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Ion Pairing agents

Part 1. Introduction to Ion Pair Chromatography

Ion pair chromatography (IPC) is one of the techniques used to separate charged substances and hydrophilic species. It is widely used to selectively analyse acids and bases, particularly with reverse phase chromatography. However, it is commonly known that setting analytical conditions for IPC can be troublesome and often a good reproducibility is difficult to obtain. Most of the problems occur due to insufficient consideration given to selecting correct ion pairing agent for your mobile phases, ions with the opposite charge of target components. To help you understand the IPC we will focus in our blog on alkyl sulfonates and perchlorates commonly used in IPC of bases and cations. Both will serve as great example to provide an overview on how to determine which ion pairing agent to use based on their respective characteristics. 

Part 2. Alkyl Sulfonates

Alkyl sulfonate is a typical ion pairing agent used in IPC of substances with a positive charge. Typically, sodium salt of alkyl sulfonate ranging from 5 to 12 carbons is used. In general, the separation mechanism used in reverse phase ion pair chromatography is explained as consisting of two processes: 

  • Ion pair distribution process – which pairs the target compound with counterions and captures them in a stationary phase (column) 
  • Ion-exchange process – which retains the target compound by ionic interaction with the counterion and hydrophobically is adsorbed to the stationary phase (column) 

When discussing alkyl sulfonates, we have to consider that the main mechanism of interaction is an ion-exchange process. For this reason, the constituent-retention effect increases with the alkyl sulfonate carbon number. In addition, for the same type of alkyl sulfonate, the retention strength is higher for lower concentrations of the organic solvent in the mobile phase. Let me explain this a bit better – if an alkyl sulfonate with a large number of carbons is used with an extremely low concentration of organic solvent, a quasi-ion-exchange mode is entered, and once equilibrium is reached, it is possible to retain the target constituent without adding counterions to the mobile phase. 

The concentration of the counterions also affects retention of the compound but with a surface-active agent such as alkyl sulfonate, there is a correlation between the concentration and the retention behaviour. This is presented in below Figure 1.  

Influence on Ion Concentration on compound retention.

Taking a closer look at the figure 1, it is clear that in the low counterions concentration range, each of the target compounds retention increases in a linear fashion, but once counterion saturation point is reached (called the “fold-over point”), the target retention level reverses direction and begins decreasing. This is explained by the alkyl sulfonate forming micelles, resulting in a secondary hydrophobic phase/layer within the mobile phase. Therefore, there is a limited range of counterion concentration that can be used for IPC. 

Part 3. Perchloric acid 

Unlike alkyl sulfonates, perchlorates used as a sodium salt are not hydrophobic. Therefore, there is no possibility of achieving any kind of ion-exchange effect. Since perchlorates have a large ionic radius, they easily form ion pairs, and the separation mechanism for IPC should be considered nearly entirely of the ion-pair distribution process. For this reason, there is no “fold over point” between the concentration of counterion and compound retention, as we saw above in case on alkyl sulfonates. Therefore, compound retention keeps gradually increasing as the perchlorate concentration increases. Furthermore, the compound retention effect is uniform, regardless of organic solvent concentration in mobile phase(s).  

However, since the retention of components by perchlorates is based on the hydrophobic properties of the components themselves that appear when their charge is cancelled by the formation of ion pairs, it may not be applicable for some components. 

Part 4. Selection of Counterions  

Based on the characteristics of respective ion pairs, the below discussion will describe practical considerations for differentiating when to use different ion pairs. The fundamental objective of using IPC and the ion pairs is to increase the retention strength of bases and cations, but they are also used to suppress peak tailing in some cases. The peak tailing is known common phenomenon when using C18 (ODS) and other silica gel columns in IPC.  

When selecting counterions and ion pairing agents we must establish the main goal of our Ion pair chromatography: 

  • Considering the retention time increase objective – alkyl sulfonates are more useful, because they can control compound retention over a wide range. 
  • Peak tailing suppression – perchlorates are preferable because they are easier to use and can be used for any compound in general. 

Therefore, we can easily conclude that alkyl sulfonates are preferred for compound retention, while perchlorates are preferred for tailing suppression. 


However, if target compounds are hydrophobic ions, there is no significant difference between alkyl sulfonates and perchlorates. Point to remember is that if an alkyl sulfonate is used, the concentration of the organic solvent in the mobile phase is extremely important when determining the number of carbons for the alkyl sulfonate. Eluting compounds at fixed positions using fixed organic solvent concentrations requires that the fewer the number of carbons, the higher the concentration. As shown in Figure 1 above, there is a limit to ion pair concentrations, so if the organic solvent concentration is high, alkyl sulfonates with few carbons should not be used. However, that does not mean that higher carbon counts are necessarily preferable. Rather, fewer carbons are an advantage in terms of column equilibrium. The lower the organic solvent concentration and the lower the ion pair concentration, the longer it takes for the column to reach equilibrium, but if an alkyl sulfonate with high carbon count is used with a low organic solvent concentration, it will take a very long time for the column to stabilize because a low concentration is being used, of course. Consequently, it is important to decide the organic solvent concentration before selecting the number of carbons when working with alkyl sulfonates. Figure 2 below shows general guidelines for determining use conditions (ion pair concentrations and organic solvent concentrations) effective for respective ion pair. Going forward you can use it as a reference for selecting ion pairs acetonitrile combination. 

Effective Ion-pairing ranges

Part 5. Organic Solvent Concentration 

Finally, it is a time to look at the last piece of the puzzle, selection of organic mobile phase content concentration. To decide the organic solvent concentration, first you must consider the original hydrophobicity of the target compound. In other words, think about how well the mobile phase retains the target compound without including any ion pairing agents in it. For example, if Ion pair chromatography is to be used to elute a compound in 10 minutes, mobile phase conditions must allow eluting the compound within 10 minutes without including any pair ions. Consequently, the lower limit for organic solvent concentration within mobile phase is the concentration that results in eluting the compound in 10 minutes, so it is necessary to set the organic concentration equal to or higher than that. In case where ion pair chromatography is being used to simply increase the retention strength of a compound, any appropriate concentration equal to or higher than that could be selected. However, if compounds with similar hydrophobicity must be separated, the organic concentration should be kept as close to the lower limit as possible, because the higher the organic solvent concentration, the smaller the relative retention, meaning peaks will coelute. On the other hand, if target compounds include not only bases and cations, but also neutral substances or acids, the concentration of organic which can adequately retain such compounds should be at the upper limit. 

Part 6. Summary

Take this opportunity, stop, and think about the ion pairing agents you are using in your methods. Furthermore, with a better understanding of the organic solvent / ion pairing concentration correlation you can better troubleshoot your methods and optimize with the basic knowledge from our blog above. I hope you enjoyed the July edition of our blog and will implement this knowledge in your future analytical challenges.  


Additional Resources and Further Reading

Product: Nexera LC-40 | Liquid Chromatography System | Shimadzu

Liquid Chromatography – Master the Basics

This article is part of our “Liquid Chromatography – Master the Basics” series, your go-to resource for comprehensive and insightful updates on the world of liquid chromatography. Each month in 2024 we will dive into a Liquid Chromatography topic, offering content that is both accessible to beginners and beneficial for experienced scientists.

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Sebastian Jurek is an application consultant with Mason Technology with specialist knowledge in the Shimadzu range of instrumentation. He holds more than 22 years experience in chromatography techniques and analytical method development, optimisation and troubleshooting.

Get in touch with Sebastian today if you would like further information on our range of Shimadzu products.

Sebastian-Jurek-application-consultant-with-Mason-TechnologySebastian Jurek
Application Consultant for Shimadzu Chromatography
E:      sjurek@masontec.ie
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