Stationary phase hydrophobic interactions with

The effects from molecular size are often related to hydrophobic interactions with nonpolar parts of the stationary phase. Hydrophobic interactions can be reduced by including 10-20% of methanol/acetonitrile in the mobile phase. This is often done when cation exchangers are used in the first dimension of two-dimensional separation of peptides, with reversed phase in the second dimension. 

Interactions between proteins and salts in the binding buffer are also a major determinant of selectivity. Salts that are strong retention promoters in HIC are excluded from protein surfaces by repulsion from their hydrophobic amide backbones and hydrophobic amino acid residues.8,9 This causes the mobile phase to exert an exclusionary pressure that favors the association of proteins with the column, regardless of stationary-phase hydrophobicity.1(W2 Because this mechanism involves the entire protein surface, the degree of exclusion is proportional to average protein hydrophobicity, regardless of the distribution of hydrophobic sites. 

Retention in RP chromatography is based on the interaction of the hydrophobic part of the analyte with the hydrophobic section of the stationary phase. This interaction can be modulated with the type and the concentration of the organic modiher in the mobile phase. The selectivity is mainly inflnenced by the interaction of the polar fnnctional gronps of the analyte with constituents of the mobile phase (bnffer, salts, etc. in the aqneons part) and with the amonnt and activity of residual surface silanols, which are, of course, also modihed by mobile phase constituents. 

Figure 21, Proposed model of adsorbed chiral selector (A-alkylproline)- Cu(U)-[free amino acid] mixed chelate complex, The lipophilized proline selector is held in position via intercalation of the alkyl chain. Case A the alkyl part of the mixed chelate complex is fixed by hydrophobic interactions with stationary phase (RP-J). Case B the complex formation is stabilized by other types of hydrophobic attraction. Chiral recognition and elution order is therefore not only dependent on the simple and isolatedly viewed chelate complex stability. In general, retention and chiral recognition in chiral LC is based on mixed-mode adsorption/dcsorption processes which act synergisticallv and also antagonistically with respect to the observed chiral resolution and intermolecular complex formation.

Nelis and De Leenheer (180) used isocratic NARP-HPLC with Zorbax ODS (a monomeric ODS stationary phase with a 20% carbon loading) and a mobile phase of acetonitrile/dichlorometh-ane/methanol (70 20 10) to separate nine carotenoids spanning a wide polarity range. This classic separation was achieved by virtue of the fact that the Zorbax ODS material supplied at that time was nonendcapped. The carotenes were retained by hydrophobic interaction with the ODS... 

The main process determining analyte retention is the hydrophobic interactions with the stationary phase and its competition with organic mobile-phase additive. This simplistic description allows for a rough estimation of the analyte retention and, in principle, is applicable only for very ideal systems, like the separation of alkylbenzenes in methanol/water mixtures. 

A recently proposed method (314) for the separation of fat-soluble vitamins by electrokinetic chromatography was further developed (315). The separation medium consisted of acetonitrile water (80 20 v/v) and contained tetradecylam-monium bromide as a pseudostationary phase. The high acetonitrile content was necessary to keep the hydrophobic vitamins in solution during electrophoresis. With the cathode placed at the capillary outlet, the fat-soluble vitamins were separated based on different hydrophobic interactions with the pseudo-stationary phase. The vitamins migrated in order of decreasing hydrophobicity prior to the electroosmotic flow. 

During the last ten years, it was realized that all organic ionizable compounds show some specific hydrophobic interactions with reversed-phase stationary phases [6-8]. These relatively weak interactions offer significant HPLC selectivity in the separation of even related compounds. pH is a primary tool for controlling this selectivity through the change of the analyte ionization state. 

While the driving force of reversed-phase retention is the interaction of the hydrophobic part of the analyte with the hydrophobic stationary phase, the interaction of the polar functional groups of the analyte with the mobile phase and with the residual silanols on a silka-based packing ate reqwnsible for the selectivity of a separatioiL This has two important consequences ... 

When templates are polar, the increase of the aqueous content in the mobile phase causes a marked decreasing of the column capacity factor, whereas templates of moderate or low polarity are increasingly retained and the column behaves as a true reverse phase. Such increase in retention can be attributed to a shift of the partition equilibrium towards the stationary phase (bulk of the polymer+binding sites) due to hydrophobic interactions with the template. 

The first situation is encoimtered when an anionic solute is eluted with an anionic surfactant, or a cationic solute is eluted with a cationic surfactant [e.g., dissociated phenol with the anionic surfactant sodium dodecyl sulfate (SDS), and protonated benzylamine with the cationic surfactant dodecyl trimethylammonium bromide (DTAB), on a C18 column] [2]. Electrostatic repulsion from the micelle should not affect the retention as the solute will still reside in the bulk solvent phase, and therefore, will move down the column. In contrast, repulsion from the surfactant-modified stationary phase should cause a decreased retention, and the solute may elute in the dead volume. However, it may be retained by hydrophobic interaction with the stationary phase, although this effect will be reduced by the electrostatic repulsion. Because due to different hydrophobic interactions, dissociated phenol and 2-naphthol are well separated with SDS. 

The second situation appears when a solute is chromatographed with an oppositely charged surfactant, where electrostatic attraction occurs between both species. Electrostatic attraction between solute and micelle will complement any hydrophobic interaction, and thus, it can be expected that the solute will remain in the mobile phase for a longer period of time, decreasing the retention. However, electrostatic and hydrophobic interactions with the stationary phase are often sufficiently large to offset micellar attraction and thus thee retention will increase. That is why dissociated phenol and 2-naphthol are retained to a greater extent with DTAB than with SDS on a C18 column [2],... 

Charges on solute and surfactant are opposite. Electrostatic and hydrophobic interactions with the stationary phase may be sufficiently large to offset the increase in micelle attraction, and retention will be strong. 

The hydrophobicity of the reversed phase indicates how strongly an analyte interacting primarily via hydrophobic interactions with the stationary phase is... 

For charged solutes eluted with ionic surfactants, two situations are possible repulsion or attraction, depending on the mutual charges of solutes and surfactant. In the case of electrostatic repulsion with the stationary phase, charged solutes cannot be retained and elute at the dead volume, unless significant hydrophobic interactions with the modified bonded layer exists. In contrast, combined electrostatic attraction and hydrophobic interactions with the modified stationary phase may give rise to strong retention. 

To better distinguish the contributions of polar interactions to retention, the LEER model was transformed into the so-called hydrophobic subtraction model (HSM) for RPLC, where the hydrophobic contribution to retention is compensated for by relating the solute retention to a standard nonpolar reference compound. This approach was applied to characterize more than 300 stationary phases for RPLC, including silica gel supports with bonded alkyl-, cyanopropyl-, phenylalkyl-, and fluoro-substituted stationary phases and columns with embedded or end-capping polar groups. The QSRR models can be used to characterize and compare the suitabihty of columns not only for reversed-phase, but also for NP and HILIC systems. 

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