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For Research Use Only. Not for use in diagnostic procedures.
Dionex CarboPac PA300-4µm
155014 Revision 02 • September 2020
User Manual
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Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 2 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
Product Manual for
Dionex CarboPac PA300-4µm Column
2 × 250 mm (Item # 303346)
Dionex CarboPac PA300-4µm Guard Column
2 × 50 mm (Item # 303347)
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Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 3 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
© 2020 Thermo Fisher Scientific Inc. All rights reserved.
All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.
Thermo Fisher Scientific Inc. provides this document to its customers with a product purchase to use in the product
operation. This document is copyright protected and any reproduction of the whole or any part of this document is
strictly prohibited, except with the written authorization of Thermo Fisher Scientific Inc.
The contents of this document are subject to change without notice. All technical information in this document is
for reference purposes only. System configurations and specifications in this document supersede all previous
information received by the purchaser.
Thermo Fisher Scientific Inc. makes no representations that this document is complete, accurate or error-free and
assumes no responsibility and will not be liable for any errors, omissions, damage or loss that might result from
any use of this document, even if the information in the document is followed properly.
This document is not part of any sales contract between Thermo Fisher Scientific Inc. and a purchaser. This
document shall in no way govern or modify any Terms and Conditions of Sale, which Terms and Conditions of
Sale shall govern all conflicting information between the two documents.
Revision History:
Revision 01, April 2020, Original Publication.
Revision 02, September 2020, Added section 6.6 (Comparison between Ag/AgCl and PdH reference electrodes)
and updated example applications.
Page 4
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 4 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
Safety and Special Notices
Make sure you follow the precautionary statements presented in this guide. The safety and other
special notices appear in boxes.
Safety and special notices include the following:
Indicates a potentially hazardous situation which, if not avoided, could result in death or
serious injury.
Indicates a potentially hazardous situation which, if not avoided, could result in damage
to equipment.
Indicates a potentially hazardous situation which, if not avoided, may result in minor or
moderate injury. Also used to identify a situation or practice that may seriously damage
the instrument but will not cause injury.
Indicates information of general interest.
IMPORTANT
Highlights information necessary to prevent damage to software, loss of data, or invalid
test results; or might contain information that is critical for optimal performance of the
system.
Tip
Highlights helpful information that can make a task easier.
SAFETY
!
WARNING
!
CAUTION
!
NOTE
!
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Contents
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 5 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
Contents
Safety and Special Notices ...................................................................................................................... 4
1. Introduction ......................................................................................................... 8
1.1 Dionex CarboPac PA300-4µm column .......................................................................................... 8
1.1.1 Resin / Latex Characteristics: ................................................................................................................ 8
1.1.2 Typical Operating Parameters: .............................................................................................................. 8
2. System Requirements and Installation ............................................................. 9
2.1 The Dionex High-Pressure Ion Chromatography Systems ............................................................. 9
2.2 System Requirements for Column Operation ............................................................................... 10
2.2.1 Installation of Disposable Electrode into a Dionex ED50 Cell, pH-Ag/AgCl
Reference Electrode or PdH Reference Electrode ....................................................................................... 10
2.2.2 System Void Volume .......................................................................................................................... 10
2.3 The Injection Loop ....................................................................................................................... 10
2.3.1 The 2 mm System Injection Loop, 2.5 µL to 10 µL ............................................................................ 10
2.4 Installing the Dionex BorateTrap Inline Trap Column ................................................................. 10
2.5 The Dionex CarboPac PA300-4µm Guard Column ..................................................................... 11
2.6 Procedure for Dionex EGC Installation and conditioning ............................................................ 11
2.7 Installing the Dionex CR-ATC Trap Column for Use with Dionex EGC .................................... 11
2.8 Eluent Storage............................................................................................................................... 12
2.9 System Start-up............................................................................................................................. 12
2.9.1 System Background Check ................................................................................................................. 12
2.9.2 Verification of Column Cleanliness .................................................................................................... 12
3. Before You Start................................................................................................13
3.1 Dionex CarboPac PA300-4µm column Operational Parameters .................................................. 13
3.1.1 The Best Operational Guidelines ........................................................................................................ 13
3.1.2 Initial Check List ................................................................................................................................. 14
4. Purity Requirements for Chemicals ................................................................15
4.1.1 Deionized Water.................................................................................................................................. 15
4.1.2 Hydroxide ............................................................................................................................................ 15
4.1.3 Sodium Acetate ................................................................................................................................... 15
5. Preparation of Eluents and Standards............................................................16
5.1 Deionized Water ........................................................................................................................... 16
5.2 0.2 M Sodium Hydroxide ............................................................................................................. 16
5.2.1 Sodium Hydroxide Eluent Concentration ........................................................................................... 16
5.3 50 mM Sodium Hydroxide / 25 mM Sodium Acetate .................................................................. 17
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Contents
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Product Manual for Dionex CarboPac PA300-4µm Columns
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5.4 0.1 M Sodium Hydroxide / 0.25 M Sodium Acetate .................................................................... 18
5.5 Sample Preparation ....................................................................................................................... 18
6. Example Applications .......................................................................................20
6.1 Analysis of Sugar Alditols: A Quality Assurance Report (QAR) ................................................ 20
6.2 Characterization of Released Glycans Using CarboPac PA300-4µm .......................................... 21
6.2.1 Bovine Fetuin N-Linked and O-Linked Glycan Alditol Profiling ....................................................... 21
6.2.2 Porcine Gastric Type III Mucin O-Linked Glycan Alditol ................................................................. 23
6.2.3 N-Linked Glycan Profile of Trastuzumab (Herceptin). ....................................................................... 25
6.2.4 N-linked Glycan Profile of Bovine Milk Proteins ............................................................................... 26
6.3 Analysis of Free Complex Carbohydrates in Food ....................................................................... 27
6.3.1 Galacto-oligosaccharides (GOS) Syrup .............................................................................................. 27
6.3.2 Free Oligosaccharides in Infant Formula with 2’-FL HMO ................................................................ 28
6.4 Structural Characterization of Complex Carbohydrates Using CarboPac PA300-4µm
Column in the IC-MS platform .................................................................................................... 29
6.4.1 Porcine Gastric Type III Mucin O-Linked Glycan Alditol by IC-MS ................................................ 29
6.4.2 Analysis of Bovine Milk Free Oligosaccharides Using IC-MS .......................................................... 31
6.5 Robustness and ruggedness of Dionex CarboPac PA300-4µm .................................................... 32
6.6 Comparison between Ag/AgCl and PdH reference electrodes ..................................................... 33
7. Troubleshooting Guide .....................................................................................35
7.1 High Back Pressure ...................................................................................................................... 35
7.1.1 Finding the Source of High System Pressure ...................................................................................... 35
7.1.2 Replacing Column Bed Support Assemblies for 2 mm column .......................................................... 36
7.1.3 Filter Eluent ......................................................................................................................................... 36
7.1.4 Filter Samples ..................................................................................................................................... 36
7.2 High Background .......................................................................................................................... 37
7.2.1 Preparation of Eluents ......................................................................................................................... 37
7.2.2 Dionex CR-ATC Column.................................................................................................................... 37
7.2.3 A Contaminated Guard or Analytical Column .................................................................................... 38
7.3 Poor Resolution ............................................................................................................................ 38
7.3.1 Loss of Column Efficiency ................................................................................................................. 38
7.3.2 Shortened Retention Times ................................................................................................................. 39
7.3.3 Loss of Resolution for early eluting peaks .......................................................................................... 40
7.3.4 Spurious Peaks .................................................................................................................................... 40
7.3.5 No Peaks, Poor Peak Area Reproducibility or too Small Peak Areas ................................................. 41
7.3.6 Large Baseline Dip in the Chromatogram ........................................................................................... 41
7.3.7 Unidentified Peaks Appear with Expected Analyte Peaks .................................................................. 41
7.3.8 Decreased Detection Sensitivity .......................................................................................................... 42
7.3.9 Excessive Gradient Drift ..................................................................................................................... 43
7.4 Reconditioning or Replacement of the Gold (conventional or disposable) Electrodes or
Replacement of the Reference Electrode ..................................................................................... 43
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Contents
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
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155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
Appendix A: Dionex CarboPac PA300-4µm Column Care .............................44
A.1 Recommended Operation Pressures ............................................................................................. 44
A.2 Column Start-Up........................................................................................................................... 44
A.3 Column Storage ............................................................................................................................ 45
A.4 Dionex CarboPac PA300-4µm Column Cleanup ......................................................................... 45
Appendix B: Quality Assurance Report ............................................................46
Page 8
1 – Introduction
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 8 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
1. Introduction
1.1 Dionex CarboPac PA300-4µm column
The Thermo Scientific™ Dionex™ CarboPac™ PA300-4µm column is the latest addition to the
CarboPac family of columns offered for carbohydrate separations. The new column was
developed for the high-resolution separations of complex oligosaccharides from biologics and
food samples. This column is packed with polymeric, state-of-the-art supermacroporous resin
coated with anion exchange latex particles and is stable over the entire range of pH 0-14. The
unique pH stability of the packing material allows eluent compositions that are conducive to
anodic oxidation of carbohydrates at the surface of gold electrodes.
High-pH anion-exchange chromatography coupled with pulsed amperometric detection (HPAEPAD) has been demonstrated to be effective in the separation and detection of carbohydrates
ranging from small monosaccharides to branched oligosaccharides, and large linear
polysaccharides. HPAE is proven to be capable of separating monosaccharides, positional linkage
and branch isomers of branched oligosaccharides, and homopolymer oligosaccharides that differ
only in length. Each class of carbohydrates has different separation characteristics requiring
different column chemistries to address the need of this complex world of glycans.
Note that Dionex™ CarboPac™ PA300-4µm column uses 4µm supermacroporous resin particles
to provide high resolution and high-capacity column. The high resolution provides better peak
identification and high capacity allows the injection of more concentrated samples without
overloading the column. The use of 4µm resin particles compared to the 6µm commonly found
in earlier Dionex CarboPac products, the Dionex CarboPac PA300-4µm will require the use of
high-pressure IC (HPICTM) systems such as the Thermo ScientificTM DionexTM ICS-5000+
HPICTM system or the Thermo ScientificTM DionexTM ICS-6000 HPICTM system.
1.1.1 Resin / Latex Characteristics:
Particle Size: 4 μm (Analytical Column*)
Particle Size: 8 μm (Guard Column**)
Particle Cross-linking: 55%
Functional Group: Quaternary ammonium functional groups
Latex Diameter: 60 nm
Ion exchange capacity: 85 µeq per 2 × 250 mm column.
1.3 µeq per 2 x 50 mm column
*Analytical Column Resin Composition: Supermacroporous polyvinylbenzyl ammonium polymer cross-
linked with divinylbenzene.
** Guard Column Resin Composition: Microporous polyvinylbenzyl ammonium polymer cross-linked with
divinylbenzene.
1.1.2 Typical Operating Parameters:
pH range: 0-14
Temperature Limit: 4-60°C
Pressure Limit: 5000 psi
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2 – System Requirements and Installation
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
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155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
2. System Requirements and Installation
Read the instrument manuals. This manual assumes that you are using Thermo Scientific
Dionex instrumentation and are familiar with the installation and operation of the Thermo
Scientific Dionex Ion Chromatograph (IC). If you do not understand the operation of the
system, take the time to familiarize yourself with the various system components before
beginning an analysis.
The proper configuration of an Ion Chromatography System (ICS) is dependent on column
format. A gradient pump is designed to blend and pump isocratic, linear, or gradient mixtures of
up to four mobile phase components at precisely controlled proportions and flow rates. An
isocratic pump is for applications not requiring gradient or proportioned eluent capabilities. For
high-pressure applications, the use of high-pressure ViperTM fittings is recommended. Please use
the following Item #’s to order Kit with Viper Fittings (4/2mm represent the ID of the separation
column).
088804 – Dionex IC PEEK Viper Fittings Kit for 4 mm Dionex ICS-6000 system with
Electrochemical Detector (ED)
302966 – Dionex IC PEEK Viper Fittings Kit for 2 mm Dionex ICS-6000 system with
Electrochemical Detector (ED)
(To order the Item # ask your local sales representative for a quote)
2.1 The Dionex High-Pressure Ion Chromatography Systems
A Dionex High-Pressure Ion Chromatography System (HPIC) is recommended when running
Dionex CarboPac PA300-4µm columns due to the higher backpressures generated at typical
operational flow rates with 4µm resins. Systems should have the capability to operate up to at
least 5000 psi. Standard IC systems, with an upper limit of 3000 psi, will be inadequate for
optimized column operation.
Care should always be taken not to exceed the maximum operating pressure of the system
component. ICS systems with lower backpressure capabilities are not recommended as
reduced flow rates will result in reduced throughput.
Contact your local representative for information on how to customize your system to
your application needs.
NOTE
!
WARNING
!
NOTE
!
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2 – System Requirements and Installation
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 10 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
2.2 System Requirements for Column Operation
Dionex CarboPac Columns are designed to run on Dionex Ion Chromatographs equipped with
electrochemical detectors. We strongly recommend the use of Thermo Scientific ViperTM tubing.
The ViperTM tubing will help ensure an easier and more secure installation. It is highly
recommended to ensure that the system used for carbohydrate analysis are metal-free. Metal ions
from a metal system will contaminate the CarboPac column and may contaminate the working
electrode. Running a CarboPac column on a metal system voids the column warranty.
2.2.1 Installation of Disposable Electrode into a Dionex ED50 Cell, pH-Ag/AgCl Reference
Electrode or PdH Reference Electrode
The 2 mil (0.002”) thick Teflon gaskets included in each package of disposable electrodes are
usually required; otherwise, the disposable electrode product warranty may be voided. A gasket
is always required because it forms the flow-through channel. In addition, the quadruple
waveform must be used for carbohydrate analysis otherwise the disposable electrodes will fail
early, and their product warranties will be void. Always wear gloves when handling electrodes.
Never touch the electrode surface. To install a disposable working electrode and reference
electrode (pH-Ag/AgCl or PdH) refer to the Product Manual for Disposable Electrodes Doc. No.
065040, ICS-6000 Ion Chromatography System Manual Doc. No. 22181-97002 and User’s
Compendium for Electrochemical Detection Doc. No. 065340.
2.2.2 System Void Volume
When using a Dionex CarboPac PA300-4µm column, it is important to minimize system void
volume. For the best 2 mm column performance, we strongly recommend the use of Thermo
Scientific ViperTM fittings installed between the injection valve and detector. Minimize the lengths
of all connecting tubing and remove all unnecessary switching valves and couplers.
2.3 The Injection Loop
2.3.1 The 2 mm System Injection Loop, 2.5 µL to 10 µL
For 2 mm column applications, a 2.5 µL injection loop typically recommended. Injecting a larger
amount can result in overloading the column or detector, which can affect detection linearity. For
low concentrations, larger injection loops can be used to increase sensitivity.
2.4 Installing the Dionex BorateTrap Inline Trap Column
Borate is a known contaminant in laboratory water supplies. Borate contamination of
chromatography eluents may be a result of degrading deionized water systems or as leachate from
borosilicate glassware. If borate is present in the eluent, it binds both to the anion-exchange
column and to the carbohydrate analytes. The carbohydrate-borate complex is less efficiently
eluted from the anion-exchanger than is the carbohydrate, resulting in peak tailing, particularly
where vicinal cis hydroxyl groups are present, such as for mannose and sugar alcohols.
For optimal performance during IC carbohydrate analysis using HPAE-PAD detection, remove
borate contamination using the Thermo Scientific™ Dionex™ BorateTrap™ Inline Trap Column
(Item # 047078). When placed between the eluent pump gradient mixture and the injection valve,
the trap does not affect the efficiencies or retention times of the carbohydrate being analyzed. The
Dionex BorateTrap column eliminates peak tailing for mannose, fructose, and sugar alcohols
resulting from borate contamination.
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2 – System Requirements and Installation
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 11 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
2.5 The Dionex CarboPac PA300-4µm Guard Column
A Dionex CarboPac PA300-4µm Guard Column is normally used with the Dionex CarboPac
PA300-4µm Analytical Column. Retention times will increase by ~5% when a guard column is
placed in-line before the analytical column while using isocratic elution. A guard column helps
prevent sample contaminants from fouling the analytical column. It is cheaper and easier to clean
or replace a guard column than an analytical column. Replacing the Dionex CarboPac PA3004µm Guard Column at the first sign of peak efficiency loss or decreased retention time will
prolong the life of the Dionex CarboPac PA300-4µm Analytical Column.
2.6 Procedure for Dionex EGC Installation and conditioning
For detailed information on how to install EGC KOH Cartridge, see Dionex Eluent Generator
Cartridges Product Manual (Document # 065018).
2.7 Installing the Dionex CR-ATC Trap Column for Use with Dionex EGC
For Dionex CarboPac PA300-4µm applications using the Dionex EGC 500 KOH cartridge, a
Dionex CR-ATC 600 Continuously Regenerated Trap Column (Item # 088662) should be
installed. See the Dionex CR-TC 600 Product Manual (Document No. 079684) for instructions.
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2 – System Requirements and Installation
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 12 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
2.8 Eluent Storage
The Dionex CarboPac PA300-4µm column is designed to be used with hydroxide eluent systems.
Storage under a helium atmosphere ensures contamination-free operation and proper pump
performance (nitrogen can be used if eluents do not contain organic solvents).
2.9 System Start-up
2.9.1 System Background Check
This procedure is performed using the conditions of the test chromatogram.
Make sure that…
A. the cell is not yet on,
B. the pump is pumping 10 mM NaOH at 0.25 mL/min
C. a length of narrow-bore tubing is installed between the injector and detector cell to
generate ~1000 psi back pressure,
D. The column(s) are not yet installed.
Confirm that the pH is 12.1 ±1 pH unit. With the pH within this range, turn on the cell using the
carbohydrate standard quad waveform (See Table 3, Section 6.3, Disposable Electrode Manual,
document number 065040) and begin background signal monitoring from the ChromeleonTM
control panel. Monitor the trace for at least 30 minutes. Confirm that the background is between
10 and 35 nC. If the background is above 50 nC or the pH is out of range, see the
“Troubleshooting” section at the end of this manual, and ensure that the vacuum degas system is
operational.
Thermo Scientific recommends sanitizing the entire system with at least 2 hours of 100mM
KOH at 1.0mL/min using the KOH Eluent Generator cartridge. If the system does not use
an eluent generator, 2M NaOH can be used prior to initial start-up and after long idle
periods.
2.9.2 Verification of Column Cleanliness
Install the Dionex CarboPac PA300-4µm column set only after the initial system test determines
a background level within the specified range. A premature installation on a contaminated system
will cause delays during the column equilibration.
The Dionex CarboPac PA300-4µm is shipped in 10 mM NaOH. Any column that is stored longterm should be stored in the same solution. To prepare the column for standard analysis, the
Dionex CarboPac PA300-4µm must be washed for at least 30 minutes (an hour preferred) with
200mM NaOH at 0.25 mL/min flow rate. Equilibrate the column set under the test chromatogram
conditions (See Quality Assurance Report; QAR) and performing two blank injections (DI water)
to ensure that DI water injection has no peaks.
Once the columns are equilibrated, inject a system suitability standard such as the column’s QAR
standard, to establish the performance of the column at start-up. This chromatogram can then be
referred to when troubleshooting your system. Once you obtain the expected chromatographic
performance, you are ready to proceed to run your application.
Thermo Scientific recommends that the system suitability standard be run whenever you reinstall
a column after long-term storage.
NOTE
!
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3 – Before You Start
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 13 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
3. Before You Start
3.1 Dionex CarboPac PA300-4µm column Operational Parameters
• pH range: 0 - 14
• Temperature limit: 4-60 °C
• Pressure limit: 5000 psi
• Organic Solvent Limit: 0-100%
• Typical Eluents: NaOH and NaOAc
• Standard Flow Rate: 2 mm: 0.25 mL/min
• Maximum Flow Rate: 2 mm: 0.30 mL/min
• Typical Back Pressure: 3000 psi, at 0.25 mL/min (analytical column)
• Typical Back Pressure: 200 psi, at 0.25 mL/min (guard column)
Using hydroxide eluent concentration below 8 mM will produce a significantly lower
detection response.
When using a high level of organic solvent, it is advised to add some base or acid to avoid
any damage to the column performance
3.1.1 The Best Operational Guidelines
ALWAYS…
NEVER…
• Use a 50% (w/w) sodium hydroxide solution (ThermoFisher
Scientific Item # 080389) as the source of sodium
hydroxide.
• Use dedicated plastic volumetric flasks and plastic
serological pipettes for eluent preparation and volume
adjustments.
• Use plastic labware and eluent bottles for all eluents
containing NaOH. NaOH dissolves borosilicate glass,
releasing borate into the solution.
• Use high purity water (>18.2 MΩ-cm resistivity) to prepare
sodium hydroxide eluents. The water should be degassed,
for best results, and prevent carbonate contamination.
• Keep your NaOH eluent blanketed with helium or nitrogen.
Prepare new NaOH eluent if left unblanketed for more than
30 minutes.
• Use EGC-KOH generated eluent when possible to avoid
any eluent preparation issues.
• Purge at least 40 mL of new eluent through the lines when
changing eluent or adding fresh eluent. This will ensure that
your fresh eluent is primed through the lines up to the pump
head.
• Verify the equilibration time necessary before injection to
avoid baseline issues or artifacts or to avoid an unnecessary
increase in total method time.
• Proceed to the next installation step if the
previous step has failed.
• Start an installation with any of the
checklist items below missing.
• Use ‘communal’ filtration units or filters
made of unknown or unsuitable materials
(e.g., cellulose derivatives).
• Use MeOH or other organic solvents as
rinse fluid in the autosampler. Use only
water, replaced daily, or use sampler wash
bottles blanketed with ~3 psi nitrogen or
helium.
• Run above 60 °C or 5000 psi.
NOTE
!
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3 – Before You Start
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 14 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
3.1.2 Initial Check List
The following items MUST be available in your lab. The absence of any of these may compromise
your analysis.
• Laboratory water unit delivering >18.2 -cm water at the installation site.
• Vacuum system for eluent vacuum filtration
• Sterile packed Nalgene Filtration units (pore size: 0.2 µm, filtered material: Nylon), 1 L
funnel size
• Inert gas cylinder (helium or nitrogen) with a regulator valve (for example, a 0-200 psi
gauge on the low-pressure side) and the appropriate size adaptors plus tubing
• Sterile-packed 10 mL and 25 mL disposable pipets and suitable pipetting bulbs or pumps.
• Disposable, plastic (PE) large-size (at least 20 mL) syringe for priming the pump
• Plastic eluent bottles with gas-tight cap-fittings.
Page 15
4 – Purity Requirements for Chemicals
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 15 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
4. Purity Requirements for Chemicals
Obtaining reliable, reproducible, and accurate results require eluents that are free from impurities
and prepared only from the chemicals recommended below. Thermo Scientific cannot guarantee
proper column performance when alternate suppliers of chemicals or lower purity water are
utilized.
4.1.1 Deionized Water
Deionized water used to feed the Eluent Generator should be Type I reagent grade water with a
specific resistance >18.2 -cm. The water should be free from ionized impurities, organics,
microorganisms and particulate matter larger than 0.2 µm. UV treatment as a part of the water
purification unit is recommended. Follow the manufacturer’s instructions regarding the
replacement of UV lamps, ion exchange, and adsorbent cartridges. All filters used for water
purification must be free from electrochemically active components, including surfactants.
Expanding their period of use beyond the recommended time may lead to bacterial contamination
and as a result, a laborious cleanup may be required. The use of contaminated water for eluents
can lead to high background signals and significant gradient artifacts.
4.1.2 Hydroxide
Use a Dionex EGC 500 KOH Eluent Generator Cartridge (Item # 075778) installed with CR-ATC
600 (Item # 088662) in the EG module. Manually prepared hydroxide eluents will absorb CO2
during and after preparation altering elution selectivity over time. Use 50% w/w sodium
hydroxide (Certified Grade, Thermo Fisher Scientific Item # 080389) for preparation.
4.1.3 Sodium Acetate
Thermo Scientific highly recommends the use of Dionex Sodium Acetate Reagent (Item #
059326) for carbohydrate analysis. Thermo Scientific cannot guarantee proper detection
performance when different grades or alternate suppliers of sodium acetate utilized.
Page 16
5 – Preparation of Eluents and Standards
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 16 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
5. Preparation of Eluents and Standards
Always sanitize the entire analyzer with 2M NaOH prior to initial start-up and after idle
periods.
Obtaining reliable, consistent and accurate results requires eluents that are free of ionic and
electrochemically active impurities. Chemicals and deionized water used to prepare eluents must
be of the highest purity available. Maintaining low trace impurities and low particle levels in
eluents also helps to protect your columns and system components. Thermo Scientific cannot
guarantee proper column performance when the quality of the chemicals, solvents, and water used
to prepare eluents is substandard.
5.1 Deionized Water
Vacuum degas the water by placing the eluent reservoir in a sonicator and drawing a vacuum on
the filled reservoir with a vacuum pump. Degas the aqueous eluent in a glass bottle before
transferring to the plastic bottle. Cap each bottle and minimize the length of time the bottle is
opened to the atmosphere. Vacuum filtration through 0.2 μm Nylon filters (Thermo Fisher
Scientific Item # 164-0020) is a good alternative to vacuum degassing under sonication and is
sufficient for most cases. On-line eluent and EG-produced eluent degassing is supported using the
Thermo Scientific pumping systems.
5.2 0.2 M Sodium Hydroxide
DO NOT prepare NaOH eluents from sodium hydroxide pellets! The pellets are coated with
a layer of carbonate.
Always store degassed NaOH eluents in plastic eluent bottles blanketed with helium or nitrogen
to avoid carbon dioxide contamination from the air. Carbonate in the eluent can significantly
reduce retention times for carbohydrates.
5.2.1 Sodium Hydroxide Eluent Concentration
Gravimetric Method
When formulating eluents from 50% sodium hydroxide, Thermo Scientific recommends weighing
out the required amount of 50% sodium hydroxide. Use the assayed concentration value from the
sodium hydroxide bottle.
Example: To make 1 L of 0.2 M NaOH use 16.004 g of 50% sodium hydroxide:
For 0.2 M:
0.2 mole/L × 40.01 g/mole
= 16.004 g diluted to 1 L
50%
NOTE
!
NOTE
!
Page 17
5 – Preparation of Eluents and Standards
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 17 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
Volumetric Method
Although it is more difficult to make precise carbonate-free eluents for gradient analysis
volumetrically, you may choose to use the following formula to determine the correct volume of
50% sodium hydroxide to be diluted.
g = dvr
Where g = weight of sodium hydroxide required (g)
d = density of concentrated solution (g/mL)
v = volume of the 50% sodium hydroxide required (mL)
r = % purity of the concentrated solution
Example: To make 1 L of 0.2 M NaOH use 10.46 mL of 50% sodium hydroxide:
For 0.2 M:
0.2 mole/L × 40.01 g/mole
= 10.46 mL diluted to 1 L
50% × 1.53 g/mL*
* This density applies to 50% NaOH. If the concentration of the NaOH solution is significantly
different from 50%, the gravimetric method should be used instead.
Sodium Hydroxide Eluents
Dilute the amount of 50% (w/w) NaOH Reagent specified below in Table 1. Prepare CarboPac
Eluents with degassed, deionized water (18.2 MΩ-cm) to a final volume of 1,000 mL using a
volumetric flask. Avoid the introduction of carbon dioxide from the air into the aliquot of 50%
(w/w) NaOH bottle or the deionized water being used to make the eluent. Do not shake the 50%
(w/w) NaOH bottle or pipette the required aliquot from the top of the solution where sodium
carbonate may have formed.
Table 1 Mass or Volume of NaOH Required to Make 1 L of Common Eluents
Eluent Concentration
(M)
NaOH (50%)
(g)
NaOH (50%)
(mL)
0.15
12.0
7.8
0.5
40.0
26.2
0.8
64.0
41.8
1.0
80.0
52.3
5.3 50 mM Sodium Hydroxide / 25 mM Sodium Acetate
Acetate has no buffering capacity at high pH, so to maintain baseline stability, it is important to
keep the sodium hydroxide concentration constant during the sodium acetate gradient. This is
achieved by making the eluents as follows:
Eluent A: x mM NaOH
Eluent B: x mM NaOH, y mM NaOAc
Page 18
5 – Preparation of Eluents and Standards
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 18 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
To make one (1) liter of 50 mM sodium hydroxide/ 25 mM sodium acetate, dispense
approximately 800 mL of vacuum-degassed water into a 1 L volumetric flask. Add a stir bar and
begin stirring. Weigh out 2.05 g anhydrous, crystalline sodium acetate (Thermo Scientific Dionex
Sodium Acetate Reagent, Item # 059326). Add the solid acetate steadily to the briskly stirring
water to avoid the formation of clumps which are slow to dissolve. Once the salt has dissolved,
remove the stir bar with a magnetic retriever. Add DI water to the flask to bring the volume to the
1 L mark.
Vacuum filter the solution through a 0.2 µm Nylon filter. This may take a while as the filter
may clog with insoluble material from the sodium acetate. Using a plastic tip volumetric pipet,
measure 4.0 g (approximately 2.6 mL) of 50% (w/w) sodium hydroxide solution. Dispense the
sodium hydroxide solution into the acetate solution about 1 inch under the surface of the acetate
solution. The eluent should be kept blanketed under helium or nitrogen at 34 to 55 kPa (5–8 psi)
at all times, and it should last about 1 week.
5.4 0.1 M Sodium Hydroxide / 0.25 M Sodium Acetate
To make one (1) liter of 0.1M sodium hydroxide/ 0.25M sodium acetate, dispense approximately
800 mL of vacuum-degassed DI water into a 1 L volumetric flask. Vacuum degas for
approximately 5 minutes. Add a stir bar and begin stirring. Weigh out 20.51 g anhydrous,
crystalline sodium acetate (Thermo Scientific Dionex Sodium Acetate Reagent, Item # 059326).
Add the solid acetate steadily to the briskly stirring water to avoid the formation of clumps which
are slow to dissolve. Once the salt has dissolved, remove the stir bar with a magnetic retriever.
Add DI water to the flask to bring the volume to the 1 L mark.
Vacuum filter the solution through a 0.2 µm Nylon filter. This may take a while as the filter
may clog with insoluble material from the sodium acetate. Using a plastic tip volumetric pipet,
measure 8.01 g (approximately 5.2 mL) of 50% (w/w) sodium hydroxide solution. Dispense the
sodium hydroxide solution into the acetate solution about 1 inch under the surface of the acetate
solution. The eluent should be always be kept a under helium or nitrogen at 34 to 55 kPa (5–8
psi), and last about 1 week.
Thermo Scientific recommends the use of plasticware, pipets and filtration apparatus for
exclusive use in the preparation of carbohydrate eluents.
5.5 Sample Preparation
The Dionex CarboPac PA300-4µm column is an anion exchange column. Thus, the normal
caveats applicable to ion exchange chromatography apply to this column. High salt concentrations
in samples should be avoided where possible. Special care should be taken with samples
containing high concentrations of strongly anionic compounds for the Dionex CarboPac PA3004µm column (e.g. chloride, carbonate, phosphate, etc.). It is best to avoid extremes of sample pH
(especially extremely acidic samples). The presence of anionic detergents (e.g., SDS) in samples
should be avoided entirely. Nonionic or cationic detergents may be acceptable in low
concentrations.
IMPORTANT
The presence of anionic detergents (e.g. SDS) in samples should be avoided entirely.
Nonionic or cationic detergents may be acceptable in low concentrations.
NOTE
!
Page 19
5 – Preparation of Eluents and Standards
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 19 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
Matrix Interferent
Effect
Possible Removal
Hydroxylated compounds
(e.g. Tris buffers, alcohols)
PED-active (interferes with
carbohydrate detection)
Dialysis, dilution
Halides
Will bind to column, may affect
retention time of analytes and
interact with gold electrode.
Dialysis, dilution, or solid-phase
extraction using Thermo
Scientific Dionex OnGuard Ag
(silver) cartridge.
Amine-containing compounds
(including proteins, peptides,
and free amino acids).
PED active
Solid-phase extraction using
Dionex OnGuard A (anionexchange). For inline use, the
Dionex AminoTrap column is
used for proteins, peptides, and
amino acids.
Lipids
May foul column
Liquid-liquid extraction or
supercritical fluid extraction.
Organic solvents
May affect analyte retention and
cause diminished electrode
response.
Solid-phase extraction using
Dionex OnGuard RP (reversephase).
Anionic detergents (such as
SDS)
Will bind irreversibly to the
column.
Solid-phase extraction using
Dionex OnGuard RP.
When using Pulsed Amperometry Detection (PAD), eliminate electrochemically active
components (e.g. TRIS buffer, alcohols, and other hydroxylated compounds) from eluents and
samples. Small amounts of organic solvents in the sample may not harm the column, although the
organics may interfere with the chromatography or detection of the analytes of interest.
Sample matrices in glycoprotein analysis can be simplified by performing a Western blot and
selectively removing the carbohydrates from the PVDF membrane-bound proteins. Please ask for
Dionex Technical Note 30, “Monosaccharide and Oligosaccharide Analysis of Glycoproteins
Electrotransferred onto Polyvinylidene Fluoride (PVDF) membranes,” or retrieve it from our Web
site at www.thermoscientific.com.
Page 20
6 – Example Applications
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 20 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
6. Example Applications
The CarboPac PA300-4µm column is designed for isocratic and/or gradient separation of glycans using sodium
hydroxide and sodium acetate eluent system. Analyte separation is highly dependent on hydroxide concentration in
HPAEC. Some separations may require only an isocratic eluent method. However, some groups of analytes will require
a step or gradient elution. Retention of carbohydrates can be varied with eluent concentration, in some cases changing
the elution order as the eluent concentration increases.
The following section provides an example of types of applications for which the Dionex CarboPac PA300-4µm
column can be used. The chromatograms in this section were obtained using columns that reproduced the Quality
Assurance Report on an optimized Ion Chromatograph. Different systems will differ slightly in performance due to
slight differences in column set, system void volumes, liquid sweep-out times of different components and laboratory
temperatures. Depending on your system, you may have to make small adjustments to your gradient conditions and/or
operating temperature to achieve the required resolution for the analytes of interest.
6.1 Analysis of Sugar Alditols: A Quality Assurance Report (QAR)
Isocratic separation of mono- and di-saccharide alditol standards is used to test the performance of the Dionex CarboPac
PA300-4µm column. While the CarboPac PA300-4µm analytical column should always be used with a CarboPac
PA300-4µm Guard Column, the QAR chromatogram shipped with the analytical column does not employ a guard
column. The addition of the Guard column will increase elution time by ~5% when compared to the Analytical Column
by itself. To guarantee that all Dionex CarboPac PA300-4µm Analytical Columns meet high quality and reproducible
performance specification standards, all columns undergo the following quality assurance testing with sugar alditol
standards at 30 ºC.
The Dionex CarboPac PA300-4µm column was developed to provide fast, high-resolution separations for of complex
oligosaccharides from biologics and food samples. Using the Dionex CarboPac PA300-4µm column and 10 mM NaOH
isocratically, the seven sugar alditols can be separated within 12 min as shown in Figure 1.
Figure 1 Dionex CarboPac PA300-4µm 2×250 mm: Without and With Guard Column
0
2
4
6
8 10 12
0
120
nC
0
120
nC
Minutes
Analytical Column
Analytical + Guard Column
Column: Dionex CarboPac PA300-4µm, 2x250mm
Flow Rate: 0.25mL/min
Temperature: 30oC
Detection: Integrated Amperometry,
Quadruple Pulse Waveform
Working Electrode: PTFE Gold (Disposable)
Ref Electrode: Ag/AgCI
Injection Volume: 2.5 µL
Standard Conc: 50 µM
Eluents: A - Milli Q Water
B - 200 mM NaOH
Eluent Program
Peaks:
1. Myo-Inositol
2. Xylitol
3. Dulcitol
4. Sorbitol
5. Mannitol
6. Perseitol
7. Maltitol
1
2
345
6
7
1
2
4
5
6
7
3
Time
%A %B
Comments
-25.0
0 100
Regeneration
-15.0
0 100
-14.9
95 5
Equilibration
0.0 95 5
Load/Inject
12.0
95 5
End
Page 21
6 – Example Applications
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 21 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
6.2 Characterization of Released Glycans Using CarboPac PA300-4µm
6.2.1 Bovine Fetuin N-Linked and O-Linked Glycan Alditol Profiling
Most secreted and cell surface proteins are glycosylated. They typically have multiple glycosylation sites, each site
containing several structures. Therefore, a protein can exist in many glycoforms. Glycans present on the protein can
have a profound effect on the protein structure and on biological function. Therefore, the study of glycans is an
important part of protein characterization. Biopharmaceuticals, including glycoproteins require thorough
characterization and analysis to meet European, US and Japanese regulatory standards for New Drug Approval as
defined by the International Conference for Harmonization (ICH) process. Even at the clinical trial stage, the national
requirements for conduct of clinical studies necessitate clear identification, quality, and purity of the investigational
drug (FDA guidance documents for IND, MCA guidelines for CTX).
The high resolution of the CarboPac PA300-4µm is demonstrated in the following example. CarboPac columns can
separate mono, oligo, and polysaccharides based on fine structural differences in branching, linkage isomerism,
anomericity and sialylation. In the fetuin oligosaccharide alditol standard shown in Figure 2, peaks are separated
according to branching, sialylation and linkage isomerism. The disialylated biantennary peaks are eluted before the
trisialylated triantennary peaks which are eluted before the tetrasialylated tetraantennary peaks. In addition, within each
grouping, the α2-6 isomer is eluted before, and well resolved from, the α2-3 isomer.
The Thermo Scientific Dionex OligoStandard, Sialylalted N-Linked Alditols, Item # 043064 contains 25 nmol
oligosaccharides released and purified from bovine fetuin. Dilute the standard prior to use, by adding a known volume
of DI water (for example 1 mL; a 25 μL injection corresponds to 625 pmol of oligosaccharide). Thermo Scientific
recommends running this standard every time a new column is installed and subsequently anytime it becomes necessary
to troubleshoot your system.
Following enzymatic deglycosylation of N-linked glycans, the remaining O-linked glycans can be cleaved from the
fetuin protein using standard β-elimination with subsequent sample cleanup. This produces two distinct populations
of glycans from the glycoprotein: N-linked alditols (Figure 2) and O-linked alditols (Figure 3).
Figure 2 N-Linked Glycan Alditol Profile of Bovine Fetuin on Dionex CarboPac
PA300-4µm
Column: Dionex CarboPac PA300-4µm (Guard + Analytical)
Flow Rate: 0.25mL/min
Temperature: 30oC
Detection: Integrated amperometry, quadruple pulse waveform
Working Electrode: PTFE Gold (Disposable)
Ref. Electrode: Ag/AgCI
Injection Volume: 2.5 µL
Eluents: A - Milli Q Water
B - 200 mM NaOH
C - 50 mM NaOH 25 mM NaOAc
D - 100 mM NaOH 250 mM NaOAc
Eluent Program
0
15
30 45 60 75
0
300
Minutes
nC
Figure 2. N-linked Glycan Alditol Profile of Bovine Fetuin on Dionex CarboPac PA300-4um
Time
%A %B %C %D
Comments
-1.0
78.5
19.5
2 0
Equilibration
0.0
78.5
19.5
2 0
Load/inject
4.0
78.5
19.5
2 0
20.0
20 20 60 0
50.0
0 0 0 100
Regeneration
59.9
0 0 0 100
60.0
78.5
19.5
2 0
Equilibration
75.0
78.5
19.5
2 0
End
Page 22
6 – Example Applications
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 22 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
Figure 3 O-Linked Glycan Alditol Profile of Bovine Fetuin on Dionex CarboPac
PA300-4µm
Column: DionexCarboPac PA300-4µm (Guard + Analytical)
Flow Rate: 0.25mL/min
Temperature: 30oC
Detection: Integrated amperometry, quadruple pulse waveform
Working Electrode: PTFE Gold (Disposable)
Ref. Electrode: Ag/AgCI
Injection Volume: 2.5 µL
Eluents: A - Milli Q Water
B - 200 mM NaOH
C - 50 mM NaOH 25 mM NaOAc
D - 100 mM NaOH 250 mM NaOAc
Eluent Program
0 15 30 45 60 75
0
77
Minutes
nC
Figure 3. O-Linked Glycan Alditol Profile of Bovine Fetuin on DionexCarboPac PA300-4um
Time
%A %B %C %D
Comments
-1.0
78.5
19.5
2 0
Equilibration
0.0
78.5
19.5
2 0
Load/inject
4.0
78.5
19.5
2 0
20.0
20 20 60 0
50.0
0 0 0 100
Regeneration
59.9
0 0 0 100
60.0
78.5
19.5
2 0
Equilibration
75.0
78.5
19.5
2 0
End
Page 23
6 – Example Applications
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 23 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
6.2.2 Porcine Gastric Type III Mucin O-Linked Glycan Alditol
Glycosylation in mammals represents a very diverse set of pre- and post-translational modifications. One of the most
diverse and complex sets of glycans is found on mammalian mucin proteins. Mucins are heavily glycosylated proteins
that are often involved in cellular recognition and signaling, and they often form chemical barriers on the extracellular
surface of cells. Regulation of this family of proteins is thought to be tightly controlled, as the overexpression of many
mucin proteins is strongly associated with many types of cancers.
The characterization of mucin proteins has been incredibly challenging largely due to the nature of mucin glycosylation.
Mucins have tandem repeat sequences of amino acids with high concentrations of serine and threonine residues, and
these often become saturated with O-linked glycans. O-linked glycosylation is non-template driven, and the glycans
themselves are often complex in structure and composition, creating a high-density, heterogenous cluster of glycans on
regions of the mucin protein. This complexity often inhibits analysis of the glycoproteins or glycopeptides, creating a
need for additional technologies to characterize the sample.
Figure 4 shows a characteristic chromatogram of porcine gastric Mucin type III O-glycan on a CarboPac PA300-4µm
column. Note the diversity of glycans present in the sample. O-linked glycans were released by β-elimination with
subsequent sample cleanup.
Figure 5 demonstrates a chromatographic comparison of Mucin type III O-glycan traces using the CarboPac PA3004µm compared to the CarboPac PA200 column. CarboPac PA300-4µm column exceeds the CarboPac PA200
performance in resolving the early eluting neutral O-glycans while maintaining the resolution of later eluted charged
glycans.
Figure 4 O-Linked Glycan Profile of Porcine Gastric Mucin Type III on Dionex
CarboPac PA300-4µm
0 15 30 45 60 75
0
300
Minutes
nC
Column: Dionex CarboPac PA300-4µm (Guard + Analytical)
Flow Rate: 0.25mL/min
Temperature: 30oC
Detection: Integrated amperometry, quadruple pulse waveform
Working Electrode: PTFE Gold (Disposable)
Ref. Electrode: Ag/AgCI
Injection Volume: 2.5 µL
Eluents: A - Milli Q Water
B - 200 mM NaOH
C - 50 mM NaOH 25 mM NaOAc
D - 100 mM NaOH 250 mM NaOAc
Eluent Program
Figure 4. O-Linked Glycan Profile of Porcine Gastric Mucin Type III on Dionex CarboPac PA300-4um
Time
%A %B %C %D
Comments
-1.0
78.5
19.5
2 0
Equilibration
0.0
78.5
19.5
2 0
Load/inject
4.0
78.5
19.5
2 0
20.0
20 20 60 0
50.0
0 0 0 100
Regeneration
59.9
0 0 0 100
60.0
78.5
19.5
2 0
Equilibration
75.0
78.5
19.5
2 0
End
Page 24
6 – Example Applications
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 24 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
Figure 5 Dionex CarboPac PA300-4µm vs. CarboPac PA200 Separation of O-glycans
Released from Porcine Mucin Type III.
Page 25
6 – Example Applications
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 25 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
6.2.3 N-Linked Glycan Profile of Trastuzumab (Herceptin).
Antibodies have become a cornerstone of numerous medical and scientific procedures, and they are therefore produced
routinely for both medical and research applications. In the medical world, they are used as diagnostics, to detect
cancers or infection by certain bacteria or viruses; as vaccines, to boost the body's immune response; and as
therapeutics, to target foreign bacteria, viruses or cancerous cells.
Figure 6 below shows a chromatographic separation of N-glycans released from Trastuzumab, a groundbreaking
monoclonal antibody used to treat HER2 positive breast, stomach, and esophageal cancers, using CarboPac PA3004µm column.
Figure 6 N-Linked Glycan Profile of Trastuzumab (Herceptin) on Dionex CarboPac
PA300-4µm
Figure 6 N-Linked Glycan Profile of Trastuzumab (Herceptin) on Dionex CarboPac PA300-4µm
0
15
30 45
60
75
0
70
Minutes
nC
Column: DionexCarboPac PA300-4µm (Guard + Analytical)
Flow Rate: 0.25mL/min
Temperature: 30oC
Detection: Integrated amperometry, quadruple pulse waveform
Working Electrode: PTFE Gold (Disposable)
Ref. Electrode: Ag/AgCI
Injection Volume: 2.5 µL
Eluents: A - Milli Q Water
B - 200 mM NaOH
C - 50 mM NaOH 25 mM NaOAc
D - 100 mM NaOH 250 mM NaOAc
Eluent Program
Time
%A %B %C %D
Comments
-1.0
78.5
19.5
2 0
Equilibration
0.0
78.5
19.5
2 0
Load/inject
4.0
78.5
19.5
2 0
20.0
20 20 60 0
50.0
0 0 0 30
59.9
0 0 0 30
60.0
78.5
19.5
2 0
Equilibration
75.0
78.5
19.5
2 0
End
Page 26
6 – Example Applications
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 26 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
6.2.4 N-linked Glycan Profile of Bovine Milk Proteins
Glycosylation of milk proteins is associated with many functions, such as passive protection from pathogen binding to
the intestinal cells, maintaining correct protein folding, protecting proteins from digestion, and facilitating the release
of encrypted bioactive peptides. Structural characterization of milk glycans is critical in understanding the biological
properties that are associated with the presence of glycans, and ultimately in determining milk's nutritional quality. Nlinked glycans released from milk proteins include oligomannose, complex, and hybrid type, with an abundance of
sialylation.
Figure 7 demonstrates N-glycan profile using CarboPac PA300-4µm column. N-glycans were released from 5 mg of
bovine milk protein by PNGase F treatment, followed by the sample purification with a HyperSepTM HypercarbTM Filter
Plate (Item # 60110-504).
Figure 7 N-Linked Glycan Profile of Bovine Milk Protein on Dionex CarboPac PA300-
4µm
Figure 7 N-Linked Glycan Profile of Bovine Milk Protein on Dionex CarboPac PA300-4µm
Column: Dionex CarboPac PA300-4µm (Guard + Analytical)
Flow Rate: 0.25mL/min
Temperature: 30oC
Detection: Integrated amperometry, quadruple pulse waveform
Working Electrode: PTFE Gold (Disposable)
Ref. Electrode: Ag/AgCI
Injection Volume: 2.5 µL
Eluents: A - Milli Q Water
B - 200 mM NaOH
C - 50 mM NaOH 25 mM NaOAc
D - 100 mM NaOH 250 mM NaOAc
Eluent Program
0
15 30
45 60
75
5
40
Minutes
nC
Time
%A %B %C %D
Comments
-1.0
78.5
19.5
2 0
Equilibration
0.0
78.5
19.5
2 0
Load/inject
4.0
78.5
19.5
2 0
20.0
20 20 60 0
50.0
0 0 0 100
Regeneration
59.9
0 0 0 100
60.0
78.5
19.5
2 0
Equilibration
75.0
78.5
19.5
2 0
End
Page 27
6 – Example Applications
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 27 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
6.3 Analysis of Free Complex Carbohydrates in Food
6.3.1 Galacto-oligosaccharides (GOS) Syrup
Galacto-oligosaccharides (GOS) are a group of non-digestible carbohydrates that are increasingly being used as
functional food ingredients. The GOS fractions comprise galactose oligosaccharides with a terminal glucose, varying
in type of glycosidic linkages between monosaccharide units and in the degree of polymerization (DP). The differences
in GOS structures can affect their roles on gut microbiota.
Figure 8 demonstrates the separation of oligosaccharides in Bimuno® GOS syrup with a CarboPac PA300-4µm
column. Samples were prepared by dissolving 20 mg of Bimuno® GOS syrup in 10 mL of water to make a stock
solution, diluting the stock to make a solution with the final concentration of 400 mg/L, and filtering through a 0.2 μm
filter. The mixture of GOS is separated mainly based on their size (DP). The early eluting peaks (RT earlier than 33
minutes) are mainly DP2 and DP3 oligosaccharides, with a presence of low-abundant DP4 molecules. Peaks eluting
between 33 and 35 minutes are mainly DP4. Peaks present between 35 and 37 minutes are mainly DP5. Peaks eluting
near 39 minutes are mainly DP6. The size of the oligosaccharides was identified by the HPAE-MS platform.
Figure 8 Oligosaccharides Profile in GOS Syrup on Dionex CarboPac PA300-4µm
Column: Dionex CarboPac PA300-4µm (Guard + Analytical)
Flow Rate: 0.25mL/min
Temperature: 30oC
Detection: Integrated amperometry, quadruple pulse waveform
Working Electrode: PTFE Gold (Disposable)
Ref. Electrode: Ag/AgCI
Injection Volume: 2.5 µL
Eluents: A - Milli Q Water
B - 200 mM NaOH
C - 50 mM NaOH 25 mM NaOAc
D - 100 mM NaOH 250 mM NaOAc
Eluent Program
Figure 8 Oligosaccharides Profile in GOS Syrup on Dionex CarboPac PA300-4µm
Time
%A %B %C %D
Comments
-1.0
78.5
19.5
2 0
Equilibration
0.0
78.5
19.5
2 0
Load/inject
4.0
78.5
19.5
2 0
20.0
20 20 60 0
50.0
0 0 0 100
Regeneration
59.9
0 0 0 100
60.0
78.5
19.5
2 0
Equilibration
75.0
78.5
19.5
2 0
End
0
15
30 45 60 75
0
750
Minutes
Response (
nC)
7
DP6
8
13 & 14
10
DP4
DP5
1
16
2-4
5
6
9
11
12
15
17
18
19
20 & 21
Peaks:
DP2: 1, 8 & 9, 13 & 14
DP3: 2 - 6, 11 & 12, 17 - 21
DP4: 7, 10, 15 & 16
Page 28
6 – Example Applications
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 28 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
6.3.2 Free Oligosaccharides in Infant Formula with 2’-FL HMO
Human milk oligosaccharides (HMOs) are a family of free glycans that contains diverse structures and are highly
abundant in human milk. Numerous publications have demonstrated the importance of HMOs not only in enhancing
the development of the intestinal microbiota of newborns, but also bolstering the immune system in breastfed infants.
Therefore, HMOs are a vital component of infant nutrition. The most abundant HMO in most mothers’ breast milk is
2′-fucosyllactose (2’-FL). Preclinical data suggests that the addition of HMOs to infant formula is safe, resulting in the
addition of 2′-FL HMO to some commercial infant formulas.
Figure 9 shows the oligosaccharide profile in a commercial infant formula product with 2’-FL. The sample containing
0.1 mL of infant formula product was diluted and passed through a HyperSepTM HypercarbTM Filter Plate (Item #
60110-504) for cleanup prior to analysis. The peak eluted at 21.275 min is 2’-FL, and the identity was confirmed with
a 2’-FL standard purchased from Biosynth Carbosynth®.
Figure 9 Oligosaccharides Profile in Infant Formula with 2’-FL on Dionex CarboPac
PA300-4µm
Peak:
1: 2’-Fucosyllactose
RT: 21.275 min
Column: Dionex CarboPac PA300-4µm (Guard + Analytical)
Flow Rate: 0.25mL/min
Temperature: 30oC
Detection: Integrated amperometry, quadruple pulse waveform
Working Electrode: PTFE Gold (Disposable)
Ref. Electrode: Ag/AgCI
Injection Volume: 2.5 µL
Eluents: A - Milli Q Water
B - 200 mM NaOH
C - 50 mM NaOH 25 mM NaOAc
D - 100 mM NaOH 250 mM NaOAc
Eluent Program
Figure 9 Oligosaccharides Profile in Infant Formula with 2’-FL on DionexCarboPac PA300-4µm
Time
%A %B %C %D
Comments
-1.0
78.5
19.5
2 0
Equilibration
0.0
78.5
19.5
2 0
Load/inject
4.0
78.5
19.5
2 0
20.0
20 20 60 0
50.0
0 0 0 100
Regeneration
59.9
0 0 0 100
60.0
78.5
19.5
2 0
Equilibration
75.0
78.5
19.5
2 0
End
0 15 30 45 60 75
0
500
Minutes
nC
2’-Fucosyllactose standard
Infant Formula with 2’-FL
1
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6 – Example Applications
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 29 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
6.4 Structural Characterization of Complex Carbohydrates Using CarboPac PA300-
4µm Column in the IC-MS platform
6.4.1 Porcine Gastric Type III Mucin O-Linked Glycan Alditol by IC-MS
High resolution separations, especially to separate populations of chemically similar but structurally heterogenous
glycans, becomes important for subsequent mass spectrometric characterization. When the CarboPac PA300-4µm
column is used in an IC-MS workflow, it enables the identification of glycan structures.
Figure 10 demonstrates the base peak chromatogram of porcine gastric mucin type III O-glycans injected into an Q
Exactive™ HF-X Hybrid Quadrupole-Orbitrap™ Mass Spectrometer. A selection of peaks are numbered and identified
below in Table 2. Peak compositional identities were confirmed with previously published data (Jin, C., Kenny, D. T.,
Skoog, E. C., Padra, M., Adamczyk, B., Vitizeva, V., Thorell, A., Linden S. K., & Karlsson, N. G. (2017). Structural
diversity of human gastric mucin glycans. Molecular & Cellular Proteomics, 16(5), 743-758).
In this application example, we demonstrate that IC-MS platform supports simultaneous separation and detection of
neutral and charged (sialylated and sulfated) glycans without the need for derivatization. High resolution MS data and
tandem MS/MS spectrum with diagnostic fragments provide a highly reliable structural annotation of heterogenous
glycans. The unique IC-MS workflow presented here provides confirmatory, orthogonal information for glycan
analysis.
Figure 11 show a characteristic chromatogram and the ability for the CarboPac PA300-4µm to resolve compositional
isomers.
Figure 10 Base Peak Chromatogram of O-Linked Glycans of Porcine Gastric Mucin
Protein on Dionex CarboPac PA300-4µm
0
100
0 15 30 45
60
75
Time (min)
Relative Abundance
1
3
2
4
5
6
7
8
9
10
Column: Dionex CarboPac PA300-4µm (Guard + Analytical)
Flow Rate: 0.25mL/min
Temperature: 30oC
Detection: Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap™ MS
Injection Volume: 20 µL
Eluents: A - Milli Q Water
B - 200 mM NaOH
C - 50 mM NaOH 25 mM NaOAc
D - 100 mM NaOH 250 mM NaOAc
Eluent Program
Time
%A %B %C %D
Comments
-1.0
78.5
19.5
2 0
Equilibration
0.0
78.5
19.5
2 0
Load/inject
4.0
78.5
19.5
2 0
20.0
20 20 60 0
50.0
0 0 0 100
Regeneration
59.9
0 0 0 100
60.0
78.5
19.5
2 0
Equilibration
75.0
78.5
19.5
2 0
End
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6 – Example Applications
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Product Manual for Dionex CarboPac PA300-4µm Columns
Page 30 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
Table 2 The Compositional Identities of Selected Peaks in Figure 10.
Figure 11 Dionex CarboPac PA300-4µm Column Provides Isomer Resolution of Both
Neutral and Sulfated O-glycans from Porcine Gastric Mucin Type III Sample
Peak RT (min) Observed m/z Theoretical m/z
Mass accuracy
(ppm)
Composition
Charge
state
1 4.85 733.2896 733. 2884 1.64 Hex Fuc [HexNAc]
2
1
2 6.08 530.2080 530.2090 1.89 Hex Fuc HexNAc 1
3 8.46 733.2892 733. 2884 1.09 Hex Fuc [HexNAc]
2
1
4 11.97 1041.4006 1041.3992 1.34 [Hex]2[Fuc]2[HexNAc]
2
1
5 15.58 1098.4233 1098.4206 2.46 [Hex]2Fuc [HexNAc]
3
1
6 17.66 1203.4506 1203.4520 1.16
[Hex]3[Fuc]2[HexNAc]
2
1
7 34.01 821.3063 821.3045
2.19
NeuAc Hex Fuc HexNAc 1
8 38.19 852.2811 852.2848 4.34 N-glycan, hybrid, sulfated 2
9 45.92
1121.3568 1121.3559 0.80
[Hex]2[Fuc]2[HexNAc]2-S 1
10 53.19 813.2474 813.2452 2.70 Hex Fuc [HexNAc]2-S 1
Hex: Hexose; Fuc: Fucose; HexNAc: N-Acetylhexosamine; NeuAc: N-Acetylneuraminic acid; S: Sulfated
0
15
30
45
60
75
Time (min)
0
100
0
100
Relative Abundance
0
100
#2: RT 15.58
m/z: 1098.4233
#1: RT 10.07
m/z: 1098.4231
#3: RT17.15
m/z:1098.4232
#2: RT 47.92
m/z: 844.2839
#2: RT 42.17
m/z:661.7144
#1: RT 40.76
m/z: 661.7151
#3: RT 48.39
m/z: 661.7147
#1: RT 46.58
m/z: 844.2835
[Hex]2Fuc [HexNAc]
3
[Hex]3[Fuc]2[HexNAc]4-S
[Hex]2[Fuc]2[HexNAc]3-S
Figure 11 Dionex CarboPac PA300-4µm Column Provides Isomer Resolution of Both Neutral and
Sulfated O-glycans from Porcine Gastric Mucin Type III Sample
Page 31
6 – Example Applications
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 31 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
6.4.2 Analysis of Bovine Milk Free Oligosaccharides Using IC-MS
Oligosaccharides are bioactive molecules that have a variety of health benefits to consumers. One of the known health
benefits is the use of oligosaccharides as prebiotics, which play an important role in establishing the intestinal
microbiota by selectively simulating the growth of beneficial bacteria. Bovine milk is a promising candidate source for
novel functional carbohydrates. It contains oligosaccharides that have structures similar to those found in human milk,
and there is a growing interest in examining the structures of those milk oligosaccharides to understand their potential
biological benefits.
Figure 12 shows the analysis of oligosaccharides extracted from 0.1 mL of a commercial, lactose-free bovine milk
sample after sample cleanup with a HyperSepTM HypercarbTM Filter Plate. The purified oligosaccharide sample was
analyzed using HPAE-PAD and IC-MS individually via two independent injections. The chromatograph clearly shows
the ability of the CarboPac PA300-4µm column to separate charged, sialylated glycan structures from neutral, nonsialylated structures. The peak identities are confirmed with tandem mass spectrometry analysis and analytical
standards purchased from Biosynth Carbosynth®. The chromatograms obtained from two different detectors are similar,
yet with some variations in peak responses. Response factors provided by the two different detection methods for each
glycan species is dependent on the glycan structures.
Figure 12 Analysis of Free Oligosaccharides from Commercial, Lactose-Free Bovine
Milk with a Dionex CarboPac PA300-4µm Column
2
3
4
1
5
PAD response
Base peak
chromatogram
Peaks:
1: [Hex]
3
2: [Hex]2 HexNAc
3: Lacto-N-neotetraose
4: 6'-sialyllactose
5: 3'-sialyllactose
Column: Dionex CarboPac PA300-4µm (Guard + Analytical)
Flow Rate: 0.25mL/min
Temperature: 30oC
PAD Detection: Integrated Amperometry, Quadruple Pulse Waveform
Working Electrode: PTFE Gold (Disposable)
Ref. Electrode: Ag/AgCI
Mass spectrometer: Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap™ MS
Injection Volume: 10 µL
Eluents: A - Milli Q Water
B - 200 mM NaOH
C - 50 mM NaOH 25 mM NaOAc
D - 100 mM NaOH 250 mM NaOAc
Eluent Program
0
15
30
45 60 75
0
300
nC
2
3
4
1
5
0
100
0 15 30 45 60 75
Time (min)
Relative Abundance
Figure 12. Analysis of Free Oligosaccharides from Commercial, Lactose-Free Bovine Milk with a Dionex
CarboPac PA300-4µm Column
Time
%A %B %C %D
Comments
-1.0
78.5
19.5
2 0
Equilibration
0.0
78.5
19.5
2 0
Load/inject
4.0
78.5
19.5
2 0
20.0
20 20 60 0
50.0
0 0 0 100
Regeneration
59.9
0 0 0 100
60.0
78.5
19.5
2 0
Equilibration
75.0
78.5
19.5
2 0
End
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6 – Example Applications
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 32 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
6.5 Robustness and ruggedness of Dionex CarboPac PA300-4µm
The Dionex CarboPac PA300-4µm column provides consistent chromatography performance and highly reproducible
separation. To demonstrate the ruggedness of the Dionex CarboPac PA300-4µm column, a mixture of seven sugar
alditols was injected three times every 10th run for a total of 100 back-to-back runs. Every 10th run is displayed here
in Figure 13, showing high reproducibility for the Dionex CarboPac PA300-4µm column.
Figure 13. Stability Test on a Dionex CarboPac PA300-4µm Column
Column: Dionex CarboPacPA300-4µm (Guard + Analytical)
Eluent: 10mM KOH
Flow Rate: 0.25mL/min
Temperature: 30oC
Detection: Integrated Amperometry,
Quadruple Pulse Waveform
Working Electrode: PTFE Gold (Disposable)
Ref Electrode: Ag/AgCI
Injection Volume: 2.5 µL
Standard Conc: 50 µM
0 3 6 9 12 15
0
450
1
2
3
4
5
6
7
Minutes
nC
Peaks:
1. Myo-Inositol
2. Xylitol
3. Dulcitol
4. Sorbitol
5. Mannitol
6. Perseitol
7. Maltitol
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6 – Example Applications
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Product Manual for Dionex CarboPac PA300-4µm Columns
Page 33 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
6.6 Comparison between Ag/AgCl and PdH reference electrodes
The Electrochemical Detector (ED) cell is a miniature flow-through amperometric detection cell that includes three
different electrodes: a titanium cell body (the counter electrode), a working electrode, and a reference electrode. For
carbohydrate applications, pulsed amperometric detection is typically performed with a series of potentials at a gold
working electrode where the analytes are detected.
A reference electrode is required to assure application of the proper working electrode potential. Both silver/silver
chloride (Ag/AgCl) electrodes and palladium hydrogen (PdH) reference electrodes have been commonly used in
carbohydrate applications. In this section, the performance of a PdH reference electrode was demonstrated and
compared to a Ag/AgCl reference electrode for two carbohydrate applications: a mixture of sugar alditols (shown in
figure 14) and N-glycans released from bovine fetuin glycoprotein (shown in figure 15). In both applications, the signal
response was similar, but slightly higher, with the PdH reference electrode.
Please refer to Thermo Scientific Electrochemical Detection User’s Compendium
(http://tools.thermofisher.com/content/sfs/manuals/Man-065340-Electrochemical-Detection-Man065340-EN.pdf) for
detailed instructions on the installation of a PdH reference electrode.
Figure 14. Comparison between a PdH and a Ag/AgCl Reference Electrode for Analysis
of Sugar Alditols on Dionex CarboPac PA300-4µm
Figure 14. Comparison between a PdH and a Ag/AgCl Reference Electrode for Analysis of Sugar Alditols
on Dionex CarboPac PA300-4µm
Column: Dionex CarboPac PA300-4µm, 2x250mm
Flow Rate: 0.25mL/min
Temperature: 30 oC
Detection: Integrated Amperometry, Quadruple Pulse Waveform
Working Electrode: PTFE Gold (Disposable)
Ref Electrode: PdH or Ag/AgCI
Injection Volume: 2.5 µL
Standard Amount: 50 µM
Eluents: A - Milli Q Water
B - 200 mM NaOH
Eluent Program
Peaks:
1. Myo-Inositol
2. Xylitol
3. Dulcitol
4. Sorbitol
5. Mannitol
6. Perseitol
7. Maltitol
Time
%A %B
Comments
-25.0
0 100
Regeneration
-15.0
0 100
-14.9
95 5
Equilibration
0.0 95 5
Load/Inject
12.0
95 5
End
0 4 8 12
0
180
0
180
Minutes
PdH
Ag/AgCl
Response (
nC)
Respone
(nC)
1
2
4
5
6
7
3
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Product Manual for Dionex CarboPac PA300-4µm Columns
Page 34 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
Figure 15. Comparison between a PdH and a Ag/AgCl Reference Electrode for Analysis
of N-linked Glycan Alditol Profile of Bovine Fetuin on Dionex CarboPac
PA300-4µm
Figure 15. Comparison between a PdH and a Ag/AgCl Reference Electrode for Analysis of N-linked
Glycan Alditol Profile of Bovine Fetuin on Dionex CarboPac PA300-4µm
nC
Minutes
Column: DionexCarboPac PA300-4µm (Guard + Analytical)
Flow Rate: 0.25mL/min
Temperature: 30 oC
Detection: Integrated amperometry, quadruple pulse waveform
Working Electrode: PTFE Gold (Disposable)
Ref. Electrode: PdH or Ag/AgCI
Injection Volume: 2.5 µL
Eluents: A - Milli Q Water
B - 200 mM NaOH
C - 50 mM NaOH 25 mM NaOAc
D - 100 mM NaOH 250 mM NaOAc
Eluent Program
Time
%A %B %C %D
Comments
-1.0
78.5
19.5
2 0
Equilibration
0.0
78.5
19.5
2 0
Load/inject
4.0
78.5
19.5
2 0
20.0
20 20 60 0
50.0
0 0 0 100
Regeneration
59.9
0 0 0 100
60.0
78.5
19.5
2 0
Equilibration
75.0
78.5
19.5
2 0
End
Response (
nC)
250
0 15 30 45 60 75
0
250
0
PdH
Ag/AgCl
Response (
nC)
Page 35
7 – Troubleshooting Guide
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 35 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
7. Troubleshooting Guide
The purpose of the Troubleshooting Guide is to help you solve operating problems that may arise
while using Dionex CarboPac columns. For more information on problems that originate with the
Ion Chromatograph (IC), refer to the Troubleshooting Guide in the appropriate operator’s manual.
Remember that some of the problems may be related to parts of your experimental protocol
(sample contamination, imprecision during sample transfer, etc.). The following text should help
you to locate and eliminate problems traceable to the carbohydrate hardware and chemistries. It
also provides a selection of cleanup and reconditioning procedures that have been found effective.
For assistance, contact Technical Support for Dionex Products. In the U.S., call 1-800-346-
6390. Outside the U.S., call the nearest Thermo Fisher Scientific office.
7.1 High Back Pressure
7.1.1 Finding the Source of High System Pressure
Column pressure, (after subtracting the system pressure) for the Dionex CarboPac PA300-4µm
Analytical Column should be close to the pressure listed in the QAR when using the test
chromatogram conditions. If a Dionex CarboPac guard and analytical column are both installed,
column pressure will increase by 5-10 % over the pressure listed in the QAR for the column. If
the total system pressure is much higher than expected, it is advisable to determine the cause of
the high system pressure.
A. Make sure that the pump is set to the correct eluent flow rate. Higher than recommended
eluent flow rates will cause higher pressure. If necessary, measure the pump flow rate
by collecting the DI H2O eluent for a specified time at operating pressure, and measure
the collected amount using an analytical balance. This data (weight/time) will give actual
flow rate.
B. Determine which part of the system is causing the high pressure. High pressure could be
due to plugged or constricted tubing, an injection valve with a clogged port or worn rotor,
a column bed support clogged with particulates, or a clogged detector cell.
C. To determine which part of the chromatographic system is causing the problem,
disconnect the pump eluent line from the injection valve and turn the pump on. Watch
the pressure; it should not exceed 200 psi. (unless a backpressure coil has been installed
between the pump outlet and the injection valve in which case, first disconnect the eluent
line from the pump to the backpressure coil). The pressure with the eluent generator
connected should be <400 psi. Continue adding system components (backpressure coil
(if present), injection valve, column(s), and detector) one by one, while monitoring the
system pressure. The pressure should increase by the sum of the measured pressures of
the individual guard and analytical columns (see product QAR) when the CarboPac
Guard and Analytical columns are connected.
D. Measure the system back pressure by attaching a short piece of new 0.010” tubing in
place of the column.
NOTE
!
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7 – Troubleshooting Guide
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Product Manual for Dionex CarboPac PA300-4µm Columns
Page 36 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
7.1.2 Replacing Column Bed Support Assemblies for 2 mm column
If the column inlet bed support is determined to be the cause of the high back pressure, it should
be replaced. To change the inlet bed support assembly, refer to the following instructions, using
one of the two spare inlet bed support assemblies included in the Ship Kit.
A. Disconnect the column from the system.
B. Carefully unscrew the inlet (top) column fitting. Use two open-end wrenches.
C. Remove the bed support. Turn the end fitting over and tap it against a benchtop or other
hard, flat surface to remove the bed support assembly. If the bed support must be pried
out of the end fitting, use a sharp pointed object such as a pair of tweezers, but be careful
that you do not scratch the walls of the end fitting. Discard the old bed support assembly.
D. Place a new bed support assembly (provided with each analytical column) into the end
fitting. Make sure that the end of the column tube is clean and free of any particulate
matter so that it will properly seal against the bed support assembly. Drop the bed
support assembly into the end fitting, making sure that the bed support assembly is
centered at the bottom of the end fitting. Tap the end fitting gently on a hard surface to
reorient the bed support assembly as necessary in order to properly situate the bed
support assembly in the end fitting.
If the column tube end is not clean when inserted into the end fitting, particulate matter
may obstruct a proper seal between the end of the column tube and the bed support
assembly. If this is the case, additional tightening may not seal the column but instead
damage the column tube or the end fitting.
E. While holding the column in an inverted configuration, tighten the end fitting back onto
the column. Tighten it finger-tight, then an additional 1/4 turn (25 in-lb). Tighten further
only if leaks are observed.
F. Reconnect the column to the system and resume operation.
7.1.3 Filter Eluent
Eluents containing particulate material or bacteria may clog the column inlet bed support. Filter
eluents through a 0.2 µm Nylon or PES (PolyEtherSulfone) filter (We recommend the Thermo
Scientific, Nalgene Sterile Disposable Filter Units with Nylon Membrane, Item # 164-0020).
DO NOT use a cellulosic filter (e.g., cellulose acetate or regenerated cellulose) as these will
introduce cellulosic polymers into your eluent, and thus many Integrated Amperometric peaks
will appear, even with blank “injections”.
7.1.4 Filter Samples
Samples containing particulate material may clog the column inlet bed support. Filter samples
through a 0.2 µm Nylon or PES (PolyEtherSulfone) filter prior to injection.
CAUTION
!
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7 – Troubleshooting Guide
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Product Manual for Dionex CarboPac PA300-4µm Columns
Page 37 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
7.2 High Background
While it may be possible to obtain reasonable performance even with elevated levels of detection
background according to some requirements, high background frequently brings about increased
gradient artifacts and can be accompanied by a presence of ghost peaks. Detection sensitivity may
also change suddenly when the detection background is too high. A background >35 nC with 10
mM hydroxide at 0.25 mL/min and 30°C using the quadruple waveform indicates one of the
following possibilities:
A. Incorrect detection parameters.
Verify that Ag/AgCl is specified as a reference electrode. Check all of the waveform
values in the program against those in the Disposable Electrode Manual. If the pH
reading with 10 mM NaOH is above 13.2 replace the reference electrode.
B. Compromised working electrode surface.
Briefly install a new working electrode and check the background as above. If the
reading remains > 35 nC, remove the new electrode within 30 minutes and continue
testing for column or system contamination. If the detector background signal is in the
10-35 nC range, continue with your work with the new electrode installed.
C. Column contamination: Remove the column set from the system first and replace it
with a length of yellow PEEK tubing, generating a pressure drop between 1000 and
2000 psi. If the background reading improves after the column is removed from the
system, go to Appendix A, “CarboPac PA300-4µm Column Care”.
D. Water contamination: Prepare eluents using a fresh ultra-pure water from another
source. If the background is reduced, investigate the source of contamination in the
original source of water.
E. System Contamination: If the background remains high even with fresh water and
without the column, carry out a 2 M sodium hydroxide rinse. In a properly working
system, the electrochemical detection (ED) background for the Dionex CarboPac
PA300-4µm QAR eluent is 10-35nC. If the background is much higher, determine the
cause of high background. Consider the possibility that the eluent filter might be
cellulosic, as that will introduce very high signal and noise.
7.2.1 Preparation of Eluents
A. Make sure that the eluents are made correctly.
B. Make sure that the eluents are made from chemicals with the recommended purity.
C. Make sure that the deionized water used to prepare the reagents has a specific resistance
of 18.2 MΩ-cm or greater
7.2.2 Dionex CR-ATC Column
A. When using eluent generator (Dionex EGC 500 KOH) to generate eluent, install a
Dionex CR-ATC 500 Anion Trap Column.
B. If the background is elevated due to contamination of the Dionex CR-ATC 500, please
refer to Sections 5.3 and 6, in the Dionex CR-ATC 500 Product Manual (Document No.
079684) for corrective action.
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Product Manual for Dionex CarboPac PA300-4µm Columns
Page 38 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
7.2.3 A Contaminated Guard or Analytical Column
A. Remove the columns from the system.
B. Install a back-pressure coil that generates approximately 2000 psi and continue to pump
eluent. If the background decreases, the column(s) is (are) the cause of the high
background.
C. To eliminate downtime, clean or replace the analytical column at the first sign of column
performance degradation. Clean the column as instructed in, “Appendix A, Dionex
CarboPac PA300-4µm Column Care”.
7.3 Poor Resolution
One of the unique features of Dionex CarboPac columns is the fast equilibration time in gradient
applications from the ending eluent (high ionic strength) to the beginning eluent (low ionic
strength). The actual equilibration time depends on the ratio of the strongest eluent concentration
to the weakest eluent concentration and application flow rate. Typically, equilibration times range
from 10 to 15 minutes at 0.25 mL/min for 2 mm ID columns.
If increased separation is needed for early eluting peaks, reduce the initial eluent concentration.
Due to different system configurations, the delivered gradient profile may not exactly match the
gradient shown in example applications in the column product manual. Gradient conditions can
be adjusted to improve resolution or to adjust retention times either by changing the gradient
timing or by changing the initial and/or final eluent concentration.
A. Keep the eluent concentrations constant and adjust the gradient time. This is the simplest
way to compensate for total system differences if resolution is the problem.
B. Change the initial and/or final eluent concentration and adjust the gradient time. This
approach requires more time to develop and more experience with methods development
work. Its advantage is that it allows a method to be tailored for a particular application,
where selectivity, resolution, and total run time are optimized. Be aware that poor peak
resolution can be due to any or all of the following factors.
7.3.1 Loss of Column Efficiency
A. Check to see if headspace has developed in the guard or analytical column. This is
usually due to improper use of the column such as exposing it to high pressures. Remove
the column’s inlet end fitting (see Section 7.1.2, “Replacing Column Bed Support
Assemblies”). If the resin does not fill the column body to the top, the column must be
replaced.
B. Extra-column effects can result in sample band dispersion or band broadening. Make
sure you are using viper fittings for all connections, including the sample loop (if
necessary, PEEK tubing with an ID of no greater than 0.005" can be used if junctions
are fitted and tightened appropriately). For PEEK tubing, cut the tubing lengths as short
as possible. Check for leaks.
C. If tubing is not connected properly from the inlet and outlet of the column, it can cause
low efficiency. When installing Dionex CarboPac columns, it is recommended to turn
off the pump while connecting the column inlet and the column outlet to the detector.
This will help avoid any slippage of the ferrule when attempting to secure the fitting
under elevated pressure conditions.
D. Bypassing the pre-column heater unit if the extra volume introduced by this unit is the
cause for peak broadening or loss of efficiency.
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Product Manual for Dionex CarboPac PA300-4µm Columns
Page 39 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
7.3.2 Shortened Retention Times
Even with adequate system and column efficiency, resolution of peaks will be compromised
if analytes elute too early.
A. Check the flow rate. See if the eluent flow rate is equivalent to the flow rate specified by
the analytical protocol. Collect the eluent for a specified time after the column and
measure the eluent flow rate gravimetrically using an analytical balance.
B. Check to see if the eluent compositions and concentrations are correct. An eluent that is
too concentrated will cause the peaks to elute earlier. If eluent concentration is too
concentrated, prepare fresh eluent.
If you are using a gradient pump to proportion components from two or three different
eluent reservoirs, the resulting eluent composition may not be accurate enough for the
application. Try using a single reservoir containing the correct eluent composition to see if
this is the problem. This is more likely to occur when one of the proportioned eluents is less
than 5%.
C. Column contamination can lead to a loss of column capacity. Highly retained
contaminants will tend to occupy anion exchange sites limiting the number of sites
available for retention of the analytes. Refer to "Appendix A, Dionex CarboPac PA3004µm Column Care,” for recommended column cleanup procedures.
Possible sources of column contamination are impurities in chemicals and in the deionized
water used for eluents or components of the sample matrix. Be especially careful to make
sure that the recommended chemicals are used. The deionized water should have a specific
resistance of > 18.2 -cm.
D. Diluting the eluent will improve peak resolution but will also increase the retention
times. If a 10% dilution of the eluent is not sufficient to obtain the desired peak
resolution, or if the resulting increase in retention times is unacceptable, try cleaning
the column (see Appendix A, Dionex CarboPac PA300-4µm Column Care).
After cleaning the column, reinstall it in the system and let it equilibrate with eluent for
about 30 minutes directing the column effluent to waste. Then connect the column to
the electrochemical detector cell. No water wash is necessary. The column is
equilibrated when consecutive injections of the standard result in reproducible retention
times. Capacity close to the original capacity should be restored by this treatment since
the contaminants should be cleared from the column.
For assistance, contact Technical Support for Dionex Products. In the U.S., call 1-800346-6390. Outside the U.S., call the nearest Thermo Fisher Scientific office.
NOTE
!
NOTE
!
NOTE
!
NOTE
!
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Product Manual for Dionex CarboPac PA300-4µm Columns
Page 40 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
7.3.3 Loss of Resolution for early eluting peaks
If poor resolution or efficiency is observed for early eluting peaks compared to the later eluting
peaks, check the following:
A. Improper eluent concentration may be the problem if retention time is less than expected.
Check the flow rate of the pump, as pump flow rate will affect the eluent concentration
in an RFIC-EG system. Ensure the Eluent Generator is set to the correct eluent
concentration.
B. Column overloading may be an issue. Reduce the amount of sample injected onto the
column by either diluting the sample or injecting a smaller volume.
C. Sluggish operation of the injection valve may also cause this, due to partially plugged
port faces. Refer to the valve manual for instructions.
D. Improperly swept volumes anywhere in the system prior to the guard and
analytical/capillary columns may reduce resolution of early peaks. Swap components,
one at a time, in the system prior to the analytical/capillary column and test for early
eluting peak resolution after every component change.
7.3.4 Spurious Peaks
A. The column(s) may be contaminated. If the samples contain an appreciable level of
polyvalent ions and the column is used with a weak eluent system, the retention times
will decrease and spurious, inefficient (broad) peaks may show up at unexpected times.
Clean the column as indicated in “Column Care”.
For assistance, contact Technical Support for Dionex Products. In the U.S., call 1-800346-6390. Outside the U.S., call the nearest Thermo Fisher Scientific office.
B. The injection valve may need maintenance. When an injection valve is actuated, the
possibility of creating a baseline disturbance exists. This baseline upset may appear as a
peak of varying size and shape. This will occur when the injection valve needs to be
cleaned or re-torqued (see injection valve manual). Check to see that there are no
restrictions in the tubing connected to the valve. Also check the valve port faces for
blockage and replace them if necessary. Refer to the Valve Manual for troubleshooting
and service procedures. Small baseline disturbances at the beginning or at the end of the
chromatogram can be overlooked as long as they do not interfere with the quantification
of the peaks of interest.
C. Another potential cause of spurious peaks or dips is amperometric reference electrode
offset. Since the reference electrode is continually exposed to hydroxide, the Cl- in the
Ag/AgCl electrode will eventually be exchanged for hydroxide (OH-). This will result in
a voltage offset and result in delivery of waveform potentials that differ from the
programmed values by the offset potential. This may result in unexpected peaks and dips
in the chromatographic carbohydrate retention window. If this is suspected, perform the
reference electrode calibration in Chromeleon. If the offset is > 25 mV, replace the ED
cell reference electrode.
NOTE
!
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7.3.5 No Peaks, Poor Peak Area Reproducibility or too Small Peak Areas
A. Check the position and filling levels of sample vials in the autosampler.
B. Check injector needle-height setting.
C. Check the injection valve orientation. If the valve is installed upside down, or if it is
plumbed 180° out of the correct orientation, switching from load to inject will bypass
the loop. If this is confirmed, re-plumb the valve in the correct orientation.
D. Check each line of the schedule for proper injector parameters. Use full-loop sampling
and appropriate loop size.
E. Check the total transfer volume (TLV) and recalibrate this volume. If the newly
calibrated volume has changed dramatically from the previously recorded value, service
the autosampler transfer line.
F. Service the injection valve (check for leaks, Tefzel fragments, or sediments inside the
valve).
G. Remove the borate trap from the system and perform a quality assurance test of the
column (Appendix B) without borate trap in-line. If the old borate trap was the cause of
loss of peaks, replace the contaminated borate trap with a new one and/or clean the
contaminated borate trap using the following protocol:
• Set up a stand-alone pump with following wash solutions:
a) 1M Methanesulfonic acid (MSA)
b) 2M NaOH
• Connect the inlet of the contaminated borate trap to the pump outlet, and direct the
outlet of the borate trap to a waste container
• Wash the borate trap at 1mL/min with 1M MSA for at least 2 hours, and then with
2M NaOH for 2 hours
• Test the cleaned borate trap under the quality assurance report for CarboPac
PA300-4µm conditions (Appendix B) and ensure QAR data is acceptable.
7.3.6 Large Baseline Dip in the Chromatogram
A large baseline dip appearing into the chromatogram is usually caused by oxygen in the sample
injected. This ‘oxygen dip’ is normal and can be reduced in magnitude with higher hydroxide
concentrations in the eluent. If the sample vial is emptied causing the sampler to “inject” air, this
dip will dramatically increase.
7.3.7 Unidentified Peaks Appear with Expected Analyte Peaks
During an acetate or hydroxide gradient, several small peaks may appear. These peaks are usually
due to trace contaminants in the water supply used to prepare eluents. The contaminants
accumulate on the column during the isocratic, or low eluent strength section of the
chromatogram, and are eluted, frequently as irregular baseline deformations or sharp spikes, with
the increasing eluent strength.
Some trace contaminants can co-elute with glycans, compromising accuracy of quantitation at
lower concentrations. If extraneous peaks are observed even after the water supply is excluded as
a possible cause, clean the autosampler lines and sample loop. The autosampler should be cleaned
using the following protocol:
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Product Manual for Dionex CarboPac PA300-4µm Columns
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155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
A. Disconnect the column and detector cell from the autosampler.
B. Set the pump to 100% deionized water.
C. Place the following solutions in the autosampler and inject in sequence. Use 25 µL full
loop injections:
1. 1 M NaOH
2. Deionized water
3. IPA
4. Deionized water
5. 1 M HCl
6. Deionized water
Sometimes multiple cycles of each solution may be required. Also, if you suspect a dirty sample
loop or injection valve rinse them with above protocol.
7.3.8 Decreased Detection Sensitivity
Always confirm the loss of response by performing at least one injection of the system suitability
standard mix as described in Section 6.1. This is to make sure that a decreased level of response
is not being caused by system problems.
Any decrease in detection sensitivity means that the working electrode surface has been affected.
The operator should install a replacement (disposable) working electrode. Spare reference
electrodes and disposable gold working electrodes should always be available in order to avoid
unnecessary delays.
Exceptions:
Check the pH reading. If the value is out of range or >13.2, install a new reference electrode and
then install a new gold disposable working electrode. The system cleanup is not necessary. The
decrease in sensitivity can be caused by a loss of surface area on the disposable electrode, or by
deposition of gold-oxide on the conventional electrode surface because the reference potential
was too high. The conventional gold working electrode can be reconditioned by polishing.
Peak heights will also increase with increasing eluent concentrations, especially between 1 and
10 mM. This is due to improvement of the kinetics in the electrode detection related to ionic
strength and pH effects. If you run the same standard at 1mM and at 12 mM, peak heights will
be significantly higher at 12mM. You can expect a peak area (and height) decrease whenever
reducing your eluent strength below 8 mM.
After installing a new working electrode (disposable or conventional, with or without the
complete system cleanup), confirm the expected detection sensitivity. Should the response be too
low, immediately remove the new working electrode from the system to minimize its
contamination.
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7.3.9 Excessive Gradient Drift
The magnitude of the gradient baseline drift can be minimized by high eluent strength column
wash steps during the times when the system is not in use for sample or standard analysis. This
will keep the column conditioned, free from buildup of carbonate and other contaminants, and
ready for analysis.
A. Make sure the gradient drift is not caused by the eluents and/or detector cell (Working or
Reference electrodes).
B. Set column temperature to 30 °C and wash the guard and analytical column with 1M NaOH
for at least four hours (preferably overnight). For high concentrations of NaOH, we
recommend bypassing the ED cell and diverting the column effluent to waste.
7.4 Reconditioning or Replacement of the Gold (conventional or disposable)
Electrodes or Replacement of the Reference Electrode
Refer to the Product Manual for Disposable Electrodes (Doc. No. 065040), Dionex ICS-6000 Ion
Chromatography System Manual (Doc. No. 22181-97002) or User’s Compendium for
Electrochemical Detection (Doc. No. 065340) for any help necessary with electrochemical
detection, working and reference electrodes.
Page 44
Appendix A – Dionex CarboPac PA210-Fast-4µm Column Care
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 44 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
Appendix A: Dionex CarboPac PA300-4µm
Column Care
A.1 Recommended Operation Pressures
Operating a column above its recommended pressure limit can cause irreversible loss of column
performance. The maximum recommended operating pressure for Dionex CarboPac PA300-4µm
column is 5,000 psi (34.47 MPa).
A.2 Column Start-Up
The Dionex CarboPac columns are shipped using sodium hydroxide (see QAR) as the storage
solution. Use manually prepared sodium hydroxide eluent employed in the Quality Assurance
Report (QAR). Install the column in the chromatography module and direct the column effluent
to waste for 60 minutes, and then connect to the ED cell. It is recommended to clean the column
for 1 to 2 hours with 200 mM NaOH at 0.25 mL/min for 2 mm column to ensure good
chromatography without baseline artifacts. Test the column performance under the conditions
described in the QAR. Continue making injections of the test standard until consecutive injections
of the standard give reproducible retention times. Equilibration is complete when consecutive
injections of the standard give reproducible retention times.
If chromatographic efficiency or resolution is poorer than the QAR, see Sections 7.3 Poor
Resolution and Section 7.3.1 Loss of Column Efficiency.
IMPORTANT
When making any tubing connections (column installation, replacing tubing etc.), it is
recommended to make these connections with the pump turned off.
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Appendix A – Dionex CarboPac PA210-Fast-4µm Column Care
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 45 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
A.3 Column Storage
Store the CarboPac PA300-4µm column in 10 mM hydroxide eluent. Flush the column for a
minimum of 20 minutes with the storage solution. Cap both ends securely, using the plugs
supplied with the column.
A.4 Dionex CarboPac PA300-4µm Column Cleanup
The Dionex CarboPac PA300-4µm can be readily cleaned by rinsing the column with ~ 60 column
volumes of 200 mM NaOH. More stubborn contamination problems may necessitate a thorough
column cleaning. Use the following steps to thoroughly clean the Dionex CarboPac PA300-4µm;
use 0.25 mL/min for 2mm Column formats to avoid over-pressurization of the column:
A. Disconnect column from the ED cell and direct the column effluent to waste. If your
system is configured with both a guard column and an analytical column, reverse the
order of the guard and analytical column in the eluent flow path, but ensure the flow
direction is as shown on the column label. Do not direct flow backward through the
column(s).
When cleaning an analytical column and a guard column in series, ensure that the guard
column is placed after the analytical column in the eluent flow path. Otherwise,
contaminants that have accumulated on the guard column elute onto the analytical column
causing irreversible damage. If in doubt, clean each column separately.
B. Wash the Dionex CarboPac PA300-4µm column with deionized water (18.2 -cm)
for about 30 minutes and then clean with 1 M MSA for one to two hours at 0.25mL/min.
C. Wash the column with deionized water for about 30 minutes.
D. Then clean the Dionex CarboPac PA300-4µm with 200 mM NaOH for at least two hours.
E. Reconnect column to the cell and equilibrate the column with the desired initial
conditions; test the column performance using the QAR standard and eluent.
CAUTION
!
Page 46
Appendix B – Quality Assurance Report
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
Page 46 of 46
155014-02 For Research Use Only. Not for Use in Diagnostic Procedures.
Appendix B: Quality Assurance Report
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