Zones, chromatographic

It is probable that the solvents given in the individual reagent monographs are not suitable for all the substances with which the reagent will react. This point should be taken into account especially for quantitative work and the user should make appropriate modifications. In particular, there must be no loss of substance or reaction product by dissolution (formation of comet tails by the chromatographic zones). 

Note Rhodamine B is a universal reagent that can be used on silica gel, talc, starch [5] and cellulose layers, just as on urea [1] or silver nitrate-impregnated [7] phases. Liquid paraffin-impregnated silica gel and RP layers are less suitable, since the background to the chromatographic zones is also intensely colored. It is often possible to increase the detection sensitivity by placing the plate in an atmosphere of ammonia after it has been sprayed or dipped, alternatively it can be oversprayed with sodium or potassium hydroxide solution. 

A detailed description of the versatility of multiple development techniques in one dimension has been given by Szabady and Nyiredy (18). These authors compared conventional TLC with unidimensional (UMD) and incremental (IMD) multiple development methods by chromatographing furocoumarin isomers on silica using chloroform as the monocomponent mobile phase. The development distance for all three methods was 70 mm, while the number of development steps for both of the "D techniques was five. Comparison of the effects of UMD and IMD on zone-centre separation and on chromatographic zone width reveals that UMD increases zone-centre separation more effectively in the lower Rf range, while IMD results in narrower spots (Figure 8.8). 

Thirdly, if it is not possible to apply the SRS technique, it can be established whether a primary, secondary or tertiary alcohol is present by oxidizing the alcohol on the chromatographic zone and then subjecting the oxidation product to a detection reaction. On oxidation primary alcohols form aldehydes, secondary alcohols ketones and tertiary alcohols are not oxidized. 

Colored or, under long-wavelength UV light (X, = 365 nm), yellow or blue fluorescent chromatograph zones appear, even after drying the chromatogram in a stream of cold air, but sometimes only after heating to 80 °C [17], to 130 C [9] or 150 C [22] for 5-10 min. 

LCEC is a special case of steady-state hydrodynamic chronoamperometry. In LCEC, the concentration changes as the chromatographic zones flow past the detector. The electrode is operated in the limiting current region for the eluted compounds, even though the concentration varies as the zones enter and leave the detector compartment. It is important to note that the volume of solution in the active region of the typical electrochemical detector ( 1 pL or even less) is very small compared to the volume occupied by the typical chromatographic zone ( — 0.1-1 mL). 

The simplest picture of chromatographic zone broadening comes from the random walk model. The random walk model for chromatography will be developed at some length in Chapter 11 here we present a synopsis. 

Despite such complications of detail, the random-walk model describes the essence of chromatographic zone spreading. It properly accounts for the way in which all major experimental parameters influence the broadening process. 

TABLE 1 Typical Values of Parameters Important in Chromatographic Zone Broadening... 

If the detector response is linearly proportional to the concentration of solute in the column effluent, there is also a linear proportionality between the peak area and the total mass of solute in the eluted chromatographic zone. Hence, for the peak areas of the compound under determination, i, calibration standard s and the reference compound r (cf., the definition of the relative specific response), Ai, As andAr, respectively, we have... 

Efficiency and selectivity are complementary chromatographic descriptors. A column with high efficiency can generate narrow chromatographic zones and allows the separation of analytes with low selectivity. On the other hand, if the chromatographic system has high selectivity for two analytes, they could be separated on the column with low efficiency, as shown in Figure 1-9. 

The process of analyte retention in high-performance liquid chromatography (HPLC) involves many different aspects of molecular behavior and interactions in condensed media in a dynamic interfacial system. Molecular diffusion in the eluent flow with complex flow dynamics in a bimodal porous space is only one of many complex processes responsible for broadening of the chromatographic zone. Dynamic transfer of the analyte molecules between mobile phase and adsorbent surface in the presence of secondary equilibria effects is also only part of the processes responsible for the analyte retention on the column. These processes just outline a complex picture that chromatographic theory should be able to describe. 

HPLC theory could be subdivided in two distinct aspects kinetic and thermodynamic. Kinetic aspect of chromatographic zone migration is responsible for the band broadening, and the thermodynamic aspect is responsible for the analyte retention in the column. From the analytical point of view, kinetic factors determine the width of chromatographic peak whereas the thermodynamic factors determine peak position on the chromatogram. Both aspects are equally important, and successful separation could be achieved either by optimization of band broadening (efficiency) or by variation of the peak positions on the chromatogram (selectivity). From the practical point of view, separation efficiency in HPLC is more related to instrument optimization, column... 

The most rigorous discussion of the formation of chromatographic zone and the mathematical description of zone-broadening is given in reference 1. Here only practically important and useful equations will be discussed. 

In the introduction section we define the term resolution as the abihty of the column to resolve two analyte in two separate peaks (or chromatographic zones). In more general form than it was given before, the resolution can be defined as the half of the distance between the centers of gravity of two chromatographic zones related to the sum of their standard deviations ... 

For symmetrical chromatographic bands, this is the ratio of the distance between peaks maxima to the peak width. The distance between peak maxima is proportional to the distance of the chromatographic zone migration, and the peak width is proportional to the square root of this distance. Figure 2-4 illustrates this relationship. 

Linear velocity of chromatographic zone as a function of the eluent composition u(c) [as derived in equation (2-38)] in the most simple model of the retention process (partitioning) is... 

Peak tailing is the most commonly observed effect of sample overloading. In essence, in most cases this effect is associated with nonlinear adsorption isotherms. In Chapter 2 the relationship of the retention volume and the derivative of the excess adsorption isotherm of the analyte on given stationary phase surface was derived. If the isotherm is linear within the injected concentration region, all components of the chromatographic zone are moving... 

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