Reversed phase chromatography concentration

Hydrophobic interaction chromatography (HIC) can be considered to be a variant of reversed phase chromatography, in which the polarity of the mobile phase is modulated by adjusting the concentration of a salt such as ammonium sulfate. The analyte, which is initially adsorbed to a hydrophobic phase, desorbs as the ionic strength is decreased. One application demonstrating extraordinary selectivity was the separation of isoforms of a monoclonal antibody differing only in the inclusion of a particular aspartic acid residue in the normal, cyclic, or iso forms.27 The uses and limitations of hydrophobic interaction chromatography in process-scale purifications are discussed in Chapter 3. 

Antia, F. D. and Horvath, Cs., Dependence of retention of the organic modifier concentration and multicomponent adsorptive behavior in reversed-phase chromatography, /. Chromatogr., 550, 411, 1991. 

Counter-ions which are frequently used include tetrabutylammonium phosphate for the separation of anions and hexane sulphonic acid for cations. The appropriate counter-ions are incorporated in the solvent, usually at a concentration of about 5 mmol 1" and the separation performed on the usual reverse phase media. This ability to separate ionic species as well as non-polar molecules considerably enhances the value of reverse-phase chromatography. 

The influence of various structural and physicochemical parameters of the stationary and mobile phases on the tailing of a cationic dye in reversed-phase chromatography has been studied in detail. Measurements were performed in a C8 reversed-phase column (80 X 4.6 mm). The isocratic mobile phase was ACN-0.01 M aqueous HC1 (90 10, v/v). Analyses were carried out at 20°C and the flow rate was 1-5 ml/min. The concentration of the cationic dye, l,l -didodecyl-3,3,3, 3 -tetramethylindocarbocyanine perchlorate (Dil) in the model solutions varied between 0.9-309 pM. The dependence of the chromatographic profile of the dye on the injected concentration is illustrated in Fig. 3.112. Calculations and mathematical modelling indicated that the peak tailing of the dye can be... 

In reverse-phase chromatography, the stationary phase is nonpolar (often a hydrocarbon) and the mobile phase is relatively polar (e.g., water, methanol, and acetonitrile). The most polar components elute first, and increasing the mobile phase polarity (i.e., decreasing the organic solvent concentration) increases elution time. 

From reversed-phase chromatography of uncharged analytes it is known that the retention factor as a function of the organic modifier concentration often follows the relation ... 

The model for ionic retention and ion-pair chromatography that are discussed in Sections 15.2 and 15.3 has been tested and applied to a number of different systems and works very well in most of the cases. From colloid and surface chemistry is known that the model has its limitations, and under certain chromatographic conditions, the presented model will not be valid. The limitations of the model when applied to reversed-phase chromatography of ions still need to be found. Some are self-evident, such as if the pairing-ion concentration is close or above the CMC or when the retention factor is very low so that the accumulation in the double layer is important in comparison to the adsorption, see Ref. [7] for a discussion concerning the accumulation in the double layer. 

Using the technique of electrophoresis on paper, Forfar et al. (F7) found that cholesterol was abnormally concentrated in the (3-lipoprotein fraction. Using reverse phase chromatography, an attempt was made to demonstrate sterols other than cholesterol, but this was unsuccessful. If they are actually present, such substances must have an Rf similar to that of cholesterol or else be present in quantities too small to be demonstrated by the technique employed. 

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