TLC of Barbiturates and Benzodiazepines

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Having carried out presumptive tests for these drugs, if a positive result occurs, then further analysis is required. The next phase in the analysis is TLC. Methanol solutions of the drugs can be used, while the standards employed should include the drug which has been suggested through physical examination of the dosage form. However, these drug groups also present some special difficulties in their TLC analysis - these are discussed below.

9.2.3.1 TLC Analysis of Barbiturates

In this case, the TLC system most commonly employed uses silica gel plates and a mobile phase of ethyl acetate/methanol/25% ammonia (85:10:5, by volume). The plates are prepared and the chromatogram developed in the standard way. After development, the plate is removed from the mobile phase, the solvent front marked, and the plate dried. Visualization of barbiturates is best achieved by the use of a mercuric chloride-diphenylcarbazone reagent. The latter is prepared as two component solutions, i.e. (i) 0.1 g of diphenylcarbazone in 50 ml of methanol, and (ii) 0.1 g of mercuric chloride in 50 ml of ethanol. These solutions should be freshly prepared and mixed just before use. The presence of barbiturates will give rise to blue-violet spots on a pink background when using this reagent system.

What are the health and safety issues associated with the use of this (spray) reagent?

Answer

All mercuric compounds are extremely toxic, and thus there are health hazards associated with the use of the mercuric chloride-diphenyl car-bazone reagent. Testing should ideally be carried out in a fume cupboard, with standard safety procedures being observed, e.g. the use of rubber gloves, etc. In addition, there is the risk of potential contamination of the working area - this should be thoroughly cleaned after use. Finally, special precautions need to made with respect to the disposal of any waste materials after testing has been carried out.

Table 9.1 below shows the data obtained for a number of different barbiturates after TLC analysis under the conditions described above. Why are the Rf values presented for the alkyl compounds so similar and what is the difficulty with this fact?

Answer

The reason that the Rf data are so similar is that straight-phase TLC is principally based on sorption-desorption processes. The only variation between the barbiturates is the nature of the substituent at the C5 position. For those compounds with alkyl chains, for example, barbitone, butobar-bitone and pentobarbitone, the length and polarity of the side-groups will affect the overall polarity of the molecule through van de Waals forces, which are relatively weak in comparison to other types of interactions which result from polarity considerations. The consequence of this is that there is very little difference in polarity to exploit for the chromatographic separation of these molecules. (Similar polarization effects are also operational in the case of the ring-substituted compounds - see below.) When these facts are coupled to the lack of resolving power of TLC in general, it becomes clear that there is insufficient difference to identify barbiturates on a definitive basis by using this chromatographic technique.

SAQ 9.1

Why is the Rf value of phenobarbitone less than cyclobarbitone (see Table 9.1)?

9.2.3.2 TLC Analysis of Benzodiazepines

The benzodiazepines are a diverse group of drugs and require a combination of different TLC solvent systems to resolve the many drugs in this group. The systems which have been used include chloroform/acetone (80:20 and 90:10, by volume) and cyclohexane/toluene/diethylamine (75:15:10, by volume). The

Table 9.1 Structures of a number of different barbiturates and corresponding chromatographic data obtained after analysis by TLC (DQ 9.2 and SAQ 9.1)

Barbiturate

Structure

Barbitone

Butobarbitone

Pentobarbitone

Phenobarbitone

Cyclobarbitone

ch3ch2x

ch3ch2

CH3CH2CH2CH2'

O

ch3ch2

ch3ch2ch2ch

ch3ch2

ch3ch2ch2ch

ch3 o

0.33

0.39

0.44

0.29

0.35

chromatographic plates are prepared, developed and dried in the same way as that used for barbiturates. However, the principle difficulty in this case lies in the fact that there is no specific development reagent for benzodiazepines. The reagents that have been used include 1 M H2SO4, which is sprayed onto the plate, and after heating is subsequently viewed under UV light (at 366 nm). Any fluorescent spots are recorded. The chromatogram is then oversprayed with acidified potassium iodoplatinate reagent, which forms purple spots. If the colour reactions observed for the samples and standards match, then further analysis is required.

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