Sampling precolumn

Bartok, T., Borsok, G., and Sagi, F., RP-HPLC separation of polyamines after automatic FMOC-C1 derivatization and precolumn sample clean-up using column switching, /. Liq. Chromatogr., 15, 777, 1992. 

A variety of pre- and postcolumn sample derivatization schemes has been developed in CE, mainly for the attachment of fluorescent labels for detection purposes. Precolumn sample modification is usually less demanding than postcolumn reactions for reasons discussed in Sect. 3.2.4. 

It is too often assumed that because precolumn sample preparation devices, such as solid-phase extraction cartridges, are simple tools, they require relatively little skill or attention to detail for successful use. Cartridges do, however, require attention to detail for successful operation in sample enrichment procedures. Two of the most important parameters to control and understand are flow-rate effects and recovery (or loadability) effects. 

Accurately assay the precolumn sample and pooled eluted samples for GOT activity using the following suggested dilutions in 0.1 M potassium phosphate buffer (pH 7.5) ... 

Trippel-Schulte, P., Zeiske, J., and Kettrup, A., Trace analysis of selected benzine and diamino diphenyl methane derivatives in urine by means of liquid chromatography using precolumn sample preconcentration, UV and electrochemical detection, Chromatographia, 22, 138-146, 1986. 

It is felt that the precolumn sampling techniques deserve much attention in future studies, as they can serve a double function in biochemical investigations (a) removal of solvents or derivatization agents and (b) protection of the analytical column from non-volatile impurities. Chemical nature of the precolumn packing can also be varied to suit a particular sample type. Further investigations aiming at the optimization and automation of the precolumn sampling techniques appear desirable. 

The sample introduction system must be capable of introducing a known and variable volume of sample solution reproducibly into the pressurized mobile phase as a sharp plug without adversely affecting the efficiency of the column. The superiority of valve injection has been adequately demonstrated for this purpose and is now universally used in virtually all modern instruments for both manual and automated sample introduction systems [1,2,7,31,32]. Earlier approaches using septum-equipped injectors have passed into disuse for a several reasons, such as limited pressure capability, poor resealability, contamination of the mobile phase, disruption of the column packing, etc., but mainly because they were awkward and inconvenient to use compared with valves. For dilute sample solutions volume overload restricts the maximum sample volume that can be introduced onto the column without a dramatic loss of performance. On-column or precolumn sample focusing mechanisms can be exploited as a trace enrichment technique to enhance sample detectability. Solid-phase extraction and in-column solid-phase microextraction provide a convenient mechanism for isolation, concentration and matrix simplification that are easily interfaced to a liquid chromatograph for fully or semi-automated analysis of complex samples (section 5.3.2). 

Rice JR, Kissinger PT. 1982. Liquid chromatography with precolumn sample preconcentration and electrochemical detection Determination of aromatic amines in environmental samples. Environ Sci Technol 1695) 263-268. 

A similar method to measure amino acid transmitter levels in microdialysates, using microbore HPLC, is presented in detail in Chapter 15. Please note that the Smolders et al method makes use of different precolumn sample derivatization procedures than used here and, therefore, step 6 of Section 3.3. will need to be altered accordingly should the Smolders et al. method be chosen. 

Tobramycin was derivatized wifli o-phthalaldehyde and analyzed on a C]g column (A == 254 nm or 340 nm, ex 450 nm, em) using a 52/48 acetonitrile/water (20 mM phosphate buffer at pH 6.5) mobile phase [1382]. A 25pL injection of each of a 12.8 pM series of standards was made. The fluorescence-based detection method was more than 40 times more sensitive than the UV method. Precolumn sample derivatization increased the recovery of tobramycin by a factor of 3. 

Precolumn Sampling (OTQ. Synonymous to selective sampling with open tubular columns. 

An on-line concentration, isolation, and Hquid chromatographic separation method for the analysis of trace organics in natural waters has been described (63). Concentration and isolation are accompHshed with two precolumns connected in series the first acts as a filter for removal of interferences the second actually concentrates target solutes. The technique is appHcable even if no selective sorbent is available for the specific analyte of interest. Detection limits of less than 0.1 ppb were achieved for polar herbicides (qv) in the chlorotriazine and phenylurea classes. A novel method for deterrnination of tetracyclines in animal tissues and fluids was developed with sample extraction and cleanup based on tendency of tetracyclines to chelate with divalent metal ions (64). The metal chelate affinity precolumn was connected on-line to reversed-phase hplc column, and detection limits for several different tetracyclines in a variety of matrices were in the 10—50 ppb range. 

A range of preparative and semipreparative soft gel systems with an improved mechanical stability and thus the chance to run them with increased flow rates were tested for their potential on the separation of starch glucans. For each of these systems a Sephacryl S-200 precolumn proved to be a perfect shock absorber for sample application, improved reproducibility of separations, and increased lifetime of soft gel systems. 

Another TSK combination (precolumn -I- PWM -I 6000 -I 5000 -I- 4000 -I-3000) was tested on differences in separation performance between individual narrow distributed samples and mixtures of several narrow distributed samples. The result is summarized in Eig. 16.31 within experimental error the summed chromatograms (theory) of four narrow distributed glucans (dextran) match perfectly with the experimentally determined chromatogram of the mixture. The (theory/experimental) ratio, plotted for quantification of the match, in-... 

In Fig. 16.32, application of a TSK PW SEC system consisting of a combination of precolumn + PWM + 6000 + 5000 + 4000 + 3000 demonstrates a possibility for analytical purposes to change from DMSO-dissolved glucans to an aqueous solution. An initially DMSO-dissolved potato starch sample was applied to the TSK PW system and because separated with an aqueous... 

In this way, the liquid can be transferred at a speed corresponding to the evaporation speed. The fraction to be analysed is contained in a loop (see Eigure 2.5), connected to a switching valve. By opening the valve, the sample in the loop is driven by the carrier gas into the GC unit (8), instead of the LC pump. An early vapour exit is usually placed after a few metres of the deactivated precolumn (9) and a short piece (3-4 m) of the main column (retaining precolumn). This valve is opened during solvent evaporation in order to reduce the amount of solvent that would reach the detector, and at the same time, to increase the solvent evaporation rate (6). 

Figure 2.6 Gas cluotnatograni of a 10 ml test sample containing C I4 C26 alkanes in -hexane (about 1 ppb each) the earner gas (H2) inlet pressure was 2.5 bar for a 22 m X 0.32 mm id separation column coupled with a 2 m X 0.32 mm id uncoated precolumn (no vapour exit). Reprinted from Journal of High Resolution Chromatography, 9, K. Grob et al., Concunent solvent evaporation for on-line coupled HPLC-HRGC , pp. 95-101, 1986, with peimission from Wiley-VCH.

Figure 2.12 Schematic representation of an on-line SPE-GC system consisting of three switching valves (VI-V3), two pumps (a solvent-delivery unit (SDU) pump and a syringe pump) and a GC system equipped with a solvent-vapour exit (SVE), an MS instrument detector, a retention gap, a retaining precolumn and an analytical column. Reprinted from Journal of Chromatography, AIIS, A. J. H. Eouter et al, Analysis of microcontaminants in aqueous samples hy fully automated on-line solid-phase extraction-gas chromatography-mass selective detection , pp. 67-83, copyright 1996, with permission from Elsevier Science.

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). 

A. Faijam, A. E. Brugman, A. Soldaat, P. Timmerman, H. Fingerman, G. J. de Jong, R. W. Frei and U. A. Th Brinkman, Immunoaffinity precolumn foi selective sample preti eatment in column liquid cliromatography immunoselective desoiption , Chromatographia 31 469-477 (1991). 

A. Farjam, R. de Vries, H. Lingeman and U. A. Th Brinkman, Immuno precolumns for selective on-line sample pretr eatment of aflatoxins in milk prior to column liquid cliro-matography , Int. J. Environ. Anal. Chem. 44 175-184 (1991). 

Another example of multi-column analysis has been demonstrated for the determination of impurities in styrene. The marked compounds in the styrene sample (Figure 12.15(a)) were solvent flushed via a splitline, with the analysis being carried out with a cryotrapping separation (CTS) (see Figure 12.15(b)). The first column, was an Ultra-2 (25 m X 0.32 mm i.d., d( = 0.25 p.m) precolumn, while the main column was a DB-WAX (30 m X 0.32 mm, d = 0.25 p.m) with an FID being employed as the detection system. 

An application of an LC-SFC system has been demonstrated by the separation of non-ionic surfactants consisting of mono- and di-laurates of poly (ethyleneglycol) (23). Without fractionation in the precolumn by normal phase HPLC (Figure 12.18 (a)) and transfer of the whole sample into the SFC system, the different homologues coeluted with each other. (Figure 12.18(b)). In contrast with prior fractionation by HPLC into two fractions and consequent analysis by SFC, the homologues in the two fractions were well resolved (Figures 12.18(c) and 12.18(d)). 

When a first column of a very short length (and therefore a low selectivity) is used (this is especially suitable for multiresidue methods), we talk about an on-line precolumn (PC) switching technique coupled to LC (PC-LC or solid-phase extraction (SPE)-LC). This is particulary useful for the enrichment of analytes, and enables a higher sample volume to be injected into the analytical column and a higher sensitivity to be reached. The sample is passed through the precolumn and analytes are retained, while water is eliminated then, by switching the valve, the analytes retained in the precolumn are transferred to the analytical column by the mobile phase, and with not just a fraction, as in the previous cases. 

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.

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