Instant Notes: Analytical Chemistry

Solute detection Solutes separated by TLCremain on the surface of the plate after development.
As the majority of solutes are colorless, their spots must be locatedusing a
chemical or physical means of visualization. Chemical methods include:

● Spraying the plate with a locating, or chromogenicreagent that reacts chemi-

cally with all solutes or those containing specific functional groups or struc-

tural features to give colored spots. The plates sometimes need to be warmed

to accelerate the chromogenic reaction and intensify the spots. Examples are

given in Table 2;

● Viewing the plate under a UV lampset at an emission wavelength of 254 nm

or 370 nm to reveal the solutes as dark spots or bright fluorescent spots on a

uniform background fluorescence. Commercial plates can be purchased with

an insoluble fluorescent substance incorporated into the stationary phase to

provide the background fluorescence, or the plate can be sprayed with a

fluorescent reagent after development;

● Spraying the plate with concentrated sulphuric or nitric acid and heating to

oxidize and char organic solutes which are revealed as brown to black spots;

● Exposing the plate to iodine vapor in a sealed chamber when many organic

solutes develop dark brown colorations;

● Scanning across the surface of the plate with a densitometer, an instrument

that measures the intensity of radiation reflected from the surface when irra-

diated with a UV or visible lamp. Solutes that absorb radiation are recorded

as peaks on a recorder trace or VDU screen;

● Radiolabelled solutes can be detected by autoradiography (blackening of a

photographic film sensitive to X-rays), liquid scintillation counting on

scraped-off areas of the stationary phase, or monitoring the surface of the

plate with a Geiger-Müller tube.

Table 2. Some examples of TLC locating reagents

Method of detection Color of solute spots Application

General reagents

Phosphomolybdic acid +heat Dark blue Many organics

Conc. sulphuric acid +heat Charred brown-black All organics

Iodine vapor Brown Many organics

Selective reagents

Ninhydrin Pink to purple Amino acids and amines

2,4-Dinitrophenylhydrazone Orange/red Carbonyl compounds

Bromocresol green/ blue Yellow Organic acids

2,7-Fluorescein Yellow-green Most organics

Vanillin/ sulphuric acid Blue, green, pink Alcohols, ketones

Rhodamine-B Red fluorescence Lipids

Anisaldehyde/antimony trichloride Various Steroids

Diphenylamine/zinc Various Pesticides

Variations of the basic development procedure and new stationary phases

aimed at improving resolution, sensitivity, speed, reproducibility and selectivity

have been developed from time to time. Two of the more significant and useful

ones are:

● Two-dimensional TLC. This is a means of improving resolution in samples

where the component solutes have similar chemical characteristics and hence

Alternative TLC
procedures

134 Section D – Separation techniques