Thin layer chromatography spectrophotometric methods

Some studies have evaluated the quantity of a specific constituent in various herbal products by a thin-layer chromatography spectrophotometric method. Of 44 feverfew products that were evaluated, 14 (32%) did not contain the minimum of 0.2% parthenolide content (active ingredient) and 10 (22%) did not contain any detectable levels of parthenolide [30]. 

Spectrophotometric deterrnination at 550 nm is relatively insensitive and is useful for the deterrnination of vitamin B 2 in high potency products such as premixes. Thin-layer chromatography and open-column chromatography have been appHed to both the direct assay of cobalamins and to the fractionation and removal of interfering substances from sample extracts prior to microbiological or radioassay. Atomic absorption spectrophotometry of cobalt has been proposed for the deterrnination of vitamin B 2 in dry feeds. Chemical methods based on the estimation of cyanide or the presence of 5,6-dimethylben2irnida2ole in the vitamin B 2 molecule have not been widely used. 

We studied conditions for the determination of tiametoxam (TM), the active component of the fungicide Actai a (Syngenta, Switzerland) by the method of thin layer chromatography with use of the Perkin-Elmer liquid chromatograph combined with spectrophotometric detector. The 250 mm-long and 4.6 mm in diameter steel column filled with Silasorb was used. 

All previous discussion has focused on sample preparation, i.e., removal of the targeted analyte(s) from the sample matrix, isolation of the analyte(s) from other co-extracted, undesirable sample components, and transfer of the analytes into a solvent suitable for final analysis. Over the years, numerous types of analytical instruments have been employed for this final analysis step as noted in the preceding text and Tables 3 and 4. Overall, GC and LC are the most often used analytical techniques, and modern GC and LC instrumentation coupled with mass spectrometry (MS) and tandem mass spectrometry (MS/MS) detection systems are currently the analytical techniques of choice. Methods relying on spectrophotometric detection and thin-layer chromatography (TLC) are now rarely employed, except perhaps for qualitative purposes. 

Dwivedi et al. used a thin-layer chromatographic densitometric and ultraviolet spectrophotometric methods for the simultaneous determination of primaquine and a new antimalarial agent, CDRI compound number 80/53 [68]. The new antimalari-al agent, compound 80/53 is unstable in acidic conditions where it is converted into primaquine. To conduct stability studies of this compound, thin-layer chromatography densitometric and ultraviolet spectrophotometric determination methods were developed. These methods are also suitable of the determination of compound 80/53 or primaquine in bulk and pharmaceutical dosage forms. 

Electrophoretic and isotachoelectrophoretic techniques are gaining in popularity in soil analysis with applications to polyaromatic hydrocarbons, polychlorobiphenyls, tetrahydrothiophene and triazine herbicides, Paraquat and Diquat and growth regulators. Other lesser-used techniques include spectrophotometric methods (five determinants), spectrofluorimetric methods (two determinants), luminescence methods (one determinant), titration methods (one determinant), thin-layer chromatography (five applications), NHR spectroscopy (two applications) and enzymic immunoassays (one determinant). 

Spengler and Jumar [90] used a spectrophotometric method and thin layer chromatography to determine carbamate and urea herbicide residues in sediments. The sample is extracted with acetone, the extract is evaporated in vacuo at 40°C and the residue is hydrolysed with sulphuric acid. The solution is made alkaline with 15% aqueous sodium hydroxide and the liberated aniline (or substituted aniline) is steam distilled and collected in hydrochloric acid. The amine is diazotized and coupled with thymol, the solution is cleaned up on a column of MN 2100 cellulose power and the azo-dye is determined spectrophotometrically at 440nm (465nm for the dye derived from 3-chloro- or 3.4-dichloroaniline) with correction for the extinction of a reagent blank. 

One milligram of microsomal protein is added to 0.1 M potassium phosphate buffer (pH 7.4) containing 50 mM NaF, 10 mM dithiothreitol, 1 mM EDTA, 20% glycerol (v/v), 150 iM 5-cholestene-3/3, 7a-diol, and 0.915% CHAPS. The reaction is initiated by 1 mM NAD+ to give a final reaction volume of 1.0 mL. After incubation at 37°C for 5 minutes, the reaction is terminated by adding 2 mL of 95% ethanol. An internal recovery standard, 4-cholesten-3-one (3 fig in methanol) is also added. The steroid products are extracted into 5 mL of petroleum ether (repeated twice). After the ether has been removed at 40°C under a stream of nitrogen, the products are dissolved in 100 fxL of mobile phase and 20 ju.L is injected into the column. The amount of product formed is linear with protein (to 1.5 mg) and with time (up to 10 min, 1 mg protein). The assay is much more sensitive than the direct spectrophotometric assay, and it avoids the use of thin-layer chromatography and radioisotopes described in other methods. 

Although TLC-MS (mass spectrometry) has been shown to be technically feasible and applicable to a variety of problems, thin-layer chromatography is generally coupled with spectrophotometric methods for quantitative analysis of enantiomers. Optical quantitation can be achieved by in situ densitometry by measurement of UV-vis absorption, fluorescence or fluorescence quenching, or after exctraction of solutes from the scraped layer. The evaluation of detection limits for separated enantiomers is essential because precise determinations of trace levels of a d- or L-en-antiomer in an excess of the other become more and more important. Detection limits as low as 0.1% of an enantiomer in the other have been obtained. 

Plant Residues. The paraquat residues in Cannabis plants at differ-ent times after application and the percent of i C extract with ethyl acetate and methanol are shown in Table I. Combustion of plant material harvested at 29 h showed a significant loss of C (69%) when compared to time 0. The amount of C extracted with methanol also exhibited a decline with time, particularly at 29 h. Vincent et al. (8) have also observed a similar loss of paraquat residues on the same collection of Cannabis, using a residue method based on sodium dithioate reduction of the extracted paraquat, followed by spectrophotometric analysis. Attempts to examine the possible production of labeled products by thin-layer chromatography on cellulose-coated plates with several solvent systems have been unsuccessful to date due to the high ratio of plant material to labeled paraquat in the samples. The plant residues measured in the present study would appear to be consistent with mean values (331 ppm) found in 20 confiscated marihuana samples reported by Turner et al. (9), although there was considerable variation (2-2264 ppm) in the confiscated samples. 

A general quantitative procedure using spectrophotometric analyses following thin-layer chromatography has been given by Lehmann et al. (1967). The final method of analysis may involve infrared spectroscopy, as was illustrated for the determination of oral contraceptives (Beyermann and Roeder, 1967). 

Benzoic acid and sorbic acid can both be extracted using modified Monier Williams distillation methods and quantified spectrophotometrically, or in the case of benzoic acid, by thin-layer chromatography. LC and GLC methodologies are available and routinely used, having the advantage of simultaneous quantification of both benzoic acid/benzoates and sorbic acid. 

Generally, methods are based on solvent extraction of the additive followed by analysis for the extracted additive by a suitable physical technique such as visible spectrophotometry of the coupled antioxidant, redox spectrophotometric methods, ultraviolet spectroscopy, infrared spectroscopy, gas chromatography, thin-layer chromatography or column chromatography. In general, direct chemical methods of analysis have not foimd favour. These include potentiometric titration with standard sodium isopropoxide in pyridine medium or reaction of the antioxidant with excess standard potassium bromide-potassium bromate (ie. free bromine) and estimation of the unused bromine by addition of potassium iodide and determination of the iodine produced by titration with sodium thiosulphate to the starch end-point. ... 

---