Barbiturate Analysis

Reagent and Chemicals
1# Chloroform mixture 9:1 (v/v)

2# HgSO4 spray
suspend, with mixing 5 gr HgO in 100 ml H2O
Add while mixing 20 ml H2SO4
Cool dilute to 250 ml H2O

3# Diphenylcarbazone spray
dssolve 5 gr in 50 ml ChCl2 store in dark bottle

4# Fluorescein spray
to 500 ml H2O add 20 gr NaOH and 20 gr sodium fluoroscein

5# KMNO4 spray
0.1% aqueous solution

6# Standards barbiturate acids
10 mg/100 ml (w/v) in CHCl2

6# Silica gel"G"
According to Stahl

Equipment
1. satndard apparatus for coating glass plates with a 250-u layer of silica gel "G"
2. rectangular jars, 8 1/2 x 4 x 8 1/2 inch with ground glass cover
3. spray bottles
4. Tuberculin syringes with size 24 1/4 inch needles
5. Mineralite ultraviolet hand lamp with 254mu filter

Method

Plates (20 x 20 cm.) were coated with Silica gel G using standard thin-layer chromatography technics. These coated plates were dried in a ventilated oven for 20 mm. at 100 celcius.The dried plates were then stored at room temperature for subsequent use. Prior to an analysis, lines 1/2 inch a part were ruled on the plate, forming a series of parallel columns for concurrent analyses of many samples.

Two drops of a chloroform solution of each sample were applied at a point 1 in. from the bottom of the plate in the center of a column. The spots were air dried and then the plate was placed in the rectangular jar containing 110 ml. of the 9:1 chloroform-acetone mixture. The mixture should beplaced in the jar at the beginning of the preparation for this analysis to insure saturating the jar with the mixture’s vapor. This jar can be reused and its solvent mixture need not be changed for 2 days.

In this solvent system, the solvent front will rise 10-12 cm. in 45-60 mm. The plate is then removed and the solvent front marked immediately. Then the plate is air dried at room temperature. When dry, the plate is sprayed first with the HgSO4, allowed to dry partially, and then sprayed again with the diphenylcarbazone. In each instance the size, position, and color intensity of each spot is noted. An excess of the diphenylcarbazone is used. The overall colored background resulting slowly disappears and purple or violet spots remain to allow accurate determination of their respective Rf values.

A second identification system that has proved effective consists of an initial spray with alkaline fluorescein. When viewed under 254 m light the barbiturates give a purple spot against a yellow-green background. After completing this observation, the plate may then be sprayed with the KMnO4). All unsaturated substituted barbiturates yield yellow spots against a purple background. The same KMnO4 spray can be used without the fluorescein to give similar data.

Since 12-14 columns can easily be ruled on one plate, the same sampie and suitable standards can be applied to consecutive columns, and then a second or third series can be placed on the next group of columns on the same plate. Several different sprays can then be used on one plate by suitably masking all but one series of columns for each spray. On each plate, known barbiturates, singly and in mixtures, should be run.

Sample Preparation
Samples for analysis are isolated by direct CHC13 extraction at pH 7. Blood and tissue samples require no p11 adjustment, but urine, stomach content, and drugs should be made alkaline or dissolved in NaOH. The alkaline solution should be filtered and a 3-5 ml. sample taken. These are then made mildly acid with dilute mineral acid and extracted with 50 ml. CHC13.

A filtered 40-ml. aliquot of the chloroform is then evaporated to dryness tInder forced air and mild heat. The residue is dissolved in 0.5 ml. CHCL, and portions of this solution are applied to the plate along with suitable standards and mixtures there of.
Useful Links:
Barbiturat Class
Barbituric Acid Chemical Structure and Properties