Thermo Fisher Scientific Dionex IonPac ICE-AS1 Analytical Column User manual

Brand
Thermo Fisher Scientific
Model
Dionex IonPac ICE-AS1 Analytical Column
Type
User manual
For Research Use Only. Not for use in diagnostic procedures.
Dionex IonPac ICE-AS1 Analytical Column
031181 Revision 07 • March 2017
User Manual
Thermo Scientific
Product Manual for Dionex IonPac ICE-AS1 Column
Page 2 of 52
031181-07 For Research Use Only. Not for use in diagnostic procedures.
Product Manual
for
Dionex IonPac ICE-AS1 Analytical Column
(9 x 150 mm, P/N 302622)
(9 x 250 mm, P/N 043197)
(4 x 250 mm, P/N 064198)
Thermo Scientific
Product Manual for Dionex IonPac ICE-AS1 Column
Page 3 of 52
031181-07 For Research Use Only. Not for use in diagnostic procedures.
© 2017 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.
For Research Use Only. Not for Use in Diagnostic Procedures.
Revision History:
Revision 04, March, 2017, Reformatted for Thermo Fisher Scientific Inc. Added information for 9 x 150 mm,
P/N 302622.
Thermo Scientific
Product Manual for Dionex IonPac ICE-AS1 Column
Page 4 of 52
031181-07 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
!
Contents
Thermo Scientific
Product Manual for Dionex IonPac ICE-AS1 Column
Page 5 of 52
031181-07 For Research Use Only. Not for use in diagnostic procedures.
Contents
Contents................................................................................................................................................................... 5
1. Introduction ..................................................................................................................................................... 7
2. Exclusion Chromatography System .................................................................................................................. 9
3. Installation ..................................................................................................................................................... 11
3.1 System Requirements ............................................................................................................................ 11
3.2 System Void Volume............................................................................................................................. 11
3.3 The Injection Loop ................................................................................................................................ 11
3.4 Eluent Storage ....................................................................................................................................... 11
3.5 Dionex ACRS-ICE 500 Suppressor Requirements .................................................................................. 11
4. Operation ...................................................................................................................................................... 12
4.1 General Operating Conditions ................................................................................................................ 12
4.2 Dionex IonPac ICE-AS1 Operation Precautions ..................................................................................... 12
4.3 Chemical Purity Requirements ............................................................................................................... 12
4.3.1 Inorganic Chemicals...................................................................................................................... 12
4.3.2 Deionized Water ........................................................................................................................... 12
4.3.3 Solvents ........................................................................................................................................ 13
4.4 Eluent Preparation ................................................................................................................................. 14
4.4.1 Acid Eluent Preparation ................................................................................................................ 14
4.4.2 Eluents Containing Solvents .......................................................................................................... 14
4.5 Anion Suppression Regenerant Preparation ............................................................................................ 14
5. Example Applications ..................................................................................................................................... 15
5.1 Preparation of Eluents ........................................................................................................................... 15
5.2 pKa Values of Selected Organic Acids ................................................................................................... 15
5.3 Dionex IonPac ICE-AS1 Elution Plots and Tables .................................................................................. 18
5.3.1 Dionex IonPac ICE-AS1 Run Time vs. Eluent Strength ................................................................. 18
5.3.2 Dionex IonPac ICE-AS1 Run Time vs. Eluent Strength (Expanded Scale)...................................... 19
5.3.3 Dionex IonPac ICE-AS1 Run Time vs. Temperature...................................................................... 21
5.4 Production Test Chromatogram ............................................................................................................. 22
5.4.1 Dionex IonPac ICE-AS1 9 x 250 mm ............................................................................................ 22
5.4.2 Dionex IonPac ICE-AS1 4 x 250 mm ............................................................................................ 24
5.5 Comparison of Dionex IonPac ICE-AS1 and Dionex IonPac ICE-AS6 for C1 - C6 Retention (9 x 250 mm)
25
5.6 Temperature Effects .............................................................................................................................. 26
5.7 UV Detection with Dionex IonPac ICE-AS1 .......................................................................................... 27
5.8 The Addition of Solvent to the Eluent to Reduce Run Time .................................................................... 28
5.9 Analysis of Aliphatic and Hydroxy Acids .............................................................................................. 29
Contents
Thermo Scientific
Product Manual for Dionex IonPac ICE-AS1 Column
Page 6 of 52
031181-07 For Research Use Only. Not for use in diagnostic procedures.
5.10 Analysis of Various Organic Acids ........................................................................................................ 30
5.11 Use of Solvent to Optimize Selectivity ................................................................................................... 31
5.12 Tracking Degradation of Acrylic Acid in an Organic Acid Mix .............................................................. 32
5.13 Analysis of Silicate in a Plating Bath Matrix .......................................................................................... 33
5.14 Analysis of Organic Acids in a 52% Nitric Acid Matrix ......................................................................... 34
5.15 Analysis of Cyanide Using the Dionex IonPac ICE-AS1 Column ........................................................... 35
5.16 Pulsed Amperometric Detection of Sulfite ............................................................................................. 36
5.17 Analysis of Carbonate and Tetraborate Using the Dionex IonPac ICE-AS1 Column ............................... 37
5.18 Analysis of Aliphatic Alcohols Using Pulsed Amperometric Detection ................................................... 38
5.19 Glycols Separation ................................................................................................................................ 39
5.20 Nitriles: Acetronitrile and Propionitrile .................................................................................................. 40
5.21 Ketones: Acetone and 2-Butatone .......................................................................................................... 41
5.22 Alkene (I): Acrylic Acid and Propionic Acid .......................................................................................... 42
5.23 Alkyne: 3-Butyn-2-one and 2-Butatone .................................................................................................. 43
6. Troubleshooting ............................................................................................................................................. 44
6.1 High Back Pressure ............................................................................................................................... 45
6.1.1 Finding the Source of High System Pressure .................................................................................. 45
6.1.2 Replacing Column Bed Support Assemblies .................................................................................. 46
6.2 High Background or Noise .................................................................................................................... 47
6.2.1 Preparation of Eluents ................................................................................................................... 47
6.2.2 A Contaminated Guard or Analytical Column ................................................................................ 47
6.2.3 Contaminated Hardware ................................................................................................................ 47
6.2.4 A Contaminated Anion Chemically Regenerated Suppressor for ICE, Dionex ACRS-ICE 500 ....... 47
6.3 Poor Peak Resolution............................................................................................................................. 48
6.3.1 Loss of Column Efficiency ............................................................................................................ 48
6.3.2 Poor Resolution Due to Shortened Retention Times ....................................................................... 48
6.3.3 Loss of Front End Resolution ........................................................................................................ 49
6.4 Spurious Peaks ...................................................................................................................................... 49
6.5 Split Peaks ............................................................................................................................................ 50
Appendix A – Column Care ................................................................................................................................... 51
A.1 Recommended Operation Pressures ....................................................................................................... 51
A.2 Column Start-Up ................................................................................................................................... 51
A.3 Column Storage ..................................................................................................................................... 51
A.4 Column Cleanup.................................................................................................................................... 51
A.5 Choosing the Appropriate Cleanup Solution ........................................................................................... 52
A.6 Column Cleanup Procedure ................................................................................................................... 52
1 – Introduction
Thermo Scientific
Product Manual for Dionex IonPac ICE-AS1 Column
Page 7 of 52
031181-07 For Research Use Only. Not for use in diagnostic procedures.
1. Introduction
Ion exclusion uses a fully sulfonated resin with a dilute solution of strong acid as eluent for the
separation of weak acid anions. The retention mechanisms are Donnan exclusion, steric exclusion,
and adsorption partition. A strong acid eluent facilitates protonation of weak organic acids. In the
neutral form, these acids are not subject to Donnan exclusion and penetrate into the pores of
negatively charged sulfonated polystyrene/divinylbenzene resin. Separation is accomplished by
differences in pKa's, size, and hydrophobicity of the acid anions. The Donnan exclusion
mechanism causes stronger acid anions to elute before weaker acid anions according to increasing
pKa. For example, acetate (pK=4.56) elutes before propionate (pK=4.67). The adsorption
mechanism causes hydrophilic acid anions to elute before hydrophobic acid anions. For example,
tartrate elutes before succinate due to its two hydroxyls. Strong organic acid anions, such as
oxalate (pK=1.04) and pyruvate (pK=2.26, which remain totally or partially ionized) are subjected
to Donnan exclusion and elute early. Strong mineral acid anions are totally excluded and elute in
the void.
Dionex IonPac ICE-AS1 columns are stable between pH 0 - 7 and they are compatible with
eluents containing 0 ‑ 50% HPLC solvents such as methanol or acetonitrile. (Do not run eluents
with cations other than hydronium ion through the Dionex IonPac ICE-AS1.)
Figure 1 The Ion Exclusion Separation
1 – Introduction
Thermo Scientific
Product Manual for Dionex IonPac ICE-AS1 Column
Page 8 of 52
031181-07 For Research Use Only. Not for use in diagnostic procedures.
The Dionex IonPac ICE-AS1 is composed of a 7.5 µm cross-linked styrene/divinylbenzene resin
that is functionalized with sulfonate groups. The ion exchange capacity of the 9 x 250 mm
analytical column is 27 meq/column. The ion exchange capacity of the 9 x 150 mm analytical
column is 16.2 meq/column. The ion exchange capacity of the 4 x 250 mm analytical column is
5.3 meq/column. The Dionex IonPac ICE-AS1 has low hydrophobicity when compared to the
ICE-AS6. The Dionex IonPac ICE-AS1 also has high solvent compatibility (50%) when
compared to the ICE-AS6 (15%).
When setting up the analytical system, observe the special precautions listed in Section 4,
“Operation.” PEEK (polyetheretherketone) is used to make column hardware. PEEK has excellent
chemical resistance to most organic solvents and inorganic solutions. However, concentrated
sulfuric acid and concentrated nitric acid will attack PEEK. Tetrahydrofuran at concentrations of
greater than 20% is not compatible with PEEK systems. The Dionex IonPac ICE-AS1 Analytical
Column has a minimum efficiency of 9,000 plates/column for acetic acid under standard operating
conditions, and it operates at a back pressure between 600-900 psi (4.14 - 6.20 MPa) at 0.8
mL/min with the test eluent(0.16 mL/min for the 4 x 250 mm). However, both Dionex IonPac
ICE-AS1 columns are capable of operating at back pressures up to 1,000 psi (6.90 MPa). Dionex
IonPac ICE-AS1 Analytical Columns have 10‑32 threaded PEEK end fittings for use with
ferrule/bolt liquid line fittings.
Assistance is available for Dionex Products. In the U.S., call 1-800-346-6390. Outside the U.S.,
contact the nearest Thermo Fisher Scientific office.
2 – Exclusion Chromatography System
Thermo Scientific
Product Manual for Dionex IonPac ICE-AS1 Column
Page 9 of 52
031181-07 For Research Use Only. Not for use in diagnostic procedures.
2. Exclusion Chromatography System
Table 1 Configuration
Configuration
4-mm
9-mm
Eluent Flow Rate
0.16 mL/min
0.8 mL/min
Dionex Anion Chemically
Regenerated Suppressor for ICE
4mm Dionex ACRS-ICE 500 (P/N
084714)
9mm Dionex ACRS-ICE 500 (P/N
084715)
Injection Loop
2 - 15 μL
10 - 50 μL
System Void Volume
Eliminate switching valves,
couplers and the GM-3 Gradient
Mixer. Use only the 2-mm GM-4
Mixer
(P/N 049135).
Minimize dead volume. Switching
valves, couplers can be used. Use
the GM-2, GM-3 or recommended
gradient mixers.
Pumps
Use a Thermo Scientific Dionex IC
system in microbore configuration.
Use a Thermo Scientific Dionex IC
system in standard bore
configuration.
Detectors
Dionex ICS-Series Variable
Wavelength Detector with PEEK
Flow Cell (7 mm, 2.5 µL) (P/N
6074.0300)
Dionex ICS-5000+ CD
Conductivity Detector (analytical)
and Integrated Cell (P/N 079829).
Dionex Integrion CD Conductivity
Detector (P/N 22153-60036).
Dionex ICS-5000+ ED
Electrochemical Detector (P/N
072042) and Cell (P/N 072044).
Recommended back pressure: 30–
40 psi
Dionex ICS-Series Variable
Wavelength Detector with PEEK
Flow Cell (10 mm, 11 µL) (P/N
066346)
Dionex ICS-5000+ CD
Conductivity Detector (analytical)
and Integrated Cell (P/N 079829).
Dionex Integrion CD Conductivity
Detector (P/N 22153-60036).
Dionex ICS-5000+ ED
Electrochemical Detector (P/N
072042) and Cell (P/N 072044).
Recommended back pressure: 30–
40 psi
2 – Exclusion Chromatography System
Thermo Scientific
Product Manual for Dionex IonPac ICE-AS1 Column
Page 10 of 52
031181-07 For Research Use Only. Not for use in diagnostic procedures.
Table 2 Tubing Back Pressures
Color
Item #
ID Inches
ID cm
Volume
mL/cm
Back
Pressure
psi/ft at 1
mL/min
Back
Pressure
psi/ft at
0.25
mL/min
Back
Pressure
psi/cm at 1
mL/min
Green
044777
0.030
0.076
4.560
0.086
0.021
0.003
Orange
042855
0.020
0.051
2.027
0.435
0.109
0.015
Blue
049714
0.013
0.033
0.856
2.437
0.609
0.081
Black
042690
0.010
0.025
0.507
6.960
1.740
0.232
Red
044221
0.005
0.013
0.127
111.360
27.840
3.712
Yellow
049715
0.003
0.008
0.046
859.259
214.815
28.642
3 – Installation
Thermo Scientific
Product Manual for Dionex IonPac ICE-AS1 Column
Page 11 of 52
031181-07 For Research Use Only. Not for use in diagnostic procedures.
3. Installation
3.1 System Requirements
The Dionex IonPac ICE-AS1 Analytical Column is designed to be run on any Thermo Scientific
Dionex Ion Chromatograph equipped with suppressed conductivity detection.
3.2 System Void Volume
For best performance, all of the tubing installed between the injection valve and detector should
be 0.005" (P/N 044221) ID PEEK tubing. Note that 0.010" ID PEEK tubing (P/N 042690) may
be used but peak efficiency will be compromised which may also result in decreased peak
resolution. Minimize the lengths of all connecting tubing and remove all unnecessary switching
valves and couplers. If you need assistance in properly configuring your system contact technical
support for Dionex Products. In the U.S., call 1-800-346-6390. Outside the U.S., contact the
nearest Thermo Fisher Scientific office.
3.3 The Injection Loop
For most applications on a 4-mm analytical system, a 2-15 µL injection loop will be sufficient.
For the 9-mm column, a 10-50 µL injection loop can be used.
3.4 Eluent Storage
The Dionex IonPac ICE-AS1 column is designed to be used with acid eluent systems and isocratic
analysis. Storage under a helium atmosphere ensures contamination free operation and proper
pump performance. (Nitrogen can be used if eluents do not contain solvents). Acidic eluents that
contain acetonitrile should be made fresh daily. Acetonitrile slowly hydrolyzes in acidic solutions.
3.5 Dionex ACRS-ICE 500 Suppressor Requirements
The Dionex ACRS-ICE 500 Anion Chemically Regenerated Suppressor for ICE (4mm ACRS-
ICE 500, P/N 084714 or 2 mm ACRS-ICE 500, P/N 084715) should be used. The Dionex AERS
500 or Dionex ACRS 500 cannot be used for ion exclusion applications that require suppressed
conductivity detection. The Dionex ACRS-ICE 500 is compatible with all solvents and
concentrations as the systems and columns. Use Dionex Cation Regenerant Solution,
Tetrabutylammonium hydroxide (TBAOH, P/N 039602). Dilute as required for the example
applications. For detailed information on the operation of the Dionex ACRS-ICE 500
suppressors, see the "Product Manual for Dionex ACRS-ICE 500 suppressors" (Document No.
032661).
4 – Operation
Thermo Scientific
Product Manual for Dionex IonPac ICE-AS1 Column
Page 12 of 52
031181-07 For Research Use Only. Not for use in diagnostic procedures.
4. Operation
4.1 General Operating Conditions
The selectivity of the Dionex IonPac ICE-AS1 Analytical Column is designed to separate an
extensive group of low molecular weight organics acids in less than 20 minutes. The Dionex
IonPac ICE-AS1 column consists of a cross-linked (8%), microporous, hydrophilic resin that has
been sulfonated. The nature of the cross-linked polymeric structure of the packing material makes
the Dionex IonPac ICE-AS1 columns compatible with pH 0 - 7 eluents (see Section 4.2, “Dionex
IonPac ICE-AS1 Operation Precautions”) and 0 - 50% organic solvent eluents. The Dionex
IonPac ICE-AS1 can be used with any suppressible ionic eluent that does not exceed the capacity
of the Dionex Chemically Regenerated Suppressor for ICE, ACRS-ICE 500.
4.2 Dionex IonPac ICE-AS1 Operation Precautions
Maximum solvent concentration is 50%.
DO NOT use hydroxide eluents.
Filter and degas eluents.
Filter samples.
Maximum recommended operating pressure is 1,000 psi (6.90 MPa).
Always run the column with fresh eluent, with suppressor disconnected,
for about 20 minutes if the column has not been used for a week or longer.
4.3 Chemical Purity Requirements
Obtaining reliable, consistent, and accurate results requires eluents that are free of ionic and
spectrophotometric impurities. Chemicals, solvents, 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 help to protect your ion exchange columns and system components. Thermo Fisher
Scientific cannot guarantee proper column performance when the quality of the chemicals,
solvents and water used to prepare eluents has been compromised.
4.3.1 Inorganic Chemicals
Reagent Grade inorganic chemicals should always be used to prepare ionic eluents. Whenever
possible, inorganic chemicals that meet or surpass the latest American Chemical Society standard
for purity should be used. These inorganic chemicals will show a lot analysis on each label. The
analyses performed in Section 5, “Example Applications,” use heptafluorobutyric acid obtained
from Fluka Chemie AG.
4.3.2 Deionized Water
The deionized water used to prepare eluents should be Type I Reagent Grade Water with a specific
resistance of 18.2 megohm-cm. The deionized water should be free of ionized impurities,
organics, microorganisms, and particulate matter larger than 0.2 µm. Bottled HPLC-Grade Water
(with the exception of Burdick & Jackson) should not be used since most bottled water contains
an unacceptable level of ionic impurities.
CAUTION
!
4 – Operation
Thermo Scientific
Product Manual for Dionex IonPac ICE-AS1 Column
Page 13 of 52
031181-07 For Research Use Only. Not for use in diagnostic procedures.
4.3.3 Solvents
Solvents can be added to the ionic eluents used with Dionex IonPac ICE-AS1 columns to modify
the ion exchange process or improve sample solubility. The solvents used must be free of ionic
impurities. However, since most manufacturers of solvents do not test for ionic impurities, it is
important that the highest grade of solvents available be used. Currently, several manufacturers
are making ultrahigh purity solvents that are compatible for HPLC and spectrophotometric
applications. These ultrahigh purity solvents will usually ensure that your chromatography is not
affected by ionic impurities in the solvent. Currently at Thermo Fisher Scientific, we have
obtained consistent results using High Purity Solvents manufactured by Burdick and Jackson and
Optima® Solvents by Fisher Scientific.
When using a solvent in an ionic eluent, column generated back pressures will depend on the
solvent used, concentration of the solvent, the ionic strength of the eluent and the flow rate used.
The column back pressure will vary as the composition of water-methanol and water-acetonitrile
mixture varies. The practical back pressure limit for the Dionex IonPac ICE-AS1 columns is 2,000
psi.
Table 4 HPLC Solvents for Use with Dionex IonPac ICE-AS1 Columns
Solvent
Maximum Operating
Concentration*
Acetonitrile
50%
Methanol
50%
2-Propanol
50%
Tetrahydrofuran
50%
* See Section 4.2, “Dionex IonPac ICE-AS1 Operation Precautions”
4 – Operation
Thermo Scientific
Product Manual for Dionex IonPac ICE-AS1 Column
Page 14 of 52
031181-07 For Research Use Only. Not for use in diagnostic procedures.
4.4 Eluent Preparation
4.4.1 Acid Eluent Preparation
The acidic eluents used with the Dionex IonPac ICE-AS1 columns are stable and require no
special storage. Always prepare eluents with Type I Reagent Grade Water (see Section 4.3.2,
“Deionized Water”) which has been properly degassed.
Eluents that contain solvents should be stored in glass eluent bottles pressurized with helium
(nitrogen is soluble in solvents).
4.4.2 Eluents Containing Solvents
When mixing solvents with water, remember to mix solvent with water on a volume to volume
basis. If a procedure requires an eluent of 10% acetonitrile, prepare the eluent by adding 100 mL
of acetonitrile to an eluent reservoir. Then add 900 mL of deionized water to the acetonitrile in
the reservoir. Using this procedure to mix solvents with water will ensure that a consistent true
volume/volume eluent is obtained. Premixing water with solvent will minimize the possibility of
outgassing.
Degas the aqueous component of the eluent and then add the solvent component. Avoid
excessive purging or degassing of eluents containing solvents if possible, since a volatile
solvent can be “boiled” off from the solution.
4.5 Anion Suppression Regenerant Preparation
The regenerant used with the Dionex ACRS-ICE 500 Suppressor when used with the Dionex
IonPac ICE-AS1 to perform the analyses in Section 5, “Example Applications,” is 5 mM
tetrabutylammonium hydroxide (TBAOH). Use Dionex Cation Regenerant Solution (P/N
039602). Dilute 50 mL of the 0.1 M Cation Regenerant Solution to 1 L with degassed Type I
Reagent Grade Water. Usually 4 liters at a time is prepared. For a guide to properly adjusting the
regenerant flow rate, see Document No. 032661, the Product Manual for the Dionex ACRS-ICE
500.
NOTE
!
5 – Example Applications
Thermo Scientific
Product Manual for Dionex IonPac ICE-AS1 Column
Page 15 of 52
031181-07 For Research Use Only. Not for use in diagnostic procedures.
5. Example Applications
The chromatograms in this section were obtained using columns that reproduced the Production Test Chromatogram
(see Section 5.3, “Production Test Chromatogram”) on optimized Ion Chromatographs (see Section 3, “Installation”).
Systems will vary slightly in performance due to slight differences in column sets, system void volumes, liquid sweep-
out times of components, and laboratory temperatures.
The Dionex IonPac ICE-AS1 may be used isocratically for chromatographing a large number of carboxylic acids.
If your sample or standard contains organic acids, adding chromate (about 10 mg/L) will help stabilize them from
bacterial degradation at room temperature.
Before attempting any of the following example applications, ensure your system is properly configured.
Ensure all of the eluents have been made from high purity reagents and deionized water. All water used in the
preparation of eluents should be degassed, deionized water. For chemical purity requirements see Section 4.2,
“Chemical Purity Requirements.”
Run synthetic standards to calibrate and confirm the operation of your system. This column has a very high loading
capacity and can handle a large number of dirty samples. If the Dionex IonPac ICE-AS1 shows signs of fouling after
running complex samples, refer to the column cleanup protocols in Appendix A, “Column Care.”
5.1 Preparation of Eluents
The standard eluent for the example applications presented in this section is 1.0 mM heptafluorobutyric acid. It was
prepared from heptafluorobutyric acid (same as perfluorobutyric acid) obtained from FLUKA Chemie AG (P/N 77249).
It is > 99% (GC) purity with a molecular weight of 214.04 and a density of 1.652. It is supplied in 10.0 mL bottles
(16.52 g).
Heptafluorobutyric Acid Stock Solution (0.0772 M):
Dilute the entire contents of one 10.0 mL bottle to 1 L.
Heptafluorobutyric Acid Eluent (1.0 mM):
Dilute 13.5 g of the stock solution (0.0772 M) to 1 L.
If you prefer to work from a 0.100 M stock solution, dilute 21.40 g of the > 99% purity heptafluorobutyric acid to 1 L.
The eluent can then be made by diluting 10.0 g of the 0.100 M stock solution to 1 L to obtain the 1.0 mM
heptafluorobutyric acid eluent. Heptafluorobutyric acid was chosen for minimum background conductivity. However,
other mineral acids such as sulfuric and hydrochloric can be used.
5.2 pKa Values of Selected Organic Acids
The following tables list the pKs of selected organic acids in alphabetical order and in ascending order of pK. Organic
acids elute in approximately the order of ascending pK, but additional hydrogen bonding and adsorption variables
modify the elution order slightly so that the elution order of the acids is not strictly in order of ascending pK. The tables
plus the example applications are designed to give the chromatographer a simple method for estimating the ability of
the Dionex IonPac ICE-AS1 to separate various combinations of organic acids.
5 – Example Applications
Thermo Scientific
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031181-07 For Research Use Only. Not for use in diagnostic procedures.
Table 5 Organic Acids with pK's in Alphabetic order
Compound pK1 pK2 pK3
Acetic (ethanoic) 4.56
Aconitic (cis-propene-1,2,3-tricarboxylic) N/A
Acrylic (propenoic) 4.26
Adipic (hexanedioic) 4.26 5.03
Anisic (4-methoxybenzoic) 4.48
Ascorbic 4.03 11.34
Azelaic (nonanedioic) 4.39 5.12
Benzoic 4.00
Bromoacetic 2.72
Butanoic 4.63
Caproic (hexanoic) 4.85
Chloroacetic 2.68
Citraconic (cis-methylbutenedioic) 2.20 5.60
Citric (2-hydroxypropane-1,2,3-tricarboxylic) 2.87 4.35 5.69
Crotonic (trans-but-2-enoic) 4.69
Cyanoacetic 2.63
Dichloroacetic 0.87
Diglycolic (oxydiacetic) 2.79 3.93
Dithiotartaric (2,3-dimercaptobutanedioic) 2.71 3.48 8.89
Fluoroacetic 2.59
Formic (methanoic) 3.55
Fumaric (trans-butenedioic) 2.85 4.10
Galacturonic 3.23 11.42
Gentistic (5-hydroxysalicylic) 2.70
Glutaric (pentanedioic) 4.13 5.03
Glyceric (dl-2,3-dihydroxypropanoic) 3.52
Glycolic (hydroxyacetic) 3.63
Guanidine 13.54
2-Hydroxyisobutyric 3.72
4-Hydroxybenzoic 4.10 9.96
Hippuric (n-benzoylglycine) 3.50
Iodoacetic 2.98
Isobutyric (2-methylpropionic) 4.63
Isocitrate (dl-1-hydroxypropane-1,2,3-tricarboxylic) 3.02 4.28 5.75
Isovaleric (3-methylbutanoic) 4.58
Itaconic (methylenebutanedioic) 3.68 5.14
Ketoglutaric (2-oxopentanedioic) 1.85 4.44
Lactic (d-2-hydroxypropanoic) 3.66
Maleic (cis-butenedioic) 1.75 5.83
Malic (l-hydroxybutanedioic) 3.24 4.71
Malonic (propanedioic) 2.65 5.28
Mandelic (l-phenylhydroxyacetic) 3.19
Mellitic (benzenehexacarboxylic) 0.70 2.21 3.52
3-Mercaptopropanoic 4.34 10.84
Mesaconic (trans-methylbutene) 2.61
Mucic 3.08 3.63
Nitroacetic 1.46
Octanoic 4.89
Orotic (uracil-6-carboxylic) 1.96 9.34
Oxalic (ethanedioic) 1.04 3.82
Phthalic (benzene-1,2-dicarboxylic) 2.75 4.93
Pimelic (heptanedioic) 4.31 5.08
Pivalic (2,2-dimethylpropanoic) 4.83
Propanoic 4.67
Pyruvic (2-oxopropanoic) 2.26
Quinic (1,3,4,5-tetrahydroxycyclohexanecarboxylic) 3.36
Salicylic (2-hydroxybenzoic) 2.81 13.40
Squaric (3,4-dihydroxy-3-cyclobutene-1,2-dione 0.40 3.10
Succinic (butanedioic) 4.00 5.24
Tartaric (d-2,3-dihydroxybutanedioic) 2.82 3.95
Terephthalic
Thioglycolic (mercaptoacetic) 3.42 10.11
Thiolactic (dl-2-mercaptopropanoic) 3.48 10.08
Thiomalic (dl-mercaptobutanedioic) 3.30 4.60 10.38
Trichloroacetic 0.66
Trimellitic (benzene-1,2,4-tricarboxylic) 2.40 3.71 5.01
Uric (2,6,8-trihydroxypurine) 5.61
Valeric (pentanoic) 4.64
Vinylacetic (but-3-enoic) 4.12
5 – Example Applications
Thermo Scientific
Product Manual for Dionex IonPac ICE-AS1 Column
Page 17 of 52
031181-07 For Research Use Only. Not for use in diagnostic procedures.
Table 6 Organic Acids with pK's in Order of Increasing pK1
Compound pK1 pK2 pK3
Aconitic (cis-propene-1,2,3-tricarboxylic) N/A
Terephthalic
Squaric (3,4-dihydroxy-3-cyclobutene-1,2-dione 0.40 3.10
Trichloroacetic 0.66
Mellitic (benzenehexacarboxylic) 0.70 2.21 3.52
Dichloroacetic 0.87
Oxalic (ethanedioic) 1.04 3.82
Nitroacetic 1.46
Maleic (cis-butenedioic) 1.75 5.83
Ketoglutaric (2-oxopentanedioic) 1.85 4.44
Orotic (uracil-6-carboxylic) 1.96 9.34
Citraconic (cis-methylbutenedioic) 2.20 5.60
Pyruvic (2-oxopropanoic) 2.26
Trimellitic (benzene-1,2,4-tricarboxylic) 2.40 3.71 5.01
Fluoroacetic 2.59
Mesaconic (trans-methylbutene) 2.61
Cyanoacetic 2.63
Malonic (propanedioic) 2.65 5.28
Chloroacetic 2.68
Gentistic (5-hydroxysalicylic) 2.70
Dithiotartaric (2,3-dimercaptobutanedioic) 2.71 3.48 8.89
Bromoacetic 2.72
Phthalic (benzene-1,2-dicarboxylic) 2.75 4.93
Diglycolic (oxydiacetic) 2.79 3.93
Salicylic (2-hydroxybenzoic) 2.81 13.40
Tartaric (d-2,3-dihydroxybutanedioic) 2.82 3.95
Fumaric (trans-butenedioic) 2.85 4.10
Citric (2-hydroxypropane-1,2,3-tricarboxylic) 2.87 4.35 5.69
Iodoacetic 2.98
Isocitrate (dl-1-hydroxypropane-1,2,3-tricarboxylic) 3.02 4.28 5.75
Mucic 3.08 3.63
Mandelic (l-phenylhydroxyacetic) 3.19
Galacturonic 3.23 11.42
Malic (l-hydroxybutanedioic) 3.24 4.71
Thiomalic (dl-mercaptobutanedioic) 3.30 4.60 10.38
Quinic (1,3,4,5-tetrahydroxycyclohexanecarboxylic) 3.36
Thioglycolic (mercaptoacetic) 3.42 10.11
Thiolactic (dl-2-mercaptopropanoic) 3.48 10.08
Hippuric (n-benzoylglycine) 3.50
Glyceric (dl-2,3-dihydroxypropanoic) 3.52
Formic (methanoic) 3.55
Glycolic (hydroxyacetic) 3.63
Lactic (d-2-hydroxypropanoic) 3.66
Itaconic (methylenebutanedioic) 3.68 5.14
2-Hydroxyisobutyric 3.72
Benzoic 4.00
Succinic (butanedioic) 4.00 5.24
Ascorbic 4.03 11.34
4-Hydroxybenzoic 4.10 9.96
Vinylacetic (but-3-enoic) 4.12
Glutaric (pentanedioic) 4.13 5.03
Acrylic (propenoic) 4.26
Adipic (hexanedioic) 4.26 5.03
Pimelic (heptanedioic) 4.31 5.08
3-Mercaptopropanoic 4.34 10.84
Azelaic (nonanedioic) 4.39 5.12
Anisic (4-methoxybenzoic) 4.48
Acetic (ethanoic) 4.56
Isovaleric (3-methylbutanoic) 4.58
Butanoic 4.63
Isobutyric (2-methylpropionic) 4.63
Valeric (pentanoic) 4.64
Propanoic 4.67
Crotonic (trans-but-2-enoic) 4.69
Pivalic (2,2-dimethylpropanoic) 4.83
Caproic (hexanoic) 4.85
Octanoic 4.89
Uric (2,6,8-trihydroxypurine) 5.61
Guanidine 13.54
5 – Example Applications
Thermo Scientific
Product Manual for Dionex IonPac ICE-AS1 Column
Page 18 of 52
031181-07 For Research Use Only. Not for use in diagnostic procedures.
5.3 Dionex IonPac ICE-AS1 Elution Plots and Tables
The following tables show the effect of eluent strength on retention time for a large group of low molecular weight
organic acids. Table 4 is an expanded scale of Table 3. These tables are useful for determining the optimum eluent
strength to minimize co-elution. Table 5 lists, in ascending order, the retention times of common organic acids using
0.4 mM, 1.0 mM, 2.0 mM, and 5.0 mM HCl as eluent.
5.3.1 Dionex IonPac ICE-AS1 Run Time vs. Eluent Strength
Figure 1 Run Time vs. Eluent Strength
BO3
HIBA
Shikimic
Lactic
Glycolic
Fumaric
Br-Acetic
Cl-Acetic
Tricarballyic
Threonic
Quinic
Malic
Malonic
Citriconic
Citric
Tartaric
Caproic
Veleric
Butyric
CO3
Acrylic
Propionic
Adipic
Acetic
Glutaric
B-OH-Butyric
Formic
Itaconic
Succinic
0.2 1.2 2.2 3.2 4.2 5.2
5
15
25
35
45
Eluent Strength: mM HCl
Retention Time (minutes)
5 – Example Applications
Thermo Scientific
Product Manual for Dionex IonPac ICE-AS1 Column
Page 19 of 52
031181-07 For Research Use Only. Not for use in diagnostic procedures.
5.3.2 Dionex IonPac ICE-AS1 Run Time vs. Eluent Strength (Expanded Scale)
Figure 2 RunTime vs. Eluent Strength (Expanded Scale)
BO3
HIBA
Shikimic
Lactic
Glycolic
Fumaric
Br-Acetic
Cl-Acetic
Tricarballyic
Threonic
Quinic
Malic
Malonic
Citriconic
Citric
Tartaric
Butyric
CO3
Acrylic
Propionic
Adipic
Acetic
Glutaric
B-OH-Butyric
Formic
Itaconic
Succinic
012 3 45
18
17
16
15
14
13
12
11
10
9
8
7
6
Eluent Strength: mM HCl
Retention Time (minutes)
5 – Example Applications
Thermo Scientific
Product Manual for Dionex IonPac ICE-AS1 Column
Page 20 of 52
031181-07 For Research Use Only. Not for use in diagnostic procedures.
Table 7 Dionex IonPac ICE-AS1 Run Times vs. Eluent Strength, Ambient
Solute 0.4 mM HCl 1.0 mM HCl 2.0 mM HCl 5 mM HCl
Tartaric 6.73 7.07 7.37 7.87
Citric 6.73 6.92 7.13 8.45
Citriconic 6.75 7.12 7.62 9.13
Malonic 7.03 7.38 7.87 8.83
Malic 7.57 7.90 8.12 8.53
Quinic 7.63 8.02 8.28 8.60
Threonic 7.70 8.18 8.45
Tricarballyic 7.70 7.97 8.20 8.42
Cl-Acetic 8.05 9.17 10.33 12.87
Br-Acetic 8.65 10.25 11.98 14.65
Fumaric 8.80 10.27 11.35 13.37
Glycolic 9.47 9.88 10.10 10.45
Lactic 9.48 10.05 10.13 10.42
Shikimic 9.48 9.80 9.83 9.98
HIBA 9.58 10.07 10.07 10.27
BO3 9.68 9.55 9.55
Succinic 9.70 10.08 9.95 10.27
Itaconic 9.78 10.22 10.48 10.90
Formic 9.90 10.90 11.32 11.60
B-OH-Butyric 10.77 10.80 11.02 10.98
Glutaric 11.50 12.00 11.77 12.03
Acetic 12.33 12.55 12.58 12.42
Adipic 13.72 13.85 13.78 14.12
Propionic 14.37 14.58 14.62 14.43
Acrylic 14.52 15.17 15.15 15.38
CO3 15.85 15.67 15.73
Butyric 17.50 17.83 17.88 17.62
Valeric 25.53 26.02 26.08 25.52
Caproic 41.02 41.70 41.55 40.32
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Thermo Fisher Scientific Dionex IonPac ICE-AS1 Analytical Column User manual

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Thermo Fisher Scientific
Model
Dionex IonPac ICE-AS1 Analytical Column
Type
User manual
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