Microcolumn applications

Higher column efficiencies are required in order to investigate the complex mixtures encountered in petrochemical, biochemical and clinical studies, 

Operating conditions A Injection volume Mobile phase Flow rate Detector 

For the practising chromatographer, often a search of the relevant literature will identify a similar application which with some minor modification will prove suitable for a particular need. The applications literature available is formidable a number of technical abstracts are published [133], technical information published by instrument manufacturers and chromatography 

The chromatographic methods most suitable for spectroscopic identification techniques are GC and HPLC although TLC and recently CZE and SFC methods have received attention. GC was the first technique to be interfaced to a mass spectrometer since it proved relatively easy to transfer the gaseous effluent and GC-MS instruments were capable of sufficiently rapid scan times even in the 1970s to acquire undiscriminated spectral data [1]. Interfacing HPLC systems proved more difficult due to 

Such large amounts of data can only be sensibly and rapidly analysed and compared with reference spectra using microprocessors such as the fast 32 bit processors in PCs. The main systems in use today are discussed below, and in addition to the above mentioned techniques the microwave induced plasma (MIP) detector, a helium microwave plasma emission source coupled to a GC and an optical emission spectrometer are reviewed. 

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Pentoney, S. L., Huang, X. H., Burgi, D. S., and Zare, R. N., Online connector for microcolumns— Application to the on-column ortho-phthaldialdehyde derivatization of amino-acids separated by capillary zone electrophoresis. Anal. Chem., 60, 2625, 1988. 

In some applications, additional components acting as reactors for specific chemical pretreatment are incorporated within the flow manifold. Typical examples are ion-exchange microcolumns for preconcentration of the analyte or removal of interferences and redox reactors, which are used either to convert the analyte into a more suitable oxidation state or to produce online an unstable reagent. Typical examples of online pretreatment are given in Table 2. Apart from these sophisticated reactors, a simple and frequently used reactor is a delay coil (see also Fig. 4), which may be formed by knitting a segment of the transfer line. This coil allows slow CL reactions to proceed extensively and enter into the flow cell at the time required for maximum radiation. The position of the reactors within the manifold is either before or after the injection port depending on the application. 

Edelmann FT (1996) Rare Earth Complexes with Heteroallylic Ligands. 179 113 -148 Edelmann FT (1996) Lanthanide Metallocenes in Homogeneous Catalysis. 179 247-276 Effenhauser CS (1998) Integrated Chip-Based Microcolumn Separation Systems. 194 51 - 82 Ehrfeld W, Hessel V, Lehr H (1998) Microreactors for Chemical Synthesis and Biotechnology -Current Developments and Future Applications. 194 233 - 252 Ekhart CW, see de Raadt A (1997) 187 157-186... 

E.W. Hooijschuur, C.E. Kientz and U.A. Brink-man, Application of microcolumn liquid chromatography and capillary electrophoresis with flame photometric detection for the screening of degradation products of chemical warfare agents in water and soil, J. Chromatogr. A, 928, 187-199 (2001). 

Epitope excision was performed by application of 2-5 xg antigen to the antibody microcolumn (Tian et al. 2005). Binding was performed for 60 min at 20°C. After washing with binding buffer, proteolytic digestion was carried out on the column with 0.2 xg protease in 200 xl PBS for 2 h at 37°C. Supernatant unbound peptides were removed using washing buffer and the epitope dissociated by addition of 500 pi 0.1 % trifluoroacetic acid (TFA). After incubation for 15 min at 20 C, the epitope eluate was lyophilised and reconstituted in 10 pi NaAc buffer. 

From Table 1, it is very clear that, as the absolute volume of a microcolumn is relatively small, the extracolumn volume will contribute significantly to disturb the separation performance of the chromatography system. Because the small extra-column volume is still a large portion of the total system volume which, in turn, is much smaller than the conventional column system, and it is hard to eliminate such small extra-column volume, even if attempts to reduce are applied, a serious problem would be produced. Most typical discussions on the applicability... 

CL Flurer, M Novotny. Dual microcolumn immunoaffinity liquid chromatography an analytical application to human plasma proteins. Anal Chem 65 817-821, 1993. 

Figure 12.3. Leaching profiles of metals in SRM 2710 soil obtained by the application of extractants according to the original SM T three-step sequential extraction protocol using (a) a flow-through microcolumn-based system (column inner volume, 0.36 mL sample, 25 mg extraction flow rate, 3 mL min subfraction volume, 5 mL), and (b) a flow-through rotating coiled column-based system (column inner volume, 20 mL sample, 500 mg extraction flow rate, 1 mL min subfraction volume, 10 mL). [(a) From Chomchoei et al., 2005b) (h) from Fedotov et al., 2005b, by permission of the Royal Society of Chemistry.)...

Lloyd JBF. 1975. Nitrogen heterocycle and polynuclear hydrocarbon fluorescence and adsorption effects in the presence of silica gel Applications in high-pressure liquid and microcolumn chromatography. Analyst 100 529-539. 

Some of the interesting applications of LOV inclnde the manipnlation of beads and microcolnmns for sample clean-up or separation. In particnlar, one version of the system has received considerable attention and is called microseqnential injection-lab-on-valve (pSl-LOV). The J,SI-LOV device can be used for sample pretreatment alone or in seqnence with analysis and detection (usually absorbance or flnorescence). It has also been employed as the front end of a miniaturised CE system. An interesting nse of J,SI-LOV has been in conjunction with suspensions of specially coated beads. These beads can be manipulated by the multiport valve to flow into the holding coil and from there into the detector flow cell to form a temporary microcolnmn. The sample is then mixed with the bead column and any reactions are detected, e.g. adsorption over time. After measurements have been made, the beads are sent to waste. The formation and removal of these temporary microcolumns has advantages over the nse of fixed colnmns snch as ... 

In one case such compositions were encapsulated and machine coated on a flexible substrate. In the second set of tests, the identical formulations were applied to microcolumn type membranes (manufactured by Nuclepore). Transparent polyester plastic sheets 25 ium thick were used to cover both sides of these samples after the application step. Laboratory steel pressure rollers that applied... 

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