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Laboratory

Objectives and Summary

The aim of this study is to evaluate the analysis of variety of oligosaccharides using HPAE-PAD with dual
eluent generation cartridges (EGC) on ICS-6000 HPIC system. The samples are maltodextrin in water, enzyme
hydrolysed wheat arabinoxylan and enzyme hydrolysed beechwood xylan. The report shows that the
maltodextrin and the enzyme hydrolysates can be very well separated on Dionex CarboPac PA200, 1x250mm
column sets using a KOH/KMSA gradient by dual EGC mode.
This report is considered proof of concept for purchasing an ICS-6000 system from Thermo Fisher Scientific.

Instrumentation
Instrument: ICS-6000 HPIC
DP: Isocratic pump or gradient pump
Eluent Generator: Duel EGC mode
EGC 400-MSA cartridge
EGC 400- KOH cartridge;
EGC 400 MSA Degasser
EGC 400 KOH Degasser.
Detector/Chromatography Compartment: DC with PAD
Autosampler: AS-AP
Data Management System: Chromeleon 7.2.9

Analytical Conditions

Column: CarboPac PA200 1x250mm (P/N: 302861)

CarboPac PA200 1x50mm (P/N:302862)
Eluent: KMSA/KOH gradient by dual EGC (see below)
Flow Rate: 0.063ml/min
Column Temp: 30°C
Compartment Temp: 30°C
Background: <45 nC at initial gradient
Detection: Pulsed Amperometry, quadruple waveform
Electrode: PTFE gold, disposable electrode, Ag/AgCl reference electrode,
1mil gasket.
Backpressure: ~3550-3700 psi at initial gradient
Injection: 0.4ul
Sample and 1) 50g/L Maltodextrin in water
preparation: 2) Enzyme hydrolysed 5g/L wheat arabinoxylan
(megazyme, P-xylnbe) in 50mM NaOAc, pH 5
3) Enzyme hydrolysed 5g/L beechwood xylan
(megazyme, P-xylnbe) in 50mM NaOAc, pH 5
Samples were filtered by 0.45um filter (customer provided)
Sample 1 was diluted in DI water prior to injection

This application report is published as a proof of principle. No ISO data available for included figures.

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Laboratory

Gradient profiles for oligosaccharides:

For Maltodextrin
Flow
Time rate KMSA KOH Curve
(min) ml/min mM mM
-15 0.063 15 50
0 0.063 15 50
35 0.063 150 50 3
45 0.063 150 50

For Enzyme hydrolysed wheat arabinoxylan

Flow
Time rate KMSA KOH Curve
(min) ml/min mM mM
-15 0.063 5 50
0 0.063 5 50
35 0.063 150 50 5
45 0.063 150 50

For Enzyme hydrolysed 5g/L beechwoodxylan

Flow
Time rate KMSA KOH Curve
(min) ml/min mM mM
-15 0.063 5 80
0 0.063 5 80
35 0.063 120 80 5
50 0.063 120 80

Plumbing Schematic for ICS-6000 system Dual EGC Mode, 1mm column

This application report is published as a proof of principle. No ISO data available for included figures.

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Laboratory

Chromatograms

Figure1. Overlaid chromatograms of maltodextrin (top) and DP2-DP5 standard (bottom)

Figure2. Zoomed-in chromatogram of Maltodextrin

This application report is published as a proof of principle. No ISO data available for included figures.

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Figure3. Stacked chromatograms of wheat hydrolysate (top) with DP2-DP5 (middle) and
Xylose (bottom)

Figure3. Zoomed-in chromatogram of wheat hydrolysate

This application report is published as a proof of principle. No ISO data available for included figures.

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Figure4. Stacked chromatograms of beechwood hydrolysate (top) with DP2-DP5 (middle)

and xylose (bottom)

Figure5. Zoomed-in chromatogram of beechwood hydrolysate

This application report is published as a proof of principle. No ISO data available for included figures.

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Laboratory

Figure6. Reproducibility of three replicated injections of 0.5g/L maltodextrin

Conclusions

1. Enzyme hydrolysates and maltodextrin can be very well separated on CarboPac

PA200, 1x250mm column set using a KOH/KMSA gradient by dual EGC mode on
ICS-6000 HPIC system within 60 minutes. Up to DP37 polysaccharides can be
identified in maltodextrin sample. Over 58 peaks are observed in enzyme
hydrolysates.
2. Three different gradient profiles are used for analysis of these oligosaccharides for
better resolution. The huge peak around 2.4 minutes in two enzyme hydrolysates
could be xylose or xylose co-eluted with other monosaccharides (figure 3 &4)
3. Dual EGC mode provides the ability to run gradient method using isocratic pump
and eliminates the manual preparation of eluents.

References:
1) “HPAE-PAD analysis of galactosyl-oligosaccharide-containing samples using dual
eluent generation cartridges” (Application note 72714, Thermo Fisher Scientific)
2) “Determination of Plant-Derived Neutral Oligo- and Polysaccharides Using the
CarboPac PA200” (Application Update 150, Thermo Fisher Scientific)
3) “Dionex ICS-6000 Ion Chromatography System Dual EGC Mode Installation”
(ThermoFisher Scientific 065760-01)
This application report is published as a proof of principle. No ISO data available for included figures.

Application Report No. XX-2011-XXX 7/7