©2009 Thermo Fisher Scientifi c Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientifi c Inc. and its subsidiaries. Litho in U.S.A. 08_1747 JH/AK-IW-1/09 BN1016087

Fisher Scientifi c Global Export, Latin America1-770-871-47251-770-871-4726www.fi shersci.com

For customer serviceTo fax an orderTo order online

Fisher Scientifi c US 1-800-766-70001-800-926-1166www.fi shersci.com

Fisher HealthCare US1-800-640-06401-800-290-0290www.fi sherhealthcare.com

Fisher Scientifi c Canada1-800-234-74371-800-463-2996www.fi shersci.ca

5) All of Acros Organics alkylsulfonate ion pair reagents are anhydrous. The concentration of the ion pair reagent in themobile phase plays an important role in the separation of com-plex mixtures. A slight change of concentration by 10-20% can alter the retention time which can lead to inconsistent resolve overlapping peaks. The anhydrous form makes the preparation of the mobile phase with the desired concentration of the ion pair reagent much more straightforward, and therefore im-proves the reproducibility of the method.

6) Pack Sizes: As IPC is becoming more and more popular as the method of choice for the separation of polar and ionic mixtures, we are now offering larger pack sizes to help our customer toreduce cost in use and save time by eliminating unnecessary method validation due to lot change of the ion pair reagent.

Acknowledgement: Thanks to Mr. Nico Vonk at Avans, Breda, the Netherlands for his contribution to the paper.

Acros Organics Ion Pair ReagentsFor Ion Pair Chromatography

Acros Organics Alkylsulfonate Ion Pair Reagent Product List

Product Number Pack Size Description

AC396660250 25GR 1-Propanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396661000 100GR 1-Propanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396640250 25GR 1-Butanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396641000 100GR 1-Butanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396650250 25GR 1-Pentanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396651000 100GR 1-Pentanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396670250 25GR 1-Hexanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396671000 100GR 1-Hexanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396680250 25GR 1-Heptanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396681000 100GR 1-Heptanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396690250 25GR 1-Octanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396691000 100GR 1-Octanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396700050 5GR 1-Nonanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396700250 25GR 1-Nonanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396710250 25GR 1-Decanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396711000 100GR 1-Decanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396720050 5GR 1-Dodecanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396720250 25GR 1-Dodecanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

more size choices • QUALITY • less variation • anhydrous - easier to control working concentration • more size choices • less variation • CONVENIENCE • anhydrous - easier to control working concentration • more size choices • RESULTS • less

It is well-known that the use of reversed phase-high performance liq-uid Chromatography (RP-HPLC) is limited for the separation and iden-tifi cation of complex mixtures having components with large variation in pKa values. Ionic suppression technique is only suitable for mixtures of which the pKa values of each component lie close together. The use of this technique is further restricted for mixtures with pKa values less than 3 or greater than 8 due to the fact that the retention cannot be affected by adjusting the pH of the mobile phase without an unwanted reduction of the column lifetime.

The limitations of ionic suppression for separation of ionized com-ponents Ied to the development of ion pair chromatography (IPC). IPC is a type of RP-HPLC in which ion pair reagents are added to the mobile phase to promote the formation of ion pairs with charged analytes, which make the reversed phase column suitable for the separation of ionic molecules. The retention/separation follows a dynamic combination of reversed phase and ion pair-ion exchange mechanisms.

IPC is often used for the separation of basic (nitrogen containing) compounds, such as biogenic amines. These compounds very often show poor peak-shape on the regular RP-HPLC. Also ion suppression technique is not suitable since it would require the use of pH>8 buffered mobile phase, which is not compatible with most of the standard reversed phase columns. In those situations ion pair chro-matography is the best option.

Nowadays IPC is widely used in pharmaceutical research and drug discovery for the separation and identifi cation of biological sub-stances since most of these compounds are ionic in nature.

Ion pair reagents are comprised of a long linear alkyl chain (from C3 up to C16) and an ionic group which can reversibly adsorb to the alkyl chains (C8 or C18) of the RP-phase, forming a dynamic ion-exchanger, at which ionic compounds can be separated.

There are two main types of ion pair reagents: anionic alkyl sol-fonates for basic compounds (e.g. biogenic amines) and cationic quaternary amines for acidic compounds (e.g. nucleotides), with the former being more widely used.

Commonly used Alkyl sulfonates IPC are 1-hexanesulfonic acid so-dium salt, 1-heptasulfonic acid sodium salt and 1-octanesulfonic acid sodium salt.

The selectivity of the system strongly depends on the choice and amount of the ion pair former in the mobile phase. Reagents with long chain lengths will considerably be better adsorbed onto the RP phase, affecting the retention in a positive way. A disadvantage is the time needed to obtain an equilibrium which is slow for longer chains ( > C12).

Ion pair reagents are usually added in low concentrations (typically between 2 and 10mmol) to the mobile phase, which consists of or-ganic eluent(s) and an aqueous buffer solution. Quite often the sepa-ration of compounds in complex mixture is performed with a solvent gradient with an increasing concentration of the organic eluents in the mobile phase (“solvent A & B”). It is good practice to add the same concentration of ion pair agent to both solvents in order to keep the concentration of the reagent constant at the service of the stationary phase during the gradient run. In this situation it is critical to use high quality solvents and additives in the mobile phase. HPLC grade solvents are adequate, but it can be diffi cult to fi nd buffer salts and ion pair reagents of the same quality.

In conclusion, Ion pair chromatography is a very selective and powerful separation technique, but needs extra attention during method development and the daily routine in the procedures for column start-up, mobile phase composition and system equilibration procedures.

The Features and Benefi ts of Acros Organics Alkylsulfonates IPC Reagents

Acros Organics offers an extensive range of typically used alkyl-sulfonate IPC reagents with outstanding quality. Each product is tested against strict specifi cations to ensure the reproducibility, reliability and accuracy of the analyses.

1) Lowest UV absorption even at high concentrations: As shown in Table 1, Acros Organics IPC reagents have very low UV absorp-tion down to 200nm even at high concentration.

Table 1: Acros Organics Alkylsulfonate IPC Reagents UV Absorption

Alkylsulfonate IPC ProductCode

Conc. (M)

UV absorptionmax λ (nm)

210 220 230 260

1-Propanesulfonic acid,sodium salt

39666 0.5 0.1 0.06 0.04 0.02

1-Butanesulfonic acid,sodium salt

39664 0.5 0.1 0.06 0.04 0.02

1-Pentanesulfonic acid,sodium salt

39665 0.5 0.1 0.06 0.04 0.02

1-Hexanesulfonic acid,sodium salt

39667 0.5 0.1 0.06 0.04 0.02

1-Heptanesulfonic acid,sodium salt

39668 0.5 0.1 0.06 0.04 0.02

1-Octanesulfonic acid,sodium salt

39669 0.4 0.1 0.06 0.04 0.02

1-Nonanesulfonic acid,sodium salt

39670 0.2 0.05 0.03 0.02 0.02

1-Decanesulfonic acid,sodium salt

39671 0.2(40°C)

0.05 0.03 0.02 0.02

1-Dodecanesulfonic acid,sodium salt

39672 0.18(hot H2O)

0.05 0.03 0.02

2) Free of dust and insoluble matter: As part of the specifi cations, products are subjected to 0.5 micro membrane fi ltration test at 0.5 M concentration to ensure they are free of dust and in-soluble matters.

3) Best performance in gradient HPLC: They went through a tough application test and have shown little base line shift in the gradient HPLC.

Column: Zorbax Eclipse Plus (C18,1.8µm) 50x4.6mm Mobile Phase: A: 0.005M Hexanesulfonic acid, sodium salt, anhydrous in water. B: Acetonitrile Flowrate: Flow: 1.0mL/min. Temperature: 35.0°C

Detection: UV, 205nmGradient: A: Acetonitrile.

B: 0.005M Hexanesulfonic acid, sodium salt, in water

Time (Min.) %A %B

0 0 100

5 0 100

10 100 0

20 100 0

4) Application/performance test: Separation of biogenic amines can be easily accomplished using Acros Organics ultrapurealkylsulfonic acid sodium salt. Figure 2 shows the separation of a mixture containing 4-amidinobenzamide hydrochloride,3-hydroxytyramine hydrochloride, histamine dihydrochloride and serotonin hydrochloride.

Column: Zorbax Eclipse Plus (C18,1.8µm) 50x4.6mm Mobile Phase: A: 90 parts acetonitrile and 10 parts 0.005M Hexanesulfonic acid sodium salt in phosphoric acid 0.01M at pH2.4. B: 0.005M 1-hexanesulfonic acid sodium salt + diluted phosphoric acid pH2.4 Flowrate: Flow: 1.5mL/min. Temperature: 20.0°C Detection: UV, 220nm Gradient:

Time (Min.) %A %B

0 0 100

10 10 90

20 10 90

Injection volume: 10µL Sample solution: 10mg/100mL, Solvent: acetonitrile/ phosphoric acid 0.01M (10/90) Retention time: - 6.9 min.: 4-amidinobenzamide hydrochloride (22440) - 7.8 min.: 3-hydroxytyramine hydrochloride (12200) - 8.8 min.: Histamine dihydrochloride (15062) - 11.2 min.: Serotonin hydrochloride (21502)

Figure 2: Separation of Biogenic Amines by 1-Hexanesulfonic Acid Sodium SaltFigure 1: Gradient test for 1-hexanesulfonic acid, sodium salt

Alkylsulfonate Ion Pair Chromatography Reagents From Acros Organics

Free of dust • low UV absorption • anhydrous - easier to control working concentration • low interference of insoluble material • ideal for ion pair chromatography • QUALITY • Free of dust • low UV absorption • RESULTS • anhydrous - easier to control working concentration • low interference of insoluble material • PERFORMANCE • ideal for ion pair chromatography • Free of dust • low UV absorption • anhydrous - easier to control working concentration • low interference of insoluble material • Ide

It is well-known that the use of reversed phase-high performance liq-uid Chromatography (RP-HPLC) is limited for the separation and iden-tifi cation of complex mixtures having components with large variation in pKa values. Ionic suppression technique is only suitable for mixtures of which the pKa values of each component lie close together. The use of this technique is further restricted for mixtures with pKa values less than 3 or greater than 8 due to the fact that the retention cannot be affected by adjusting the pH of the mobile phase without an unwanted reduction of the column lifetime.

The limitations of ionic suppression for separation of ionized com-ponents Ied to the development of ion pair chromatography (IPC). IPC is a type of RP-HPLC in which ion pair reagents are added to the mobile phase to promote the formation of ion pairs with charged analytes, which make the reversed phase column suitable for the separation of ionic molecules. The retention/separation follows a dynamic combination of reversed phase and ion pair-ion exchange mechanisms.

IPC is often used for the separation of basic (nitrogen containing) compounds, such as biogenic amines. These compounds very often show poor peak-shape on the regular RP-HPLC. Also ion suppression technique is not suitable since it would require the use of pH>8 buffered mobile phase, which is not compatible with most of the standard reversed phase columns. In those situations ion pair chro-matography is the best option.

Nowadays IPC is widely used in pharmaceutical research and drug discovery for the separation and identifi cation of biological sub-stances since most of these compounds are ionic in nature.

Ion pair reagents are comprised of a long linear alkyl chain (from C3 up to C16) and an ionic group which can reversibly adsorb to the alkyl chains (C8 or C18) of the RP-phase, forming a dynamic ion-exchanger, at which ionic compounds can be separated.

There are two main types of ion pair reagents: anionic alkyl sol-fonates for basic compounds (e.g. biogenic amines) and cationic quaternary amines for acidic compounds (e.g. nucleotides), with the former being more widely used.

Commonly used Alkyl sulfonates IPC are 1-hexanesulfonic acid so-dium salt, 1-heptasulfonic acid sodium salt and 1-octanesulfonic acid sodium salt.

The selectivity of the system strongly depends on the choice and amount of the ion pair former in the mobile phase. Reagents with long chain lengths will considerably be better adsorbed onto the RP phase, affecting the retention in a positive way. A disadvantage is the time needed to obtain an equilibrium which is slow for longer chains ( > C12).

Ion pair reagents are usually added in low concentrations (typically between 2 and 10mmol) to the mobile phase, which consists of or-ganic eluent(s) and an aqueous buffer solution. Quite often the sepa-ration of compounds in complex mixture is performed with a solvent gradient with an increasing concentration of the organic eluents in the mobile phase (“solvent A & B”). It is good practice to add the same concentration of ion pair agent to both solvents in order to keep the concentration of the reagent constant at the service of the stationary phase during the gradient run. In this situation it is critical to use high quality solvents and additives in the mobile phase. HPLC grade solvents are adequate, but it can be diffi cult to fi nd buffer salts and ion pair reagents of the same quality.

In conclusion, Ion pair chromatography is a very selective and powerful separation technique, but needs extra attention during method development and the daily routine in the procedures for column start-up, mobile phase composition and system equilibration procedures.

The Features and Benefi ts of Acros Organics Alkylsulfonates IPC Reagents

Acros Organics offers an extensive range of typically used alkyl-sulfonate IPC reagents with outstanding quality. Each product is tested against strict specifi cations to ensure the reproducibility, reliability and accuracy of the analyses.

1) Lowest UV absorption even at high concentrations: As shown in Table 1, Acros Organics IPC reagents have very low UV absorp-tion down to 200nm even at high concentration.

Table 1: Acros Organics Alkylsulfonate IPC Reagents UV Absorption

Alkylsulfonate IPC ProductCode

Conc. (M)

UV absorptionmax λ (nm)

210 220 230 260

1-Propanesulfonic acid,sodium salt

39666 0.5 0.1 0.06 0.04 0.02

1-Butanesulfonic acid,sodium salt

39664 0.5 0.1 0.06 0.04 0.02

1-Pentanesulfonic acid,sodium salt

39665 0.5 0.1 0.06 0.04 0.02

1-Hexanesulfonic acid,sodium salt

39667 0.5 0.1 0.06 0.04 0.02

1-Heptanesulfonic acid,sodium salt

39668 0.5 0.1 0.06 0.04 0.02

1-Octanesulfonic acid,sodium salt

39669 0.4 0.1 0.06 0.04 0.02

1-Nonanesulfonic acid,sodium salt

39670 0.2 0.05 0.03 0.02 0.02

1-Decanesulfonic acid,sodium salt

39671 0.2(40°C)

0.05 0.03 0.02 0.02

1-Dodecanesulfonic acid,sodium salt

39672 0.18(hot H2O)

0.05 0.03 0.02

2) Free of dust and insoluble matter: As part of the specifi cations, products are subjected to 0.5 micro membrane fi ltration test at 0.5 M concentration to ensure they are free of dust and in-soluble matters.

3) Best performance in gradient HPLC: They went through a tough application test and have shown little base line shift in the gradient HPLC.

Column: Zorbax Eclipse Plus (C18,1.8µm) 50x4.6mm Mobile Phase: A: 0.005M Hexanesulfonic acid, sodium salt, anhydrous in water. B: Acetonitrile Flowrate: Flow: 1.0mL/min. Temperature: 35.0°C

Detection: UV, 205nmGradient: A: Acetonitrile.

B: 0.005M Hexanesulfonic acid, sodium salt, in water

Time (Min.) %A %B

0 0 100

5 0 100

10 100 0

20 100 0

4) Application/performance test: Separation of biogenic amines can be easily accomplished using Acros Organics ultrapurealkylsulfonic acid sodium salt. Figure 2 shows the separation of a mixture containing 4-amidinobenzamide hydrochloride,3-hydroxytyramine hydrochloride, histamine dihydrochloride and serotonin hydrochloride.

Column: Zorbax Eclipse Plus (C18,1.8µm) 50x4.6mm Mobile Phase: A: 90 parts acetonitrile and 10 parts 0.005M Hexanesulfonic acid sodium salt in phosphoric acid 0.01M at pH2.4. B: 0.005M 1-hexanesulfonic acid sodium salt + diluted phosphoric acid pH2.4 Flowrate: Flow: 1.5mL/min. Temperature: 20.0°C Detection: UV, 220nm Gradient:

Time (Min.) %A %B

0 0 100

10 10 90

20 10 90

Injection volume: 10µL Sample solution: 10mg/100mL, Solvent: acetonitrile/ phosphoric acid 0.01M (10/90) Retention time: - 6.9 min.: 4-amidinobenzamide hydrochloride (22440) - 7.8 min.: 3-hydroxytyramine hydrochloride (12200) - 8.8 min.: Histamine dihydrochloride (15062) - 11.2 min.: Serotonin hydrochloride (21502)

Figure 2: Separation of Biogenic Amines by 1-Hexanesulfonic Acid Sodium SaltFigure 1: Gradient test for 1-hexanesulfonic acid, sodium salt

Alkylsulfonate Ion Pair Chromatography Reagents From Acros Organics

Free of dust • low UV absorption • anhydrous - easier to control working concentration • low interference of insoluble material • ideal for ion pair chromatography • QUALITY • Free of dust • low UV absorption • RESULTS • anhydrous - easier to control working concentration • low interference of insoluble material • PERFORMANCE • ideal for ion pair chromatography • Free of dust • low UV absorption • anhydrous - easier to control working concentration • low interference of insoluble material • Ide

©2009 Thermo Fisher Scientifi c Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientifi c Inc. and its subsidiaries. Litho in U.S.A. 08_1747 JH/AK-IW-1/09 BN1016087

Fisher Scientifi c Global Export, Latin America1-770-871-47251-770-871-4726www.fi shersci.com

For customer serviceTo fax an orderTo order online

Fisher Scientifi c US 1-800-766-70001-800-926-1166www.fi shersci.com

Fisher HealthCare US1-800-640-06401-800-290-0290www.fi sherhealthcare.com

Fisher Scientifi c Canada1-800-234-74371-800-463-2996www.fi shersci.ca

5) All of Acros Organics alkylsulfonate ion pair reagents are anhydrous. The concentration of the ion pair reagent in themobile phase plays an important role in the separation of com-plex mixtures. A slight change of concentration by 10-20% can alter the retention time which can lead to inconsistent resolve overlapping peaks. The anhydrous form makes the preparation of the mobile phase with the desired concentration of the ion pair reagent much more straightforward, and therefore im-proves the reproducibility of the method.

6) Pack Sizes: As IPC is becoming more and more popular as the method of choice for the separation of polar and ionic mixtures, we are now offering larger pack sizes to help our customer toreduce cost in use and save time by eliminating unnecessary method validation due to lot change of the ion pair reagent.

Acknowledgement: Thanks to Mr. Nico Vonk at Avans, Breda, the Netherlands for his contribution to the paper.

Acros Organics Ion Pair ReagentsFor Ion Pair Chromatography

Acros Organics Alkylsulfonate Ion Pair Reagent Product List

Product Number Pack Size Description

AC396660250 25GR 1-Propanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396661000 100GR 1-Propanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396640250 25GR 1-Butanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396641000 100GR 1-Butanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396650250 25GR 1-Pentanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396651000 100GR 1-Pentanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396670250 25GR 1-Hexanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396671000 100GR 1-Hexanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396680250 25GR 1-Heptanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396681000 100GR 1-Heptanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396690250 25GR 1-Octanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396691000 100GR 1-Octanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396700050 5GR 1-Nonanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396700250 25GR 1-Nonanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396710250 25GR 1-Decanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396711000 100GR 1-Decanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396720050 5GR 1-Dodecanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

AC396720250 25GR 1-Dodecanesulfonic acid, sodium salt, anhydrous, Ion pair chromatography, 99+%

more size choices • QUALITY • less variation • anhydrous - easier to control working concentration • more size choices • less variation • CONVENIENCE • anhydrous - easier to control working concentration • more size choices • RESULTS • less