lining the walls with filter-paper soaked in the appropriate solvent or solvent mixture. The tank should
be prepared about 15 minutes before the chromatogram is to be developed.
The optimum amount of sample required to produce detectable spots with a minimum of spreading due
to overloading is about 0.01 μg to 50 μg. With samples much larger than this, isotherms become non-
linear, the Rf values alter significantly with sample size and resolution suffers because of increased
tailing or fronting.
Sample Application
The process of placing a sample on the thin layer is called spotting. The sample should be dissolved in
up to 10 μl of a solvent which should be volatile so as to prevent undue spreading of the spot.
Accurately measured amounts of sample can be spotted using a micropipette or microsyringe but for
qualitative purposes a melting-point tube drawn out to a fine tip will suffice. A faint pencil line can be
drawn about 1–2cm from one edge of the plate and the sample spots placed along this line 1 cm apart
and not closer than 1.5 cm to the sides. A ruler or raised Perspex template can be used to aid spotting
the samples on to the plate accurately.
Development Procedures
For convenience, the ascending method is used mostly although there is no reason why descending or
horizontal development cannot be used for special purposes. An alternative method consists of forming
a sandwich with a glass plate the same size as the chromatographic plate. The two are clamped together
with a thin spacer to protect the surface of the thin layer and the bottom side is immersed in a narrow
trough containing the solvent. The small volume of air trapped between the two plates is rapidly
saturated with the developing solution.
Two-dimensional Development
More effective separation of complex mixtures and those consisting of components with similar Rf
values can be achieved by successive developments at 90° to each other. The sample is spotted at one
corner and developed with the first solvent. After thorough drying, the plate is turned through 90° and
developed with the second solvent. The two-way chromatogram (Figure 4.49) can be compared with a
'standard map' obtained from chromatographing known mixtures.
Detection of Separated Components
Coloured substances can be observed visually in situ after development of the chromatogram, but those
which are colourless must be visualized by physical or chemical means. For many samples, and
especially for