RPLC

Figure 9.9 Schematic illustration of the instmmental setup used for three-dimensional SEC-RPLC-CZE.

A. W. Moore-Jr and J. W. Jorgenson, Rapid comprehensive two-dimensional separations of peptides via RPLC-optically gated capillary zone electiophoresis . Anal. Chem. 67 3448-3455 (1995). 

Other bioanalytical applications of systems in which the eluate of a first LC column is sampled in continuous and repetitive intervals and subjected to a second LC dimension are, for example, described by Wheatly et a/. (11) and Matsuoka et al. (12). Wheatly coupled gradient affinity LC with RPLC for the determination of the isoenzymatic- and subunit composition of glutathione 5-transferses in cytosol... 

In LC-LC and SPE-LC, the presenee of water is eommonly no problem at all. Aetually, the reverse is true beeause eluents in RPLC are typieally water-methanol or water-aeetonitrile mixtures, and a high water eontent is mandatory during traee enriehment in order to ensure strong retention. However, when sueh an SPE pre-eolumn or analytieal eolumn is eoupled to a GC system, the introduetion of water should be avoided eompletely or, at best, be permitted under strietly eontrolled eondi-tions (see above). It will be elear that on-line traee enriehment (and elean-up) by SPE... 

Figure 12.11 Coupled SEC-RPLC separation of compound Chemigum mbber stock (a) SEC ti ace (b) RPLC trace of fraction 1, dibutylphthalate (c) RPLC trace of fraction 2, elemental sulfur. Coupled SEC conditions MicroPak TSK 3000H (50 cm) X 2000H (50 cm) X 1000 H (80 cm) columns (8 mm i.d.) eluent, THE at a flow rate of 1 mL/min UV detection at 215 nm (1.0 a.u.f.s.) injection volume, 200 p-L. RPLC conditions MicroPak MCH (25 cm X 2.2 mm i.d.) column flow rate, 0.5 mL/min injection volume, lOpL gradient, acetonitrile-water (20 80 v/v) to 100% acetonitrile at 3% acetonitrile/min UV detection at 254 nm (0.05 a.u.f.s.). Reprinted from Journal of Chromatography, 149, E. L. Jolmson et al., Coupled column cliromatography employing exclusion and a reversed phase. A potential general approach to sequential analysis , pp. 571-585, copyright 1978, with permission from Elsevier Science.

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 13.6 Direct RPLC analysis of a blank ground water sample spiked with 4.5 (p-g 1 ETU, (a) with and (b) without column-switching. A 60 X 4.6 mm i.d. column and a 150 X 4.6 mm i.d. column were used for C-1 and C-2, respectively, with pure water as M-1 and methanol-0.025 M ammonium acetate (pH, 7.5) (5 95, v/v) as M-2 S-1 and S-2 aie the interfering peaks. Reprinted from Chromatographia, 31, E. A. Hogendoom et at., Columnswitching RPLC for the trace-level determination of ethylenetlaiourea in aqueous samples , pp. 285-292, 1991, with permission from Vieweg Publishing.

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.

In multiresidue analysis, where more analytes with a wide polarity range need to be determined, large transfer volumes are required, and consequently, the selectivity is lower. However, since the major interferences in water analysis are the polar humic and fulvic acids, removing this early eluting interference in coupled-column RPLC will also be feasible in multiresidue methodology. 

Figure 13.11 Column-switcliing RPLC trace of a surface water sample spiked with eight chlorophenoxyacid herbicides at the 0.5 p-g 1 level 1, 2,4-dichlorophenoxyacetic acid 2, 4-chloro-2-methylphenoxyacetic acid 3, 2-(2,4-diclilorophenoxy) propanoic acid 4, 2-(4-cliloro-2-methylphenoxy) propanoic acid 5, 2,4,5-trichlorophenoxyacetic acid 6, 4-(2,4-dichlorophenoxy) butanoic acid 7, 4-(4-chloro-2-methylphenoxy) butanoic acid 8, 2-(2,4,5-tiichlorophenoxy) propionic acid. Reprinted from Analytica Chimica Acta, 283, J. V. Sancho-Llopis et al., Rapid method for the determination of eight chlorophenoxy acid residues in environmental water samples using off-line solid-phase extraction and on-line selective precolumn switcliing , pp. 287-296, 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.

An alternative way of eliminating water in the RPLC eluent is to introduce an SPE trapping column after the LC column (88, 99). After a post-column addition of water (to prevent breakthrough of the less retained compounds), the fraction that elutes from the RPLC column is trapped on to a short-column which is usually packed with polymeric sorbent. This system can use mobile phases containing salts, buffers or ion-pair reagents which can not be introduced directly into the GC unit. This system has been successfully applied, for example, to the analysis of polycyclic aromatic hydrocarbons (PAHs) in water samples (99). 

Another interface for RPLC-GC is the programmed-temperature-vaporization (PTV) system, an interesting application of which is the determination of phthalates... 

The experimental results obtained in the laboratory by the researchers can be monitored using computer programs with help of empirical equations or models. Most of the computer-assisted procedures have been developed for HPLC separations and mainly for RPLC, and some of them are commercially available. 

Relationships between lipophilicity and retention parameters obtained by RPLC methods using isocratic or gradient condition are reviewed. Advantages and limitations of the two approaches are also pointed out, and general guidelines to determine partition coefficients in 1-octanol-water are proposed. Finally, more recent literature data on Hpophilicity determination by capillary electrophoresis of neutral compounds and neutral forms of ionizable compounds are compiled. Quotation is restricted to key references for every method presented - an exhaustive listing is only given for the last few years. 

RPLC methods were largely used for the determination of log as illustrated by... 

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