Column-switching —

Column switching in HPLC offers an alternative for the processing of liquid samples such as biological fluids or water. Samples are injected directly onto one HPLC column and the analytes retained. After a set time a second solvent elutes the analytes onto a second analytical column for separation and detection. There are several alternatives in column switching such as the back-flushing of retained components. It is also possible to use more than two columns. Columns can be of the same type or contain different stationary phases. The valves involved in column switching are automated so the technique offers the possibility of complete automation. 

Column switching provides an alternative to gradients elution, the various parts of the chromatogram being separated over two or more columns. The less significant parts of the chromatogram are often discarded the/rowf, heart or end cuts are obtained and separated further. 

Columns and valves can be arranged in whatever manner the analyst cares to devise, provided that the main objective of maximum selectivity is kept in mind. Some possible column combinations are as follows  

The last alternative, which generally involves more than one mobile phase, is known as multidimensional chromatography and appears to be the most promising. All arrangements should attempt to provide a peak compression effect so that unavoidable extra-column volumes become insignificant and the highest possible separation performance is achieved. 

Equipment for column switching is available commercially but equally good results can be obtained from a do it yourself system. Six- or ten-port valves can be used, one ten-port valve being capable of replacing two six-port valves. 

There is a certain risk of system peaks (Section 19.9) with column-switching methods. System peaks can be invisible but they alter the shape of co-eluted peaks. 

Column switching enables analyses to be carried out which are either impossible to achieve on a single column system or do not yield the required information using conventional methods. Switching of the carrier gas is achieved using multiport gas switching valves. These are used to switch the 

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An ion chromatographic system that included column switching and gradient analysis was used for the deterrnination of cations such as Na", Ca ", Mg ", K", and NH" 4 and anions such as Cf, NO, NO , and in fog water samples (72). Ion-exchange chromatography compares very well with... 

C. Wang, E. Haitmut, G. Wang, L. Zhou, E. Bayer and P. Lu, Determination of amino acid enantiomers by two-column gas cliromatography with valveless column switching , J. Chromatogr. 262 352-359 (1983). 

A practical method for enhancing the peak capacity, and thus the resolution of analytes in multicomponent complex mixtures, can be achieved by changing the mode of the separation during the chromatographic analysis, employing a column switching system in order to optimize a separation. 

Figure 5.2 Schematic representation of the final column-switching system (a) foi ward-flush position (b) back-flush position (further details are given in the text). Reprinted from Journal of Chromatography, A 828, A. K. Sakhi et al. Quantitative determination of endogenous retinoids in mouse embiyos by high-performance liquid cliromatography with on-line solid-phase exti action, column switcliing and electi ochemical detection , pp. 451 -460, copyright 1998, with permission from Elsevier Science.

Figure 5.3 Analysis of 100 ml of (a) surface water and (b) drinking water sample spiked with 0.1 pig/ml of microcystins, using column-switching HPLC 1, microcystin-RR 2, microcystin-YR 3, microcystin-LR. Reprinted from Journal of Chromatography A, 848, H. S. Lee et al, On-line trace enrichment for the simultaneous determination of microcystins in aqueous samples using high performance liquid chromatography with diode-array detection , pp 179-184, copyright 1999, with permission from Elsevier Science.

Figure 5.4 Schematic diagrams of a heait-cut valve configuration system. Reprinted from Journal of Chromatography, 602, S. R. Villasenor, Matrix elimination in ion cliromatography by heart-cut column switching techniques , pp. 155-161, copyright 1992, with permission from Elsevier Science.

M. A. J. Bayliss, P. R. Baker and D. Wilkinson, Determination of the two major- human metabolites of tipredane in human urine by liigh performance liquid cliromatogr-aphy with column switching , J. Chromatogr. 694 199-209 (1997). 

Z. Yu and D. Westerlund, Char-acterization of the precolumn biortap 500 C g for direct injection of plasma samples in a column-switching system , Chromatographia, 47 299-304(1998). 

M. Cavalleri, W. Pollini and L. Colombo, Determination of ramoplanin in human urine by high performance liquid cliromatography with automated column switching ,... 

M. Tanaka and H. Yamazaki, Dkect detemination of pantoprazole enantiomers in human serum by reversed-phase liigh peifomance liquid chi omatography using a cellulose-based cliiral stationaiy phase and column-switching system as a sample cleanup procedure , Aim/. Chem. 68 1513-1516(1996). 

L. Liu, H. Cheng, J. J. Zhao and J. D. Rogers, Determination of montelukast (MK-0476) and its 5-enantiomer in human plasma by stereoselective high performance liquid cliromatography with column-switching , ]. Pharm. Biomed. Anal. 15 631-638 (1997). 

K. MacNamara and A. Hoffmann, Simultaneous nitr Ogen, sulphur and mass spectr omet-rtc analysis after multi-column switching of complex whiskey flavour extracts , in... 

R. M. Mader, B. Rizovski, G. G. Steger, H. Rainer, R. Proprentner and R. Kotz, Determination of methoti exate in human urine at nanomolar levels by high-performance liquid cliromatography with column switching , 7. Chromatogr. 613 311-316 (1993). 

K. Yamashita, M. Motohashi and T. Yashiki, Column-switching techniques for high-performance liquid cliromatography of ibuprofen and mefenamic acid in human serum with shoit-wavelength ultraviolet detection , J. Chromatogr. 570 329-338 (1991). 

K. Yamashita, M. Motohaslii and T. Yashiki, High-performance liquid cliromatograpliic determination of phenylpropanolamine in human plasma and urine, using column switching combined with ion-pair chromatography , J. Chromatogr. 527 103-114 (1990). 

T. Miyabayashi, K. Yamashita, E Aoki, M. Motohashi, T. Yashiki and K. Yatani, Determination of manidipine and pyridine metabolite in human serum by liigh-perfor -mance liquid cliromatography with ultraviolet detection and column switching , J. Chromatogr. 494 209 - 217 (1989). 

Y. Oda, N. Asakawa, T. Kajima, Y. Yoshida and T. Sato, On-line determination and resolution of verapamil enantiomers by high-performance liquid chromatography with column-switching , Pharm. Res. 8 997-1001 (1991). 

T. Seki, K. Yamaji, Y. Orita, S. Moriguchi and A. Sliinoda, Simultaneous determination of uric acid and creatinine in biological fluids by column-switching liquid cliromatogra-phy with ulti aviolet detection , 7. Chromatogr. A 730 139-145 (1996). 

J. R K. Huber and G. Lamprecht, Assay of neopterin in serum by means of two-dimensional high-performance liquid chromatography with automated column switching using tliree retention mechanism , 7. Chromatogr. B 666 223-232(1995). 

T. Okuda, Y. Nakagawa and M. Motohashi, Complete two-dimensional separation for analysis of acidic compounds in plasma using column-switching reversed-phase liquid clrromatography , 7. Chromatogr. B126 225-236 (1999). 

G. Ziircher and M. Da Prada, Simple automated high-performance liquid cliromato-graplric column-switching tecnique for the measurement of dopa and 3-O-methyldopa in plasma , 7. Chromatogr. 530 253-262 (1990). 

R. A. Coe, E. S. DeCesare and J. W. Eee, Quantitation of efletirizine in human plasma and urine using automated solid-phase exti action and column-switching high-performance liquid clrromatography , 7. Chromatogr. B 730 239-247 (1999). 

Multi-column switching can be an effective approach for the determination of high and low concentrations of sample components in complex mixtures. This is a very powerful technique for the analytical and preparative separation of components... 

This method can quantify levels of 0.1 p.g 1 in real samples and reproducibility values are good the total analysis time was 8 min. Eigure 13.8 compares the chromatogram obtained by using this method with one obtained without column switching. 

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.

The experimental conditions are shown in Table 13.1, while Figure 13.9 shows the chromatogram of a soil sample extract spiked with fenpropimorph obtained by this method, plus the chromatogram from the two columns connected in series without column switching. 

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.

E. A. Hogendoorn, P. van Zoonen and U. A. Th Brinkman, Column-switching RPEC for the trace-level determination of ethylenethiourea in aqueous samples , Chromatographia 31 285-292 (1991). 

E. A. Hogendoorn, C. E. Goewie and P. van Zoonen, Application of HPEC column switching in pesticide residue analysis, Eresenius , ]. Anal. Chem. 339 348-356 (1991). 

E. R. Brouwer and U. A. Th Brinkman, Determination of phenolic compounds in surface water using on-line liquid chr omatographic precolumn-based column-switching tecniques , J. Chromatogr. 678 223-231 (1994). 

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