Liquid RPLC

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.

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 13.13 On-line trace eniicliment-RPLC-diode-aiTay detection (DAD) cliromatogram (at 230 nm) obtained from 200 ml of tap water spiked with various pesticides at levels of 1 p.g L. Reprinted from Chromatographia, 43, C. Aguilar et al., Deteimination of pesticides by on-line ti ace emicliment-reversed-phase liquid clrromatography-diode-aiTay detection and confirmation by paiticle-beam mass specti ometi y , pp. 592-598, 1996, with permission from Vieweg Publisliing.

The mechanism of reversed phase chromatography can be understood by contrast with normal phase chromatography. Normal phase liquid chromatography (NPLC) is usually performed on a polar silica stationary phase with a nonpolar mobile phase, while reversed phase chromatography is performed on a nonpolar stationary phase with a polar mobile phase. In RPLC, solute retention is mainly due to hydrophobic interactions between the solutes and the nonpolar hydrocarbon stationary surface. The nonpolar... 

Miller and Hawthorne [416] have developed a chromatographic method that allows subcritical (hot/liquid) water to be used as a mobile phase for packed-column RPLC with solute detection by FID, UV or F also PHWE-LC-GC-FTD couplings are used. Before LC elution the extract is dried in a solid-phase trap to remove the water. In analogy to SFE-SFC, on-line coupled superheated water extraction-superheated water chromatography (SWE-SWC) has been proposed [417]. On-line sample extraction, clean-up and fractionation increases sensitivity, avoids contamination and minimises sources of error. 

Normal-phase (NP) and reversed-phase (RP) liquid chromatography are simple divisions of the LC techniques based on the relative polarities of the mobile and stationary phases (Figure 4.10). Both NPLC and RPLC analysis make use of either the isocratic or gradient elution modes of separation (i.e. constant or variable composition of the mobile phase, respectively). Selection from these four available separation techniques depends on many variables but basically on the number and chemical structure of the compounds to be separated and on the scope of the analysis. 

Also, subcritical (hot/liquid) water can be used as a mobile phase for packed-column RPLC with solute detection by means of FID [942]. In the multidimensional on-line PHWE-LC-GC-FTD/MS scheme, the solid sample is extracted with hot pressurised water (without the need for sample pretreatment), and the analytes are trapped in a solid-phase trap [943]. The trap is eluted with a nitrogen flow, and the analytes are carried on to a LC column for cleanup, and separated on a GC column using the on-column interface. The closed PHWE-LC-GC system is suitable for many kinds of sample matrices and analytes. The main benefit of the system is that the concentration step is highly efficient, so that the sensitivity is about 800 times better than that obtained with traditional methods [944]. Because small sample amounts are required (10 mg), special attention has to be paid to the homogeneity of the sample. The system is... 

SEC in combination with multidimensional liquid chromatography (LC-LC) may be used to carry out polymer/additive analysis. In this approach, the sample is dissolved before injection into the SEC system for prefractionation of the polymer fractions. High-MW components are separated from the additives. The additive fraction is collected, concentrated by evaporation, and injected to a multidimensional RPLC system consisting of two columns of different selectivity. The first column is used for sample prefractionation and cleanup, after which the additive fraction is transferred to the analytical column for the final separation. The total method (SEC, LC-LC) has been used for the analysis of the main phenolic compounds in complex pyrolysis oils with minimal sample preparation [974]. The identification is reliable because three analytical steps (SEC, RPLC and RPLC) with different selectivities are employed. The complexity of pyrolysis oils makes their analysis a demanding task, and careful sample preparation is typically required. 

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. 

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