HPAEC analysis of monosaccharides
Standard Operational Procedure
Jagger
HPAEC analysis of monosaccharides
Introduction
The chromatographic principle described in the following is based on anion exchange, which is
appropriate for analyzing carbohydrates. This SOP describes monosaccharide analysis of arabinose,
galactose, glucose, xylose and mannose which are the major monomer components in biomass cellulose
and hemicelluloses. Other monosaccharides can also be separated by HPAEC, but this will require
adjustments to the methods presented here. For development of modified programs for other
monosaccharides please consult Dionex application note: Determination of monosaccharides in acidhydrolyzed corn stover using HPAEC-PAD (#73820) or the super users. Some general principles are
determining the separation of monosaccharides:
Anion exchange is performed at high pH to promote deprotonation of the analytes hydroxyl and
carboxyls groups. Typically for carbohydrates, this results in one negative charge per hydroxyl group.
The columns stationary phase is positively charged and will thereby interact with the negative
counter-ions in the mobile phase.
Generally the more negative charges per molecule, the stronger the affinity to the stationary phase
and thereby the longer the retention time, but for monosaccharides in particular the basis for
separation (differences in chemical properties) is very small and therefore also alignment/spatial
configuration of the hydroxyl groups are important for separation.
As a result of this, eluting conditions needs to be very weak to distinguish the minor differences
between analytes.
Traditionally, xylose and mannose are particularly difficult to separate and if both are present in a
sample, even weaker elution conditions needs to be applied. In this SOP two methods are described,
one for good resolution of all five (see above) monosaccharides (a longer and less sensitive method)
and one where xylose and mannose will be co-eluting if present in the sample (faster and higher
sensitivity). In case of doubt, run a few key samples at the first method to clarify whether both
xylose and mannose are present. Both runs are isocratic.
pH is very important for detection (pulsed amperometric detection) which at the correct conditions
creates high sensitivity compared to other detection principles. Eluents for HPAEC is always 0.1 M
NaOH (A), 1 M NaOAc in 0.1 M NaOH (B) and Milli-Q water (C) and is prepared according to the
procedure described in the Instrument SOP. For monosaccharides sensitivity is somewhat
compromised because of the weak eluting conditions needed. Optimal sensitivity (which is not the
case for monosaccharide analysis) is observed at NaOH concentrations above 25 mM. Poor
sensitivity may be partly overcome by post column addition of 200 mM NaOH if necessary (in rare
cases).
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HPAEC analysis of monosaccharides
Standard Operational Procedure
Jagger
HPLC system
The analysis is performed on the Dionex ICS 3000 system with PAD detection. See relevant instrumental
SOP forsystem operation.
Analytical column
The analytical column applied is CarboPac™ PA1 from Dionex, Thermo Scientific. For this SOP a 250x2
mm column (with 10 µm particles) is used, equipped with guard column of the same brand, 50x2 mm.
The resin consists of pellicular polystyrene/divinyl benzene substrate agglomerated quaternary
ammonium functionalized latex microbeads and is pH stable throughout the pH scale.
Column precautions
Operational temperature should not go above 55 °C and no more than 2% organic solvents can be
present in the mobile phase. Normal operating pressure for this column including guard at 0.25 mL/min
is approx. 14-1700 psi when a 500 psi pressure restrictor is installed and maximum pressure is 3000 psi.
Operating conditions
Column temperature: 30 °C
Flow conditions: 0.25 mL/min
Eluents: 0.1 M NaOH (A), Milli-Q water (C). Consult Instrument SOP for eluent preparation procedure.
Injection volume: 2 µL (note that the autosampler spends additional volume during sampling, so one
injection in total is no less than 50 µL)
Instrumental method:
For separation of xylose/mannose: Monosaccharides_Iso_99% (Table 1)
For separation of arabinose, galactose, glucose and either xylose or mannose:
Monosaccharides_Iso_7.5mM (Table 1).
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HPAEC analysis of monosaccharides
Standard Operational Procedure
Jagger
Table 1: Instrument method: Monosaccharides_Iso_7.5mM and Monosaccharides_Iso_99%.
Monosaccharides_Iso_7.5mM
Monosaccharides_Iso_99%
Time (min)
A (%)
C (%)
A (%)
C (%)
0:00
7.5
92.5
1
99
22:00
7.5
92.5
1
99
30:00
-
-
1
99
The eluting is in both cases isocratic, but to compensate for lower NaOH concentration in method for
separating xylose and mannose longer run time is needed. This method will also result in a decreased
sensitivity compared to the other.
Chromatographic run
30.0
nC
1
2
3
25.0
4
20.0
15.0
5
10.0
5.0
0.0
min
-5.0
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
20.0
22.0
24.0
26.0
28.0
30.0
Figure 1: Chromatographic run at 1 mM NaOH. 1: Arabinose, 2: Galactose, 3: Glucose, 4: Xylose, 5: Mannose
Figure 1 shows a chromatogram of a sample containing five monosaccharides, arabinose, galactose,
glucose, xylose and mannose. The 1 mM analytical run will effectively separate all five species.
The retention time of each component is greatly dependent on the state of the eluents and shortened
retention times are common when eluents get older. This type of chromatography is affected by
dissolved carbonate in the eluents, which originates from eluent exposure to atmospheric CO2.
Carbonate binds strongly to the stationary phase’s active sites diminishing the column capacity and
reducing analyte affinity. Heavy carbonate contamination has large effects on the separation and will
ruin the resolution (but does not damage the column). For optimal performance, the eluents should not
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HPAEC analysis of monosaccharides
Standard Operational Procedure
Jagger
be more than 48 hours old. Monosaccharide analysis is particular labile to carbonate contamination
since the eluting conditions are weak. During a 10-15 sample run, decreases in retention time will be
significant even though separation will normally still be sufficient. For quantification and automatic
assignment of component names (see SOP for quantification in Chromeleon) it may be a trick to divide a
longer sequence into several shorter once (10-15 injections in total), each containing a set of standards.
These can then be queued. Automatic assignment may otherwise be difficult since drifts in retention
time causes complete shift of retention time for glucose and eventually xylose, leading to wrong
annotation galactose or glucose, respectively (figure 2). This can be overcome by assigning new
retention times in each short sequence.
30,0
nC
28,0
1
1
26,0
2
24,0
2/3
3
15,0
16,0
4
4
22,0
20,0
18,0
16,0
14,0
12,0
10,0
8,0
6,0
4,0
2,0
0,0
-2,0
-4,0
min
-5,0
0,0
1,0
2,0
3,0
4,0
5,0
6,0
7,0
8,0
9,0
10,0
11,0
12,0
13,0
14,0
17,0
18,0
19,0
20,0
Figure 2: Monosaccharide run (7.5 mM NaOH). 1: Arabinose, 2: Galactose, 3: Glucose, 4: Xylose. Black
chromatogram corresponds to time zero after eluent change. Pink chromatogram corresponds to plus 14 hours
after eluent change. Note the shift in retention time where pink glucose overlaps black galactose.
Sample preparation
Samples must be absolutely clear of particulate matter, and if not they should be centrifuged extensively
or filtered (0.2 /0,45 µm filter). Dilution of samples should be done in water or preferably in 15 mM
NaOH to knock-out any buffer capacity and to mimic the initial conditions of the eluting run. If samples
are strongly acidic or has high buffering capacity, higher NaOH concentrations may be necessary to
make pH alkaline. For new sample types check pH in the final dilution to ensure that it is above 12.
However, note that if pH and/or ionic strength are significantly different from the running conditions in
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HPAEC analysis of monosaccharides
Standard Operational Procedure
Jagger
the analysis this may affect the run. Low pH may not affect separation but will decrease the sensitivity,
whereas high ionic strength in the form of hydroxide ions may increase the sensitivity but result in
reduced resolution.
Samples should be dispensed in appropriate HPLC vials. NB! Make sure no air bubbles are present in the
bottom of the vial as the injection needle will sample from here. Flick the vial with a finger to remove air
bubbles. The autosampler spends more sample than the actual injection volume (2 µL) and it is
therefore important to keep a minimum volume of 50 µL in each vial.
Calibration
As already mentioned the detection principle is sensitive but due to the weak eluting conditions the
sensitivity will be lower than what can maximally be obtained. When running at 7.5 mM NaOH elution
the upper limit for calibration is 0.1 g/L of each of the monosaccharides at. Sensitivity will effectively be
lower for the 1 mM NaOH elution. Table 2 gives LOD (limit of detection) and LOQ (limit of quantification)
values for all five monosaccharides (note that LOD/LOQ for mannose is determined at 1 mM elution). If
increased sensitivity is necessary, increasing loop size or post column addition of 200 mM NaOH may be
the solution.
Table 2: LOD/LOQ values for five monosaccharides. *Mannose determined with 1 mM NaOH elution. Others
determined with 7.5 mM elution. Due to a dip in the baseline immediately before glucose, LOD for glucose
should be considered the same as LOQ.
LOD (ng/inject) LOQ (ng/inject)
Arabinose
0.193
0.64
Galactose
0.17
0.57
Glucose
-
0.51
Xylose
0.08
0.27
Mannose*
0.85
2.82
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HPAEC analysis of monosaccharides
Standard Operational Procedure
Jagger
180
Xylose
y = 1546,8x
R² = 0,9996
160
Area (nC*min)
140
120
Glucose
y = 814,86x
R² = 0,9996
100
Arabinose
80
Galactose
Glucose
60
Xylose
40
Galactose
y = 737,52x
R² = 0,9993
20
0
0
0,02
0,04
0,06
0,08
0,1
0,12
Concentration (g/L)
Arabinose
y = 651,17x
R² = 0,9999
Figure 3: Calibration curve for monosaccharides arabinose, galactose, glucose and xylose determined with 7.5
mM NaOH elution.
16
14
Mannose
y = 148,28x
R² = 0,9997
Area (nC*min)
12
10
8
Mannose
6
4
2
0
0
0,02
0,04
0,06
0,08
0,1
0,12
Concentration g/L
Figure 4: Calibration curve for mannose determined with 1 mM NaOH
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HPAEC analysis of monosaccharides
Standard Operational Procedure
Jagger
Always provide a new set of standards for the analytes of interest in every sequence and perform a
recalibration in the processing method. The calibration curves provided here are meant as inspiration. An
existing processing method may be found on the instrument and is named “Monosaccharides-7.5 mM”.
Trouble shooting
Identify if any immediate precautions should be taken and then call instrument super users (currently
Jane W. A. or Bjørge W.).
Column regeneration
In general the column needs a minimum of maintenance, but for every change of eluent the first run
must be a regeneration run to remove accumulated carbonate. The program for this is called “Start after
eluent change” and contains a longer run period with high NaOAc concentrations ended by a long
reconditioning to 0.1 M NaOH, followed by a reconditioning step of 30 minutes with initial conditions.
Because the elution conditions are weak for monosaccharide analysis a stronger cleaning needs to run
frequently to remove non-eluted compounds (like glucuronic acid etc). This is done by running a
cleaning method “Monosaccharides – cleaning” as the last method in every monosaccharide sequence.
This includes a steep gradient with NaOAc and a long reconditioning step (30 minutes) to initial
conditions (7.5 mM NaOH).
The guard column is installed to protect the much more expensive analytical column and if bad column
performance is observed it could be due to accumulation of contamination on the guard. Testing’s
should be done to sort out the cause but may be solved by replacing the guard column.
The most important parameters to monitor for overall column performance are peak shape and
pressure. If pressure increases consult super users to resolve the reason for pressure increase. If no
obvious blocking is present in the system (rotor seal destruction) it may be caused by blocking of the
guard column inlet frit and this should be changed. This should only be done by super users.
Column storage conditions
The column is normally not stored but continuous flow is applied. The eluents have high salt
concentrations and these may precipitate during unintended flow stop. If the column is to be stored (fx.
during holiday periods), exchange all buffers with 0.1 M NaOH and cap the column carefully to avoid
drainage. In such cases it is important to store the system correctly and therefore consult the
Instrument SOP.
Editorial log
First edition. 2012.03.08
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