THE RETARDATION FACTOR (Rf) – Concept & calculation in TLC/HPTLC:
Concept of Rf value in TLC/HPTLC:
In TLC/HPTLC, basically, two phases are important i.e. stationary phase and mobile phase. The gas phase also plays an important role if we are giving saturation.
Nowadays stationary phase is mostly silica gel and mobile phase selection depends on the analyte of interest. Usually, it could be a mixture of binary, tertiary or quaternary solvents.
The compound/Analyte of interest is dissolved in a suitable diluent, and applied onto the stationary phase in the form of spots/bonds and the plate is developed in a suitable mobile phase. The analyte and the mobile phase move over the stationary phase due to the capillary action.
The differential mobility of different compounds is achieved by the relative affinity of compounds towards the stationary phase and mobile phase. The compounds with a higher affinity towards the stationary phase move slowly as the stationary phase retains the respective compounds.
Since the stationary phase is polar in TLC, polar compounds move slowly. However, compounds with a higher affinity towards the mobile phase move faster through the stationary phase. The mobile phase is relatively less polar and, non-polar compounds, which have less affinity towards the stationary phase, move faster through the stationary phase.
After completion of the development step, separated compounds can be seen as spots/bands in short UV (254nm), long UV (366nm) or in white light depending on the response of the analyte, with the help of suitable TLC photo documentation device like CAMAG visualizer. CAMAG TLC scanner can be used for quantification, data like Rf values, area under the peak etc.
Response/Migration of analyte is expressed in terms of retardation factor/ratio to front). The retardation factor (RF) in planar chromatography, also known as the retention factor, is a term used to describe the relative positions of zones in the chromatogram.
It is calculated based on the ratio of the distances travelled by the centre of a zone “A” (b) and the distance travelled by the mobile phase front (a) with respect to the application position as shown in the above diagram.
By definition, Rf values are always less than one, and they are displayed with two decimals. An Rf value of 1 or too close to it means that the spot and the solvent front travel close together and are therefore unreliable.
IUPAC defines hRf as Rf multiplied by 100, but most pharmacopoeias and publications use RF.
The Rf values for a given compound should stay constant if you are following same chromatographic conditions every time you are repeating the analysis.
What are the factors which affect Rf values in TLC/HPTLC?
The Rf values depends on the sample application position but also the level of developing solvent in the chamber which should be kept constant. Most significantly Rf values may show variation due to humidity in the air or the development which is carried out in different humid conditions.
This problem is often encountered while doing TLC. However, with High Performance Thin Layer Chromatography, humidity can be controlled by using saturated solution of magnesium chloride hexahydrate solution.
As HPTLC is an official technique in United States Pharmacopoeia (USP), its guidelines suggest development in a controlled environment of 33 % humidity which is achieved a by a saturated solution of magnesium chloride.
CAMAG ADC2 (Automatic development Chamber) is used for isocratic development. Steps like plate development, monitoring and plate drying is completely automatic and under controlled humidity.
Humidity can be controlled at 33% by using saturated solution of magnesium chloride. This complies with the USP guidelines for plate development and it ensures reproducible Rf values every time analysis is repeated.