Thermo Scientific PA300-4µm Product Manual
For Research Use Only. Not for use in diagnostic procedures.
Dionex CarboPac PA300-4µm
155014 Revision 02 • September 2020
User Manual
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)
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
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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.
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
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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
!
Contents
Thermo Scientific
Product Manual for Dionex CarboPac PA300-4µm Columns
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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
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
Contents
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Product Manual for Dionex CarboPac PA300-4µm Columns
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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
1 – Introduction
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Product Manual for Dionex CarboPac PA300-4µm Columns
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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
2 – System Requirements and Installation
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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
!
2 – System Requirements and Installation
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Product Manual for Dionex CarboPac PA300-4µm Columns
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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.
2 – System Requirements and Installation
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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.
2 – System Requirements and Installation
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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
!
3 – Before You Start
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Product Manual for Dionex CarboPac PA300-4µm Columns
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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
!
3 – Before You Start
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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.
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