Barbiturates background

The analysis of clinical samples is often complicated by the complexity of the sample matrix, which may contribute a significant background absorption at the desired wavelength. The determination of serum barbiturates provides one example of how this problem is overcome. The barbiturates are extracted from a sample of serum with CHCI3, and extracted from the CHCI3 into 0.45 M NaOH (pH 13). The absorbance of the aqueous extract is measured at 260 nm and includes contributions from the barbiturates as well as other components extracted from the serum sample. The pH of the sample is then lowered to approximately 10 by adding NH4CI, and the absorbance remeasured. Since the barbiturates do not absorb at this pH, the absorbance at pH 10 is used to correct the absorbance at pH 13 thus... 

In the case of organophosphorus insecticides [7] and usually also in the case of barbiturates and other pharmaceuticals [1, 3] white chromatogram zones are produced on a lilac background. Barbiturates sometimes appear — especially after the use of basic mobile phases — as blue-violet colored zones on a pink background [2, 5]. 

Barbiturates and their metabolites always appear as red-violet chromatogram zones on a white background with this variant of the reagent [6]. 

Note The reaction for barbiturates according to variant I is increased in sensitivity if the chromatogram is exposed to direct sunlight or UV light after it has been sprayed this causes the background coloration to fade almost completely and the blue zones stand out more distinctly [4]. 

Cover columns 3 to 8 with a glass plate and spray columns 1 and 2 with mercuric chloride-diphenylcarbazone reagent White spots on a violet background indicate the presence of barbiturates or related compounds (e.g. glutethimide). Cover columns 1 and 2 and 5 to 8 with glass plates. 

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. 

Sabatino51 applied the barbiturate in acid form in a suitable solvent about 1 inch from the edge of an 8" x 8" Whatman No. 1 sheet of paper. A standard barbiturate solution (0.001 ml. of a 20 mg./lO ml. alcohol) was spotted along with the sample. The paper was mist sprayed with 0.5 M sodium carbonate and placed in the chromatograph tank containing redistilled ethylene chloride as the mobile solvent. After development of 4-6" above the spotting line, the paper was removed and dried at 100°C. in a forced draft oven. It was then sprayed with 0.05 N silver nitrate in alcohol solution and placed in the oven until tan spots on a brown background appeared. 

Detection of barbituric acids is possible non-specifically by chromatography on so-called fluorescent layers, e. g., silica gel GF254. If not present in too small amounts, they appear in short wave UV light as spots of quenched fluorescence. A popular method of detection has been with mercury compounds, especially mercurous nitrate (Rgt. No. 157) which yields insoluble barbiturates [10, 34, 43, 56, 77, 140, 166]. The sensitivity of response varies with the barbituric acid. A combined spray reagent of a mercuric salt and diphenylcarbazone solutions (No. 156) has been often used the spots then stand out better against the background [32, 93, 160]. Cobalt salts have been used with various bases for detection (Zwikker-reaction and modifications, Rgt. No. 51) [56, 152, 170, 174]. N-methylated barbituric acids do not react since the second nitrogen atom cannot take part in enolisation [98]. A further, non-specific method... 

---