Reversed-phase chromatography advantage

Hydrophobic interaction chromatography (HIC) occupies a unique niche in the field of analytical chromatography. A particular advantage of HIC is its unique selectivity. Whereas ion-exchange chromatography (IEC) principally reveals differences based on the surface charge of native proteins, HIC reveals differences based principally on their surface hydrophobicity. HIC is complementary to reversed-phase chromatography (RPC) in a different sense. Whereas HIC discriminates primarily on the basis of surface hydrophobicity, RPC principally reveals differences based on total hydrophobicity of all the hydrophobic residues of denatured proteins. 

Recent advances in chromatography have made it possible to employ microbore HPLC for the determination of NOC. Its main advantage is that it uses a very low mobile-phase flow (20-100 /rl/min). This might make the TEA compatible with a reversed-phase system. Massey et al. (73), in fact, have successfully used reversed-phase chromatography for the HPLC-TEA determination of V-nitroso-V, 7V -di methylpiperazinium iodide. A 500-mm X 1-mm microbore ODS column and a mobile phase consisting of 0.1 M ammonium heptane-sulfonate in methanol water (70 30) (flow rate 20 /zl/min) was used for the HPLC separation. In another study, Riihl and Reusch (74) used a microbore Spherisorb 3 SW column for HPLC-TEA determination of volatile V-nitrosamines. The mobile phase was a mixture of 2-propanol and n-hexane (2.5 97.5). Further application of such techniques for the determination of various polar NOC, especially A-nitrosamides, in foods is desirable. 

The popularity of reversed-phase chromatography can be explained by its unmatched simplicity, versatility, and scope.12 Although reversed-phase chromatography is used routinely for separating non-polar, non-ionic compounds, it is also possible and practical to separate ionic compounds on standard reversed-phase stationary-phase materials by using secondary equilibria, such as ion suppression, ion-pair formation, metal complexation, and micelle formation. To take advantage of these secondary equilibria,... 

The growing popularity of reversed phase chromatography in particular has prompted polymer manufacturers to investigate the use of polymeric media for this mode of operation. Macroporous copolymers of styrene and divinylbenzene have similar properties to silica based stationary phases bonded with alkyl chains. However, the absence of leachables and stability at high pH can offer advantages under certain circumstances. High quality, mechanically stable macroporous polymeries are now manufactured at much larger scales than the... 

The selection of acetonitrile versus methyl alcohol has several considerations even though their flammabilities and toxicities are relatively close. The advantages of using acetonitrile over methanol are (1) its lower UV absorbance cut-off. (2) its lower viscosity, (3) and its smaller viscosity dependence on temperature. The advantages of using methanol over acetonitrile are (1) its lower cost and lower cost fluctuation in the market place, and (2) its ease to recycle with water as a co-solvent (e.g. in reversed-phase chromatography) since methanol-water does not have an azeotrope as does acetonitrile-water. At a boiling point of 76.5°C, the azeotrope composition of acetonitrile-water is 83.7 16.3 [78]. 

An advantage of reversed-phase chromatography in the analysis of alkaloids in biological fluids is that an analysis can be carried out directly without any laborious sample clean-up procedure. However, the use of a precolumn to avoid a too rapid deterioration of the HPLC column is advisable (see Chapter 11). When using aqueous salt solutions in reversed-phase chromatography, one has to be aware of the risk of corrosion of stainless steel columns (see Table 1.3)38. 

The six chromatographic modes available in HPLC for the isolation and purification of natural products are described in more detail in Section 9.02.2. Since the groups of natural products differ in their molecular properties, certain chromatographic modes have been shown to work better with particular natural product groups. However, in order to take full advantage of a specific HPLC mode for a separation task and to effectively utilize time and resources, comprehensive method development should be performed. An example of such method development from the analytical to the preparative stage is described for reversed-phase chromatography (HP-RPC), the most frequently employed mode in natural product purification, in Section 9.02.3. 

---