Reversed Phase Ion Pair Chromatography in Amino Acid Analysis

What is Reversed Phase Ion Pair Chromatography?

Reversed Phase Ion Pair Chromatography (RP-IPC) is a widely applied technique in amino acid analysis, offering distinct advantages in simplicity of operation and high separation efficiency. RP-IPC is a subset of ion pair chromatography, and its extensive use can be attributed to its simplicity and effectiveness without the need for derivatization or specialized columns.

The method excels in its ability to operate without the requirement for derivatization and specialized chromatographic columns, making it a convenient choice for amino acid analysis. The inherent advantages of reversed-phase chromatography, coupled with the simplicity of operation, contribute to the popularity of RP-IPC in the field of amino acid analysis. This method is particularly prevalent due to its compatibility with mass spectrometry (MS/MS) techniques, offering high sensitivity and selectivity in the analysis of complex samples. RP-IPC is commonly employed for trace-level determination of amino acids, showcasing its versatility in analytical applications.

Common Ion-Pair Reagents in Amino Acid Analysis

In ion-pair reversed-phase ion chromatography (IP-RPIC), the versatile ionic characteristics of amino acids lead to the formation of ion pairs, where the carboxyl and amino groups of amino acids ionize and bind with cationic and anionic surfactants, respectively. Anionic surfactants, particularly alkyl sulfonates like sodium pentanesulfonate, sodium hexanesulfonate, and sodium heptanesulfonate, are widely used in this method. Alternatively, perfluorocarboxylic acids, such as trifluoroacetic acid (TFA), heptafluorobutyric acid (HFBA), nonafluoropentanoic acid (NFPA), tridecafluorohexanoic acid (TDFHA), and perfluorooctanoic acid (PDFOA), are preferred for compatibility with evaporative light scattering detection (ELSD) or mass spectrometry (MS) detectors.

While TFA is commonly used due to its high purity, water solubility, and high transparency at 220 nm, it exhibits lower sensitivity and selectivity for strongly polar amino acids. Longer carbon-chain perfluorocarboxylic acids, such as HFBA, have proven to be better choices than TFA. HFBA is extensively used in ion pair chromatography for amino acid analysis, contributing to increased retention times. NFPA, compared to HFBA, offers improved separation efficiency and reduced background noise in mass spectrometry detection, making it more suitable for amino acid mass spectrometry analysis.

The equilibration time for TFA is proportional to the dead time and exhibits low adsorption on porous graphitic carbon (PGC) columns. In contrast, TDFHA or PDFOA significantly adsorb onto the PGC surface, forming a system resembling a dynamic ion-exchange model. As the carbon chain length of perfluorocarboxylic acids increases, so does their retention capacity.

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What is Amino Acid?

Ion-Pair Reagent Concentrations

In the development of a method for quantitatively analyzing 22 amino acids in plasma, researchers used a mobile phase containing 0.5 mmol·L-1 HFBA. Another study identified that the optimal selectivity of NFPA is within the concentration range of 20 mmol·L-1 to 25 mmol·L-1. Beyond this range, both retention trends and efficiency tend to decrease. Confirming previous findings, PDFOA with the longest carbon chain (5 mmol·L-1) was found to be the most suitable ion-pair reagent on a classical RP column. Interestingly, increasing the concentration did not enhance selectivity, a phenomenon also observed with TDFHA.

Mobile Phase pH

The pH values of PDFOA aqueous solutions at various concentrations were measured: 0.01%, pH 4.4; 0.02%, pH 3.1; 0.06%, pH 2.9; and 0.08%, pH 2.8. For comparison, pH values for 0.1% TFA (pH 2.05) and 0.1% HFBA (pH 2.55) were reported. The selection of an appropriate perfluorocarboxylic acid solution as a mobile phase depends on the pH tolerance range of the chromatographic column and the separation efficiency of amino acids.

Chromatographic Columns

The most commonly used chromatographic column for ion-pair chromatography analysis of amino acids is the classical reversed-phase C18 column. In a comparative study, two next-generation chromatographic columns based on high-purity silica gel and one suitable for analyzing polar compounds were investigated. The optimal ion-pair reagent condition for one column was found to be 0.5 mmol·L-1 PDFOA, while the other two columns achieved optimal separation under 0.75 mmol·L-1 PDFOA conditions. This is attributed to the exceptionally hydrophobic nature of one column, possessing the highest retention factor values and consistently delivering superior separation results under all conditions.

Detectors

Evaporative Light Scattering Detector (ELSD)

ELSD offers the advantage of complete evaporation of the mobile phase during gradient elution, resulting in a remarkably stable baseline. However, using ELSD as a detector comes with potential challenges, such as the impact of temperature on the physical form of perfluorocarboxylic acids, affecting the detection process. For instance, PDFOA is a solid at room temperature, but studies have demonstrated that when detected using ELSD, PDFOA exhibits sufficient volatility without adding background noise. Despite its stability, ELSD is known for its lower sensitivity and issues like co-elution or induced peaks during gradient elution, which can obscure certain amino acids. Therefore, the use of a Mass Spectrometry (MS) detector becomes essential to overcome these limitations and enhance sensitivity in amino acid analysis.

Mass Spectrometry

Mass Spectrometry (MS) offers high sensitivity and exceptional selectivity, making it well-suited for analyzing amino acid content in complex matrices. Its ability to detect co-eluted amino acids enhances its specificity, making it preferable for intricate amino acid analyses.

In MS, the non-volatile nature of the mobile phase restricts the use of certain ion-pair reagents. Commonly used cationic reagents include hexylamine, dibutylamine, and tripropylamine, while perfluorocarboxylic acids serve as popular anionic reagents. The latter, such as trifluoroacetic acid (TFA), heptafluorobutyric acid (HFBA), nonafluoropentanoic acid (NFPA), trifluorohexanoic acid (TDFHA), and perfluorooctanoic acid (PDFOA), demonstrate compatibility with MS detectors and accommodate the increasingly diverse species analyzed in polar compound studies.

Despite the advantages, MS in amino acid analysis faces challenges due to potential interference from ion-pair reagents. To address this, researchers have developed a rapid, highly sensitive, and structurally specific LC-MS/MS method. This technique, utilizing TDFHA dilution and dual-column separation, effectively removes impurity interference and minimizes ion suppression, making it more advanced compared to other amino acid analysis methods.

To maintain effective chromatographic separation and prevent sensitivity loss under Electrospray Ionization (ESI) conditions, the use of atmospheric pressure ionization techniques insensitive to solution ionization, such as Microwave-Induced Plasma Ionization (MIPI), is recommended. Integration of HFBA as an ion-pair reagent with atmospheric pressure ionization is suggested as a promising direction for amino acid analysis via HPLC-MS. Recent developments, such as MIPI sources, exhibit potential for broader application in HPLC/MIPI-MS, offering advantages in maintaining high sensitivity across a wide flow rate range.

Reversed-phase ion-pair HPLC chromatogram of a mixed standard containing anserine (ANS), carnosine (CAR), and balenine (BAL) (Kumagai et al., 2021).

Reference

Kumagai, Momochika, et al. "Quantification of Histidine-Containing Dipeptides in Dolphin Serum Using a Reversed-Phase Ion-Pair High-Performance Liquid Chromatography Method." Separations 8.8 (2021): 128.

* For Research Use Only. Not for use in diagnostic procedures.

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