Reversed-phase chromatography cleaning columns

The three isomers of cresol are not as readily separated by HPLC, although recent techniques have been developed to accomplish this task. Reversed-phase chromatography columns have been used for the analysis of cresols with limited success. Recently, a new reversed-phase support has been developed that allows complete separation of the three cresol isomers (Bassler and Hartwick 1989). Inclusion complexes of the cresols with p-cyclodextrin cleanly separate the three isomers on commercially available columns (Yoshikawa et al. 1986). Detection limits down to 1 ppm can be obtained by this method. 

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. 

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.

Figure 13.9 Coupled-column RPLC-UV (215 nm) analysis of 100 p.1 of an extract of a spiked soil sample (fenpropimoiph, 0.052 mg Kg ). LC conditions C-1, 5 p.m Hypersil SAS (60 m X 4.6 mm i.d.) C-2, 5 p.m Hypersil ODS (150 m X 4.6 mm i.d.) M-1, acetonitrile-0.5 % ammonia in water (50 50, v/v) M-2, acetonitrile-0.5 % ammonia in water (90 10, v/v) flow-rate, 1 ml min clean-up volume, 5.9 ml transfer volume, 0.45 ml. The dashed line represents the cliromatogram obtained when using the two columns connected in series without column switcliing. Reprinted from Journal of Chromatography A, 703, E. A. Hogendoom and R van Zoonen, Coupled-column reversed-phase liquid cliromatography in envir onmental analysis , pp. 149-166, copyright 1995, with permission from Elsevier Science.

Figure 13,12 Illusti ation of the clean-up method, showing the analysis of an air sample (a) with and (b) without column switching. Details of the analytical conditions are given in the text. Reprinted from Journal of Chromatography, A 697, R R. Kootsti a and H. A. Herbold, Automated solid-phase exti action and coupled-column reversed-phase liquid cltromatogra-phy for the trace-level determination of low-molecular-mass carbonyl compounds in ak , pp. 203-211, copyright 1995, with permission from Elsevier Science.

Different resins, including acid alumina, silica gel, C-18 reverse phase, and florisil, as well as different solvents, were evaluated for their ability to clean-up the heptachlor-spiked samples for an immunoassay. The data obtained using acid alumina and C-18 reverse phase resin as the solid support and elution with 20 mL of the solvent indicated are siunmarized in Table n. It may be noticed that many of the resin/elution solvent combinations were able to give >9S% recoveries of the heptachlor (based on 1 C-heptachlor recovery), but when non-spiked beef fat samples were analyzed in the cELISA inhibition values of from 18 to 7S% were observed. The inhibition, observed in nonspiked samples represents unknown interfering compounds that coeluted with heptachlor. In the case of chromatography on an acid alumina column followed by elution with 20 mL of 75% acetonitrile in water, only 18% inhibition in nonspiked samples was observed. Material from identical experiments using nonspiked and spiked beef fat were collected as 3 mL fractions. Each fraction was then analyzed... 

A molecularly imprinted column for liquid chromatography can be used not only to separate analytes, but also to selectively extract analytes from complex samples. This technique is called on-line Molecularly Imprinted Solid Phase Extraction (on-line MISPE), and it combines the high extraction efficiency of reverse phase SPE for aqueous samples with the high selectivity of the molecular-imprinted polymers. Examples of successful selective extraction and clean-up are reported in Figs. 9 and 10. 

The first publication that reported the use of LC—MS for quantification of IsoPs in urine used reversed-phase LC coupled with ESI/MS. The method used only 1 mL urine and the clean-up procedure using solid phase extraction (SPE) columns gave quantitative recovery of the IsoP. The chromatographic runs and SPE purification methods are short, and this results in a very easy and user-friendly procedure (Li et al., 1999). A comprehensive review by Tsikas et al. describes sample preparation techniques and compares GC—MS methods with the most recent LC—MS/MS methods (Tsikas et al., 2003). The focus is primarily on 8-f5o-PGF2ci and highlights the difficulty to detect only one IsoP isomer without immunoaffinity chromatography preparation. The large concentration differences for the various IsoP classes in urine are also addressed. 

There are two different modes in which on-line extraction/clean-up by turbulent flow chromatography is performed (Zhou 2005). In the simpler mode, single column extraction, the analytical sample or primary extract is injected directly onto the extraction column and extracted directly onto the analytical column by the turbulent flow mobile phase that has high eluent strength (Section 4.4.2a), i.e., high organic content in the case of analytes to be analyzed by reverse phase HPLC. This... 

High-performance affinity chromatography has recently been reported with trypsin-modified avidin supported on 5 pm silica. While the separations were successful and a wide range of foods were studied, elution times were 80 minutes and ADAM post-column reactions were still required (Hayakawa et al. 2009). However, such affinity columns within a solid-phase extraction (SPE) platform make realistic choices for sample preparation, whereby the biotin can be purified and concentrated prior to reversed-phase HPLC. R-Biopharm has recently developed a commercially available antibody-based immunoaffinity column to bind biotin from aqueous extracts, providing an excellent technique to clean up complex samples. 

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