Chromatography problems from interference

These photometric and enzymatic methods suffer from interferences by endogenous and exogenous compounds and can lead to inaccurate results. To avoid these problems, in recent years, CE, gas chromatography-mass spectrometry (GC-MS), and HPLC methods have been developed. Reversed-phase, normal phase, ion exchange, ion parr, column-switching, MEKC, and size exclusion chromatography were used for purine derivative determination. 

Sample collection, preservation, and purification are problems common to TLC and all other chromatographic methods. For complex samples, the TLC development will usually not completely resolve the analyte (the substance to be determined) from interferences unless a prior purification is carried out. This is most often done by selective extraction and column chromatography. In some cases, substances are converted, prior to TLC, to a derivative that is more suitable for separation, detection, and/or quantification than the parent compound. 

One of the major problems lies in the extent of interference from other constituents of a sample. This can often be obviated by a prior separation using chromatography or solvent extraction (p. 55) or by the use of masking agents (p. 40), pH control or changes in oxidation state. Standards should always be matched to the gross composition of the sample as closely as possible, and calibration curves frequently checked. The precision of absorption measurements has already been discussed (p. 361). 

If interference is a major problem the sample must be partially purified before analysis. This breaks the analysis into preparatory and quantitative stages. In order to reduce the technical difficulties resulting from such two-stage methods much work has gone into the development of analytical techniques such as gas and liquid chromatography in which separation and quantitation are effected sequentially. 

One solution to the above problems would be to purify either the substrate or the enzyme. For example, in the case of ECB, the substrate of the enzyme could be extracted from the cells and then purified by chromatography to remove any impurities that would interfere with the bioconversion processes. The ECB could be left as a solution in a solvent or dried to a powder form. ECB deacylase could also be purified to either a concentrated enzyme solution or solid form. Another option is to immobilize the enzyme or substrate on a suitable support. In the case of ECB deacylase, the advantage would be that the enzyme could be reused and the savings gained would obviously have to be compared with the extra costs of immobilization. The next section looks in more detail at the novel approach of immobilizing the ECB substrate. 

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