Page 373 - Academic Press Encyclopedia of Physical Science and Technology 3rd Analytical Chemistry

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Encyclopedia of Physical Science and Technology EN0011A-541 July 25, 2001 17:27

476 Organic Chemistry, Compound Detection

compares to typical values for conventional TLC plates of detection reagent iodine can be used as a 1% alcoholic so-
some 2000 theoretical plates over 12 cm in 25 min. lutionspray,butmorefrequently,theplateissimplyplaced
in a closed container containing a few iodine crystals. The
iodine vapor forms weak charge-transfer complexes with
B. Impregnated Layers
most organic compounds which show up as brown spots
The range of applications of both TLC and HPTLC is con- on a pale yellow background within a few minutes. Sen-
siderably expanded by means of different impregnation sitivities in the 0.1–0.5-µg range are often obtained with
agents such as acids, bases, or salts added to layers in var- iodine.
ious concentrations. Stable hydrophilic stationary phases Charring reagents (H 2 SO 4 ) are suitable for glass-
are formed by treatment with agents such as formamide, backed layers with inorganic (e.g., gypsum) binders only.
DMF, ethylene glycol, and various buffers. Lipophilic sta- Many charring reagents produce colored zones when heat-
tionary phases for reversed-phase TLC are obtained by im- ing is carried out at relatively low temperature; they form
pregnation with liquid paraffin, undecane, and mineral and black zones at higher temperatures.
silicone oils. Impregnation with specific reagents aids the Spraying of a chromatogram with a 5% solution of
separation of certain types of compounds such as AgNO 3 phosphomolybdic acid followed by a brief heating at
forcompoundswithdoublebonds,boricacidorsodiumar- 110 C gives dark blue spots against a yellow background
◦
senite for vicinal dihydroxy isomers; sodium bisulfite for with a large variety of organic compounds.
carbonyl compounds, and trinitrobenzene or picric acid A solution of Rhodamine B produces violet spots on a
for polynuclear aromatic compounds. pink background. Antimony trichloride or pentachloride
solution in carbon tetrachloride produce spots of different
characteristic colors with many organic compounds.
C. Detection and Visualization
Over 300 spray reagents are known to react more or
Following development, chromatograms are removed less specifically with different functional groups to reveal
from the chamber and are air- or oven-dried to remove natural products and organic or biochemicals as colored or
the mobile phase, zones are detected by various means. fluorescent zones. Table IV contains a selection of specific
Colored substances may be viewed in daylight without detection reagents. Methods for the quantitation of thin-
any treatment. Detection of colorless substances is sim- layer chromatograms can be divided into two categories.
plest if compounds show self-absorption in the short- In the first, solutes are assayed directly on the layer, either
wave ultraviolet (UV) region (254 nm) or if they can by visual comparison, area measurement or densitometry.
be excited to produce fluorescence by short-wave and/or In the second, solutes are eluted from the sorbent before
by long-wave (365 nm) UV radiation. Otherwise, detec- being examined further.
tion can be achieved by means of chromogenic reagents
(producing colored zones), fluorogenic reagents (pro-
D. Radiochemical Techniques
ducing fluorescent zones), or by biological enzymatic
methods. Radioactive isotopes are widely used as tracers or labels
Enzymatic reactions can be monitored on the plate, for substances separated by TLC for following the causes
and the end products can be detected. Biological test of chemical and biochemical reactions, determining the
procedures are used in the specific detection of biolog- distribution of substances in a reaction mixture, elucidat-
ically active compounds. Thus, detection of hemolyz- ing metabolic pathways of drugs, pesticides, pollutants,
ing compounds such as saponins is achieved by casting and natural substances in human, animal and plant tis-
a blood–gelatin suspension on the layer and observing sues, and assessing the purity of isotopes. The most widely
3
hemolytic zones that are transparent and nearly color- used labelled substances in TLRC contain H (tritium)
14
less on the turbid red gelatin layer background. An- and C.
other means of detection is the use of Geiger or flow The detection of radioactive substances on TLC plates
counters or other specialized means to locate radioactive is carried out by liquid scintillation, film registration or
solutes. autoradiography, and by direct scanning. A thermomicro
Detection reagents may be impregnated into the procedure for rapid extraction and direct application in
layer prior to sample application and development. TLC is the thermomicro application of substances (TAS)
Chromogenic reagents are of two types: (1) general method.
reagents that react with a wide variety of different com- The TAS method is a procedure for the isolation and
pound types and can totally characterize an unknown sam- separation of many substances from solid materials and
ple, and (2) specific reagents that indicate the presence of their direct transfer to the starting line on a TLC plate. This
a particular compound or functional group. The universal is performed by application of heat, either by distillation