This method invulving the measurement of a change in the capacitance of a condenser is not new (1-3). The dielectric constant of water vapor is greater than that of oxygen, nitrogen, helium, carbon dioxide, carbon monoxide, sulfur dioxide, hydrogen sulfide, methane, etc. Thus water vapor may be readily determined in the presence of the above gases. A simple circuit was designed and the reference cell of the detector filled with the gases to be normalized at their existing temperature and pressure. Upon changing the water vapor
(1) J. Griffith and A. Phillips, Analyst (London), 77, 897 (1952), (2) D. Turner, Nature, 181, 1265 (1958). 33,515 (1961). (3) D. Winefordner, ANAL.CHEM.,
concentration in the sensing condenser, correlation of the percentage humidity was determined. The detector was separated from the electronic circuit and could be placed in a process stream at temperatures up to 300 “C and pressures up to 100 psi gauge. The detector exhibited a linear dynamic range of 0.5 to 90.0% humidity (at 25 “C, 1 atm) as typified in Figure 1. A response time of less than one second was recorded for the drift-free amplifier unit shown in Figure 2. The analysis is nondestructive, and the detector shown in Figure 3 is independent of changes in temperature and flow of the stream. RECEIVED for review December 3, 1968. Accepted January 29, 1969.
An Improved Procedure for Solid Scintillation Fluorography of Tritium Labeled Compounds Kurt Randerath The John Collins Warren Laboratories of the Huntington Memorial Hospital of Harvard University at the Massachusetts General Hospital, Boston, Mass. 02114
AUTORADIOGRAPHY of paper and thin layer chromatograms is an important analytical tool in many chemical and biochemical studies involving compounds labeled with a2P,35S, and particularly 14C. The standard autoradiographic technique is, however, 500-1000-fold less sensitive for 3H than for 14C. Because of their short range, only a few per cent of the primary @-particles of tritium emerge from the surface of the chromatogram. Further losses occur not only in the gap between chromatogram and film, but also in the film itself because the range of the 3H @-particles in the film is shorter than the average distance between the silver halide grains. Organic and inorganic scintillators have been used to convert the energy of the 3H @-particles to light, which in turn produces an image in the film (liquid or solid scintillation fluorography) (1-4). Although most literature data are not directly comparable, the sensitivity reported for procedures of tritium autoradiography and fluorography that permit the subsequent recovery of the compounds is 0.1-0.3 pc/cm2/day (5, 6). Sensitivity is increased to 0.03 pc/cm2/day if the chromatogram itself is treated with an X-ray emulsion, a procedure that precludes the recovery of the labeled compounds (6). This note describes a novel simple procedure for solid scintillation fluorography of 3H-labeled compounds. Its sensitivity is 0.006-0.008 pc/cm2/day. The technique has been applied so far only to 3H-labeled nucleotides and their derivatives separated on cellulose (Eastman 6064 sheets) (1) A. T. Wilson, Biochim. Biophys. Acta, 40,522 (1960). (2) E. V. Parups, I. Hoffman, and H. R. Jackson, Tahnta, 5, 75 (1960). (3) U. Liithi and P. G. Waser, Nature, 205, 1190 (1965). (4) S. Prydz and K. S. Skammelsrud, J. Chromatogr., 32, 732 (1968). ( 5 ) G. S. Richardson, I. Weliky, W. Batchelder, M. Griffith, and L. L. Engel, ibid., 12, 11 5 (1963). ( 6 ) J. Chamberlain, A. Hughes, A. W. Rogers, and G . H. Thomas, Nature, 201, 774 (1964).
and PEI-cellulose (7) thin layers, but there is no reason why its applicability should be restricted to this class of compounds or to cellulose-based thin layers. EXPERIMENTAL
A 7% (w/v) solution of 2,5-diphenyloxazole (PPO) in diethyl ether is rapidly poured over the entire chromatographic area and is distributed evenly by tilting. This operation should take no longer than 2-5 seconds depending on the size of the layer. The chromatogram is then immediately brought into a vertical position and allowed to dry for a few minutes. The volume of the PPO solution used for this treatment is 40 pl/cm2--i.e., 16 ml for a 20 X 20-cm layer. The layer is covered with Kodak RB-54 Royal Blue Medical X-Ray film and placed between two glass plates which are held together firmly with adhesive tape. Exposure is carried out in an insulated box over dry ice (-78.5 “C) in the dark for a few hours to a few days. The film is developed with Kodak Liquid X-Ray Developer and fixed with Kodak Liquid X-Ray Fixer according to the specifications of the manufacturer. RESULTS AND DISCUSSION
The sensitivity of this procedure was found to be 0.0060.008 pc3H/cm2/day. On one-dimensional thin-layer chromatograms (spot area 0.3 cmz) 0.002-0.003 pc3H can be clearly visualized after exposure for 24 hours. The sensitivity depends on (a) the concentration of the scintillator, (b) the solvent in which the scintillator is applied to the chromatogram, (c) the mode of application of the scintillator, (d) the temperature during the exposure, and (e) the film material. Because a systematic study of these factors except for (d) (3),has not been reported in the literature, we have (7) K. Randerath and E. Randerath in “Nucleic Acids,” vol. XI1 A of “Methods in Enzymology,” S. P. Colowick and N. 0. Kaplan, Eds., Academic Press, New York, N. Y., 1968, p 323. VOL. 41, NO. 7, JUNE 1969
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briefly summarized our results pertinent to the factors influencing the over-all sensitivity of the procedure. (a) 7-10% PPO was found to be the optimum concentration range; at lower concentrations sensitivity is lower. Increasing the scintillator concentration to >lo% results in irregular blackening of the photographic emulsion and in decreased sensitivity. Addition of a wavelength shifter like 2-(a-naphthyl)-5-phenyloxazole does not improve the final image. The autoradiographic sensitivity (without PPO treatment) for 3H is 0.15 pc/cm*/day on Kodak Royal Blue film and 0.4-0.5 pc/cm2/day on Kodak No-Screen Medical X-Ray film. (b) Application of the scintillator in diethyl ether results in homogeneous distribution of PPO over the entire chromatographic area. Substituting less volatile solvents for ether may lead to uneven distribution of the scintillator, which in turn produces uneven blackening of the film. Methanol is suitable, but aromatic hydrocarbons are not. (c) It is difficult to apply a scintillator solution of sufficient concentration by spraying, because the jet tends to get blocked by scintillator crystals. Incorporation of the scintillator into the chromatographic support prior to carrying out the separation, as has been described for anthracene (3), may cause separation problems (artifacts, inadequate resolution, etc.) (d) Exposure at low temperature [cf. (3)] is essential; at room temperature sensitivity of scintillation fluorography of 8H is 15-20 times lower than at -78.5 O C . No such temperature dependence was noticed in autoradiography-Le., without PPO treatment. (e) Kodak Royal Blue Medical X-Ray film, a blue-sensitive screen-type X-ray film, was found to be best suited for
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the fluorographic procedure described; its replacement by Kodak No-Screen Medical X-Ray film results in an approximately 10-fold loss in sensitivity. The sensitivity of GAF-X Medical X-Ray film (Ansco) is only slightly lower than that of Royal Blue fi'm. Kodak Blue Brand Medical X-Ray film, another screen-type blue-sensitive film, appears to be less sensitive to the light emitted by the scintillator than either Royal Blue or GAF-X film. The radioactive nucleotides, nucleosides, or nucleic acid bases may be recovered by extraction with aqueous solutions after removal of the scintillator by soaking the chromatograms in ether. In the case of IC-labeled compounds the fluorographic procedure described in this note was found to be of approximately the same sensitivity as autoradiography (lower limit of detection 0.0005 pc/cm2/day). The fact that neither a scintillator effect nor a temperature effect is observed in this case appears to indicate that the contribution of the emitted light to the over-all IC-sensitivity is negligible. Although, for the detection of IC,no-screen-type X-ray film appears to be as sensitive as blue-sensitive screen-type film, we prefer the latter because of its less dense background. ACKNOWLEDGMENT
The author thanks G. Litt, E. Randerath, and P. C. Zamecnik for helpful discussions. RECEIVED for review January 17, 1969. Accepted March 7, 1969. Work supported by grants-in-aid from the U. S. Atomic Energy Commission (AT(30-1)-2643) and the American Cancer Society (P-516).
