Chromatography general elution problem

The general elution problem in chromatography. Improving the resolution of the overlapping bands in chromatogram (a) results in a longer analysis time for chromatogram (b). 

Separation scientists speak of a general elution problem when asked to develop a universal separation method using chromatography. What is the problem (Hint you may need to consult works by Snyder or Heftmann in the library.)... 

Figure 6.1 Illustration of the general elution problem in chromatography. Chromatograms a and b constant elution conditions. Chromatogram c (opposite page) programmed analysis.

General elution problem The compromise between elution time and resolution addressed through gradient elution (for liquid chromatography) or temperature programming (for gas chromatography). 

In the bed-development separation of sample mixtures which are not subject to the general elution problem (e.g., a two-component sample), solvent demixing is usually an unwelcome complication. The preceding relationships for describing the effect of the solvent on separation [e.g., Eqs. (8-4), (8-5), (8-10)] become inapplicable, and it is difficult to predict satisfactory separation conditions or to define the relative strength of a binary solvent. As in the case of polyzonal chromatography, there may be a tendency for two or more sample components to travel with one of the solvent fronts and hence remain unseparated. In extreme cases a satisfactory separation of a particular sample may be impossible with a given binary A-B of any composition, even where sample K° values are... 

A number of alternative techniques have been developed to tackle the general elution problem, such as flow and temperature programming, and especially column switching techniques. Coupled column chromatography involves the use of three and sometimes four different stationary phases. The sample is introduced to the primary column for a preliminary separation and then the various polar fractions are switched to the appropriate secondary columns and then to the detector. Coupled column chromatography provides enhanced resolution, and as isocratic elution is used the technique is particularly useful for the routine assay of samples with a wide range of capacity factor values. Further details on these various techniques may be found in a review by Koenigbauer and Majors [96]. 

In all forms of chromatography, one must be aware of the so-called general elution problem (illustrated in Fig. 21.20) when dealing with isocratic solvent systems and multi-component samples with widely differing k values. If a strong isocratic mobile phase is selected that will adequately elute strongly retained compounds, then the weakly retained ones will be eluted too quickly and will be poorly separated... 

In the chromatography of plant extracts on an enlarged scale, there are a few main problems general elution because of the differentiated polarity of complex mixture components being separated the structural and chemical analogy of compounds and resolution decrease due to band broadening. 

Removal of analytes from a tube is an elution problem analogous to frontal chromatography and has been discussed in detail. In general, if the desorption temperature of a gas chromatogram is high and thin coatings are used, then all the analytes are in the gas phase as soon as the coating is placed in the injector. The desorption time then corresponds to the elution of two void volumes of the capillary. For liquid desorption, the desorption volume can be even smaller since the analytes can be focused at the front of the desorption solvent. 

Chromatography. A number of HPLC and TLC methods have been developed for separation and isolation of the brevetoxins. HPLC methods use both C18 reversed-phase and normal-phase silica gel columns (8, 14, 15). Gradient or iso-cratic elutions are employed and detection usually relies upon ultraviolet (UV) absorption in the 208-215-nm range. Both brevetoxin backbone structures possess a UV absorption maximum at 208 nm, corresponding to the enal moeity (16,17). In addition, the PbTx-1 backbone has an absorption shoulder at 215 nm corresponding to the 7-lactone structure. While UV detection is generally sufficient for isolation and purification, it is not sensitive (>1 ppm) enough to detect trace levels of toxins or metabolites. Excellent separations are achieved by silica gel TLC (14, 15, 18-20). Sensitivity (>1 ppm) remains a problem, but flexibility and ease of use continue to make TLC a popular technique. 

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