template instead of a sharp pencil to mark the point of sample application. Apparently the sample became layered in the depression and failed to reach equilibrium conditions with the developing solvent. The chromatogram f’or the analysis of Tuasal bacteriostat is illustrated in Figure 1. Methanol/acetic acid (19:l) was the best solvent system for the determination of all three impurities using a singlc development. A smaller acetic acid concentration would fail to resolve 3,5-dibromosalicylanilide and 2 ’,3,4’,5-tribromosiilicylanilide, while a greater concentration of acetic acid would cause the 4’,5-dibromosalicylanilide to proceed into the secondary solvent front, reducing spot uniformity. Because 2 ’,3,5-tribro nosalicylanilide has an R f value near that of 3,4’,5-tribrornci- and 2’,3,4’,5-tetrabromosalicylanilide, it would not be detected using the 19:l methanol/acetic acid solvent system. However, by multiple development with this solvent system or a moderately higher one, resolution of this impurity can be realized. A comparison of thc TLC method with that of the ion exchange procedure for the determination of 4’,5-dibromosalicylanilide is given in Table 11. The ion exchange method by its very nature has Iexcellent accuracy and precision and would be considered the standard method in this comparison. Generally the thin layer results agree to within =kO.lxabsolute of the amount present in the 0.1 to 0.5 range.
x
Table 11. Comparison of 4’,5-Dibromosalicylanilide Determination in 3,4’,5-Tribromosalicylanilide Ion exchangeultraviolet method ( 7 ) Thin layer method
