Ion Pair Chromatography – How IPC Works, Strengths, Limitations and Applications
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.
- Retention mechanism
- Factors affecting retention
- Role of the ion pair effect
- Environmental analysis
- Pharmaceutical and food analysis
- Biological analysis
- Metal analysis
What is ion pair chromatography?
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.
Figure 1: Structure of soap.
How does ion pair reversed phase chromatography differ to other types of ion chromatography?
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).
Figure 2: Mechanism of separation in 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.
Figure 3: Mechanism of separation in IEC.
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.
How does ion pair chromatography work and what is the role of the ion pair effect?
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
Figure 4: Instrumentation for ion pair reversed phase chromatography.
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).
Figure 5: The ion pairing model of IPC separation, showing an ion pair complex forming in the mobile phase.
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).
Figure 6: The ion exchange model for IPC separation, showing interaction between an analyte ion and ion pairing reagent ion adsorbed on the stationary phase.
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.
Factors affecting retention
In IPC, several parameters can be modified to achieve the desired separation. Let’s consider some of the variables affecting analyte retention.
- Concentration of ion pairing agent – A high concentration of ion pairing reagent in the mobile phase leads to excessively strong binding of the analytes to the column, making their elution difficult. On the other hand, low concentrations can cause inadequate binding of the analytes to the column. Hence, optimizing the ion pairing reagent concentration in the mobile phase is crucial for IPC. Ion pairing reagents have no effect on the retention of neutral molecules, while they decrease the retention of similarly charged analytes and increase the retention of oppositely charged analytes. Typically, their concentration in the mobile phase is maintained between 0.5 to 20 mM.
- Choice and concentration of organic modifier – Compounds like methanol and acetonitrile are added to the mobile phase to adjust the retention of the compounds as these modifiers compete with the ion pairing reagents and the analytes for the active sites on the column. Organic modifiers impact the retention of neutral molecules as well as the adsorption of ion pairing reagents, which in turn affects the retention of charged analytes. They also reduce the polarity of the mobile phase and help dissolve the hydrophobic ion pairing reagents.
- Column equilibration – The column has to be sufficiently equilibrated with the ion pairing reagent to ensure its adsorption on the stationary phase.
- Column temperature – The viscosity of the mobile phase is reduced at higher column temperatures which results in faster separation.
- Mobile phase pH – The pH of the mobile phase has to be carefully controlled to ensure a) ionization of analyte as well as the ion pairing agent, and b) their optimal interaction and retention on the column. pH adjustment is also important while using conductivity detection.
- Mobile phase additives – Inorganic ions, such as carbonate, have been shown to reduce the retention of analytes in IPC.
Role of the ion pair effect
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.
Choosing an ion pair reagent for your ion pairs
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.
- Ammonium or tetraalkyl ammonium ions (R4N+) are used for pairing with anions. Alkylsulfates (ROSO2O-) and alkylsulfonates (RSO3-) are commonly used as ion pairing agents for the analysis of cations.
- The chain lengths of the alkyl groups, aryl groups or alkyl substituted aryl groups that constitute the lipophilic part of the reagent impact the separation and have to be optimized for the analytes under investigation.
- Hydrophilic compounds such as fluorinated organic acids, e.g., trifluoroacetic acid (TFA), ammonium hydroxide, sodium hydroxide, boric acid, hydrochloric acid and perchloric acids, are used as ion pairing reagents for hydrophobic anions.
- Reagents must be soluble in the organic modifier.
Strengths, limitations and common problems of ion pairing
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.
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
Applications of ion pair chromatography
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.
- Cationic surfactants, such as cetyltrimethylammonium ion, and anionic surfactants, such as linear alkylbenzene sulfonates, are used in the manufacture of detergents, fabric softeners and cleansing agents as well as cosmetics. Both cationic and anionic detergents have been simultaneously determined in environmental water samples by ion pair high-performance liquid chromatography (HPLC) using di-n-butylammonium ions as the ion pairing agent followed by electrospray ionization mass spectrometric detection.6
- Ionic liquids that were once considered to be environmentally safe alternatives to organic solvents have been proven to be toxic to a variety of plants, animals and microorganisms. Hence, sensitive methods for their detection in environmental samples have been developed. Baseline separation of 1‑hydroxyethyl‑3‑methyl imidazolium ([HOEtMIm]+) and 1‑hydroxypropyl‑3‑methyl imidazolium ([HOPrMIm]+) cations was achieved using an ion pair reversed phase C18 column and sodium octanesulfonate as the ion pairing reagent. This method has been applied for the detection of hydroxyl functionalized imidazolium ionic liquid cations in river water samples.7
Pharmaceutical and food analysis
- To study the safety, efficacy and stability of drugs, they are analyzed along with their metabolites, often in biological matrices. Antisense oligonucleotide drugs and their metabolites have been analyzed by ion pair reversed phase chromatography coupled with mass spectrometric detection.8
- A simple, stability-indicating HPLC method for an antipsychotic drug, asenapine, has been developed using heptane sulfonic acid as the ion pairing agent and UV detection at 220 nm. This method has been applied for the determination of low concentrations of asenapine in tablets.9
- Processed foods are fortified with iodine in the form of iodide and iodate ions. QC tests are needed to ensure the desired levels of these salts are present in consumer products. These ions have been separated and detected in medicines and beverages with reversed phase HPLC by using 1-hexyl-3-methylimidazolium tetrafluoroborate and imidazolium ionic liquid as ion pair reagents as well as UV absorbing chromophores.10
- As a quality control test, an ion pair chromatographic method with potentiometric detection has been used to detect biogenic amine content in fresh tomatoes, canned chopped tomatoes and tomato pulp.11
- Aminopolycarboxylic acids (APCA) are used as chelating agents in pharmaceuticals, cosmetics and food products to remove metal contaminants and increase the shelf life of these products. An ion pairing HPLC method has been used to determine the presence of APCAs such as ethylenediaminetetraacetic acid (EDTA) in a small molecule drug, by complexing it with copper. The presence of another APCA, diethylenetriaminepentaacetic acid (DTPA), in a monoclonal antibody was determined by complexing it with iron and using tetrabutyl ammonium ion as the ion pairing reagent.12
- To understand the biological role of the melanin pigments eumelanin and pheomelanin, carboxylic acids obtained by their oxidation have been separated and analyzed using tetra n-butylammonium bromide as the ion pairing reagent.13
- Amino acid condensation reactions are carried out to study the origin of life. Simple analytical methods are required to quantify the products of these reactions. Campbell et al. have developed an ion pair HPLC method to analyze glycine and its oligomers (Glyn), up to 14 residues long.14
- Chelates of 3 heavy metals — Co(II), Cr(III) and Ni(II) — with 2-pyridylazoresorcinol (PAR) have been analyzed within 15 minutes using ion pair HPLC.15
- Radioactive lutetium, Lu177, is used for research, and manufacture of radiopharmaceuticals. It is produced from irradiation of ytterbium, Yb177, or its compounds followed by separation from the parent Yb molecule. Ion pair reversed phase HPLC has been used to purify Lu177 from 10 mg of neutron-irradiated YB2O2 target using a silica gel column containing polar stationary phases such as nitrile or alkyl siloxane.16
- Thiosulfate is used for the extraction of gold from its ore. Thiosulfate and its degradants present in the leachate, after the recovery of metal, are estimated to determine the efficiency of the process. Sulfate, thiosulfate and polythionates have been determined in different gold extract solutions by reversed phase IPC with suppressed conductivity detection.17
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.
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