Reversed phase HPLC organic modifiers

In HPLC, a sample is separated into its components based on the interaction and partitioning of the different components of the sample between the liquid mobile phase and the stationary phase. In reversed phase HPLC, water is the primary solvent and a variety of organic solvents and modifiers are employed to change the selectivity of the separation. For ionizable components pH can play an important role in the separation. In addition, column temperature can effect the separation of some compounds. Quantitation of the interested components is achieved via comparison with an internal or external reference standard. Other standardization methods (normalization or 100% standardization) are of less importance in pharmaceutical quality control. External standards are analyzed on separate chromatograms from that of the sample while internal standards are added to the sample and thus appear on the same chromatogram. 

G. Teshima and E. Canovadavis, Separation of oxidized human growth hormone variants by reversed-phase HPLC-effect of mobile phase pH and organic modifier, J. Chromatogr., 625 201 (1992). 

Figure 8. Separations of rhGH from its methionyl analog by reversed-phase HPLC. The chromatography was done at the indicated pHs using phosphate-containing mobile phases with propanol organic modifier on a Vydac C4 column. Elutions were run in an isocratic mode.

The above discussions have shown how selected analytical techniques can be applied to vastly different proteins to solve a myriad of problems. These include routine assays amino acid and sequencing analyses specialized techniques FAB-MS and IEF conventional techniques refined to improve their utility reversed-phase HPLC using different pHs, organic modifiers, and temperatures and chemical and enzymatic modifications. The latter two procedures have been shown to be effective not only in elucidating primary structure but also in probing the conformation of proteins. 

The reverse-phase mode is used for all the separations performed in this experiment. Reverse phase is the term used when the stationary phase is more nonpolar than the mobile phase with regard to the polarity of the sample. The isopropanol/water and isopropanol/vinegar mobile phases are typical of reverse-phase mobile phases, which generally are composed of water mixed with polar organic modifiers. The bonded Cig column used is a very nonpolar surface and is the most popular stationary phase for reverse-phase HPLC. In this experiment the silica column when used in the reverse-phase mode provides a very weak nonpolar surface in comparison to C g. Silica is normally thought of as a highly polar surface and is most commonly used in the normal-phase mode. The use of silica in the normal-phase mode, with a nonpolar mobile phase is the subject of Chapter 9 (Experiment 2). 

In reversed-phase HPLC the binary mixture of water and organic components is the common eluent. It is obvious that organic modifier would be preferentially adsorbed on the surface of hydrophobic stationary phase, and this adsorption has been studied for more than 30 years [24-26]. 

Mobile phases commonly used in reversed-phase HPLC are hydro-organic mixtures. The most common reversed-phase organic modifiers include methanol and acetonitrile and/or combinations of these two modifiers. Other mobile-phase modifiers such as tetrahydrofuran, IPA, and DMSO [32] have been also used for minor selectivity adjustment however, they are not common due to their high backpressure limitations and/or high background UV absorbance. 

S.6.2 Effect of Organic on Modifier lonization-pH Shift. Typically, most reversed-phase HPLC methods use monoprotic or polyprotic acidic buffers. The determination of pK values of acids in acetonitrile/water mixtures and methanol/water mixtures have been reviewed in the literature [61-65,67,77] Several excellent reviews have been published on this topic by Roses and Bosch. [74,75] The pH can be determined directly from pH by the following relationship as shown in equation (4-19). 

In spite of many efforts, the relationships between retention and mobile phase composition are approximate. The fact is that often the values of logA extrapolated from several isocratic measurements in water-oiganic modifier eluents of varying compositions to the pure water eluent (the intercepts in Eq. (11.4)) are different from those determined experimentally (when it is possible). Besides. logA. data from the reversed-phase HPLC log A. data are often different when derived from aqueous systems modified with different organic solvents 117j. 

The polarity index or the solubility parameter may be used as a measure of solvent strength, which would be a measure of polarity in those cases. For reversed phase HPLC, solvent strength parameters have been proposed for the four most common solvents used, i.e. water (Si = 0), methanol (Si = 2.6), acetonitrile (Si = 3.2) and THF (Si = 4.5). Using these values water makes no contribution to the eluting power of the mobile phase and the solvent strength is measured by the volume fraction of organic modifier. 

Reverse phase, as its name implies is the reverse of normal phase operation the stationary phase is nonpolar and the eluent is polar. The eluent normally comprises mixtures of water and organic cosolvents (or modifiers). Other additives, such as buffers, acids, bases, and ion-pair reagents may be incorporated into the eluting solvent These are discussed in Subheading 2.1.3. The stationary phases most commonly used in reverse phase HPLC are known as... 

Figure 5.4-4. Scheme of an offline MDLC setnp. The separation with this two-dimensional HPLC separation is divided into two steps. At the first step, the analyte is separated by an ion-exchange separation (lEX) and the eluted fractions are collected. Before the second step, the samples are concentrated and desalted and organic modifiers are removed if present. The second dimension is performed by a normal reversed phase-HPLC separation (see Figure 5.4-1). If the required capacities are not available, this type of MDLC run enables the separation of both dimensions on the same HPLC after a setup change of column and solvents.

The addition of an organic solvent to modify the mobile phase is often used to alter the selectivity of the stationary phase. Methanol, acetonitrile, ethanol and dioxane at concentrations of up to 10% are used for this purpose. The effects resulting from such an addition are similar to those observed in reversed phase HPLC and generally result in a reduction in the retention of the solutes. 

Reversed phase HPLC is only rarely used for the chromatographic resolution of saccharides however, in instances where only occasional separations are required the wide availability of reversed phase columns in many laboratories saves the purchase of a specialised carbohydrate column. Reversed phase HPLC has been used primarily for the separation of oligosaccharides and separation is dependent on the degree of polymerisation (Cheetham et al., 1981). Organic phase modifiers such as -alkylamines can be used to provide increased capacity and selectivity for saccharides (Lochmuller and Hill, 1983). 

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. 

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