Bonded stationary phases hydrophobic effect

Reversed-phase stationary phases are more or less hydrophobic, and the degree of this property is characterized by their hydrophobicity H. As a general rule, retention times are longer the more C atoms the bonded stationary phase contains. (The reason is that the volume taken up by the bonded nonpolar groups, i.e. that required by the actual stationary phase, is greater with long chains than it is with shorter chains retention is directly proportional to the volume ratio between the stationary and mobile phases see Section 2.3.) Figure 10.7 demonstrates this effect. 

The separations occur due to the interactions of the alkyl (-CH2-) groups on the analytes within the mixture and the functional groups on the surface of the silica. These interactions are known as Van der Waals forces (see Chapter 2 for further explanation), but the mechanisms of retention of analytes are complex and it is possible that more than one mechanism is operating at the same time. For example, hydrophobic interactions may be taking place on the surface of the bonded stationary phase, which can cause partitioning effects with solutes as well as adsorption effects on unreacted silanol (-Si-OH-) groups. 

Soon, Knox thought to use the hydrophobic effect to form ion-pairs in aqueous phases. He developed the first use of reversed-phase ion-pair chromatography in 1976 [6]. He termed it soap chromatography. He added ionic surfactants to the polar hydro-organic mobile phase. They adsorb on the alkyl-bonded stationary phase. They also associate with the hydrophobic and ionic analytes. 

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... 

The basic model for the separation of peptidic solutes on nonpolar stationary phases assumes that reversible interactions of the solute molecules S, S2,. . . , S occur with the hydrocarbonaceous ligand L and that the interactions are due to hydrophobic associations and not to electrostatic or hydrogen bonding effects. Conceptually, the sorption of peptides to alkyl-bonded reversed phases under these conditions can be based either on partition or on adsorption processes. In a partition pro-... 

Determinations of the adsorption isotherms for a number of organic solvent-water systems in contact with hydrocarbonaceous stationary phases have shown that a layer of solvent molecules forms on the bonded-phase surface and that the extent of the layer increases with the concentration of the solvent in the mobile phase. For example, methanol shows a Langmuir-type isotherm when distributed between water and Partisil ODS (56). This effect can be exploited to enhance the resolution and the recoveries of hydrophobic peptides by the use of low concentrations, i.e., <5% v/v, of medium-chain alkyl alcohols such as tm-butanol or tert-pentanol or other polar, but nonionic solvents added to aquo-methanol or acetonitrile eluents. It also highlights the cautionary requirement that adequate equilibration of a reversed-phase system is mandatory if reproducible chromatography is to be obtained with surface-active components in the mobile phase. 

Similarly, reversed-phase HPLC can be used as an Eilternative to the racemization test for amino acids as developed by Manning and Moore (115). Rivier and Burgus (109) have suggested the use of L-phenylalanine, coupled via the N-carboxyanhydride method to a hydrolysate, to monitor racemization during synthesis, although other hydrophobic L-amino acids should also prove equally effective. The use of /eri-butyloxycarbonyl-L-amino acid-Af-hydroxysuccinimide esters in the separation of enantiomeric amino acids and diastereoisomeric peptides has been described (110). Ultimately, these methods may not prove as versatile as the use of chiral stationary phases made by stereoselective control of the bonding process or, alternatively, with surface-active reagents similar to the D-... 

Whereas the overall hydrophobic nature of the stationary phase is the most important factor in determining retention, bonded-phase structure can also influence k values. This effect can be observed in the separation of polycyclic aromatic hydrocarbons (PAHs). For stationary phases with a high bonding density and/or a high degree of association between adjacent bonded organic moieties, molecules that are more planar are preferentially retained. The National Institute of Standards and Technology (NIST) has developed reference mixtures to measure this effect. 

Methanol is usually utilized to prewet the Cjg Bond-Elut columns and opens the hydrophobic chains to increase the effective surface area. Water samples are also fortified with at least 1% methanol to continuously wet the stationary phase. This can improve recovery rates for a large number of herbicides, including triazines. By contrast, degradation products, which are often more polar than parent compounds, may not be retained as effectively in the presence of a modifier. Ground and surface water must always be filtered prior to the extraction of pesticides with the SPE technique. Prefiltering will not affect the determination of herbicides and their degradation products, since these compounds exhibit a log ATqc near 2 and consequently they are largely (99.5%) distributed in water in the dissolved phase. ... 

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