Figure 4.49
Two-dimensional chromatogram; standard map for amino acids- taurine 2. glutamic acid 3. serine 4. glycine 5. threonine 6. lysine
- alanine 8. α-amino-n-butyric acid 9. arginine 10. hydroxyproline
11. tyrosine 12. valine 13. leucine 14. β-phenylalanine 15. proline
unknowns, the chromatogram should first be examined under a UV lamp. Many organic compounds
show up as fluorescent spots when irradiated at 370 or 254 nm. An alternative method is to incorporate
a fluorescent indicator into the thin layer when non-fluorescing materials show as dark spots on a
fluorescent background. Radioactive counting techniques can be used for the detection of labelled
compounds.
The most widely used detection methods are those in which spots are visualized chemically by spraying
with a chromogenic reagent or by dipping into a reagent solution. Dipping provides a more even cover
with the reagent, but may dissolve some of the separated components or cause spreading of the spots by
diffusion. Solvents used in both methods should be reasonably volatile to facilitate drying after
treatment. Acetone, ethanol and chloroform are commonly used. Reagents may be general in that they
produce coloured spots with a wide range of compounds or they may be selective in reacting only with
compounds containing certain functional groups. Metals can be visualized by using a reagent with
which they form coloured insoluble salts or complexes. Some examples of chromogenic reagents and
their applications are given in Table 4.15(a). A general and very sensitive method for detecting organic
substances separated by TLC is to spray the chromatogram with concentrated sulphuric acid and heat
the plate to about 200°C for several minutes. The charred organic matter shows up as brown or black
spots.
Chromatograms can be stored easily for future reference unless, as sometimes happens, the spots fade.
More permanent less bulky records can be made by photographing the chromatogram, spraying it with a
lacquer or photocopying it.