Reverse-phase liquid separation

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.

A. W. Moore, Jr and J. W. Jorgenson, Comprehensive three-dimensional separation of peptides using size exclusion chromatography/reversed phase liquid chromatography/ optically gated capillary zone electrophoresis . Anal. Chem. 67 3456-3463 (1995). 

F. J. Senorans, J. Tabera and M. Herraiz, Rapid separation of free sterols in edible oils by on-line coupled reversed phase liquid chr omatography-gas chromatography , 7. Agric. Food. Chem. 44 3189-3192 (1996). 

T. Okuda, Y. Nakagawa and M. Motohashi, Complete two-dimensional separation for analysis of acidic compounds in plasma using column-switching reversed-phase liquid clrromatography , 7. Chromatogr. B126 225-236 (1999). 

Figure 13.5 Schematic presentation of the procedure involved in coupled-column RPLC AS, autosampler C-1 and C-2, first and second separation columns, respectively M-1 and M-2, mobile phases S-1 and S2, interferences A, target analytes HV, high-pressure valve D, detector. Reprinted from Journal of Chromatography, A 703, E. A. Hogendoom and R van Zoonen, Coupled-column reversed-phase liquid cliromatography in environmental analysis , pp. 149-166, copyright 1995, with permission from Elsevier Science.

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. 

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... 

Solvent optimization in reversed-phase liquid chromatography is commenced by selecting a binary mobile phase of the correct solvent strength to elute the seuaple with an acceptable range of capacity. factor values (1 < k <10 in general or 1 < k < 20 when a larger separation capacity is required). Transfer rules (section 4.6.1) are then used to calculate the composition of other isoeluotropic binary solvents with complementary selectivity. In practice, methanol, acetonitrile and tetrahydrofuran are chosen as the selectivity adjusting solvents blended in different... 

Figure 4.34 Peak-pair resolution naps and an overlapping resolution nap for the separation of nine substituted naphthalene compounds by reversed-phase liquid cbrosatography illustrated in Figure 4.33. (Reproduced with permission from ref. 545. Copyright Elsevier Scientific Publishing Co.)...

Figure 8.43 Separation of enantiomers using complexation chromatography. A, Separation of alkyloxiranes on a 42 m x 0.2S mm I.O. open tubular column coated with 0.06 M Mn(II) bis-3-(pentafluoro-propionyl)-IR-camphorate in OV-ioi at 40 C. B, Separation of D,L-amino acids by reversed-phase liquid chromatography using a mobile phase containing 0.005 M L-histidine methyl ester and 0.0025 M copper sulfate in an ammonium acetate buffer at pH 5.5. A stepwise gradient using increasing amounts of acetonitrile was used for this separation.

Lochmiiller, C. H., Moebus, M. A., Liu, Q., and Jiang, C., Temperature effect on retention and separation of poly(ethylene glycoljs in reversed-phase liquid chromatography, /. Chromatogr. Sci., 34, 69, 1996. 

Funasaki, N., Hada, S., and Neya, S., Conformational effects in reversed-phase liquid chromatographic separation of diastereomers of cyclic dipeptides, Anal. Chem., 65, 1861, 1993. 

Mohammad, J, Jaderlund, B., and Lindblom, K., New polymer-based prepacked column for the reversed-phase liquid chromatographic separation of peptides over the pH range 2-12, J. Chromatogr. A, 852, 255, 1999. 

Figure 1 Electrochemical detection of catechol, acetaminophen, and 4-methyl catechol, demonstrating the selectivity of differential pulse detection vs. constant potential detection. (A) Catechol, (B) acetaminophen, and (C) 4-methylcatechol were separated by reversed phase liquid chromatography and detected by amperometry on a carbon fiber electrode. In the upper trace, a constant potential of +0.6 V was used. In the lower trace, a base potential of +425 mV and a pulse amplitude of +50 mV were used. An Ag/AgCl reference electrode was employed. Note that acetaminophen responds much more strongly than catechol or 4-methylcatechol under the differential pulse conditions, allowing highly selective detection. (Reproduced with permission from St. Claire, III, R. L. and Jorgenson, J. W., J. Chromatogr. Sci. 23, 186, 1985. Preston Publications, A Division of Preston Industries, Inc.)...

Wise, S. A. and Sander, L. C. 1985. Factors affecting the reversed-phase liquid chromatographic separation of polycyclic aromatic hydrocarbon isomers. J. High Resolut. Chromatogr. Commun. 8 248-255. 

The column methods are much faster and are automated so that a much larger number of samples can be processed per unit time. An example of this technology, described in more detail in Chapter 10 by Lubman and coworkers, is shown in Figure 1.2, where the first dimension is from a chromatofocusing column, which gives separations in pI much like isoelectric focusing, only here the p/ axis is in bands instead of continuous pI increments. The second dimension is by reversed-phase liquid chromatography (RPLC). 

Dolan, J.W., Snyder, L.R., Djordjevic, N.M., Hill, D.W., Waeghe, T.J. (1999). Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time I. Peak capacity limitations. J. Chromatogr. A 857, 1-20. 

Gray, M.J., Dennis, G.R., Slonecker, P.J., Shalhker, R.A. (2004). Comprehensive two-dimensional separations of complex mixtures using reversed-phase liquid chromatography. J. Chromatogr. A 1041, 101. 

Others have examined the necessary parameters that should be optimized to make the two-dimensional separation operate within the context of the columns that are chosen for the unique separation applications that are being developed. This is true for most of the applications shown in this book. However, one of the common themes here is that it is often necessary to slow down the first-dimension separation system in a 2DLC system. If one does not slow down the first dimension, another approach is to speed up the second dimension so that the whole analysis is not gated by the time of the second dimension. Recently, this has been the motivation behind the very fast second-dimension systems, such as Carr and coworker s fast gradient reversed-phase liquid chromatography (RPLC) second dimension systems, which operate at elevated temperatures (Stoll et al., 2006, 2007). Having a fast second dimension makes CE an attractive technique, especially with fast gating methods, which are discussed in Chapter 5. However, these are specialized for specific applications and may require method development techniques specific to CE. 

A comprehensive 2D HPLC can be carried out with two very similar columns in reversed-phase liquid chromatography (Ikegami et al., 2005). A mixture of water and tetrahydrofuran was used as a mobile phase in the lst-D separation, and a mixture of water and methanol (CH3OH) in the 2nd-D separation with a common Ci8 stationary phase. 

Minakuchi, H., Nakanishi, K., Soga, N., Ishizuka, N., Tanaka, N. (1996). Octadecylsilylated porous silica rods as separation media for reversed-phase liquid chromatography. Anal. Chem. 68, 3498-3501. 

Holland, L.A., Jorgenson, J.W. (2000). Characterization of a comprehensive two-dimensional anion exchange-perfusive reversed phase liquid chromatography system for improved separations of peptides. J. Microcolumn. Sep. 12, 371-377. 

Chen, J., Lee, C.S., Shen, Y., Smith, R.D., Baehrecke, E.H. (2002). Integration of capillary isoelectric focusing with capillary reversed-phase liquid chromatography for two-dimensional proteomics separation. Electrophoresis 23, 3143-3148. 

Mao, Y., Zhang, X.M. (2003). Comprehensive two-dimensional separation system hy coupling capillary reverse-phase liquid chromatography to capillary isoelectric focusing for peptide and protein mapping with laser-induced fluorescence detection. Electrophoresis 24, 3289-3295. 

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