Sheila A. Schuette

Characterization of a scanning densitometer for high-performance thin-layer chromatography

Abstract Optimum conditions to maximize the observed resolution and signal-to-noise ratio of the Shimadzu CS-910 scanning densitometer for high-performance thin-layer chromatography (HPTLC) are described. Resolution is shown to depend only on the slit width and is constant for slit widths in the range 0.05–0.80 mm. Signal-to-noise ratios depend on the size of the sampling beam which is fixed by the dimensions of the slit. At small slit dimensions the noise component dominates the signal-to-noise ratio. For scanning HPTLC plates dual-beam single-wavelength operation, linear scanning, scan speeds of 24 or 48 mm min −1 , slit widths of 0.5–0.8 mm and slit height ≈ 3.0 mm are recommended for general use.

Unidimensional, sequential separation of PTH-amino acids by high-performance thin-layer chromatography

Abstract Eighteen of the twenty common protein PTH-amino acids derivatives are separated by continuous multiple development high-performance thin-layer chromatography. The separation is performed on silica gel plates using five development steps with four changes in mobile phase. The derivatives are identified by scanning densitometry, and the total analysis requires less than 1 h. The unseparated derivatives of alanine and tryptophan are baseline resolved in an alternative solvent system. Thus, all twenty of the common PTH-amino acid derivatives may be identified with the proposed method. By using the unidimensional method of development, the high sample capacity of the high-performance thin-layer chromatographic plate is preserved; samples and standards can be run simultaneously to improve the accuracy of identification. The detection limit for the PTH-amino acid derivatives determined by in situ reflectance scanning densitometry at 270 nm was found to be ca. 0.5 ng per spot.

Instrumental Evaluation of Thin-Layer Chromatograms

Recent changes in the practice of thin-layer chromatography (TLC) have created a renaissance of interest in this technique and led to its wider acceptance as a powerful tool for qualitative and quantitative analysis of mixtures. The performance breakthrough in TLC, referred to as high performance thin-layer chromatography (HPTLC), was not a result of any specific advances in instrumentation or materials, but was rather a culmination of improvements in practically all of the operations comprising TLC, [1–6]. Improvements in the quality of the sorbent layer, methods of sample application, new development techniques, and the availability of scanning densitometers for in situ quantitative analysis were all important developments in the evolution of HPTLC. The new HPTLC plates were prepared from sorbents of smaller average particle diameter and of narrower particle size distribution than those used to prepare conventional TLC plates. The performance of these plates improved an order of magnitude over those used for conventional TLC. Because of the lower sample capacity of the HPTLC sorbent layer, the amount of sample applied to it had to be scaled down from those commonly used in conventional TLC. Typical sample volumes applied to the plates are in the range 100–200 nl, sample amounts less than ca. 0.1 μg, and starting spot sizes of 1.0–2.0 mm.

Chapter 9 – HIGH PERFORMANCE THIN-LAYER CHROMATOGRAPHY

Analysis was performed on 10 × 10 cm aluminium plates precoated with 0.2 mm layers of silica gel 60 F254 (Merck, Mumbai). Before use plates were washed with methanol, activated in an oven at 105C for 20 min, then left to cool at room temperature. Standard solutions were applied to prewashed activated plates, as 6 mm bands, 6 mm apart, under a stream of nitrogen, by means of a Camag (Muttenz, Switzerland) Linomat V automated spray-on band applicator equipped with a Hamilton 100 μl syringe (Reno, Nevada, USA). The plates were developed with 10 ml of mobile phase, in a Camag twin trough chamber previously saturated with mobile phase vapour for 20 min.