columns
Anion-exchange columns for the
analysis of mono- and disaccharides
in glycoprotein therapeutics, foods,
and beverages:
• Simple, direct approach using pulsed electrochemical detection
• Disposable gold electrodes
• Glycoprotein monosaccharide
compositional analysis
• No derivatization required
• Specialized traps for interferencefree quantification
• Rapid and rugged methods
CarboPac PA20 Column
Simple, Direct Approach Using Pulsed
Electrochemical Detection
The CarboPac® PA20 column
has been developed to give fast,
efficient separations of simple sugars
without compromising resolution.
Carbohydrates, without derivatization,
are separated by anion-exchange
chromatography at high pH and detected
by pulsed electrochemical detection.
Electrochemical detection is
used to measure the current resulting
from oxidation or reduction of analyte
molecules at the surface of a working
electrode. During oxidation reactions,
electrons are transferred from
molecules of electroactive analytes,
such as carbohydrates, to the working
Passion. Power. Productivity.
electrode in the electro­chemical cell.
Detection is sensitive and highly
selective for electroactive species,
because many potentially interfering
species cannot be oxidized or reduced
and are not detected.
When a single potential is applied
to the working electrode, the detection
method is dc amperometry. Pulsed
amperometry, which uses a repeating
sequence of potentials, is the technique
employed for carbohydrate analysis,
and is a reproducible and sensitive
method for the detection of all
carbohydrates of molecular weight up
to 10,000.
Rapid and Rugged Methods
The CarboPac PA20 column has
been designed to provide excellent
resolution between the six underivatized
monosaccharides commonly found in
mammalian glycoproteins under a variety
of sodium hydroxide concentrations.
These conditions can be optimized
depending on the goal of the
separation. Figure 1 illustrates the
effect of increasing the hydroxide
concentration on the elution time.
Innovative Resin Technology
The CarboPac PA20 column uses
6.5 µm pellicullar resin technology to
provide high resolution and efficiencies
for the six common monosaccharides
found in glycoprotein hydrolysates. The
smaller resin particle is agglomerated with
an optimized latex that further improves
the column performance by imparting a
unique selectivity. This selectivity results in
excellent resolution between galactose and
glucosamine, analytes whose separation
has historically been problematic. This
optimized anion-exchange material is
packed in 3 × 150 mm and 0.4 × 150 mm
PEEK™ hardware formats which provide
fast separations (mannose elutes in 9 min).
The recommended low flow rates
reduce eluent consumption and enable
system operation over extended periods
without user intervention. The CarboPac
0.4 × 150 mm capillary column offers
the additional advantages of higher mass
sensitivity, lower sample amounts, and
significantly lower solvent use.
Glycoprotein Monosaccharide
Compositional Analysis
Many mammalian proteins have
carbohydrates attached to them. In many
cases, the presence of the carbohydrate
controls the biological activity of the
protein or the rate at which it is cleared
from the system. For example, certain
glycosylated forms of tissue plasminogen
activator (tPA) have more enzymatic
activity than others. Thus, the study of
protein glycosylation is important to
Column:
Eluent:
Flow Rate:
Detection:
60
Waveform:
CarboPac PA20 (3 x 150 mm)
8–20 mM NaOH
0.5 mL/min
Pulsed electrochemical,
gold electrode
Quadruple potential
Peaks:
1. Fucose
2. Galactosamine
3. Glucosamine
4. Galactose
5. Glucose
6. Mannose
2
1
3
45
6
20 mM NaOH
nC
16 mM NaOH
12 mM NaOH
8 mM NaOH
0
0
5
10
15
Minutes
20
25
30
18770
Figure 1. Effect of hydroxide concentration on elution times.
Column:
Gradient:
Flow Rate:
Detection:
Waveform:
Sample:
750
CarboPac PA20 and guard
12 mM NaOH, 200 mM NaOH regeneration for 6 min
0.5 mL/min
Pulsed electrochemical detection, Au electrode
Quadruple potential
30 µL reconstituted hydrolyzed serum
Peaks:
1. Galactosamine
2. Glucosamine
3. Galactose
4. Glucose
5. Mannose
nC
2
1
0
5
0
4
3
5
10
15
Minutes
20
Figure 2. Analysis of monosaccharides from hydrolyzed rabbit serum.
many scientists, including those making a
recombinant protein for therapeutic use.
Figure 2 shows the analysis of
underivatized monosaccharides from
hydrolyzed rabbit serum using the
CarboPac PA20 column.
2
25
30
18771
Sialic Acid Analysis
Sialic acids are a family of N- and
O-substituted neuraminic acids that
play an important role in physiology.
They occupy terminal positions on
many glycoproteins and serve as
markers for protein removal from
blood circulation. The amino group of
neuraminic acid is linked to either an
N-acetyl or an N-glycolyl group. These
yield N-acetylneuraminic acid (NANA)
or N-glycolylneuraminic acid (NGNA),
respectively. Figure 3 shows the separation of NANA and NGNA from
bovine fetuin hydrolyzed with 0.1 M
HCl. Using a sodium acetate gradient,
NANA and NGNA are well resolved
in 12 min.
400
Column:
Gradient:
Flow Rate:
Detection:
Waveform:
Samples:
NANA
nC
CarboPac PA20 (3 × 150 mm)
20–200 mM NaOAc in 0.1 M NaOH over 10 min
0.5 mL/min
Pulsed electrochemical, disposable Au electrode
Quadruple potential
A. 0.1 M HCl fetuin hydrolysate
(10 µg injected, 80 ˚C, 1 h)
B. NANA and NGNA standard
NGNA
A
NGNA
standard
NANA
standard
B
0
0
2
4
6
8
10
12
14
16
20
18
Minutes
18772
Figure 3. Sialic acid analysis of bovine fetuin.
Food Applications
Monosaccharides important in
food analysis are typically separated at
eluent concentrations lower than those
used for glycoprotein mono­saccharides.
Coffee sugars, such as mannitol,
arabinose, galactose, glucose, xylose,
mannose, and fructose, can be separated
using 2 mM sodium hydroxide. High
hydroxide concentrations can be used
to acclerate the analyis of well-resolved
sugars. Figure 4 shows the separation
of glucose, fructose, and sucrose from
chocolate syrup in less than 6 min
using 50 mM hydroxide.
Monosaccharides important in
dietary fiber analysis require higher
concentrations of sodium hydroxide for
timely elution and are readily eluted in
< 12 min with 52 mM sodium hydroxide.
Figure 5 shows the separation of mono­
saccharides, important in dietary fiber
analysis, with good resolution between
the sugar alcohols and sugars in a single
isocratic run.
60
Column:
Eluent:
Flow Rate:
Temperature:
Detection:
1
Waveform:
Sample:
Peaks:
nC
1. Glucose
2. Fructose
3. Sucrose
4. Unidentified
5. Unidentified
3
2
CarboPac PA20 and guard
50 mM NaOH
0.5 mL/min
30 ˚C
Pulsed electrochemical,
Au electrode
Quadruple potential
1:10,000 dilution of chocolate syrup
5
4
0
0
2
4
6
8
10
Minutes
12
14
16
18
Figure 4. Analysis of chocolate syrup using the CarboPac PA20 column.
3
20
18773
Pharmaceuticals
The United States Food and
Drug Administration (U.S. FDA) and
regulatory agencies in other countries
require that pharmaceutical products be
tested for composition to verify their
identity, strength, quality, and purity.
Recently, attention has been given to
inactive ingredients as well as active
ingredients. Many of these ingredients
are nonchromophoric and cannot be
visualized by absorbance detection.
However, carbohydrates, glycols,
sugar alcohols, and sulfur-containing
compounds can be oxidized and therefore
detected by electro­chemical detection.
Figure 6 shows the analysis of a
pediatric acetominophen elixir using the
CarboPac PA20 column. Using these
conditions, glycols, sugar alcohols, and
carbohydrates are all separated in a single
run. Figure 7 shows the analysis of a
pediatric oral decongestant under the
same conditions.
Fermentation Broths
Fermentation broth cultures have
the best chance of producing optimum
yield when the concentration of nutrients
and amino acids are determined
on-line and continuously monitored. The
CarboPac PA20 column can be used online to monitor fermentation broths and
cell cultures. Alternatively, if monitoring
the amino acid content is of interest, the
AminoPac® PA10 column should be
considered for direct detection on-line.
Figure 8 shows an analysis of a yeast
extract-peptone-dextrose (YPD) broth
supernatant. As illustrated, a large
concentration of glucose is present with
low concentrations of other sugars.
Column:
Gradient:
Flow Rate:
Detection:
50
5
CarboPac PA20 (3 × 150 mm)
52 mM NaOH
0.5 mL/min
Pulsed electrochemical,
disposable gold electrode
Quadruple potential
Waveform:
Peaks:
nC
2 3
1
1. Glycerol
2. Xylitol
3. Sorbitol
4. Mannitol
5. Glucose
6. Fructose
7. Sucrose
8. Lactose
8
4
7
6
0
0
5
10
Minutes
15
18774
Figure 5. Separation of monosaccharides important in dietary fiber analysis.
3
250
Column:
Eluent:
CarboPac PA20 and Guard
10 mM NaOH followed by
200 mM NaOH for 10 min to
regenerate the column
0.5 mL/min
30 ˚C
Pulsed electrochemical,
Au electrode
Quadruple potential
1:10,000 dilution pediatric
acetominophen elixir
Flow Rate:
Temperature:
Detection:
Waveform:
Sample:
2
nC
Peaks:
6
1. Propylene glycol
2. Glycerol
3. Sorbitol
4. Unidentified
5. Glucose
6. Sucrose
7. Fructose
1
4
5
7
10
0
2
4
6
Minutes
8
10
12
14
18775
Figure 6. Analysis of a pediatric acetominophen elixir.
Column:
Eluent:
Flow Rate:
Temperature:
Detection:
250
Waveform:
CarboPac PA20 and Guard
10 mM NaOH then 200 mM
NaOH regeneration
0.5 mL/min
30 ˚C
Pulsed electrochemical,
Au electrode,
Quadruple potential
Sample:
Peaks:
1:10,000 dilution of decongestant
1. Sorbitol
2. Glucose
3. Fructose
2
1
nC
3
0
0
2
4
6
8
Minutes
Figure 7. Analysis of a pediatric oral decongestant.
4
10
12
14
18776
Specialized Borate Trap for
Interference-Free Quantification
Borate is one of the first ions to
break through a water deionization
system. Its presence in the carbohydrate
eluents can cause a significant loss of
peak efficiency, especially for mannose
and reduced mono­saccharides. Borate
can affect monosaccharide peak
symmetry, even when present in the
low-µg/L concentration range. Figure
9 illustrates the use of the BorateTrap™
column with a CarboPac PA10 column.
This product can also be used with
CarboPac PA20 and CarboPac PA1
columns. The BorateTrap column,
installed immediately before the
injection valve, removes borate from
the eluent.
Specialized Amino Acid Trap for
Interference-Free Quantification
If the samples are glycoprotein
hydrolysates with a high ratio of
amino acids to carbohydrates, the
AminoTrap™ column is the optimal
guard column and replaces the standard
guard column. Some amino acids, such
as lysine, foul the working electrode.
They elute as a severely tailing peak
and affect the symmetry of later eluting
carbohydrates. As illustrated in
Figure 10, the AminoTrap column
delays amino acids out of the
carbohydrate elution window. This
displacement of amino acid peaks
results in cleaner chrom­atography of
monosaccharides.
Column:
Eluent:
Flow Rate:
Temperature:
Detection:
4
28
Waveform:
Sample:
3
Peaks:
1.
2.
3.
4.
5.
6.
7-9
1
nC
2
5
7
6
10
5
Glycerol
Inositol
Trehalose
Glucose
Sucrose
Fructose
Unidentified
9
8
18
0
CarboPac PA20 and Guard
10 mM NaOH followed by 200 mM NaOH
for 10 min to regenerate the column
0.5 mL/min
30 ˚C
Pulsed electrochemical,
Au electrode
Quadruple potential
1:1,000 dilution of YPD broth supernatant
15
20
25
30
Minutes
35
40
18775
Figure 8. Analysis of yeast extract-peptone-dextrose (YPD) broth supernatant.
Without Borate Trap
Column:
2
90
Eluent:
Flow Rate:
Detection:
3
1
nC
5
4
Waveform:
6
-5
0
Peaks:
10
5
With Borate Trap
CarboPac PA10
with and without AminoTrap
18 mM NaOH
1.5 mL/min
Pulsed electrochemical,
Au Electrode
Quadruple potential
1. Fucose
2. Galactosamine
3. Glucosamine
4. Galactose
5. Glucose
6. Mannose
2
100
3
1
nC
4
5
6
-5
0
5
10
12234
Figure 9. Effect of borate and the BorateTrap column on monosaccharide peak symmetry.
5
Disposable Gold Electrodes
Although carbohydrates can be
oxidized at a gold working electrode,
the products of the oxidation reaction
foul the surface of the electrode,
inhibiting further analyte oxidation.
Electrode fouling with oxidation
byproducts or sample components may
reduce response, requiring polishing to
restore the surface.
Dionex disposable gold electrodes
(Figure 11) eliminate the need for
electrode reconditioning. Disposable
electrodes are economical, and thus can
be replaced frequently.
Frequent replacement of
working electrodes renders electro­
chemical detection more predictable
and reproducible. The peak area
reproducibility for five disposable
electrodes is shown in Table 1.
Column:
CarboPac PA20 ( 3 × 150 mm)
+/- AminoTrap (3 × 30 mm)
Eluent:
12 mM NaOH
Flow Rate: 0.5 mL/min
Detection: Pulsed electrochemical,
Au electrode
Waveform: Quadruple potential
80
With AminoTrap
4
Peaks:
3
nC
1.
2.
3.
4.
No AminoTrap
2
1. Fucose
2. Galactosamine
3. Glucosamine
4. Galactose
5. Glucose
6. Mannose
Mix of 6 standard with no AminoTrap
Protein hydrolysate with no AminoTrap
Mix of 6 standard with AminoTrap
Protein hydrolysate with AminoTrap
1
-50
0
5
10
15
Minutes
20
25
30
18778
Figure 10. Profiling MAb hydrolysate on the CarboPac PA20 column, with and without an
AminoTrap column.
Figure 11. Illustration of the disposable gold electrode.
Table 1. Area Reproducibility:
Electrode-to-Electrode for Five Disposable Electrodes
Electrode
Average
Fuc
GalN
GlcN
1
n = 12
3.09
7.05
5.58
2
n = 12
3.13
7.49
3
n = 12
3.35
4
n = 12
5
n = 12
Average
S.D
Elec-to-elec
Perm. Elec
6
Gal
Glc
Man
4.92
5.82
4.22
5.95
4.68
6.24
4.25
7.65
6.04
5.32
6.23
4.52
3.12
6.76
5.33
4.70
5.52
4.18
3.12
6.93
5.46
4.83
5.54
4.20
3.16
7.18
5.67
4.89
5.87
4.27
0.11
0.38
0.31
0.26
0.36
0.14
RSD
3.41
5.30
5.44
4.35
6.05
3.28
n = 12
2.91
5.79
4.36
4.84
4.51
3.64
ORDERING IN FORMATION
To order in the U.S., call (800) 346-6390 or contact the Dionex Regional
Office nearest you. Outside the U.S., order through your local Dionex office or
distributor. Refer to the following part numbers.
CarboPac PA20 Fast Monosaccharide Columns
CarboPac PA20 Analytical Column (3 × 150 mm)................................... 060142
CarboPac PA20 Guard Column (3 × 30 mm)........................................... 060144
CarboPac PA20 Capillary Column (0.4 × 150 mm)................................. 072072
CarboPac PA20 Guard Column (0.4 × 35 mm)........................................ 072073
AminoTrap (3 × 30 mm)........................................................................... 060146
BorateTrap (4 × 50 mm)........................................................................... 047078
Electrochemical Cells and Electrodes
Electrochemical Detector (ED) without Cell............................................ 061718
ED with Reference Electrode and Spacer Block (no working electrode).061756
Disposable Gold Electrodes, 6 Electrodes (6 pack).................................. 060139
Disposable Gold Electrodes, 24 Electrodes (4 bundled 6 pack)............... 060216
AAA-Direct™ Disposable Gold Working Electrode (6 pack) ................. 060082
Gold on PTFE Disposable Electrode (6 pack).......................................... 066480
Gold Conventional Working Electrode..................................................... 061749
AAA-Certified™ Conventional Gold Working Electrode......................... 063722
Palladium Hydrogen Reference Electrode (PdH)..................................... 072075
pH-Ag/AgCl Reference Electrode............................................................ 061879
CARBOPAC PA20
column SPECIFICATIO N S
Resin Composition:
6.5 µm diameter substrate (ethylvinylbenzene
55% cross-linked with
divinylbenzene; agglomerated
with 130 nm micro­­bead latex
with difunctional quaternary
ammonium ion, 5% cross-linked)
Anion-Exchange Capacity:
65 µeq/column (3 × 150 mm
column)
1.16 µeq/column (0.4 × 150 mm
column)
Maximum operating pressure:
3500 psi (21 MPa)
Chemical Compatibility:
pH 0–14, 100% compatible with
common HPLC solvents
Temperature Range:
4–60 ˚C
Test Procedure:
Baseline resolution of fucose,
galactosamine, glucosamine,
galactose, glucose, mannose
Recommended Operating
Temperature:
30 ˚C
Recommended Flow Rate:
0.5 mL/min for 3 × 150 mm column
0.01 mL/min for 0.4 × 150 mm column
Ionic Form Eluents:
Sodium hydroxide, sodium
acetate
7
AminoPac and CarboPac are registered trademarks,
and AminoTrap, BorateTrap, AAA-Direct, and
AAA-Certified are trademarks of Dionex Corporation.
PEEK is a trademark of Victrex PLC.
Passion. Power. Productivity.
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America
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LPN 1501-01 PDF 02/10
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