High-performance thin-layer chromatography sample

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. 

The method of Denda et al.14 was used to measure ceramides. After SC was stripped with adhesive cellophane tape, it was removed from the tape and washed several times with hexane, followed by drying in a vacuum desiccator. Lipids were extracted from the SC sample in a mixture of chloroform and methanol (2 1). Ceramides were separated with a silica gel column (Bond Elut SI, Analytichem International, United States) and purified for measurement by gas chromatography (GC-14A, Shimazu Manufacturing Co., Ltd., Japan). The composition of ceramides was obtained by high-performance thin layer chromatography and scanned on a recording photodensitometer (TLC Scanner CS930, Shimazu, Japan). 

High-performance thin layer chromatography HPTLC is an improvement of the technique where the sorbent material (e.g. silica gel 60) has a finer particle size and a narrower particle size distribution than conventional TLC. HPTLC plates have an improved surface homogeneity and are thinner. The resolution is improved, analysis times are shorter and it is sufficient to apply nanolitres or nanograms of sample (Nano-TLC). 

High performance thin-layer chromatography (HPTLC) has also been used for the determination of carbamate pesticides.Thus, TLC methods provide increased selectivity through silica derivatization, as well as higher analytical precision and sensitivity with high-performance plates. Butz and Stan reported an HPTLC system with automated multiple development (AMD-HPTLC) to screen water samples for pesticides. The method was applied to the determination of 265 pesticides in drinking water spiked with 100 ng/1 of each analyte. 

High-performance thin-layer chromatography (HPTLC) presents better resolution, sensitivity, and precision than classical TLC, and allows determination of the analytes with sufficient accuracy (e.g., determination of preservatives in creams and lotions) besides this, sample preparation may be simple. However, due to the high versatility of other techniques, this is probably the less commonly used of the chromatographic techniques in this area. 

Hgure 1 Separation of ink s colored material by high-performance thin-layer chromatography. Tracks 1, 9, and 18 are reference dyes ladders tracks 2-8 and 10-15 are ink samples track 16 is a blank paper extraction, track 17 is an extractor blank. 

High-performance silica is used in high-performance thin-layer chromatography (HPTLC). HPTLC differs from normal TLC in that the size of the absorbent (usually silica) is only 5 pm, with a narrow distribution. This enables HPTLC to give better separations compared with TLC, which uses a standard silica, and, moreover, HPTLC requires a smaller sample size and has a lower detection limit compared with conventional TLC. HPTLC plates of varying sizes are commercially available and of late have found considerable applications in the field of lipids. Weins and Hauck (1995), in their survey of TLC, conclude that the use of HPTLC plates increased by 30% over the period 1993-95. An excellent application of HPTLC is illustrated in Fig. 1.1 for the separation of neutral and complex lipids. Yao and Rastetter (1985) have achieved separation of more than 20 lipid classes of tissue lipids on HPTLC plates using four developing solvents. 

Walworth, M.J., Stankovich, J.J., Van Berkel, G.J., Schulz, M., and Minarik, S. 2012. High-performance thin-layer chromatography plate blotting for liquid microjunction surface sampling probe mass spectrometric analysis of analytes separated on a wettable phase plate. Rapid Commun. Mass Spectrom., 26 31-42. 

Ruh, H., Sandhoff, R., Meyer, B., Gretz, N., and Hopf, C. 2013. Quantitative characterization of tissue globotetraosylceramides in a rat model of polycystic kidney disease by primadrop sample preparation and indirect high-performance thin layer chromatography-matrix-assisted laser desorption/ionization-time-of flight-mass spectrometry with automated data acquisition. Anal. Chem., 85 6233-6240. 

TLC is a sensitive technique, and it is important to think small with respect to sample size. The highest resolution will always be obtained with the smallest sample commensurate with the ability to visualize the analyte. This is also one of the prerequisites for reproducibility, especially if densitometry is to be used for quantitation (see Chap. 13.8). Zones that are too concentrated re.sult in integrated curve areas that behave in a nonlinear way on regression plots. Depending on the nature of the analyte, sample. sizes as small as picogram amounts are not uncommon, especially in high-performance thin layer chromatography (HPTLC). This is especially true in the analysis of fluorescent substances. Thus, for ultimate sensitivity it is sometimes desirable that a fluorescent derivative of the analyte be prepared. 

Kupke, I. R., and Zeugner, S. (1978). Quantitative high-performance thin-layer chromatography of lipids in plasma and liver homogenates after direct application of 0.5 pi samples to the silica gel layer. J. Chromatogr. 146 261-271. 

Butler, H. T., Coddens, M. E., Khatib, S., and Poole, C. F. (1985). Determination of polycyclic aromatic hydrocarbons in environmental samples by high performance thin layer chromatography and fluorescence scanning densitometry. J. Chromatogr. Sci. 23 200-207. 

HPTLC has been used for the separation of vitamin D metabolites using microparticulate silica gel as the stationary phase and a chloroform-ethyl acetate (1 1) mobile pha,se (Thierry-Palmer and Gray, 1983). They made a thorough study of HPTLC for the separation of a large variety of vitamin D metabolites. They used silica gel HPTLC-HLF plates (Analtech) and reported Rp values of the metabolites in the following three mobile phases dichloromethane-isopropanol (90 10) chloroform-ethyl acetate (1 1) and hexane-isopropanol (85 15). Justova et al. (1984) used high-performance thin-layer chromatography (HPTLC) to determine 1,25-dihydroxy vitamin D, in human plasma. Jones et al. (1992) used TLC for the prepurification of saponified samples for... 

K. Tiefenbacher and H. Woidich, Determination of the Pattern of Polycyclic Aromatic Hydrocarixms (PAH) in Food and Environmental Samples. Cleanup, TLC, in Situ-Fluorescence Characterization, Proc. 3rd Int. Symp. Instrumental High Performance Thin-Layer Chromatography, Wttrzburg, West Germany, 1985, pp. 357-366. 

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