Thin Layer Chromatography - Determination of Caffeine Using Both Densitometry Measurements and an Image Analyzing System 1

 

Determination of Caffeine Using Both Densitometry Measurements and an Image Analyzing System

 

Another method for scanning TLC plates that has been reported involves the use of an image analyzing system. This procedure employs a digital camera to photograph the plate, the image is then stored in digital form in a computer and then processed by appropriate software. Vovk et al. (16) compared the relative precision obtainable from the image analyzing system to those obtained by normal scanning densitometry. The authors chose caffeine as the model substance and the separation was carried out on a 10 cm x 20 cm glass backed plate carrying a coated layer of silica gel 0.2 mm thick (Merk, Darmstadt, Germany). The samples were place on the plate with a Linomat IV applicator (Camag, Muttenz, Switzerland) that was fitted with a 100 ml syringe. The band was 10 mm wide, and 5 ml of sample was applied at a rate of 4 ml/s. Ten or twelve bands were applied per plate 15 mm apart and 15 mm from the lower edge. The plates were developed with a solvent mixture consisting of dichloro-methane:methanol (90:10 v/v) in an unsaturated glass twin trough chamber. The caffeine solute migrated 7.5 cm and the migration time was approximately 10 min. The plates were subsequently dried in a stream of warm air for about 5 min. A chromatogram of a separation monitored by densitometric scanning is shown in figure 29.

 

Courtesy of J. Planar Chromatogr. [ref. 6]

 

Figure 29. Chromatogram Obtained by Densitometer Measurements at 270 nm of a Caffeine Standard