Improved Mono-, Di-, and Oligosaccharide
Separation on CarboPac Columns with
Lower Eluting Hydroxide Concentration
Ting Zheng, Jim Thayer, Srinivasa Rao, Chris Pohl, Dionex Corporation, Sunnyvale, CA, USA
Introduction
Columns
High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) has been widely used for separation
and detection of carbohydrates ranging from simple monosaccharides to
branched oligosaccharides, and large linear polysaccharides.1,2
CarboPac® columns are a family of high-resolution, strong anionexchange columns developed for carbohydrate separations with
HPAE-PAD under alkaline conditions. They have been proven to be
capable of separating monosaccharides; positional, linkage, and branch
isomers of oligosaccharides; and homopolymer oligosaccharides that
differ only in length.3,4 Sodium hydroxide or potassium hydroxide is
commonly used to elute carbohydrates on these columns. Sodium
acetate is sometimes added to elute multiply-charged carbohydrate
species that strongly bind to these columns. Here we demonstrate that
the separation of carbohydrates on the CarboPac PA20 and CarboPac
PA200 columns can be improved by adjusting the concentration of the
eluting hydroxide solution.
CarboPac PA20 analytical column (3 × 150 mm, Dionex, P/N 060142)
EXPERIMENTAL
Instrumentation
Analyses were performed on a Dionex ICS-3000 chromatography
system, which includes an ICS-3000 DP gradient pump, EG Eluent Generator with EluGen® EGC-KOH cartridge and continuously-regenerated
anion trap column (CR-ATC), AS Autosampler, and DC Detector/Chromatography compartment with an electrochemical cell. The carbohydrates were detected by pulsed amperometry with a gold electrode and
an Ag/AgCl reference electrode using the standard quadruple waveform
developed at Dionex.5
Chromatography was controlled by Chromeleon® chromatography
management software (Dionex Corporation).
CarboPac PA20 guard column (3 × 30 mm, Dionex, P/N 060144)
CarboPac PA200 analytical column (3 × 250 mm, Dionex, P/N 062896)
CarboPac PA200 guard column (3 × 50 mm, Dionex, P/N 062895)
Reagents and Standards
Mono- and disaccharide standards (Sigma)
Oligosaccharide standards (Dextra labs)
PNGase F (Bio-rad)
Deionized water, Type 1 reagent grade, 18.2 MΩ-cm resistivity
Sodium acetate, HPLC grade (Dionex, P/N 053926)
Sodium hydroxide, 50% (w/w) (Fisher Chemicals, P/N SS254-500)
RESULTS
Separation of Mono- and Disaccharides on the
CarboPac PA20 Column
The CarboPac PA20 column was developed to provide fast, efficient
separations for mono- and disaccharides. Rapid separation of common
monosaccharides is usually achieved by isocratic elution with a
12–20 mM NaOH or KOH solution on this column. Here we demonstrate
that the separation of a mix of eight mono- and disaccharides, commonly found in plant hydrolysates, can be improved by using lower
hydroxide concentrations than commonly employed (Figure 1). At
30 ˚C with a flow rate of 0.5 mL/min, optimal separation was achieved
by isocratic elution with 3.5 mM KOH produced by the EG. At 25 ˚C
and 0.4 mL/min, the eight sugars can be baseline resolved by isocratic
elution with 2 mM KOH (Figure 2). The EG eluent regenerator produces
carbonate-free hydroxide solution, and therefore column washing with
high concentration hydroxide and reconditioning are not needed. This
can reduce the analysis time for each cycle from ~45 min to ~25 min,
which greatly improves the throughput.
1
Separation of Neutral Oligosaccharides on the
CarboPac PA200 Column
Peaks: 1. Fucose
2. Arabinose
3. Galactose
4. Glucose
5. Sucrose
6. Xylose
7. Mannose
8. Fructose
Column:
CarboPac PA20
(3 × 150 mm)
with guard
Flow Rate:
0.5 mL/min
Column Temperature: 30 °C
Injection Volume:
20 µL
Sample Concentration: 5–10 µM
1 mM KOH
1
2
4
3
2 mM KOH
6
5
8
7
3 mM KOH
5 mM KOH
6 mM KOH
nC
9 mM KOH
The CarboPac PA200 column is designed to provide high-resolution separations for oligosaccharides. In many applications the CarboPac PA200
column produces better resolution than the CarboPac PA100 column.6 A
common gradient used for the PA200 and PA100 columns is 0–200 mM
sodium acetate in 100 mM sodium hydroxide. However, this gradient has
proven incapable of resolving many neutral oligosaccharides, especially
neutral N-linked glycans. Improved separation of neutral N-glycan
oligosaccharides was achieved by decreasing the NaOH concentration.
Figure 3 shows the effect of NaOH concentration on the separation of a
mixture of seven neutral N-glycan oligosaccharides.
12 mM KOH
15 mM KOH
CarboPac PA200
(3 × 250 mm) with guard
Flow Rate:
0.5 mL/min
Column Temp.: 30 °C
Gradient:
0–5 mM NaOAc
in different concentrations
of NaOH from 0–40 min
Inj. Volume:
20 µL
Sample Conc.: 5–10 µM
0.1
2.5
5.0
7.5
10.0
12.5
Minutes
15.0
17.5
20.0
25.0
22.5
Figure 1. Separation of eight mono- and disaccharides on the CarboPac PA20
column by isocratic elution with different concentrations of KOH.
40
Peaks: 1.
2.
3.
4,5.
6.
7.
1
Column:
Flow Rate:
CarboPac PA20 (3 × 150 mm) with guard
(A) 0.5 mL/min
(B) 0.4 mL/min
Column Temp.: (A) 30 °C
(B) 25 °C
Eluent:
(A) Isocratic elution with 3.5 mM KOH
(B) Isocratic elution with 2 mM KOH
1
4
2
1
60 mM NaOH
8
2
4
6
8
10
12
14
16 18 20
Minutes
16
20
24
Minutes
28
32
36
22
24
Figure 3. Separation of a mix of neutral oligosaccharides on the CarboPac
PA200 column.
8
5
B
12
6
3
7
26
28
30
32
34
Figure 2. Optimized separation of eight mono- and disaccharides on the
CarboPac PA 20 column. Optimizing KOH concentration, column temperature,
and flow rate allows baseline separation of all eight saccharides.
2
100 mM NaOH
0
4
2
7
nC
8
nC
4 5
6
80 mM NaOH
5
A
2 3
50 mM NaOH
6
3
7
0
40 mM NaOH
Peaks: 1. Fucose
2. Arabinose
3. Galactose
4. Glucose
5. Sucrose
6. Xylose
7. Mannose
8. Fructose
G0
Man5
G0-F
Two isomers of G1
Man6
G2
Oxygen dip
Column:
18 mM KOH
Improved Mono-, Di-, and Oligosaccharide Separation on
CarboPac Columns with Lower Eluting Hydroxide Concentration
Decreasing NaOH concentration resulted in longer retention time and
under certain conditions sample peaks eluted in the “oxygen dip”
(~32 min, Figure 3). The oxygen dip is due to oxygen dissolved in the
sample being introduced into the column upon injection. In certain
cases, this can be minimized by using a full-loop injection. By selection
of appropriate adjustments to the flow rate and hydroxide concentration, we moved the peaks away from the oxygen dip. Figure 4 shows the
separation of the mixed oligosaccharides with the optimized gradient,
which is overlaid with individual oligosaccharide standards. The two
fucosylated Man3GlcNAc4Gal linkage isomers (G1 isomers, Table 1)
were well resolved with this gradient.
Structure
Standard
GlcN
Man 5
G
M Mannnose
N-acetyl-glucosamine
M α1–6
M α1–6
M α1–4 GlcN
M α1–3
M α1–3
M
α1–6 M
Man 6
Column:
CarboPac PA200
(3 × 250 mm) with guard
Flow Rate:
0.35 mL/min
Column Temp.: 30 °C
Gradient:
1–8 mM NaOAc in 50 mM
of NaOH from 0–60 min
Inj. Volume:
20 µL
Sample Conc.: 5–10 µM
Peaks: 1.
2.
3.
4,5.
6.
7.
G0
Man5
G0-F
Two isomers of G1
Man6
G2
M
α1–3
M α1–2
GlcN
G0-F
GlcN
1
nC
6
3
4
7
Oxygen dip
2
15
5
α1–6
M α1–4 GlcN
M
F
Galactose
Fucose
β1–4 GlcN
β1–4 GlcN
α1–3
β1–2 M α1–6
M β1–4 GlcN
β1–2 M α1–3
β1–4 GlcN
F
G2
Man6
G β1–4 GlcN
G β1–4 GlcN
β1–2 M α1–6
α1–6
M β1–4 GlcN β1–4 GlcN
β1–2 M α1–3
G β1–4 GlcN
β1–2 M α1–6
Man5
F
G1
0
10
15
20
25
30
35
Minutes
40
45
50
55
60
GlcN
α1–6
M β1–4 GlcN β1–4 GlcN
β1–2 M α1–3
G1
Figure 4. Optimized separation for the mixed neutral oligosaccharides on the
CarboPac PA200 column.
F
GlcN
G β1–4 GlcN
β1–2 M α1–6
α1–6
M β1–4 GlcN β1–4 GlcN
β1–2 M α1–3
F
G0
GlcN
GlcN
β1–2 M α1–6
α1–6
M β1–4 GlcN β1–4 GlcN
β1–2 M α1–3
Table 1. Structures of the oligosaccharide standards.
3
CONCLUSIONS
Profiling of N-glycans Cleaved from Two
Glycoproteins on CarboPac PA200
N-glycans were cleaved from two glycoproteins with PNGase F. After
digestion, proteins were removed by ethanol precipitation. The released
N-glycans were vacuum dried and then resuspended in deionized water
before separation on a CarboPac PA200 column with the optimized
gradient. The major species were well resolved and correlated well with
the standards (Figure 5).
Column:
CarboPac PA200
(3 × 250 mm) with guard
Flow Rate:
0.35 mL/min
Column Temp.: 30 °C
Gradient:
1–8 mM NaOAc in 50 mM
of NaOH from 0–60 min
Inj. Volume:
20 µL
Sample Conc.: 5–10 µM
Peaks: 1.
2.
3.
4,5.
6.
7.
G0
Man5
G0-F
Two isomers of G1
Man6
G2
50
nC
Protein 2 glycans
Protein 1 glycans
Glycan standard mix
0
0
5
10
15
20
References
6
1 23
25
7
45
• Separation of a mixture of eight mono- and disaccharides on the
CarboPac PA20 column was improved by reducing KOH concentration from the commonly used range of 12–20 mM down to 3.5 mM.
The mixture was baseline resolved with 2 mM KOH isocratic elution
at 25 ˚C. This method may be useful for biofuel research and food
industries that are interested in mono- and disaccharide analysis.
• We developed a method optimized for separation of a mixture of
seven neutral N-glycan type oligosaccharides on the CarboPac
PA200 column. The method achieves near baseline resolution by
reducing the NaOH concentration to 50 mM. We also adjusted the
flow rate and NaOAc concentration gradient to move the N-glycan
elution positions away from the oxygen dip. This method is useful
in the biotechnology and pharmaceutical industries for analysis of
glycans released from glycoproteins during development and quality
control of glycoprotein therapeutics.
• Selectivity of carbohydrate retention on CarboPac columns is controlled by adjusting the eluting NaOH concentration. The concentration of the eluting NaOH is an important factor to consider when
optimizing carbohydrate separations on these columns.
30
35
Minutes
40
45
50
55
60
Figure 5. Profiling of the glycans cleaved from two glycoproteins, separated on a
CarboPac PA200 column using the optimized gradient.
1. Lee, Y.C. Carbohydrate Analyses with High-Performance AnionExchange Chromatography. J. Chromatogr. A, 1996, 720, 137–149.
2. Hardy, M.R.; Townsend, R.R. Separation of Positional Isomers of
Oligosaccharides and Glycopeptides by High-Performance AnionExchange Chromatography with Pulsed Amperometric Detection.
Proc. Natl. Acad. Sci. USA 1988, 85 (10), 3289–3293.
3. Dionex Corporation. Product Manual for the CarboPac PA20 Guard
Column and Analytical Column. Doc. No. 031884-03, Sunnyvale,
CA, 2005.
4. Dionex Corporation. Product Manual for the CarboPac MA1, PA1,
PA10, and PA100 Guard Columns and Analytical Columns.
Doc. No. 031824-06, Sunnyvale, CA, 2005.
5. Dionex Corporation. Optimal Settings for Pulsed Amperometric
Detection of Carbohydrates Using the Dionex ED40 Electrochemical
Detector. Technical Note 21, LPN 034889-03, Sunnyvale, CA, 1998.
6. Dionex Corporation. CarboPac PA200 Column Solutions for
Oligosaccharide Analysis Data Sheet. LPN 1618-03, Sunnyvale,
CA, 2006.
Note: All Dionex product manuals, data sheets, and technical notes are
available at www.dionex.com.
CarboPac, Chromeleon, and EluGen, are registered trademarks of Dionex Corporation.
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