Hydrophobic interactions - Aqueous mobile phases

When increasing the aqueous content in the mobile phase polar templates usually become less retained on MIPs, whereas templates of moderate to low polarity become more retained. The latter increase in retention is due to the hydrophobic effect [32, 145]. Thus, in contrast to the behaviour of other types of 

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Adsorption. Hydrophobic interactions, which may occur using aqueous mobile phases, usually can be eliminated by the addition of an organic modifier to the aqueous mobile phase (30,33) or by a reduction of ionic strength (3A 25.)- Recently, Haglund and Marsden (36-AO) have undertaken a systematic study on the chromatographic behavior of low molecular weight solutes on Sephadex packings and explained these results in terms of hydrophobic interactions. 

In hydrophobic interaction chromatography (HIC)> nonpolar components are selectively expelled from an aqueous mobile phase due to the cohesive forces induced in water by hydrogen bonding. These forces can be modulated by the concentration of dissolved salts. The nonpolar species adsorb on or partition into a nonpolar stationary phase largely as a result of these forces. The nonpolar phase is often a bonded phase as noted above. 

The organic solvents often used as modifiers in the aqueous mobile phase consist of n-propanol, isopropanol, methanol, and acetonitrile. They are efficient agents for modulating the hydrophobic interaction between the analytes and the protein stationary phase [144]. An increase in the organic modifier in the aqueous organic mobile phase will decrease the retention, but will have minimal effect on the overall enantioselectivity [141]. 

Fig. 9.37. Typical model for sorption complexes of proline enantiomers on (.S )-proline- or (S)-hydroxyproline-derived poly.styrene-type sorbents. Retention of (5)-Pro is diminished by the steric interaction with the water molecule crdinatcd in the axial position of the Cu(ll) ion. Retention of (R)-Pro is enhanced by the (favourable in the aqueous mobile phase) hydrophobic interaction with the non polar polystyrene chain (reprinted with permis.sion from Ref. 1403]).

Large biomolecules, while being charged under most aqueous mobile-phase conditions, still have significant hydrophobic portions that interact with the... 

HILIC uses a polar stationary phase to separate proteins and peptides. Samples are loaded onto a column using a low aqueous mobile phase and are eluted by increasing the water content of the mobile phase (152). HILIC is very suitable for separation of polar species such as sialic acid-containing glycopeptides, which have limited retention on most RP materials (153). A variant of HILIC is electrostatic repulsion-hydrophobic interactions chromatography and it utilizes IEX stationary phase and a high organic mobile phase (152,154). 

Pure aqueous mobile phases are only suitable for separations on weakly hydro-phobic stationary phases hence materials containing one tenth to one hundredth of the carbon load of classical reversed phases have been developed. This is achieved by low coverages of short-chain groups such as butyl or phenyl. Proteins can then be retained when the eluent has a relatively high salt content (e.g. 1 M or more) and eluted when the salt content drops. This mild method of protein separation, which is a variant of reversed-phase chromatography, is known as hydrophobic interaction chromatography (HIC Figure 10.16). 

In conventional reversed phase HPLC, differences in the physicochemical interactions of the eluate with the mobile phase and the stationary phase determine their partition coefficients and, hence, their capacity factor, k. In reversed-phase systems containing cyclodextrins in the mobile phase, eluates may form complexes based not only on hydrophobicity but on size as well, making these systems more complex. If 1 1 stoichiometry is involved, the primary association equilibrium, generally recognized to be of considerable importance in micellar chromatography, can be applied (11-13). The formation constant, Kf, of the inclusion complex is defined as the ratio of the entrance and exit rate constants between the solute and the cyclodextrin. Addition of organic modifiers, such as methanol, into the cyclodextrin aqueous mobile phase should alter the kinetic and thermodynamic characteristics of the system. This would alter the Kf values by modifying the entrance and exit rate constants which determine the quality of the separation. 

Characterization of partially hydrolyzed PVA by viscometry is complicated by secondary, hydrophobic interaction effects. These effects can be minimized using a suitable aqueous mobile phase with an organic modifier such as acetonitrile. 

Khaledi et al. [6, 7] were concerned with the similarities and differences in retention behavior between the mode of RPLC which employs micellar eluents and that with aqueous-organic solvents. These techniques share the basic components of an RPLC system, that is, a nonpolar stationary phase and a polar aqueous mobile phase. The hydrophobicity of solutes should play an important role in governing the retention in both systems, which is easily controlled by adjusting solute-mobile phase interactions. However, the differences in interaction mechanism can cause significant differences in retention behavior. 

Partition chromatography and reversed-phase LC usually lead to the opposite result, i.e., a heavier iso-topomer is eluted first (a>l). It has been suggested that a major contribution to the isotope effect is a hydrophobic interaction. The fact that the separation factors are higher (see Table 2) when the mobile phase contains more water demonstrates that they are affected by more restricted motion of C-H bonds (in solute), caused by tighter solvation of C-H bonds within the aqueous mobile phase relative to the hydrophobic stationary phase. On the other hand, a less restricted motion of the C-H bonds in the stationary phase would tend to favor protium over deuterium, and this could contribute to the observed isotope effect. 

In hydrophobic-interaction chromatography, nucleic acids are adsorbed to the hydrophobic surface by salting out from the aqueous mobile phase. The adsorbed nucleic acids are eluted by a decreasing salt gradient that redissolves the nucleic acids in the aqueous mobile phase. The hydrophobic interaction between a DNA molecule and the matrix is enhanced by high ionic strengths. This makes hydrophobic interactions an ideal tool for purification of DNA molecules that have been prepared in a high salt concentration. 

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