Thermo Fisher Scientific Dionex IonPac ICE-AS1 Analytical Column 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.

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

Product Manual for Dionex IonPac ICE-AS1 Column

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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

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

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