Chromatographic separation techniques liquid column chromatography

Liquid chromatography can also be used for the quantitative analysis of the separated compounds. In column chromatography, the detector response is normally related to the amount of sample in the effluent. Thus, the area under a chromatographic peak is useful for quantitative analysis in Fig. 21.2, the darkened area under peak C represents the peak area of that component. The peak height (distance h in Fig. 21.2) can also be used. In thin-layer or paper chromatography, the area of the spot is related to the amount of substance. The separated component can also be eluted from the plate or paper and measured externally by another technique (for instance, spectrophotometry). 

In Chapter 3, we have alluded to chromatographic separation as the most important separation technique to TEQA. Indeed, much of the innovative sample prep techniques for trace organics described in Chapter 3 are designed to enable a sample of environmental interest to be nicely introduced into a chromatograph. A chromatograph is an analytical instrument that has been designed and manufactured to perform either gas or liquid column chromatography. 

The second drawback of NMR is that it cannot be easily interfaced with modern chromatographic separation techniques. Thus GC (gas chromatography), HPLC (high performance liquid chromatography), SFC (supercritical fluid chromatography), and CZE (capillary zone electrophoresis) are all routinely interfaced with MS but not with NMR, although the major instrument manufacturers JEOL (Japan), Varian (USA), and Bruker (Germany) have now developed NMR probes compatible with HPLC columns. If NMR spectra of individual components of a mixture are required then the mixture usually has to be... 

The earliest of the chromatographic methods to be applied to trace analysis was paper chromatography, which in the later 1940s was developed as an advance on the original purely qualitative column chromatographic separations. Gas-liquid chromatography followed in due course, together with many variations of the two-dimensional techniques. 

High-pressure liquid chromatography (HPLC) is a variant of the simple column technique, based on the discovery that chromatographic separations are vastly improved if the stationary phase is made up of very small, uniformly sized spherical particles. Small particle size ensures a large surface area for better adsorption, and a uniform spherical shape allows a tight, uniform packing of particles. In practice, coated Si02 microspheres of 3.5 to 5 fxm diameter are often used. 

Normal-phase liquid chromatography is thus a steric-selective separation method. The molecular properties of steric isomers are not easily obtained and the molecular properties of optical isomers estimated by computational chemical calculation are the same. Therefore, the development of prediction methods for retention times in normal-phase liquid chromatography is difficult compared with reversed-phase liquid chromatography, where the hydrophobicity of the molecule is the predominant determinant of retention differences. When the molecular structure is known, the separation conditions in normal-phase LC can be estimated from Table 1.1, and from the solvent selectivity. A small-scale thin-layer liquid chromatographic separation is often a good tool to find a suitable eluent. When a silica gel column is used, the formation of a monolayer of water on the surface of the silica gel is an important technique. A water-saturated very non-polar solvent should be used as the base solvent, such as water-saturated w-hexane or isooctane. 

A certain amount of qualitative information can be obtained by means of so-called multidimensional chromatography (1). Tlris is a combination of different chromatographic techniques in which fractions from a primary separation step are transferred online to a secondary separation step. Multidimensional gas chromatography (GC), for example, involves coupling of GC columns of different selectivities so that the primary column isolates the fraction of interest, and the secondary column takes care of the final separation of that fraction. Using multidimensional liquid chromatography (LC), determination of androgen hormone residues in cattle liver has been possible (2). 

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