Page 260 - Vogel's TEXTBOOK OF QUANTITATIVE CHEMICAL ANALYSIS

8 COLUMN AN0 THlN-lAVER LlilUlO CHROMATOCRAPHV

for known quantities of the solute in the chosen solvent. Quantitative thin-layer
elution techniques have been reviewed by
It should be noted that to obtain the best results in any of these quantitative
TLC methods, the spots being used should have R, values between 0.3 and 0.7;
spots with Rf values <0.3 tend to be too concentrated whereas those with Rf
values >0.7 are too diffuse.

8.7 HlGH PERFORMANCE THIN-LAYER CHROMATOGRAPHY (HPTLC)
Recent developments in the practice of thin-layer chromatography have resulted
in a breakthrough in performance which has led to the expression 'high
performance thin-layer chromatography'. These developments have not been
the result of any specific advance in instrumentation (as with HPLC), but rather
the culmination of improvements in the various operations involved in TLC.
The three chief features of HPTLC are summarised below, but for a
comprehensive account of the subject the reader is recommended to consult a
more specialised te~t.~'
Quality of the adsorbent layer. Layers for HPTLC are prepared using specially
purified silica gel with average particle diameter of 5-15 pm and a narrow
particle size distribution. The silica gel may be modified if necessary, e.g.
chemically bonded layers are available commercially as reverse-phase plates.
Layers prepared using these improved adsorbents give up to about 5000
theoretical plates and so provide a much improved performance over
conventional TLC; this enables more difficult separations to be effected using
HPTLC, and also enables separations to be achieved in much shorter times.
Methods of sample application. Due to the lower sample capacity of the HPTLC
layer, the amount of sample applied to the layer is reduced. Typical sample
volumes are 100-200 nL which give starting spots of only 1.0-1.5 mm diameter;
after developing the plate for a distance of 3-6cm, compact separated spots
are obtained giving detection limits about ten times better than in conventional
TLC. A further advantage is that the compact starting spots allow an increase
in the number of samples which may be applied to the HPTLC plate.
The introduction of the sample into the adsorbent layer is a critical process
in HPTLC. For most quantitative work a platinum-iridium capillary of fixed
volume (100 or 200 nL), sealed into a glass support capillary of larger bore,
provides a convenient spotting device. The capillary tip is polished to provide
a smooth, planar surface of small area (CU 0.05 mm2), which when used with a
mechanical applicator minimises damage to the surface of the plate; spotting
by manual procedures invariably damages the surface.
The availability of scanning densitometers. Commercial instruments for in-situ
quantitative analysis based on direct photometric measurement have played an
important role in modern thin-layer chromatography. Although double beam
instruments are available, single beam single wavelength operation is mainly
used in HPTLC since the quality and surface homogeneity of the plates are
generally very good.
High performance thin-layer chromatography has found its greatest application
in the areas of clinical (e.g. analysis of drugs in blood) and environmental
analysis.