Chromatography of Ionic Compounds

The retention factor of partially ionized compounds can be predicted in reversed-phase liquid chromatography by Equation (4.1) 2 

The retention factor of an amino acid is given by the following equation, as the amino acid has two ionizable groups (a carboxyl group and an amino group, with Ka and Ka2, respectively)  

The maximum retention factor (kQ) is related to the log P value and k and k are the retention factors of the cationic and anionic forms, respectively. The pKa values are known, and the retention factor in a given eluent can therefore be predicted in reversed-phase liquid chromatography using an alkyl-bonded silica gel or polystyrene gel column. The separation conditions can be adjusted according to their logP and pKa values by the selection of a suitable organic modifier concentration and the pH of the eluent.3,4 

Column, octadecyl-bonded vinyl alcohol copolymer gel, 10 cm x 6 mm i.d. eluent, 0.05 M sodium phosphate solution in 20% acetonitrile flow rate 1 ml min-1 temperature, 30 °C. Compounds 3C1, 3-chlorobenzoic acid and 4Et, 4-ethylbenzoic acid. 

The selection of the counter-ion and its concentration are important for the separation of ionic compounds in reversed-phase and ion-exchange liquid chromatography. The addition of hydrophobic ions is an especially powerful method and several surfactants can be used as hydrophobic counter-ions. The theoretical column efficiency of ion-pair liquid chromatography is much better than that of an ion-exchange column, and the regeneration of a column is much faster. Thus, if we can control ion-pair liquid chromatography, we can solve a separation problem. (The important background sources in this area are listed at the end of the chapter.) 

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R Szucs, J Vindevogel, P Sandra, LC Verhagen. Sample stacking effects and large injection volumes in micellar electrokinetic chromatography of ionic compounds direct determination of iso-a-acids in beer. Chromatographia 36 323-329, 1993. 

J Vindevogel, P Sandra. Micellar electrokinetic chromatography of ionic compounds The analysis of hop bitter acids. J High Res Chromatogr 14 795-801, 1991. 

Reversed-phase liquid chromatography of ionic compounds... 

This paper describes the use of poly(styrene-divinylbenzene) copolymer, PRP-1, as a reverse-phase adsorbent in the assay of the antibiotic aztreonam and related compounds. Comparisons are also made for similar assays using silica-based columns. None of the shortcomings described earlier, associated with bonded phase columns, is observed. In addition to the reverse-phase mode, the PRP-1 columns are tested in ion-pair as well as in size exclusion modes of separation. Superior resolutions are obtained in the reverse-phase chromatography of ionic compounds without the use of lon-palring agents. In addition to the normal adsorption and/or partitioning,... 

The analysis of hydrophihc substances by RP-HPLC has some limitations, which are mainly due to the restricted retention of these analytes on the hydrophobic stationary phase. Ion exchange chromatography of ionic compounds and derivatization of the molecules of interest to render them more hydrophobic are two options to overcome this problem. Not all compounds, however, have ionic character, and derivatization is only specific for a given functional group consequently, each functional group requires time-consuming and tedious derivatization. 

Ion-pair chromatography separates ionic compounds using traditional RP stationary phases. A so-called counter-ion of opposite charge is added to the mobile phase. It forms a neutral ion-pair which can be easily separated under RP conditions. The mobile phase generally consists of water or buffer mixed with an organic modifier such as methanol or ACN. 

The elucidation of the retention mechanism in ion-pair reversed-phase chromatography using alkyl amines or alkyl sulfonates as hetaerons has evoked significant interest not only for the great potential of the method in the separation of ionic compounds but also for theoretical reasons. 

Gradient Separations of Ionic Compounds 5.4.3.1 Reversed-Phase Chromatography... 

Reversed-phase chromatography is often used to separate both neutral and ionic organic compounds. In this section, some important aspects for the understanding of the behavior of ionic compounds in reversed-phase chromatography are discussed. The important concepts introduced here are the electrical double layer and the electrostatic surface potential. It will be shown that they are essential for the understanding of the elution profile of ionic compounds. These concepts are further explored in the next section where theoretical models for ion-pair chromatography are discussed. 

BA Bidlingmeyer. Separation of ionic compounds by reversed-phase liquid chromatography an update of ion-pairing techniques. J Chromatogr Sci 18 525-39, 1980. 

A special application of LLC is ion pair partition chromatography. In this procedure, the ionic form of the solute (analyte) is paired with an appropriate counter ion of decreased polarity, e.g. tetra-tertiary-butyl amine. This ion pair is then partitioned between selected mobile and stationary phases to achieve the desired separation. In practice, ion pair chromatography is commonly conducted by utilizing a mobile phase comprised of a miscible aqueous/organic mixture containing a relatively high concentration of counter ion. The technique is applicable to analysis of many types of ionic compounds (10). 

CD s dissolved in the mobile phase solutions have been used in thin-layer chromatography with polyamide stationary phase (29,30) and 2) CD complexation equilibria of ionic compounds were studied by a chromatographic method using an ion exchanger as stationary phase and mobile phase solutions containing CD s in various concentrations (31). 

Bidlingmeyer, B.A. Separation of ionic compounds by reversed-phase liquid chromatography an update of ion-pairing techniques. J. Chmmatogr. 1980,18, 525-539. Sarzanini, C. et al.. Retention model for anionic, neutral and cationic analytes in reversed-phase ion interaction chromatography. AnaL Chem. 1996, 68,4494-4500. 

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). 

As regards the layers, ready-to-use alkyl-bonded silica plates were found to have many advantages over the previously employed home-made plates. An appropriate term proposed for this chromatographic technique is surely dynamic ion-exchange chromatography. The method can be applied to separation of a wide variety of ionic compounds and classes of compounds (see Table 2). 

Ion-pair chromatography may also be used for the separation of ionic compounds and... 

Capillary electrophoresis (CE) is a powerful separation technique. It is especially useful for separation of ionic compounds and chiral mixtures. Mass spectrometry has been coupled with CE to provide a powerful platform for separation and detection of complex mixtures such as combinatorial libraries. However, the full potential of CE in the application of routine analysis of samples has yet to be realized. This is in part due to perceived difficulty in the use of the CE technique compared to the more mature techniques of HPLC and even SFC. Dunayevskiy et al. [136] analyzed a library of 171 theoretically disubstituted xanthene derivatives with a CE/ESI-MS system. The method allowed the purity and makeup of the library to be determined 160 of the expected compounds were found to be present, and 12 side products were also detected in the mixture. Due to the ability of CE to separate analytes on the basis of charge, most of the xanthene derivatives could be resolved by simple CE-MS procedures even though 124 of the 171 theoretical compounds were isobaric with at least one other molecule in the mixture. Any remaining unresolved peaks were resolved by MS/MS experiments. The method shows promise for the analysis of small combinatorial libraries with fewer than 1000 components. Boutin et al. [137] used CE-MS along with NMR and MS/MS to characterize combinatorial peptide libraries that contain 3 variable positions. The CE-MS method was used to provide a rapid and routine method for initial assessment of the construction of the library. Simms et al. [138] developed a micellar electrokinetic chromatography method for the analysis of combinatorial libraries with an open-tube capillary and UV detection. The quick analysis time of the method made it suitable for the analysis of combinatorial library samples. CE-MS was also used in the analysis... 

Ion-pair chromatography may also be used for the separation of ionic compounds and overcomes certain problems inherent in the ion-exchange method. Ionic sample molecules are masked by a suitable counter ion. The main advantages are, firstly, that the widely available reversed-phase system can be used, so no ion exchanger is needed, and, secondly, acids, bases and neutral products can be analysed simultaneously. 

In addition to environmental apphcations, ion chromatography is now routinely being used for the analysis of ionic compounds in diverse areas such as power plant chemistry, semiconductor industry, food and beverages, household products and detergents, pharmaceuticals, biotechnology, and agriculture. In this entry, the application of ion chromatography in environmental research is described. 

Environmental analytical chemistry can be regarded as the study of a series of factors that affect the distribution and interaction of elements and substances present in the environment, the ways they are transported and transferred, as well as their effects on biological systems. " An important job for analytical chemistry today is environmental analysis. This task can be performed using modern analytical techniques and methods. In the range of ionic compounds, the most important one is ion chromatography. 

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