Zonal Analysis

The basic procedure involves removing areas of chromatographic adsorbent from a TLC plate

Table 1 Approximate lower detection limits on TLC plates for various exposure methods (dpm cm 2 with a 24-h exposure)

Exposure method 3H 14C 32P 125I

Direct exposure (autoradiography) 2.6-13 x105a 220-650a 500d 1600d

Direct exposure with intensifying screen 50de 100de

Fluorographic exposure (fluorography) 2.0-6.6 x103 bc 50-450 bc aAverage range for direct exposure of film at temperatures between —78.5 and 25°C.

b Treated with a 7% solution of 2 , 5-diphenyloxazole (PPO) in diethyl ether and exposed at a temperature of — 78°C. cTreated with a mixture of 0.5% 2 , 5-diphenyloxazole (PPO) in methyl anthranilate at —80°C with Kodak X-OMAT AR film. d Exposed at a temperature of —78°C.

e Preexposed Kodak X-OMAT R film with a calcium tungstate X-ray intensifying film. (Reproduced with permission from Clark and Klein , 1996.)

followed by measuring the associated radioactivity with each spot or zone. The zones are removed either by scraping the adsorbent from the plate (plate scraping) or by cutting pieces from flexible-backed plates and transferring the segments into counting vials. In an alternative procedure, which allows isolation of the radiolabelled sample, the plates are segmented and the radioactive components are eluted from the adsorbent with solvents and counted. To ensure maximum recovery of radioactivity by elution with solvent, the adsorbent should first be crushed to a fine powder. Measurement of radioactivity is generally accomplished using a liquid scintillation counter for the weak ^-emitters. For the y-emitters, the sectioned zones are counted without further sample preparation in an appropriate y-counter.

This technique is relatively sensitive and provides quantitative detection for samples containing low levels of radioactivity. Single peaks containing 100 d.p.m. can be readily detected. Zonal analysis has been reported to be both as sensitive and specific as gas chromatographic-mass spectrometric analysis in the assay of [14C]-labelled clinical samples. When the radiochromatograms are cut into sections and quantified using a y-counter for the analysis of y-emitting isotopes, the method is as precise as TLC scanning.

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