Separated Substances

Detection on the layer is easy if the substances are coloured or fluoresce in UV light. 

In this way, azo dyes [638], dinitrophenylhydrazones [470], 2,4-dinitrophenyl-hydrazides of fatty acids [800], Kauwolfia alkaloids [607], aristolochia acids [507] and various polynuclear aromatic hydrocarbons [599] could be localised. 

This is riot possible with the vast majority of substances. They must be detected with the help of reference chromatograms or reagents since localisation through i /-values is most unreliable [223]. 

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A detector is needed to sense when the separated substances are emerging from the end of the column. A mass spectrometer (MS) makes a very good, sensitive detector and can be coupled to either GC or LC to give the combined techniques of GC/MS or LC/MS, respectively. 

Silica gel, per se, is not so frequently used in LC as the reversed phases or the bonded phases, because silica separates substances largely by polar interactions with the silanol groups on the silica surface. In contrast, the reversed and bonded phases separate material largely by interactions with the dispersive components of the solute. As the dispersive character of substances, in general, vary more subtly than does their polar character, the reversed and bonded phases are usually preferred. In addition, silica has a significant solubility in many solvents, particularly aqueous solvents and, thus, silica columns can be less stable than those packed with bonded phases. The analytical procedure can be a little more complex and costly with silica gel columns as, in general, a wider variety of more expensive solvents are required. Reversed and bonded phases utilize blended solvents such as hexane/ethanol, methanol/water or acetonitrile/water mixtures as the mobile phase and, consequently, are considerably more economical. Nevertheless, silica gel has certain areas of application for which it is particularly useful and is very effective for separating polarizable substances such as the polynuclear aromatic hydrocarbons and substances... 

Detection methods, whieh provide real information eonceming the separated substances, are necessary in order to be able to analyze the separation result and separation performance achieved by such a system. 

In spite of numerous advances in the field of detection there are not and never have been any genuinely substance-specific chemical detection reactions. This means that, unlike the spectrometric methods, the methods of detection normally employed in chromatography cannot be employed for an unequivocal identification of compounds, they can only provide more or less definite indications for the characterization of the separated substances. Universal reagents are usually employed for a first analysis of the separation of samples of unknowns. This is then followed by the use of group-specific reagents. The more individual the pieces of information that can be provided from various sources for a presumed substance the more certainly is its presence indicated. However, all this evidence remains indicative it is not a confirmation of identity. 

Photomultipliers are appreciably more sensitive sensors than the eye in their response to line or continuum sources. Monochromators are fitted to the light beam in order to be able to operate as substance-speciflcally as possible [5]. Additional filter combinations (monochromatic and cut-off filters) are needed for the measurement of fluorescence. Appropriate instruments are not only suitable for the qualitative detection of separated substances (scanning absorption or fluorescence along the chromatogram) but also for characterization of the substance (recording of spectra in addition to hR and for quantitative determinations. 

Detector noise The detection limit for the recording of chromatographically separated substances is determined by... 

The reaction conditions can be selected so as to be able to separate substances with the same or similar chromatographic properties (critical substance pairs) by exploiting their differing chemical behavior, thus, making it easier to identify them. Specific chemical derivatization allows, for example, the esterification of... 

In general care should be taken in the choice of solvent to ensure that neither the chromatographically separated substances nor their reaction products are soluble in the solvent of the dipping reagent. 

The exploitation of specific adsorbent properties can also lead to the same goal of homogeneous denvatization of separated substances... 

After a chromatogram has been developed the TLC plate is removed from the developing chamber and the status quo is fixed by removing the mobile phase remaining in the layer as quickly as possible. This is properly performed in the fume cupboard so as not to contaminate the laboratory with solvent fumes. If possible the TLC plate should be laid horizontally because then as the mobile phase evaporates the separated substances will migrate evenly to the surface where they can be the more readily detected. A fan or hair dryer (hot or cold air stream)... 

Manual transfer of the chromatographically separated substance to the detector . These include, for example, the detection of antibiotically active substances, plant and animal hormones, mycotoxins, insecticides, spice and bitter principles and alkaloids. The frequency distribution of their employment is shown in Figure 54 [295]. 

Every chromatographic investigation begins with the preparation of the sample and the chromatographic system. This is followed by the crux of the separation process (development of the chromatogram) which is in turn followed by the visualization of the separated substances and the preservation of the chromatogram and finally by the analysis of the results. 

Electrophoresis (Section 27.3) Method for separating substances on the basis of their tendency to migrate to a positively or negatively charged electrode at a particular pH. 

A chemist is often interested in separating substances in a solution mixture. Such a problem is... 

It is possible to separate a soap-LSDA dispersion by ultrafiltration through a polymeric membrane [33]. The filtrate contained sodium and some magnesium ions but no calcium soaps or LSDA. The separated substances on the membrane could be readily dispersed in water in which they retained a high degree of surface activity. 

Two of the fundamental concepts of thermodynamics are heat and work. People once thought that heat was a separate substance, a fluid called caloric, which flowed from a hot substance to a cooler one. The French engineer Sadi Carnot... 

Separation processes are based on some difference in the properties of the substances to be separated and may operate kinetically, as in settling and centrifugation, or by establishing an equilibrium, as in absorption and extraction. Typical separation processes are shown in Table 6.1. Better separations follow from higher selectivity or higher rates of transport or transformation. The economics of separation hinges on the required purity of the separated substance or on the extent to which an unwanted impurity must be removed (Figure 6.13). 

However, the reactants are present as solutions, and once mixed do not in any real sense contain silver nitrate and sodium chloride as separate substances (even in the hypothetical instant before precipitation occurs). Example 4 might best be rewritten... 

The supply of thermal energy by a heater or IR lamp is a second method of converting the separated substances into fluorescent derivatives. Here too, at about their decomposition temperatures, many substances react to form fluorescent derivatives, generally with the catalytic participation of the active sorbent . These fluorescent derivative often provide speciflc evidence concerning the nature of the substances being detected (cf. Chapter 1.2). 

The application of high tension (e.g. 20 kV, 0.5 MHz) in an evacuated system (0.2. .. 8 torr) causes the residual gas to form a highly ionized mixture of positive and negative ions, electrons, photons and neutral gas molecules. In the presence of active sorbents this plasma reacts with the chromatographically separated substances to eld reactive ions and radicals. 

Thin-layer chromatography has the great advantage that the result of the separation is stored — usually invisibly — on the TLC/HPTLC plate as on a diskette. In such cases it needs developing or detecting, rather like an exposed film. This can now be done online or off-line so that the analyst can decide which method to use to detect the separated substances. 

Physical methods, some of which can be applied in the on-line mode (Fig. 8) and physiological methods providing information concerning the effectivity of the separated substance can be used. A later volume will treat these bioautographic methods which can be subdivided into bioassays (off-line) and bioautogrammes (on-line). 

In addition to the reagent sequences with clearly detectable reaction mechanism, which have already been described, many sequences of reagents not covered by any of the reaction types described have also found application in thin-layer chromatograpl. The reaction sequences that remain to be described were all designed to provide as speciflc a detection of the separated substances as possible. 

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