Melander and Horvath

The influence of the bonded organic moiety on solute retention has not yet been elucidated and only a very small number of papers discuss the properties and use of such phases so far. The numerous advantages of chemically bonded phases make the application of polar chemically bonded phases with nonpolar eluents quite attractive even if the standardization of these phases may pose problems 106) similar to those encountered in the standardization of aidsorbents as well as of polymeric liquid phases in gas chromatography. A detailed discussion of the properties and chromatographic use of bonded stationary phases is given by Melander and Horvath (this volume). 

In literature there are many discussions on properties required for an optimal RP. According to Melander and Horvath [2], the ideal stationary phase for RP chromatography (RPC) should have the following properties ... 

Recently, Melander and Horvath (7, 10) have proposed a single theory to account for the effects of neutral salts on the electrostatic and the hydrophobic interactions in the salting out and the chromatography of proteins. In simplified terms, the theory accounts for the solubility of proteins in terras of two contributions, electrostatic and hydrophobic in nature. 

The electrostatic term, AFgiggtrostatiC is the change in electrostatic free energy when the protein goes from the crystalline state to the solution state. Melander and Horvath have expressed this term by combining the proper terms from the Debye-Huckel and Kirkwood theories (7). This term is always positive, and is responsible for salTing in. 

The failure of the theory indicates that soy protein behavior is more complex than Melander and Horvath s (7, ) model. In deriving their simple theory, Melander and Horvath assumed that the changes in hydrophobic surface area, in the dipole moment, and in the net charge of the protein upon solubilization are invariant with respect to salt species or salt concentration. In other words, they assumed that the soluble proteins have the same thermodynamic state regardless of salt species or salt concentration. The results for soy proteins can be treated in the framework of the Melander and Horvath s theory if it is expanded to allow the exposed surface area, the dipole moment, and the net protein charge to be functions of the salt species and the salt concentration. 

Melander and Horvath [329] have suggested a five-parameter hyperbolic equation to describe k itself as a function of

[Pg.61]

Remarkable number of different names was introduced to these methods. The technique has been called soap chromatography [113], solventgenerated ion-exchange [114], ion-interaction [115], and ion-pair [116]. Researchers introduced a similar number of different theories for the description of the effect of ionic mobile-phase additives on the retention of charged analytes essentially, each specific name for this technique corresponds to its own distinct retention theory. Melander and Horvath [116] divided existing theories into two main groups stoichiometric [113,114,117-119] and nonsto-ichiometric [120-133]. 

Phenomenologically, two different mechanisms could be envisioned (a) the formation of the ion pair between the analyte and amphiphilic counterion with subsequent adsorption of this complex on the stationary phase and (b) adsorption of the amphiphilic counterion itself on the stationary phase surface and subsequent retention of charged analyte in essentially an ion-exchange mode. Melander and Horvath [117] concluded that, in reality, probably both mechanisms coexist in the chromatographic system. 

HPLC is another convenient method for measurement of the NCE pKa values. As was shown by Melander and Horvath [13], the retention of any ionizable analyte closely resembles the curve shown in Figure 12-3. Chromatographic determination of the pKa could be accurately performed with very limited amount of sample. Fast HPLC method with optimum analyte retention is suitable for this purpose, but the influence of the organic mobile-phase modifier on the mobile phase pH and analyte Ka should be accounted for in order to provide the accurate calculation of the respective Ka value. Detailed discussion of the HPLC-based methods for the Ka determination is given in Chapter 4. 

The conception that the hydrophobicity of a protein is the property of its surface is used as the basis of the technique suggested by Melander and Horvath 30). This technique consists of an analysis of the effects of inorganic salts on the aqueous solubility of proteins30). According to the model considered by Melander et al.30), the free energy of solvation of a protein macromolecule in aqueous solution is described by Eq (3) (see above). The presence of a salt alters the protein solubility due to the concentration-dependent effect of the salt on the free energy of formation... 

Protein solubility has been mechanistically explained by changes in bulk water surface tension and binding to water or ions versus protein-protein (Frommen-hagen, 1965 Melander and Horvath, 1977 Bull and Breeze, 1980 Arakawa and... 

Deviations from classical Van t Hoff behaviour in reversed phase chromatography have been described (Snyder, 1979 Melander and Horvath, 1979) in which an increased retention of specific chemical types occurs at higher temperatures this phenomenon may be observed when molecules adopt a compact, near spherical configuration compared with their molecular conformers and are retained for longer times. Also, where the extent of ionisation of either the buffer or the solute molecules is affected by changes in column temperature an altered retention time may be obtained and may result in improved peak symmetry (Knox and Vasvari, 1973 Melander et al., 1979). 

The third mechanism, proposed by Melander and Horvath (1980b), is that of dynamic complex exchange where counter-ions are present both in the mobile phase and at the surface of the stationary phase. Sample molecules which have formed an ion-pair with the counter-ion in the mobile phase can then transfer to the bound counter-ions to form a complex at the surface of the stationary phase. A modification of this process envisages that the complex is initially formed at the stationary phase surface. 

W.R. Melander and C. Horvath, High Performance Liquid Chromatography, Advances and Perspectives, Vol. 2, Academic Press, New York, 1980 P. 114. 

W.R. Melander and Cs. Horvath, Mechanistic study of ion-pair reversed-phase chromatography , J. Chromatogr., 1980, 201, 211. 

C. Horvath, W. Melander and I. Molnar, Solvophobic interactions in liquid chromatography with nonpolar stationary phases. Journal of Chromatography, 125 (1976) 129. 

Melander, W. and Horvath, C. 1977. Salt effects on hydrophobic interactions in precipitation and chromatography of proteins An interpretation of the lyotropic series. Arch. Biochem. Biophys. 183 200-215. 

---