Reversed-phase ion-pair liquid chromatography

Example ion-pair liquid chromatography of amino acids. Amino acids are zwitterions. The amino group can form an ion-pair with an alkanesulfonate ion (such as octanesulfonate), and the carboxyl group can form an ion-pair with a tetrabutylammonium ion, depending on the pH of the solution. 

In reversed-phase liquid chromatography, the ionization of the solute decreases the retention. The addition of counter-ion under these conditions forces the formation of an ion-pair between the ionized solute and counter-ion, and then the retention of the analyte increases as the paired-ion is retained. 

Strong and weak bases Alkyl-, allyl-sulfonic acids 

Column, octadecyl-bonded silica gel, 15 cm x 4.6 mm i.d. eluent, 0.01 M sodium acetate buffer containing 0.4 M copper acetate and 1.0 mM sodium alkanesulfonate (pH 5.6) flow rate, 1 ml min-1 detection, UV 230 nm. Counter-ion C6, sodium hexanesulfonate Cl, sodium heptanesulfonate and C8, sodium octane sulfonate. Compounds Ser, serine Gly, glycine Glu, glutamic acid and Asp, aspartic acid. 

The retention of acids in reversed-phase liquid chromatography can be predicted by Equation 4.1. The same equation can be applied to predict the 

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Felice, L. Felice, J. and Kissinger, P. Determination of catecholamines in rat brain parts by reverse-phase ion-pair liquid chromatography. 

Two types of system are used for ion-pair liquid chromatography. When polar stationary phase materials, such as silica gel, are used an ion-pair partition mechanism is applied. When non-polar stationary phase materials, such as octadecyl-bonded silica gel and polystyrene gel, are employed a paired-ion adsorption mechanism is involved. The former is called normal-phase ion-pair partition liquid chromatography, and the latter is called reversed-phase ion-pair liquid chromatography. 

Increasing the concentration of the counter-ion further increased the retention, but the retention reached a plateau, as seen in Figure 4.14. A total separation of the amino acids by reversed-phase ion-pair liquid chromatography could be performed.11 A column switching technique reduced the total analysis time (Figure 4.15). 

Two mechanisms for retention in reversed-phase ion-pair liquid chromatography have been considered. One is the adsorption of the hydrophobic paired ion on the hydrophobic surface of stationary phase material. In the second mechanism, the hydrophobic counter-ion is held on the surface of the hydro-phobic stationary phase, and the analyte ion is retained by ion-ion interactions, as shown in Figure 4.16. In the latter case, of a dynamic ion-exchange... 

Figure 4.16 Model of reversed-phase ion-pair liquid chromatography in which counterions are held on the stationary-phase surface.

Ion-pair liquid chromatography can be applied to compounds separated by ion-exchange liquid chromatography, and mixtures of ionic and non-ionic compounds are easily separated. The latter separation is difficult by ion-exchange liquid chromatography. Anions can be separated by reversed-phase ion-pair liquid chromatography (Figure 4.18). 

B. A. Bidlingmeyer, S.N. Deming, W.P. Price, Jr., B. Sachok, and M. Petrusek, Retention mechanism for reversed-phase ion-pair liquid chromatography , J. Chromatogr., 1979,186, 419. 

Jen and Chen [10] determined metal ions at pg L 1 concentrations in non saline waters using reversed phase ion pair liquid chromatography. 

Varvaresou, A. et al. Development and validation of a reversed-phase ion-pair liquid chromatography method for the determination of magnesium ascorbyl phosphate and melatonin in cosmetic creams. Anal. Chim. Acta 2006, 573-574, 284—290. 

Nakazawa, H. et al. Determination of dithiocarbamate fungicides by reversed-phase ion-pair liquid chromatography with chemiluminescence detection. /. Liq. Chromatogr. Rel. Technol. 2004, 27, 705-713. 

Wolf, R.E., Morrison, J.M., and Goldhaber, M.B. Simultaneous determination of Cr(iii) and Cr(vi) using reversed-phased ion-pairing liquid chromatography with dynamic reaction cell inductively coupled plasma mass spectrometry. J. Anal. Atom. Spectrom. 2007, 22, 1051-1060. 

S.6.4.2 Reversed-phase ion-pair liquid chromatography. Reversed-phase ion-pair chromatography is an alternative approach for controlling the retention of ionic compounds. This approach is particularly useful for the separation of amines on silica-based columns and it has had a profound effect on the analysis of this class of compounds. In particular, the combination of reversed-phase ion-pair liquid chromatography and electrochemical detection revolutionized the analysis of neurotransmitters in the brain (Tomlinson et al., 1978 and refs, therein). 

Christofides, J.A. Fry, D.E. Measurement of anticonvulsants in serum by reversed-phase ion-pair liquid chromatography. Clin.Chem., 1980, 26, 499-501... 

Shaikh, B. Jackson, J. Determination of neomycin in milk by reversed phase ion-pairing liquid chromatography. J.Liq.Chromatogr., 1989, 12, 1497-1515... 

Wong, Y.W. Davis, P.J. Analysis of warfarin and its metabolites by reversed-phase ion-pair liquid chromatography with fluorescence detection. J.Chromatogr., 1989, 469, 281-291... 

See also Liquid Chromatography Overview Principles Reversed Phase Ion Pair Liquid Chromatography-Mass Spectrometry. 

Takeda, A. Shinohara, T. Simultaneous analysis of tiar-amide metabolites in horse urine and plasma by sohd-phase extraction and reversed-phase ion-pair liquid chromatography. J. Anal. Toxicol. 1995,19 (6), 435-442. 

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