Thin-layer chromatography validation

From this expression (Kubelka Munk function) it follows that, within the range of validity of the theory, q,/ depends only on the ratio of the absorption coefficient to the scattering coefficient, and not on their individual values. The equation has been most useful where reflectance measurements are used to obtain information about absorption and scattering (e.g., in textile dyeing, thin layer chromatography, and IR spectroscopy). 

Reina, R.J., White, K.D. and Jahngen, E.G.E. (1997) Validated method for quantitation and identification of 4,4-desmethylsterols and triterpene diols in plant oils by thin-layer chromatography-high resolution gas chromatography-mass spectrometry. J. AOAC Intern., 80(6), 1272-1280. 

It is best to purify the methyl esters by thin-layer chromatography of the sample on another silica gel G (250 p,m) plate using this solvent system, but without any spray reagent being used. A comparable plate is run and the methyl ester band is detected by the sulfuric acid/char reaction. Then the unsprayed plate is scraped at the methyl ester area and the silica gel is extracted with petroleum-diethyl ether (80 20, v/v) or with chloroform-methanol-water (1 2 0.8, v/v). It is always prudent to spray the latter plate (after removing the desired area by scraping) with sulfuric acid and then charring. This will validate whether the apparent removal of all of the methyl esters has been accomplished. 

Baer et al. during 1986 to 1988. Urine was screened by methods in effect in the federal probation system THC metabolite and opiates were screened by EMIT and confirmed by high performance liquid chromatography (HPLC) at cutoffs of 100 and 500 ng/mL, respectively. Cocaine metabolites and PCP were screened by thin-layer chromatography (TLC) at cutoffs of 2000 and 500 ng/mL, respectively, and confirmed by GC. Hair testing was by RIA. The RIA procedures, validated by GC/MS, showed no false positives due to cross-reactivity effects. 

Beck et al. show the advantages of using simpler, less expensive methods like FTIR and thin layer chromatography (TLC). These techniques provide speed and ease of analysis. When used to investigate complex mixtures like the natural products examined by Beck et al., more extensive methods may be required to validate the findings. 

Years of doing things the same way does not validate correctness. Many chemical tests have been proven incorrect over a period of time. Newer techniques like thin layer chromatography (TLC), high performance liquid chromatography (HPLC), capillary electrophoresis (CE) and mass spectrometry (MS) have revolutionized our standards of purity. So, too, should this testing procedure prove to be. 

Displacement thin-layer chromatography (D-TLC) also stemmed from the activity of Horvath s group at Yale University. Experimental work with D-TLC has proven the validity of the rules of displacement chromatography, found by using high-performance liquid chromatography (HPLC). Kalasz et al. continued the research on D-TLC, mainly with the separation of steroids. 

In conclusion, CE is a valuable analytical tool that offers a number of possibilities for the analysis of a wide spectrum of forensicaUy interesting compounds. Practically all compounds which have been traditionally analyzed by GC, high-performance Uquid chromatography, thin-layer chromatography, or slab-gel electrophoresis, can be assayed by capillary electrophoretic procedures. AU methods of capillary electrophoresis can be validated and can meet the demands of good laboratory practice. 

In the Martin-Synge relationship (1), C , and are molar concentration of the solute in the mobile and stationary phase, respectively, and V, and V , are the volumes of these two phases. VJV, is numerically equal to AJA , the ratio of the phase cross section normal to the direction of the solvent flow, which better describes the local conditions in thin-layer chromatography. The validity of the equation is limited because the amount of solvent on the layer decrease going toward the solvent front and, therefore, the phase ratio changes. 

As an example, Urakova et al. compared the analysis of chlorogenic acid in green coffee bean extracts by thin layer chromatography on silica (i.e. normal-phase liquid chromatography) and by reversed-phase HPLC The validation data (LOD, LOQ, repeatability, and various precision parameters), the recoveries and the quantitative results were totally comparable. It can be assumed that both methods find the true value. Either method can be used, depending on the preference or instrumentation of a laboratory. 

A registration application should include documented evidence that the analytical procedures are validated and suitable for the detection and quantification of impurities (see ICH Q2A and Q2B on Analytical Validation). Technical factors (e.g., manufacturing capability and control methodology) can be considered as part of the justification for selection of alternative thresholds based on manufacturing experience with the proposed commercial process. The use of two decimal places for thresholds does not necessarily reflect the precision of the analytical procedure used for routine quality control purposes. Thus, the use of lower precision techniques (e.g., thin-layer chromatography) can be appropriate where justified and appropriately validated. Differences in the analytical procedures used during development and those proposed for the commercial product should be discussed... 

Assay of theophylline in effervesoerit tablets using high performance thin-layer chromatography and further densitometrio measurement. This preliminary testing procedure was worked out acc. to literature [144] "Validation of analytical procedures in pharmaceutical quality control" from B, Renger, H. Jehle, M. Fischer und W, Funk. 

The set-up is the same in these two standard tests and is very similar to that of liquid tests (Fig. lb). The samples are intensively composted in a static reactor over a 45-day period. A continuous air flow free of C02 is provided to the test vessels. The carbon dioxide produced in the test and blank vessels is either continuously monitored (by gas chromatography or infrared) or measured at regular intervals (titration). The bio degradability of the sample is also reported as a percentage of the bio degradability of a reference positive substance (e.g., cellulose for the thin-layer chromatography). The tests are valid if more than 70 % of the positive reference is degraded. 

The first proof of the validity of this approach was given by Gankina et al. [19] for the analysis of block copolymers by thin layer chromatography. Column liquid chromatography was used by Zimina et al. [20] for the analysis of poly(styrene-b/ock-methyl methacrylate) and poly(styrene-Wock-terr-butyl methacrylate). However, the critical conditions were established only for the polar part of the block copolymers, i.e. PMMA and PtBMA, respectively. Thus, only the polystyrene block was analyzed. 

This article covers various aspects of development and validation of methods in thin-layer chromatography. A detailed treatment of the underlying theory is given elsewhere in this encyclopedia. 

Thin-layer chromatography (TEC) is an offline process comprising several individual steps, which are independent in time and location. Each step, such as sample application, chromatogram development, detection (including derivatization if necessary), and evaluation is associated with a number of important parameters that can affect the final result of the analysis. The development and validation of a TEC method must not only involve a clear specification of those parameters but also an evaluation of their suitable ranges. Eor a given analytical task it is usually the separation step that is particularly emphasized during method development however, the other steps may become equally important if repeatability of the qualitative result or detection limits, accuracy, and precision of quantitation are of importance. 

Berny, P. Jaussaud, P. Durix, A. Ravel, C. Bony, S. Rapid determination of the mycotoxin lolitrem B in endophyte-infected perennial ryegrass by high-performance thin-layer chromatography A validated assay. J. Chromol. 1997, 769, 343. 

Petrovic, M. Kastelan-Macan, M. Lazaric, K. Babic, S. Validation of thin layer chromatography quantitation determination with a CCD camera and slit scanning densitometer. J. AO AC Int. 1999, 82 (1), 25-30. 

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