Reversed-phase liquid chromatography column

Kele, M. and Guiochon, G., Repeatability and Reproducibility of Retention Data and Band Profiles on Reversed-phase Liquid Chromatography Columns. 1. Experimental Protocol./. Chromatogr. A, 830 41-54, 1999. 

Le Mapihan, K., Vial, J., and Jardy, A. (2004). Reversed-phase liquid chromatography column testing robustness study of the test.. Chromatogr. A 1061, 149—158. 

Hultman et al. [130] developed a LC/MS/MS method for the quantitative determination of esomeprazole and its two main metabolites 5-hydro-xyesomeprazole and omeprazole sulfone in 25 /il human, rat, or dog plasma. The analytes and their internal standards were extracted from plasma into methyl ferf-butyl ether-dichloromethane (3 2). After evaporation and reconstitution of the organic extract, the analytes were separated on a reversed-phase liquid chromatography column and measured by atmospheric-pressure positive ionization mass spectrometry. 

An aqueous solution of NaCl, NaN03, and Na2S04 was passed through a Cig-silica reversed-phase liquid chromatography column eluted with water. None of the cations or anions is retained by the Cjg stationary phase, so all three salts were eluted in a single, sharp band with a retention time of 0.9 min. Then the column was equilibrated with aqueous 10 mM pentylammonium formate, whose hydrophobic tail is soluble in the C18 stationary phase. 

SKELLY N.E. 1982, Separation of inorganic and organic anions on reversed-phase liquid chromatography columns. Analytical Chemistry,... 

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). 

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

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.

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. 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. 

Reversed-phase Cig chromatography column. Keystone Scientific Betasil, 100 x 2.0-mm i.d., 5-pm particle size, 100 A, Part No. 105-701-2-CPF TSQ 7000 LC/MS/MS system with electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) interface and gradient high-performance liquid chromatography (HPLC) unit, or equivalent Vacuum manifold for use with SPE cartridges (Varian Vac Elut 10 or equivalent)... 

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.

Warren, Jr., F. V. and Bidlingmeyer, B. A., Influence of temperature on column efficiency in reversed phase liquid chromatography, Anal. Chem., 60, 2821, 1988. 

Crego, A. L., Diez-Masa, J. C., and Dabrio, M. V., Preparation of open tubular columns for reversed-phase liquid chromatography, Anal. Chem., 65, 1615, 1993. 

Irth, H., De Jong, G. J., Brinkman, U. A. Th., and Frei, R. W., Determination of disulfiram and two of its metabolites in urine by reversed-phase liquid chromatography and spectrophotometric detection after post-column com-plexation, /. Chromatogr., 424, 95, 1988. 

Yamaki, S., Isobe, T., Okuyama, T., Shinoda, T., Reversed-phase liquid chromatography on a microspherical carbon column at high temperature, /. Chromatogr. A, 728(1 2), 189, 1996. 

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). 

Although relatively unknown, the instrumentation for 2DLC was conceived and implemented by Emi and Frei (1978). They reported the valve configuration presently used in most comprehensive 2DLC systems. However, they automated neither the valve nor the data conversion process to obtain a contour map or 2D peak display. They used a gel permeation chromatography (GPC) column in the first dimension and a reversed-phase liquid chromatography (RPLC) column in the second dimension and studied complex plant extracts. 

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. 

Ikegami, T., Hara, T., Kimura, H., Kobayashi, H., Hosoya, K., Cabrera, K., Tanaka, N. (2005). Two dimensional reversed-phase liquid chromatography using two monolithic silica C18 columns and different mobile-phase modifiers in the two dimensions. J. Chromatogr. A, Forthcoming. 

Minakuchi, H., Nakanishi, K., Soga, N., Ishizuka, N., Tanaka, N. (1997). Effect of skeleton size on the performance of octadecylsilylated continuous porous silica columns in reversed-phase liquid chromatography. J. Chromatogr. A 762, 135-146. 

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