Location of Components— The spots produced by paper or thin-layer chromatography may be located by:(1)direct inspection if the compounds are visible under white or either short-wavelength (254nm)or long-wavelength (360nm)UVlight,(2)inspection in white or UVlight after treatment with reagents that will make the spots visible (reagents are most conveniently applied with an atomizer),(3)use of a Geiger-Müller counter or autoradiographic techniques in the case of the presence of radioactive substances,or (4)evidence resulting from stimulation or inhibition of bacterial growth by the placing of removed portions of the adsorbent and substance on inoculated media.
In open-column chromatography,in pressurized liquid chromatography performed under conditions of constant flow rate,and in gas chromatography,the retention time,t,defined as the time elapsed between sample injection and appearance of the peak concentration of the eluted sample zone,may be used as a parameter of identification.Solutions of the substance to be identified or derivatives thereof,of the reference compound,and of a mixture of equal amounts of these two are chromatographed successively on the same column under the same chromatographic conditions.Only one peak should be observed for the mixture.The ratio of the retention times of the test substance,the reference compound,and a mixture of these,to the retention time of an internal standard is called the relative retention time RRand is also used frequently as a parameter of identification.
The deviations of RR,RF,or t values measured for the test substance from the values obtained for the reference compound and mixture should not exceed the reliability estimates determined statistically from replicate assays of the reference compound.
Chromatographic identification by these methods under given conditions strongly indicates identity but does not constitute definitive identification.Coincidence of identity parameters under 3to 6different sets of chromatographic conditions (temperatures,column packings,adsorbents,eluants,developing solvents,various chemical derivatives,etc.)increases the probability that the test and reference substances are identical.However,many isomeric compounds cannot be separated.Specific and pertinent chemical,spectroscopic,or physicochemical identification of the eluted component combined with chromatographic identity is the most valid criterion of identification.For this purpose,the individual components separated by chromatography may be collected for further identification.