Plate radioactive zones

Figure 2. Thin-layer radiochromatogram of urine (100 il) from rats injected with labeled PbTx-3. TLC plates were developed in two sequential solvent systems chloroform ethyl acetate ethanol (50 25 25 80 10 10). Radioactive zones were scraped and counted in a liquid scintillation counter. Native PbTx-3 runs at 13 cm.

A phosphor screen can be used like an autoradiography film to detect radioactive zones on PLC plates. The available screens are sensitive to x-rays and beta and gamma emissions from isotopes such as H, bij 32p 33p -pjjg screen captures... 

For LSC, radioactive zones are scraped from a glass-backed layer or cut from a layer with flexible backing (plastic or aluminum) and transferred into a vial, mixed with a scintillation solution (commercial cocktails are available), and indirectly quantified based on measurement of the light energy produced in a scintillator counter with a photomultiplier tube (PMT). As an alternative, scraped or cut zones can be eluted with solvent into a vial to isolate the radiolabeled sample and then counted. The number of counts for each zone can be plotted as a histogram profile of the radioactivity in the chromatogram along the lane of a TLC plate. 

Prior to working with radioactive materials, the scientist should attend a pertinent training course or consult a standard source of information on radioisotope methodology to become familiar with the precautions used in the safe handling of radiochemicals in the laboratory (e.g., DuPont, 1988a Shapiro, 1972 Stewart, 1981). As much as possible, experiments should be carried out, and plates with radioactive zones stored, in a fume hood. 

TLC coupled with spectrometry and other analytical methods, video documentation, and computer imaging (Chapter 9) quantification by video densitometry (Chapter 10) validation of quantitative results (Chapter 11) and in situ instrumental measurement of radioactive zones (Chapter 13). The fomth edition continues to provide extensive coverage of sample preparation in Chapter 3. We believe that this coverage is unique and differs from that found in other available treatises on TLC. Because of the primary importance of commercial precoated plates, detailed instructions for preparing layers were removed (Chapter 3), and description of documentation by contact printing has been eliminated (Chapter 9). 

Instead of scraping and manual collection of the adsorbent, the band can be sucked off the plate with a Vacuum-cleaner -type apparatus. Dekker [50] described an apparatus for the isolation of compounds from layers by elution and direct Millipore filtration, and Platt [51] designed a zone collector that used vacuum to transfer separated zones from layers direcdy to vials for hquid scintillation counting of radioactivity. 

The procedure of autoradiography itself is very simple. Dried gel, cellulose acetate or filter paper sheet is covered with X-ray film, clamped between two metal plates and allowed to stand for an appropriate period of time. Then, the X-ray film is developed and zones of the radioactive material are visualized (see also Ref. 232). Also, Polaroid land film can be used for this purpose [223]. 

Preparative TLC is an ideal quantitative technique for radioactive and toxic substances and for feasibility studies of reactions of expensive pharmaceutical mixtures. A recent innovation has been the introduction of taper plates (Analtech) for use in preparative TLC work. The dimensions and features of the taper plate are illustrated in Figure 3.14. Sample concentration prior to separation occurs in the pre-adsorbent zone. The tapered adsorbent layer causes low R bands to separate further than on a preparative plate of constant thickness. A more uniform mobile phase flow pattern and reduced vertical band spreading, further enhance the performance. 

The duration of measurement can be appreciably reduced by regulating the plate movement. The Radio-Chromatograph (Fig. 87) permits scanning of radioactive spots at the optimum speed and of inactive zones at higher speed. 

Over the last 30 years or so the detection of radioactivity directly on TLC plates has taken dramatic leaps forward. Prior to the introduction of radiation detectors, the classical method used for the detection and quantitation of radioactivity on a plate involved exposure to x-ray film as the first step. This could take from a few hours up to one or two months, and this technique only located the radioactivity. The second step after location was quantitation, which was achieved by removing the zone of interest, either by scraping the silica gel off or by cutting away if the plates were aluminum- or plastic-backed, followed by liquid scintillation counting. Such a procedure is extremely labor intensive and is limited in terms of accuracy and resolution (see above). 

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