Reverse phase liquid chromatography gradient

Comprehensive multidimensional liquid chromatography is a relatively new development and has yet to develop a diverse application base. For the time being applications are dominated by the separation of proteins and synthetic polymers. For proteins the first dimension separations are usually based on ion exchange and the second dimension separations on reversed-phase liquid chromatography. Gradient elution was often used for both separation modes with a separation time less than 2 minutes for the second dimension separation and from 30 minutes to several hours for the first dimension separation. Current trends include the use of non-porous particles and perfusive stationary phases for the second dimension separation to reduce the total separation time and wider internal diameter columns in place of packed capillary columns to simplify interface construction and instrument operation and to allow the loading of larger sample sizes. 

The rapid separations offered by capillary electrophoresis have made it amenable as a detector in hyphenated techniques. For LC-CE, the total analysis time is usually governed by the LC separation, which generally takes minutes. However, capillary electrophoresis detection adds more peak capacity because of a second and orthogonal dimension for separation, and shorter separation conditions for LC can often be tolerated. For example, a 2.5 min reversed-phase liquid chromatography gradient was used in conjunction with 2.5 s CE separations for the detection of a tryptic digest of cytochrome c. ... 

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.

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 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.

Stoll, D.R., Cohen, J.D., Carr, P.W. (2006). Fast, comprehensive online two-dimensional high performance liquid chromatography through the use of high temperature ultrafast gradient elution reversed-phase liquid chromatography. J. Chromatogr. A 1122,... 

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. 

G. Vivo-Truyols, J.R. Torres-Lapasio and M.C. Garcia-Alvarez-Coque, Enhanced calculation of optimal gradient programs in reversed-phase liquid chromatography. J. Chromatogr.A 1018 (2003) 183-196. 

D. L. Warner and J. G. Dorsey, Reduction of Total Analysis Time in Gradient Elution, Reversed-Phase Liquid Chromatography, LCGC 1997,15, 254. 

Reverse phase liquid chromatography has typically been used for the separation of PFCs, employing either Cg or Cig columns [96], although the use of perfluoroalkyl columns has also been reported [115]. Mobile phases are typically mixtures of methanol-water or acetonitrile-water and are often modified with ammonium acetate to improve chromatographic separation and MS sensitivity. Both isocratic and gradient elution methodologies have been employed [96]. LC-MS/MS methods [116, 117] have also been developed for the separation of PFSA and PFCA isomers and generally employ linear perfluorooctyl stationary phases and acidified mobile phases. 

Shoenmakers, P.J., BiUiet, A.H., and De Galan L. Inflnence of organic modifiers on the retention behaviour in reversed-phase liquid chromatography and its consequences for gradient elution./. Chromatogr. 1979, 185, 179-195. 

Reverse phase liquid chromatography is very versatile, fast and highly reproducible. Aqueous solutions are normally used and the modifiers used are very cheap and highly pure. Separation is predictable based on the polarity, pH profile, solubility and other physicochemical characteristics of the solute molecules. Analysis time is rather short and reequilibration is generally fast. Multiple components with minor differences in polarity can be separated by appropriate choice of gradient profiles. 

Schoenmakers, P.J. Billiet, H.A.H. Tijssen, R. de Galan, L. Gradient selection in reversed-phase liquid chromatography. J. Chromatogr. 1978, 149, 519. 

Hutta, M. and Gora, R., Novel stepwise gradient reversed-phase liquid chromatography separation of humic substances, air particulate humic-hke substances and lignins, J. Chromatogr. A, 1012, 61-19,... 

Together with some other metals such as Cu, Hg, and Pb, bismuth may be identified chromatographically as the dithiocarbamate using reversed-phase liquid chromatography. The chelate is formed in a precolumn packed with Cig-bonded silica and loaded with a centrimide-dithiocarbamate ion pair. The metal complexes are preconcentrated on the precolumn and eluted with an aceto-nitrile/water gradient. The dithiocarba-mates are detected with a UV-Visible diode array detector with a detection limit in the subnanogram range (Iffland 1993). 

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