Drying of developed plates

In the first version with a mobile phase of constant composition and with single developments of the bilayer in both dimensions, a 2-D TLC separation might be achieved which is the opposite of classical 2-D TLC on the same monolayer stationary phase with two mobile phases of different composition. Unfortunately, the use of RP-18 and silica as the bilayer is rather complicated, because the solvent used in the first development modifies the stationary phase, and unless it can be easily and quantitatively removed during the intermediate drying step or, alternatively, the modification can be performed reproducibly, this can result in inadequate reproducibility of the separation system from sample to sample. It is therefore suggested instead that two single plates be used. After the reversed-phase (RP) separation and drying of the plate, the second, normal-phase, plate can be coupled to the first (see Section 8.10 below). 

Procedure Spot 0.1 mL of the Sample Solution in a line across a 20- x 20-cm glass plate coated with a 0.25-mm layer of Silica Gel G, approximately 3 cm from the bottom edge. Allow the plate to dry for about 20 min in the dark, then develop with the Solvent System in an unlined tank equilibrated for at least 20 min before the plate is inserted. Allow the solvent front to reach within about 3 cm of the top of the plate. Dry the developed plate in the dark. 

Here we must distinguish between the effect of temperature both before and after the development (drying of the plate) and the effect of heat during chromatography. In the first case, the choice of a more suitable solvent system can prevent loss or decomposition of volatile or temperature-sensitive test substances. In the second case, development should be performed at lower temperatures, e.g. in a TLC Thermo-Box (see Section 4.2.3 Effect of Temperature in Chromatography ). 

Betanine can be analysed from its uv absorption maximum at 535nm (at pH 3) while betaxanthine absorbs at 480nm. Preparation for chromatographic examination (ref. 11) by TLC on cellulose is effected by firstly homogenisation of the beetroot sample (50g) with aqueous methanol (1 1), filtration and concentration at less than 40°C. The residual solution is applied to the plate as a streak and doubly developed with isopropanol-ethanol-water-acetic acid (30 35 30 5). After drying of the plate under nitrogen, betanine appeared as a violet zone at Rf 0.27 with four other bands present. 

It is ommon practice in thin-layer chromatography, after application of a sample to a given plate, to develop the plate two or more times with drying of the plate between developments. This general technique will be referred to as multiple development. Three types of multiple development... 

In TLC, data acquisition is one, independent, off-line operation in a sophisticated analytical procedure that starts with application, continues with development (and occasionally postrun plate treatment), and finishes with scanning of developed plates. As a developed and dried plate can no longer be changed, the operator must find and select the best scanning conditions. It is necessary, in the name of correctness, to prepare SOPs (system operating procedures) so that operator is allowed, according to his skill and feel, to select freely the correct scanning conditions, since the best choice of conditions very often depends on the results of previous operations. The purist in CLP may take issue with this tactic, but this is the only way to eliminate systematic errors. 

A TLC system using cellulose plates (0.5 mm) has been reported for the preparative separation of betalains from beetroot (65). High sample load (200 mg pigment) required a prerun in the polar solvent, 2-propanol-ethanol-water-acetic acid (6 7 6 1). After development to 10 cm followed by extensive drying of the plate, betanin was finally separated from the betaxanthins using two successive developments (15 cm) with the same solvent components in different proportions (10 4 4 1). 

The U.S. domestic capacity of ammonium perchlorate is roughly estimated at 31,250 t/yr. The actual production varies, based on the requirements for soHd propellants. The 1994 production ran at about 11,200 t/yr, 36% of name plate capacity. Environmental effects of the decomposition products, which result from using soHd rocket motors based on ammonium perchlorate-containing propellants, are expected to keep increasing pubHc pressure until consumption is reduced and alternatives are developed. The 1995 price of ammonium perchlorate is in the range of 1.05/kg. Approximately 450 t/yr of NH ClO -equivalent cell Hquor is sold to produce magnesium and lithium perchlorate for use in the production of batteries (113). Total U.S. domestic sales and exports for sodium perchlorate are about 900 t/yr. In 1995, a solution containing 64% NaClO was priced at ca 1.00/kg dry product was also available at 1.21/kg. 

The chromatogram is freed from mobile phase and evenly sprayed with the spray solution or immersed for 1 s in the dipping solution. After drying the TLC plate is heated to 85 —120°C normally for 10 to 15 min but in exceptional cases for 60 min. It is advisable to observe the chromatogram during the reaction period, because the temperature and duration of heating strongly affect color development. 

HPTLC plates Silica gel 60 (Merck), which had been prewashed by single development of the plate with chloroform — methanol (50-1-50) and then dried at 110°C for 30 min. 

Repeated chromatography in a third dimension after completion of two-dimensional development. Here, development in the first, second, and third dimensions can be envisaged as occurring on three plates arranged in the form of a cube the plate is again dried between developments. 

Procedure. Pour the developing solvent into the chromatographic tank to a depth of about 0.5 cm and replace the lid. Take a prepared plate and carefully spot 5 pL of each indicator on the origin line (see Section 8.6, under Sample application) using a micropipette. Allow to dry, slide the plate into the tank and develop the chromatogram by the ascending solvent for about 1 h. Remove the plate, mark the solvent front and dry the plate in an oven at 60 °C for about 15 min. Evaluate the R value for each of the indicators using the equation... 

HPTLC plates Silica gel 60 F254 (Merck) that were prewashed before application of the sample, by developing once to the upper edge of the plate with chloroform - methanol (50+ 50), and then dried at 110 Cfor30 min. In the case of example A. the layer was conditioned to 0% rel. humidity in a conditioning chamber (over cone, sulfuric acid) after sample application. 

---