High performance thin layer chromatography sample application

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. 

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. 

The separation and identification of natural dyes from wool fibers using reverse-phase high-performance liquid chromotog-raphy (HPLC) were performed on a C-18 column. Two isocratic four-solvent systems were developed on the basis of the Snyder solvent-selectivity triangle concept (1) 10% acetonitrile, 4% alcohol, and 2% tetrahydrofuran in 0.01 M acetic acid and (2)7% acetonitrile, 8% alcohol, and 5% tetrahydrofuran in 0.01 M acetic acid. Samples were also eluted in 30% acetonitrile. Spot tests and thin-layer chromatography were performed on all samples to confirm HPLC results. The systems also were found to be potentially useful in the identification of early synthetic dyes. A system of sample preparation that minimizes the reaction of samples was discussed. The application of this HPLC separation technique to samples from 20th century Caucasian rugs and American samples unearthed from the foundation of Mission San Jose was examined. 

The method of diffuse transmittance (DT) is based on measurement of the radiation component 7dt (Fig. 1.22) that passes diffusely through an inhomogeneous layer. This method was first applied to the IR spectroscopic analysis of thin films on samples in powder form by Tolstoy in 1985 [116, 117], who obtained DT spectra of water adsorbed onto silica gel. When used in conjunction with a FTIR spectrometer, the method is called diffiise-transmittance infrared Fourier transform spectroscopy (DTIFTS). DTIFTS is the most recently developed IR spectroscopic methods for studying powder surfaces and has already found application in high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) [118, 119]. Of increasing popularity are DTIFTS measurements of powders that use an IR microscope to collect radiation [112, 119] (Section 4.3). 

This entry describes the general considerations, procedures, and instruments that are important for the correct application of sample and standard zones in thin-layer chromatography (TLC) and high-performance TLC (HPTLC). The application of spots and bands manually and by semiautomated and completely automated instrumental techniques is covered. 

---