Liquid chromatography reverse-phase

Reverse-phase Uquid chromatography (RP-HPLC) is the most important and most widely appUed version of LC. It is well suited to separate both apolar and polar compounds, but less well suited for studying permanently ionized molecules. It is easy to couple with MS. 

In RP-HPLC the stationary phase is less polar than the mobile phase and the interaction between analyte and the stationary phase has a predominantly hydrophobic (apolar) character. The most commonly used stationary phase in RP-HPLC is silica gel in which octadecyl silica chains are covalently bound to the free hydroxyl groups, indicated as a C18 phase. The typical surface of such a phase is shown in Fig. 8. 

The surface of C18 phases always contains unreacted silanol groups, which may form secondary polar interactions with the analyte. This is generally disadvantageous in RP-HPLC as it often causes peak broadening [33,40]. An important improvement is the introduction of the so-called end-capping procedure The residual silanol groups in the C18 phase are reacted with monofunctional chlorosi-lane, which decreases surface polarity. This very popular stationary phase is called C18ec, where the notation ec stands for end-capped. 

RP-HPLC is widely apphcable, although pH control must often be applied. Most important application areas include peptide and protein analysis (proteomics), drugs and their metabolites, fatty acids, and also volatile compounds such as aldehydes and ketones, although these require derivatization. 

Numerous recent reviews and guidelines have been published in this field [5,84,92]. Therefore, in this part, we summarize the basic principles only. Briefly, lipophilicity determination by RP-LC is based on the partitioning of the solute between an apolar stationary phase and a polar mobile phase. The experimental retention factor (log k) 

In practice the regression coefficients a and b have to be determined for every couple of stationary and mobile phases, by measuring the log k values for a series of reference compounds with known log P values. The calibration equation obtained then allows the determination of log P of new compounds. 

Eurthermore it was shovm that the addition of 1-octanol in mobile phase often enhances the correlation between log and log k [97-99]. In the same way the coating of a 0.8 cm HiChrom H5SAS Cl cadridge with 1-octanol recently proved its interest for the determination of log Poet of neutral pharmaceutical compounds ranging from 1 to 4 vfith a flow gradient mode [100]. 

To speed up the lipophilicity determination it was also proposed to use gradient elution procedures (for a review and guidelines, see references [5,101]). This generic approach is particularly useful when series of compounds with a broad lipophilicity range have to be tested since both polar and non-polar solutes can be retained with a reasonable elution time. 

As in isocratic mode, the variation in the percentage of organic modifier in the mobile phase can be described by the linear Soczevfinski-Snyder model 

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OXIDATIVE LUMINESCENCE OF UV ABSORBING CHEMICALS. APPLICATION TO THEIR DETERMINATION IN SUNSCREEN PRODUCTS BY REVERSED PHASE LIQUID CHROMATOGRAPHY WITH CHEMILUMINESCENCE... 

J. E. MacNair, K. C. Lewis and J. W. Jorgenson, Ultraliigh-pressure reversed-phase liquid chromatography in packed capillaiy column . Anal. Chem. 69 983 (1997). 

THREE-DIMENSIONAL SIZE EXCLUSION CHROMATOGRAPHY-REVERSE PHASE LIQUID CHROMATOGRAPHY-CAPILLARY ZONE ELECTROPHORESIS... 

Figure 9.10 Three-dimensional representation of the data volume of a tryptic digest of ovalbumin. Series of planar slices through the data volume produce stacks of disks in order to show peaks. Reprinted from Analytical Chemistry, 67, A. W. Moore Jr and J. W. Jorgenson, Comprehensive three-dimensional separation of peptides using size exclusion chromatogra-phy/reversed phase liquid chromatography/optically gated capillary zone electrophoresis, pp. 3456-3463, copyright 1995, with permission from the American Chemical Society.

E. A. Hogendoom and P. van Zoonen, Coupled-column reversed-phase liquid chromatography in envhonmental analysis (review) , ]. Chromatogr. 703 149-166 (1995). 

R. J. Senorans, J. Villen, J. Tabera and M. Heiraiz, Simplex optimization of the direct analysis of free sterols in sunflower oil by on-line coupled reversed phase liquid chromatography-gas clnomatography , 7. Agric. Food Chem. 46 1022-1026 (1998). 

Figure 13.7 Selectivity effected by employing different step gradients in the coupled-column RPLC analysis of a surface water containing 0.40 p-g 1 bentazone, by using direct sample injection (2.00 ml). Clean-up volumes, (a), (c) and (d) 4.65 ml of M-1, and (b) 3.75 ml of M-1 transfer volumes, (a), (c) and (d), 0.50 ml of M-1, and (b), 0.40 ml of M-1. The displayed cliromatograms start after clean-up on the first column. Reprinted from Journal of Chromatography, A 644, E. A. Hogendoom et al, Coupled-column reversed-phase liquid chromatography-UV analyser for the determination of polar pesticides in water , pp. 307-314, copyright 1993, with permission from Elsevier Science.

However, for water analysis, reverse-phase liquid chromatography is more suitable but its coupling with GC has some drawbacks because of the partly aqueous effluent. Several systems have been developed (88, 89) and applied to determine pollutants in water. 

One of the first examples of the application of reverse-phase liquid chromatography-gas chromatography for this type of analysis was applied to atrazine (98). This method used a loop-type interface. The mobile phase was the most important parameter because retention in the LC column must be sufficient (there must be a high percentage of water), although a low percentage of water is only possible when the loop-type interface is used to transfer the LC fraction. The authors solved this problem by using methanol/water (60 40) with 5% 1-propanol and a precolumn. The experimental conditions employed are shown in Table 13.2. 

Emenhiser C., Sander L.C., and Schwartz, S.J., Capability of a polymeric C30 stationary phase to resolve cis-trans carotenoid isomers in reversed-phase liquid chromatography, J. Chromatogr. A, 101, 105, 1995. 

Emenhiser, C. et al.. Separation of geometrical carotenoid isomers in biological extracts using a polymeric Cjq column in reversed-phase liquid chromatography, J. Agric. Food Chem., 44, 3887, 1996. 

Radke et al. [28] described an automated medium-pressure liquid chromatograph, now commonly called the Kohnen-Willsch instrument. At present, the method is widely used to isolate different fractions of soluble organic matter (for instance, as described in Reference 29 to Reference 31). A combination of normal phase and reversed-phase liquid chromatography has been used by Garrigues et al. [32] to discriminate between different aromatic ring systems and degrees of methylamine in order to characterize thermal maturity of organic matter. 

P. W. High speed gradient elution reversed-phase liquid chromatography. 

The popularity of reversed-phase liquid chromatography (RPC) is easily explained by its unmatched simplicity, versatility and scope [15,22,50,52,71,149,288-290]. Neutral and ionic solutes can be separated simultaneously and the rapid equilibration of the stationary phase with changes in mobile phase composition allows gradient elution techniques to be used routinely. Secondary chemical equilibria, such as ion suppression, ion-pair formation, metal complexatlon, and micelle formation are easily exploited in RPC to optimize separation selectivity and to augment changes availaple from varying the mobile phase solvent composition. Retention in RPC, at least in the accepted ideal sense, occurs by non-specific hydrophobic interactions of the solute with the... 

Figure 4.29 An example of the use of ternary solvents to control mobile phase strength and selectivity in reversed-phase liquid chromatography. A, methanol-water (50 50) B, tetrahydrofuran-water (32 68) C, methanol-tetrahydrofuran-water (35 10 55). Peak identification 1 - benzyl alcohol 2 phenol 3 3-phenylpropanol 4 2,4-dimethylphenol 5 benzene and 6 -diethylphthalate. (Reproduced with permission from ref. 522. Copyright Elsevier Scientific Publishing Co.)...

Figure 4.27 Flow chart for coluwi selection based on sample type (m - molecular weight). PLC precipitation-liquid chromatography SEC = size-exclusion chromatography lEC - ion-exchange chromatography HIC hydrophobic interaction chromatography LSC liquid-solid chromatography RPC - reversed-phase liquid chromatography BPC (polar) bonded-phase chromatography and IPC - ion-pair chromatography.

Solvatochromic pareuaeters, so called because they were Initially derived from solvent effects on UV/visible spectra, have been applied subsequently with success to a wide variety of solvent-dependent phenomena and have demonstrated good predictive ability. The B jo) scale of solvent polarity is based on the position of the intermolecular charge transfer absorption band of Reichardt s betaine dye [506]. Et(io> values are available for over 200 common solvents and have been used by Dorsey and co-%rarkers to study solvent interactions in reversed-phase liquid chromatography (section 4.5.4) [305,306]. For hydrogen-bonding solvents the... 

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