EVALUATION OF THE CHROMATOGRAM
After completing the detection procedure the various separated solutes on the TLC plate are marked with the help of a sharp needle (e.g., pithing needle) ; subsequently, their evaluation may be carried out either qualitatively or quantitatively, as stated below :
The Rf value (Retention Factor) various separated solutes is determined accurately. The Rf value repre-sents the differences in rate of movement of the components duly caused by their various partition coefficients i.e., their different solubility in the mobile and stationary phases. In order words, the Rf value (relate to front) is-‘the ratio between the distance starting point-centre of spot and distance starting point-solvent front’, thus it may be expressed as :
Important Points :
(i) Due to the always longer path of the solvent front, the Rf value is invariably lesser than 1.
(ii) Rf value is always constant for each component only under identical experimental parameters, and
(iii) Rf value depends upon a number of governing factors, such as : quality of the layer material ; activation grade of the layer ; thickness of layer ; quality of solvent ; equilibration of chamber ; chromatographic technique employed (e.g., ascending, descending) ; presence of impurities ; and conc. of simple applied ; and
(iv) All possible anomalies in (iii) above may be eliminated by performing a co-chromatogram of a standard substance along with that of a sample. Thus, the distance traversed by a substance is compared with that of the standard (or reference). This ‘new’ relation is usually designated as Rst-value. Therefore, in short, it is expressed as follows :
Unlike the Rf value, the Rst value may be more than 1.00 because here the substance under investiga-tion (i.e., sample) usually travels further than the standard.
In TLC, the qualitative evaluation is solely based on the determination of Rf values of unknown spots vis-a-vis Rf values of standard substances preferably on the same TLC plate so as to avoid any possible error whatsoever.
The quantitative analysis of chromatographically separated constituents may be carried out with high degree of accuracy and precision in two manners, namely :
(i) Direct Method : i.e., the quantitative determinations is performed directly on the adsorbent layer, and
(ii) Indirect Method : i.e., the separated constituents are quantitatively removed from, the adsorbent and subsequently estimated after elution.
The various methods under this category are, namely :
(i) Measurement of Spot-areas : This method is solely based on a mathematical relationship existing between the prevailing spot area and the amount of component present. It is not quite accurate due to high random errors.
(ii) Densitometry : The intensity of the colour of a component is measured on the chromatogram using a densitometer.
(iii) Spectrophotometry : Characterization of the separated spots by reading the absorption or fluores-cence curves directly from TLC plates is carried out with the help of Chromatogram Spectrophotometer devised by Zeiss, Stahl and Jork.
Besides, IR-spectroscopy, reflectance spectroscopy, spark chamber method etc., may also be employed for the direct evaluation of chromatograms.
These methods are based on elution techniques, followed by micro-analysis of the resultant eluate by adopting one or more of the undermentioned known methods, namely :
Colorimetry ; Fluorimetry ; Radiometry ; Flame-photometry ; UV-Spectrophotometry ; Gravimetry ; Polarography ; Vapourphase Chromatography ;