Sample thin layer chromatography

Thin-layer chromatography can be successfully applied to separate the compounds, closely related in chemical structure, or used in combination therapy, as well as the metabolites from biological samples. Thin-layer chromatography may also be used to control the chemical purity of the compounds and to predict the HPLC behavior of the drugs and related metabolites. High performance TLC (HPTLC) can be used as an effective quantitative alternative to other chromatographic techniques. Its accuracy and precision are satisfactory for routine use in a pharmaceutical analysis. 

Paper chromatography may be used as one step for separation and/or isolation of anthocyanins from complex samples. Thin-layer chromatography, performed on cellulose or silica gel plates, was in the first period of use a convenient extension of paper chromatography. The introduction of new stationary phases and the development of instrumentation offering the possibility of quantitative analysis provided new possibilities for method developments. The advantages of TLC are the simplicity in terms of instrumentation, sample preparation and data evaluation. 

Thin-Layer Chromatography. Chiral stationary phases have been used less extensively in tic as in high performance Hquid chromatography (hplc). This may, in large part, be due to lack of avakabiHty. The cost of many chiral selectors, as well as the accessibiHty and success of chiral additives, may have inhibited widespread commerciali2ation. Usually, nondestmctive visuali2ation of the sample spots in tic is accompHshed using iodine vapor, uv or fluorescence. However, the presence of the chiral selector in the stationary phase can mask the analyte and interfere with detection (43). 

The total phosphoms content of the sample is determined by method AOCS Ja 5-55. Analysis of phosphoUpid in lecithin concentrates (AOCS Ja 7-86) is performed by fractionation with two-dimensional thin-layer chromatography (tic) followed by acid digestion and reaction with molybdate to measure total phosphorous for each fraction at 310 nm. It is a semiquantitative method for PC, PE, PI, PA, LPC, and LPE. Method AOCS Ja 7b-91 is for the direct deterrnination of single phosphoHpids PE, PA, PI, PC in lecithin by high performance Hquid chromatography (hplc). The method is appHcable to oil-containing lecithins, deoiled lecithins, lecithin fractions, but not appHcable to lyso-PC and lyso-PE. 

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. 

Fig. 24. A thin layer chromatography (TLC) image, documenting detection of impurities in a series of dye intermediate samples under near-UV illuinination. Columns 1 and 7 represent reference materials. Photographed with Polaroid Type 339 film in a CU-5 closeup camera.

One of trends of development of thin-layer chromatography implies that replacement of aqueous-organic eluents by micellar surfactants solution. This is reduces the toxicity, flammability, environmental contamination and cost of the mobile phases, reduce sample prepar ation in some cases. 

The course of the fermentation is tested by removal of samples which are extracted with methyl isobutyl ketone. The extracts are analyzed by thin layer chromatography using a system of benzene/ethyl acetate (4 1). 

One-dimensional thin-layer chromatography. This method, based on the work of Wolf and McPherson, will determine more than 0.1% terminal 8-sultones in the neutral oil. This implies that if the AOS contains 1% neutral oil, greater than 30 ppm (active matter basis) of terminal 5-sultones can be determined. Some samples contain a compound having an R( of approximately 0.03 U less than the 5-sultones. This should not be reported as terminal 8-sultones. C14 and C16 terminal 8-sultones have the same retention (R 0.35-0.55, depending on the humidity) and therefore appear as one spot. 

Two-dimensional thin-layer chromatography. This method is used to verify the presence of terminal 5-sultones, terminal unsaturated y-sultone, and terminal 2-chloro-y-sultone, if they are detected in the gas chromatographic determination. After extraction of the neutral oil from the AOS sample, the neutral oil is made up volumetrically to at least a 10% solution in hexane. Of this solution 3 pi is spotted onto a silica gel TLC plate together with standard sultones. It is twice developed with 2-propyl ether and then after turning the plate 90° twice developed with 60/40 n-butyl chloride/methylene chloride. The... 

This volume is the second of a series of practice-orientated TLC/HPTLC books published in excellent quality by VCH Publishers. As in the first volume, a series of reagents and detection methods have been reviewed with the intention of helping the practical analyst increase the detection specificity of routine samples separated by thin-layer chromatography. 

Gupta, A.P., Gupta, M.M., Kumar, S., Simultaneous determination of curcuminoids in curcuma samples using high performance thin layer chromatography, J. Liq. Chromatogr. Rel. TechnoL, 22, 1561, 1999. 

PLC is used for separations of 2 to 5 mg of sample on thin-layer chromatography (TLC) plates (0.25-nun layer thickness) or high-performance TLC (HPTLC) plates (0.1-mm thickness). In these instances, the method is termed micropreparative TLC. The isolation of one to five compounds in amounts ranging from 5 to 1000 mg is carried out on thicker layers. PLC is performed for isolation of compounds to be used in other tasks, i.e., further identification by various analytical methods, such as ultraviolet (UV) solution spectrometry [1] or gas chromatography/mass spectrometry (GC/MS) [2], obtaining analytical standards, or investigations of chemical or biological properties [3]. 

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