Autoradiography of Tritium-Containing Thin Layer Chromatograms

Detection of tritium on paper and thin-layer chromatograms ... An Evaluation of the Available Techniques for the Autoradiography of Tritium on Chromat...
0 downloads 0 Views 153KB Size
grinding and finally, suspension aiid counting ( R , 4 ) . By going directly from precipitation and filtration to suspension and counting, most of the objectionable precipitate handling is eliminated, and the possibility of sample loss is greatly reduced. The most important features of the glass filter suspension method are the speed of preparation and elimination of sample handling steps. Additional advantages are as follows. The method is uasble over a wide range of specific activities. I t is especially suitable for samples of low specific

activity because the suspension does not quench the scintillation. Particle size of the suspended precipitate is small and uniform. Crystal size of the initial precipitate is preserved without the necessity of grinding. The glass filter suspending medium is inert and stable, and can be used with any liquid scintillator. The suspension refuses to settle or separate during the usable life of the scintillator. Gel preparation and handling is eliminated.

LITERATURE CITED

(1) Helf, S., “Suspension Counting,” pp.

96-100 in “Liquid Scintillation Counting,” E. G. Bell, F. N. Hayes, eds. Pergamon Press, New York, 1958. ( 2 ) Helf, S., White, C. G., Shelley, R. N., AXAL.CHE3.f.32, 238 (1960). (3) Peng, C. T., Zbrd., 32, 1292 (1960). (4) Radin, N. S., “Methods of Counting Acids and Other Substances by Liquid Scintillation,” pp. 108-14 in “Liquid Scintillation Counting,” E. G. Bell, F. N. Hayes, eds. Pergamon Press, New York, 1958. Reference to trade names is made to facilitate understanding and does not imply endorsement by the Bureau of Mines.

Autoradiography of Tritium-Containing Thin layer Chromatograms Herbert Sheppard and Wen Hui Tsien, Research Department, ClBA Pharmaceutical Co., Summit,

A

smpm AND sensitive method of preparing autoradiograms from thin layer chromatograms containing tritiumlabeled substances has been developed. The chromatograms are exposed to photographic plates prepared with Kodak nuclear track emulsion, type NTB, which is widely used for autoradiography of tissue sections ( 2 ) . Clean glass plates of appropriate size are dipped in a 0.05% aqueous solution of CrK(SO&. 12Hz0 (Chrome Alum) and air dried. The emulsion is melted by placing the bottle of gel in a water bath set a t 40’ to 45’ C. It is then diluted by adding two volumes of a 0.05y0 solution of ‘‘Dupanol C” in distilled water. The glass plates are heated on a slide-warming table for a few minutes. The melted emulsion is spread over the plate with a prewarmed camel hair brush 1 to 1.5 inches wide. The plate is tilted from side to side to help the emulsion flow evenly over the area, and is then returned to the warming table for a few seconds to help eliminate ridges in the surface. TO facilitate hardening of the emulsion, the plate is moved to a cool level area. The finished plates may then be stored in a light-tight box in a refrigerator. All of this work is done in a dark room where the only illumination is from a lamp containing a 15-watt bulb fixed with a Wrattan filter No. 2 to give off red light. During the period of exposure, the thin layer chromatogram is clamped in contact with the emulsion side of the plate with banker’s clips. The plates

1992

0

ANALYTICAL CHEMISTRY

Figure 1. Autoradiograms of tritiated materials on TLC plates coated with silica gel

are then placed in a light-tight box with desiccant (Drierite) and stored a t 4’ C. in a refrigerator, After an appropriate period of time, the boxes are removed from the refrigerator and allowed to come to room temperature. In a dark room illuminated as described above, the plates are removed from the boxes and separated. The plate with the NTB emulsion is developed in a horizontal position with the emulsion side up in Kodak D-72 developer (one part of D-72 to two parts of water) a t 20’ c. for 3 minutes, passed through distilled water, and placed in Kodak acid fixer until clear. Finally the plates are washed for 5 minutes in running tap water and dried in air. The degree of blackening of the emul-

N. J.

sion produced by exposure to 0.01 to 5 pc. of tritiated material on silica gel plates for a period of one week may be seen in Figure 1A. As little as 0.01 IC. of H3 is detectable in a compact spot. An autoradiogram of an actual thin layer chromatogram containing 10 IC. of a tritiated steriod plus products of degradation is seen in Figure 1B. Some of the spots are very compact and the dimensions found in Figure 1A suggest that the very faint ones may contain about 0.01 pc. of H3. The exposure time in this case was one week; longer exposures will yield darker spots. This same procedure has been applied to paper chromatograms with a glass plate placed behind the paper strip so that the paper is forced into intimate contact with the emulsion. It should be possible to quantitate the results by photographing the autoradiograms and measuring the degree of blackening of the negative with a densitometer. Thin glass plates would permit direct densitometry but these may be too fragile for handling. The technique described here constitutes a useful addition to those reviewed recently (1) for thin-layer chromatograms containing C1*and other isotopes that emit energetic particles. LITERATURE CITED

(1) Mangold, H., “Dunnschicht Chromat,ographie,” p. 62, E. Stahl, SpringerVerlog, Berlin, Germany, 1962. (2) Messier, B., Leblond, C. P., Proc. SOC, Ezpl. Bzol. Med. 97, 7 (1957).