Chromatography sample volume injected

As the analytes to be assayed are nearly always present in minute amounts, preconcentration steps are almost always necessary. The techniques used for this purpose have been mostly adopted from analytical procedures using gas or liquid chromatography as the separation step [5]. This fact is emphasized here because the appropriate adjustment of the sample preparation is necessary because the original procedures do not respect the fact that the sample volume injected into a CE system represents a few nanoliters only and requires a relatively high concentration of analytes assayed. However, in selected types of analysis, direct sample application is also possible (for a review, see Ref. 3). 

Fig. 3. High performance liquid chromatographic (HPLC) separation of components in active fraction from flash chromatography of estrous urine extract. Biologically active fraction was eluted between 65-86% of reagent B. 4-methylphenol was eluted at <0.5% reagent B. Total sample volume injected was 1 mL.

The amounts of sample analysed by gas chromatography are small. For packed columns where the sample is vaporised in the injection port or injected directly onto the column, the sample volume injected is of the order of 1 microlitre. For a 1% solution of the sample in a suitable solvent this corresponds to a mass of about 10 microgram sample. The normal means of sample introduction is through a septum using a... 

The main aims in environmental analysis are sensitivity (due to the low concentration of microcontaminants to be determined), selectivity (due to the complexity of the sample) and automation of analysis (to increase the throughput in control analysis). These three aims are achieved by multidimensional chromatography sensitivity is enhanced by large-volume injection techniques combined with peak compression, selectivity is obviously enhanced if one uses two separations with different selectivi-ties instead of one, while on-line techniques reduce the number of manual operations in the analytical procedure. 

Figure 13.10 LC-LC chromatogram of a surface water sample spiked at 2 p.g 1 with ati azine, and its metabolites (registered at 220 nm). Conditions volume of sample injected, 2 ml clean-up time, 2.60 min ti ansfer time, 4.2 min The blank was subtracted. Peak identification is as follows 1, DIA 2, HA 3, DEA 4, atrazine. Reprinted from Journal of Chromatography, A 778, F. Hernandez et al, New method for the rapid detemiination of triazine herbicides and some of thek main metabolites in water by using coupled-column liquid cliromatography and large volume injection , pp. 171-181, copyright 1997, with permission from Elsevier Science.

In addition to the analytical columns (columns used mainly for analytical work), so-called preparative columns may also be encountered. Preparative columns are used when the purpose of the experiment is to prepare a pure sample of a particular substance (from a mixture containing the substance) by GC for use in other laboratory work. The procedure for this involves the individual condensation of the mixture components of interest in a cold trap as they pass from the detector and as their peak is being traced on the recorder. While analytical columns can be suitable for this, the amount of pure substance generated is typically very small, since what is being collected is only a fraction of the extremely small volume injected. Thus, columns with very large diameters (on the order of inches) and capable of very large injection volumes (on the order of milliliters) are manufactured for the preparative work. Also, the detector used must not destroy the sample, like the flame ionization detector (Section 12.6) does, for example. Thus, the thermal conductivity detector (Section 12.6) is used most often with preparative gas chromatography. 

The peak focusing allows injections of the extremely large sample volumes, which may easily reach 10% of the total volume of the LC LC columns. This is an important advantage considering the two-dimensional liquid chromatography. For example, the LC LC column effluent can be directly forwarded into an online SEC column for further separa-tion/characterization. The LC LC principle can be applied not only for polymer separations but also for reconcentration of polymer solutions, for example, of (diluted) effluents leaving other columns applied in polymer HPLC. This may be utilized in the multidimensional polymer HPLC. 

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