Solvophobic retention

Retention in HIC can be described in terms of the solvophobic theory, in which the change in free energy on protein binding to the stationary phase with the salt concentration in the mobile phase is determined mainly by the contact surface area between the protein and stationary phase and the nature of the salt as measured by its propensity to increase the surface tension of aqueous solutions [331,333-338]. In simple terms the solvopbobic theory predicts that the log u ithn of the capacity factor should be linearly dependent on the surface tension of the mobile phase, which in turn, is a llne2u function of the salt concentration. At sufficiently high salt concentration the electrostatic contribution to retention can be considered constant, and in the absence of specific salt-protein interactions, log k should depend linearly on salt concentration as described by equation (4.21)... 

The solvophobic theory has been successfully applied to treat the effect of solute ionizatiqn as well as the effect of salts on the retention of both neutral and ionized species. There is ample experimental evidence that retention of a spfeies decreases upon ionization according to the theoretical prediction. Addition of salts to aqueous eluents increases surface tension and consequently. the retention of neutral eluites on nonpolar stationary phases. With ioni d solutes, however, the solvophobic theory predicts a minimum at low ionic strength in plots of retention versus ionic strength and this phenomenon has also been experimentally demonstrated. 

Factors that influence the retentive powers and selectivity of such bonded phases include the surface concentrations of hydrodartenaceous ligates and free silanol groups. The thermodynamic aspectitm solute interactions with the hydrocarbonaceous ligates at the surface, which are hydrophobic interactions in the case of aqueous eluents, are discussed later in this chapter within the framework of the solvophobic theory. In practice, however, solute interactions with surface silanol which may be termed silanophilic interactions can also contribute ]to retention (71, 75, 93), particularly in the case of amino compounds. Consequently the retention mechanism may be different from that which would be ol served with an ideal nonpolar phase. Therefore, increasing attention is paid to the estimation of the concentration of accessible sianols and to their elimination from the surface of bonded phases. 

If the retention enthalpies of the two sites differ, curvature may be observed in the plots. Moreover, if the enthalpies are opposite in sign, a minimum will occur in the van t Hoff plot at a temperature where the ratio of the retention foctors for the two mechanisms equals the absolute value of the reciprocal of the ratio of the corresponding enthalpies. Most frequently, however, less dramatic curvature would be expected. Such behavior may be anticipated in the RPC of amines with- arge nonpolar moieties which could be retained by silmiophilic interactions with surface silanols and by solvophobic interactions with nonpolar ligates of a reversed phase with low surface coverage. Recently an lihalysis of this behavior has been reported 93). 

In spite of the preceding observation that eluite retention in RPC with hydrocarbonaceous bonded phases may not occur by partitio ng of the eluite between two liquid phases, theoretical considerations based on the solvophobic treatment of solvent effects shows that it might be possible to relate the observed retention factors to partition coefficients between water and an organic solverit. Such a relationship would be quite useful in light of the scale developed by Hansch and his co-workers (2/12, 283) to characterize hydrophobic properties of drugs and other biologically active... 

The theoretical treatment of the hydrophobic effect is limited to pure aqueous systems. To describe chromatographic separations in RPC Horvath and Melander developed the solvophobic theory [47]. In this theory, no special assumptions are made about the properties of solute and solvent, and besides hydrophobic interaction electrostatic and other specific interactions are included. The theory has been valuable to describe the retention of nonpolar [48], polar [49], and ionizable [50] solutes in RPC. The modulation of selectivity via secondary equilibria (variation of pH, ion pair formation [51]) can also be described. On the other hand, it is not a problem to find examples of dispersive interactions in literature, e.g., separation of carotinoids with a long chain (C30) RP gives a higher selectivity compared to standard RP C18 cyclohexanols are preferentially retarded on cyclohexyl-bonded phases compared to phases with linear-bonded alkyl groups. 

As is evident from the above discussion, the composition and primary structure of the peptide as well as the characteristics of the mobile and stationary phases all play critical roles in determining the magnitude of retention in RPC. Solvophobic theory 17 39 40 47 56 69-71 can be... 

The main reason for chromatographic retention on reversed phases is solvophobic interaction, but under certain conditions silanophilic interaction and additional effects contribute to, or even govern, the chromatographic process. A concise review of physicochemical phenomena contributing to retention in RPC has been recently given by Horvath54). 

The physical phenomena causing retention in HIC are basically the same as in solvophobic RPC. In HIC, the moderate binding power of the packing is complemented by the salting-out effect of the mobile phase. The mild conditions of HIC provide the best chance for recovering proteins of biological activity. 

The solubility of polymers is, for thermodynamic reasons, more restricted than the solubility of low-molecular compounds and, consequently, the choice of solvents is limited. Potential solvents for most synthetic polymers are of moderate polarity. Alcohols and liquids of similar polarity are precipitants for many synthetic polymers. The search for a mobile phase that enables RPC through solvophobic interactions between the polymer and the nonpolar stationary phase requires attempts to make the mobile phase an unfavorable environment for the solute. This easily conflicts with the narrow limits of solubility of the polymer under investigation. Solubility effects are known to occur even in low-molecular RPC 92 94), but in polymer RPC they even may govern retention. 

The retention mechanism is not yet fully understood. The solvophobic theory does not account for any interaction in the stationary phase, which plays a passive role. The partition mechanism as described by Dill and Dorsey (27) is generally accepted. The most relevant feature is the linear plot of In k versus carbon number in a homologous series (Fig. 10), which is similar to what is observed in isothermal gas-liquid chromatography. Retention is governed mainly by hydro-... 

Two main theories, the so-called solvophobic and partitioning theories, have been developed to explain the separation mechanism on chemically bonded, non-polar phases, as illustrated in Figure 2.4. In the solvophobic theory the stationary phase is thought to behave more like a solid than a liquid, and retention is considered to be related primarily to hydrophobic interactions between the solutes and the mobile phase14-16 (solvophobic effects). Because of the solvophobic effects, the solute binds to the surface of the stationary phase, thereby reducing the surface area of analyte exposed to the mobile phase. Adsorption increases as the surface tension of the mobile phase increases.17 Hence, solutes are retained more as a result... 

Figure 2.4 (a) Solvophobic and (b) partitioning models of solute retention. 

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