature,
etc. can be selected from a wider range, when compared to post-column derivatization.
Even if the reagents are detected, no problem occurs (provided that they are separated
from the derivatives of the target compounds). One requirement for routine analysis,
however, it that the sample pretreatment be made simply and rapidly.
For the pre-column deriva-tization of saccharides, both the esterification method and
fluorescing derivatization meth-ods, using dansylhydrazine and 2-aminopyridine as the
reagents, are used effectively. The 2-aminopyridine reagent is especially effective for the
study of oligosaccharide chains of sugar proteins.
4.3.2. Post-Column Derivatization
In post-column derivatization, target compounds are derivatized after separation in the
column. This method has several requirements. Since the derivatizing reagent(s) are
continuously added to the mobile phase, they must not be detectable. To prevent peaks
from broadening, the reaction time must be as short as possible. Finally, to make the
system design simple and the operation easy, the number of reagents must be as low as
possible. Despite these requirements, the post-column derivatization method has certain
noteworthy advantages; it shows better reproducibility and linearity, and it is suitable for
routine analyses because the derivatizing reaction can be performed in an automated
sequence.
The conventional post-column derivatization detection method for saccharides is to use
oricinol sulfate or phenol sulfate as the reaction reagent and to detect derivatives by
visible absorption spectrophotometry. The drawback to this method is that strong acid
must be hand-led. Blue tetrazolium, 2,2’-bicinchoninic acid, p-hydro-xybenzoic acid
hydrazide, etc. react with reducing sugars under basic conditions to form derivatives
detectable by visible absorption spectrophotometry.

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 In anion exchange
chromatography with boric acid
buffer solution (see Section
3.4), the column effluent is
heated to convert the eluting
compounds into UV-absorbing
substances, without using any
reagent. This method cannot
provide the same level of
sensitivity as derivatization,
however.
Derivatization of sample Apparatus
components into fluorescing 1: pumps, 2: mobile phase, 3: sample injector, 4: column,
5: column oven, 6: pump, 7: reagent, 8: reaction coil,
compounds provides higher
9: reaction chamber, 10: fluorescence detector,
sensitivity than any other
11: data processor, 12: resistance coil, 13: waste.
method. Ethylenediamine,
Fig. 15. Flow diagram of HPLC with post-column
ethanolamine, 2-cyanoaceto-
derivatization using arginine.
amide, 2-aminopropylnitrile
fumarate, taurine, arginine, and
p-methoxybenzamidine are the
reagents now widely used in the
post-column derivatization of
saccharides.
Figure 15 shows the flow
diagram of the Shimadzu
saccharide analysis system (for
a gradient elution system),
which uses the post-column
fluorescence-derivatization
method, with arginine as the Conditions
column: Shim-pack ISA-07 (250mm × 4.0mm i.d.)
reaction reagent. mobile phase: 300mM (potassium)borate buffer <pH8.5>
Saccharides can be analyzed flow rate: 0.6 mL/min
by any combination of normal temperature: 65˚C
detection: post-column derivatization with arginine,
phase chromatography, size fluorescence at Ex:320nm, Em:430nm.
exclusion chromatography, and Peaks
ligand exchange chromato- 1: sucrose, 2: maltose, 3: rhamnose, 4: mannose,
5: fructose, 6: galactose, 7: xylose, 8: glucose, each
graphy. Combining anion 100pmol except sucrose (1nmol).
exchange chromatography with Fig. 16. Fluorescence detection with post-column
the saccharide/ boric acid derivatization for determination of saccharides.
complexes provides the best
results. Figure 16 shows an
example of high sensitivity

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 detection of saccharides. Figure
17 presents an example of the
gradient elution HPLC analysis
of a multi-component sample.
Electrochemical detectors are
also used for the HPLC analysis
of saccharides.
5. Conclusion
We have detailed the typical methods
of separation and detection for the HPLC
analysis of saccharides. We expect that
higher performance column packing
materials and new methods of detection
will be developed, so that the HPLC
analysis of saccharides will play an even
more important role in numerous fields.
Conditions
column: Shim-pack ISA-07 (250mm × 4.0mm i.d.)
mobile phase: A: 100mM (potassium)borate buffer <pH8.0>
B: 400mM (potassium)borate buffer <pH9.0>
gradient elution from A to B in 50min
flow rate: 0.6 mL/min
temperature: 65˚C
detection: post-column derivatization with arginine,
fluorescence at Ex:320nm, Em:430nm.
Peaks
1: sucrose, 2: cellobiose, 3: maltose, 4: lactose,
5: rhamnose, 6: ribose, 7: mannose, 8: arabinose,
9: galactose, 10: isomaltose, 11: xylose, 12: glucose.
Fig. 17. Chromatogram of saccharides obtained
by post-column derivatization.
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