Ion Pair Chromatography

Mar 13, 2023

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Chromatography of charged analytes is often challenging as they are not retained effectively on the commonly used reversed phase columns and elute in the dead volume. On columns with polar or aqueous stationary phases, the peak shapes may be poor due to excessive retention. Ion exchange columns that use charged stationary phases to separate oppositely charged ions are expensive, suitable for a small range of analytes and have limited separation efficiency. Ion pair chromatography (IPC) is a suitable alternative for chromatography of polar or ionic species.

What is ion pair chromatography?

How does ion pair reversed phase chromatography differ to other types of ion chromatography?

- Retention mechanism

- Factors affecting retention

- Role of the ion pair effect

- Environmental analysis

- Pharmaceutical and food analysis

- Biological analysis

- Metal analysis

Conclusion

IPC is a type of ion chromatography that is used to separate hydrophilic or charged analytes on columns using reversed phase or "neutral" stationary phases that do not carry charges. It involves modifying the polarity of the charged analytes through their interaction with an ion-pairing reagent that is added to the mobile phase. These reagent molecules carry charges opposite to that of the analyte ions with which they are able to form electrostatic bonds. The pairs formed between the analytes and reagent ions behave like neutral, hydrophobic moieties that can be separated on C18 or C8 columns. IPC is used for the separation of polar organic acids, bases and zwitterions as well as inorganic ions.

Ion pairing reagents are also known as ion pairing additives or hetaerons. As these molecules have a polar head group and hydrophobic hydrocarbon chains, they resemble a soap (Figure 1). Consequently, this technique was called "soap chromatography" when it was introduced by Göran Schill in 1973.1,2 It is also referred to as ion interaction chromatography, as the reagent ion interacts with the stationary phase to regulate the retention of ions present in the sample.

Ion chromatography (IC) broadly refers to the separation of ions and includes three distinct mechanisms, namely, ion exchange, ion exclusion and ion pairing. When separation is brought about by competitive interaction between the analyte ions and eluent ions for the oppositely charged sites on the stationary phase (Figure 2), the type of chromatography is called ion exchange chromatography (IEX).

A mix of undissociated, partially dissociated and fully dissociated analytes can be separated by ion exclusion chromatography (IEC), which also uses charged stationary phases (Figure 3). Mobile phase accumulates on the surface and inside the pores of the stationary phase leading to the formation of an "occluded phase". The neutral analytes are strongly retained in the occluded phase and elute last from the column. The partially dissociated analytes that have slightly lesser interaction with the adsorbed mobile phase elute earlier. Fully dissociated and charged analytes that are repelled by the similarly charged ions of the stationary phase, the "Donnan membrane", elute first, altogether in the column void volume.

Ion pair reversed phase chromatography is carried out on non-polar "reversed phase" columns by adding ion pairing reagents to the mobile phase. For instance, trifluoroacetic acid is used for pairing with positively charged peptides, while trialkylamines are used for ion pairing with anions such as carboxylates or oligonucleotides.

Mobile phase ion chromatography (MPIC) is a term often used in the context of separation of small inorganic ions by IPC, followed by their detection by suppressed conductivity measurement. This technique is suitable for the analysis of molecules with localized charges.

A few microliters of sample solution are injected into a reversed phase column. When the mobile phase containing the ion pairing reagent flows through the column, the analyte ions interact with the oppositely charged reagent ions and forms neutral complexes that can be separated on a nonpolar stationary phase. The separation of analytes is controlled by the ion pairing reagent, organic modifiers and any salts added to the mobile phase.

Ultraviolet (UV) and fluorescence spectroscopic methods are the most commonly used detection techniques for IPC. The use of other techniques such as mass spectrometry (MS) and inductively coupled plasma–mass spectrometry (ICP-MS)3 have also been reported. Conductivity measurement is a well-established method for the detection of inorganic ions by IC but less frequently used for IPC.4

Different models have been proposed to explain the mechanism of separation observed in IPC.5 In the ion pairing model, also called the partition model, interaction between the analyte ions and ion pairing reagent ions is considered to occur in the mobile phase. The analyte ions along with the counter ions form a non-polar moiety that can adsorb on the hydrophobic stationary phase. The ion pair complex is subsequently eluted by increasing the organic modifier concentration in the mobile phase (Figure 5).

The ion exchange model, also called the adsorption model, involves the adsorption of the lipophilic alkyl chain of the ion pairing reagent molecules on the stationary phase. The free polar head groups of the adsorbed molecules act as a pseudo ion-exchanger to the oppositely charged analyte ions (Figure 6).

In the ion interaction model, also called the electrostatic model, an electrical double layer is thought to be formed when a column is equilibrated with the mobile phase containing an ion pairing agent. The non-polar hydrocarbon chain of these moieties binds to the column. The polar head groups form a stationary layer of charges while the counter ions of the ion pairing reagent in the mobile phase form the oppositely charged layer. The analyte ions experience an electrostatic attraction to the stationary charges and are able to penetrate the double layer. Coulombic interaction between an analyte molecule and the "charged" layer leads to an apparent decrease in the charge on the surface of the stationary phase. Consequently, a molecule of the ion pairing agent is adsorbed on the surface of the stationary phase to restore the charge. Taken together, this can be considered as adsorption of two opposite charges – that of the analyte and of the ion pairing agent – on the stationary phase.

Figure 7: The ion interaction model for IPC separation, showing dynamic interaction between the analyte ion, ion pairing reagent ions and the stationary phase. A) The ion pairing reagent binds to the stationary phase. B) Analyte ions penetrate the double layer. C) Analyte ions bind to the polar head group. D) A new ion pairing reagent molecule binds to the stationary phase.

In IPC, several parameters can be modified to achieve the desired separation. Let's consider some of the variables affecting analyte retention.

When electrolytes are added to a solvent, they dissociate into their constituent ions and are surrounded by the solvent molecules. But as the polarity of the solvent decreases, solvation of ions decreases and due to electrostatic attraction, the interaction between them increases. As the size of the ions increases, the charge density decreases, which also leads to lesser solvation and greater interaction with the oppositely charged ions. The greater the charge on the ions, the stronger is the coulombic force of attraction between the oppositely charged ions.

Ion pairing reagents have long alkyl chains or aryl groups and are not as solvated as small ions with high charge densities. As a result, they can attract and pair with oppositely charged analyte ions. In addition, when ion pairing reagents are adsorbed on the non-polar stationary phase, their solvation is further reduced. This enables them to form "tight" or "intimate" ion pairs with analyte molecules. These ion pairs do not have any solvent molecules between the two ions. The greater the charge on the analyte and the reagent ions, the stronger the bond between them.

The choice of ion pairing reagent is perhaps the most important parameter when using IPC. The reagent type, its concentration and compatibility with the mobile phase and detector, all play an important role in the effective separation of the analytes. The following considerations must be borne in mind when selecting an ion pairing reagent for the sample under investigation.

Although ion pairing chromatography provides certain advantages over other techniques such as IEX and normal phase chromatography, it suffers from limitations as well (Table 1). These pros and cons must be carefully considered before opting for this mode of separation.

Table 1: Strengths and limitations of IPC.

Strengths

Limitations

Analysis of a mixture of polar, non-polar and ionic compounds that are difficult to separate by other techniques can be achieved by choosing an appropriate ion pairing reagent and adjusting its concentration

Long equilibration times are needed for the ion pairing reagent to adsorb onto the surface of the stationary phase

IPC can be done on C18 or C8 columns that are commonly available in most labs

Gradient analysis may be difficult for IPC as changes to mobile phase composition could lead to longer column equilibration

Both cations and anions can be separated using the same column

The ion pairing reagent should not absorb wavelengths in which the UV or fluorescence detection are carried out. Presence of light-absorbing reagents in the mobile phase can cause ghost peaks and negative peaks due to lower absorbance by the analyte compared to that of the mobile phase containing the ion pairing reagent

Ion pairing reagents help to improve retention, peak shapes and resolution

Commonly used ion pairing agents that have limited volatility have limited compatibility with mass spectrometers. TFA, when used as a modifier, causes signal suppression in mass spectrometers

Lower run times and detection limits can be obtained in the presence of ion pairing reagents

Dedicated columns may have to be used when working with IPC reagents

IPC has been applied for the analysis of a wide variety of analytes, ranging from environmental samples, pharmaceuticals and food to biological samples and metals.

Several modes of chromatography, such as hydrophilic interaction chromatography (HILIC) and IEX, and different types of stationary phases, such as ion exchange or polar, have been used for the separation of charged and polarizable molecules. Yet, IPC continues to be used for their analysis due to its simplicity and customizability. Parameters including the choice of ion pairing reagent, its concentration, organic modifiers and mobile phase additives can be optimized to achieve the desired separation.

References

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