Figure 4.48
Thin-layer chromatogram.Factors Affecting Rf Values and Resolution
Basically, the Rf value of a solute is determined by its distribution ratio which in turn is dependent on
relative solubilities for partition systems or relative polarities for adsorption systems. For example, if
adsorption TLC is used to separate a mixture of squalene (a hydrocarbon), methyl oleate, cholesterol
and α-tocopherol (vitamin E), then squalene, being the least polar, will move furthest and the
cholesterol, being the most polar, will remain close to the origin. Methyl oleate is less polar than α-
tocopherol and will therefore be found between it and the squalene. The role of polarity is discussed
more fully on p. 82.
The effect of temperature on distribution ratios has already been mentioned on page 91. Although the
separation proceeds more quickly at elevated temperatures, resolution suffers because of increased rates
of diffusion. However, in adsorption TLC only small increases in Rf values are observed even with a
20 °C rise. Strict temperature control is not necessary if samples and standards are run at the same time,
although large fluctuations should be avoided. The quality of the thin-layer materials, and in particular
the presence of impurities in them, determine the extent to which partition, adsorption, ion-exchange
and exclusion participate in the sorption process. These factors affect Rf values in an unpredictable
manner. Thin layers should be of uniform thickness, between 0.2 and 0.3 mm; with thinner layers, local
variations in thickness can result in appreciable variations in Rf values.
A stable atmosphere saturated with the vapour of the mobile phase is required to ensure reproducible Rf
values. Unless saturation conditions prevail, solvent will evaporate from the surface of the thin layer
causing an increased solvent flow but slower movement of the solvent front; Rf values consequently
increase. In practice, chromatograms are best developed in a sealed glass tank in which a saturated
atmosphere has been produced by