Summary of the State of the Art in Radiochromatography Svein Prydz B i o p h y s i c s D e p a r t m e n t , Physics Institute, U n i v e r s i t y of Oslo, Blindern, Oslo 3, N o r w a y
Film registration in radiochromatography is discussed. Low-temperature fluorography gives the lowest detection limits, 3.0 and 0.06 nCi.day/cmz for tritium and radiocarbon, respectively. Various detectors for one- and twodimensional scanning, comprising windowless gas-flow GM detectors, solid state detectors, and vacuum-operated channel electron multipliers are treated. The luminescence detection method, applying scintillators in the chromatogram and a scanned photon detector, is discussed. For tritium, the latter method is about 30 times more efficient than GM detection. Two radio-labels may be counted simultaneously using pulse height discrimination. The beta camera, the spark chamber, and the possibility of using image intensifiers are also discussed. The application of a computer to increase the signal-to-noise ratio and, hence the sensitivity, of scanners is mentioned. A comparison of the sensitivities of the available detection techniques and an appendix giving prices and technical details of the commercially available scanners are given.
Chromatography constitutes a method for both the analytical and the preparative separation of chemical compounds. Thin-layer chromatography (TLC) has become one of the most important techniques for the separation of amino acids, peptides, nucleotides, nucleosides, nucleic acid bases, and related compounds (1-5). Some general references texts on TLC may also be given (6-12). However, additional techniques of identification have been developed to take care of special problems. The use of radiolabels is such a method. The use of radioactive labels provides a handsome tool in biophysics and, above all in biochemistry and clinical medicine where it can, for example, be applied to the study of the pathways of both the synthesis and degradation of biological molecules. High specific activities in radionuclides can sometimes result in molecular disintegration t o a degree which may be acceptable in experiments where, for instance, bioG. Pataki, J. Borko, H. Ch. Curtius, and F. Tancredi, Chromatographia, 1 , 406 (1968). G. Pataki, "Dunnschichtchromatographie in der Aminosaure- und Peptid-chemie," W. de Gruyter, Berlin 1966. G. Pataki, "Techniques of Thin-Layer Chromatography in Amino Acid and Peptide Chemistry," Ann Arbor Sci. Publ., Ann Arbor, Mich., 1968. G. Pataki, Chromatogr. Rev., 9, 23 (1967). K. Randerath and E. Randerath, Methods Enzymol., 12, part A, 322 (1967). A. A. Akhrem and A. I. Kuznetsova. "Thin Layer Chromatography: A Practical Laboratory Handbook," Daniel Davey & Go., New York, N.Y., 1965. J. M. Bobbitt, "Thin-Layer Chromatography," Reinhold, New York, N.Y., 1963. J. G. Kirchner. "Thin-Layer Chromatography in Technique of Organic Chemistry," Vol. X I I , Interscience, New York, N.Y. 1967. F. 6. Padley, in "Thin-Layer Chromatography," Symposium Proceedings, lstituto Superiore de Sanita, Rome, May 1963, pp 87115, Elsevier, N.V. Uitgevers Mij., Amsterdam-C, 1964. K. Randerath, "Thin-Layer Chromatography," Academic Press, New York, N.Y., 1963. "Thin-Layer Chromatography; A Laboratory Handbook," E. Stahl, Ed., Academic Press, New York. N.Y., 1965; second edition, 1969. R . Stock and C. B. F. Rice, "Chromatographic Methods," 2nd ed., Chapman and Hall. London, 1967.
chemical pathways are studied. Moreover, in many cases the amount of the labeled substances recovered and chromatographically separated is very small. Thus, extremely sensitive methods for radiotracer detection are required. The sensitivity of the various types of detectors is strongly related to the energies of the particles from the nuclides considered. ,Because of the low energies of the Pparticles from tritium (E,,, = 18.5 keV, Ea, = 5.7 keV), their range is very short, only about 0.7 mg/cm2 corresponding to a distance of about 1 mm in standard air or 1.9 pm in a dry Ilford film emulsion (13). This fact renders the detection of 3H much less efficient than the detection of radiocarbon (14C) and the other main radionuclides in use. The great potential usefulness of 3H labeling in biochemistry has made the improvement of 3H detection methods a challenging task. This is even more so following the introduction of the thin-layer radiochromatography (TLRC) techniques, which greatly expand the range of the applications. After a development period of only a few years, a further breakthrough may still be expected. This will probably be mainly on the detection side of the problem. The better established procedures of radionuclide detection are already well covered by books and review articles (14-20). In this paper, the newer types of detectors, and some of the possible principles of detection as yet not fully tested, will be discussed. The detector systems might be grouped together and discussed from several points of view, for example from that of being mainly useful for measurements in one-dimensional distributions or of also being applicable to twodimensional ones. Further, one might distinguish between detectors for scanning and detector systems for simultaneous measurement over a larger area of activity distributions. Apart from film registration, most of the detection principles may furthermore be distinguished as using either a P-particle detector (as most do) or a luminescence detector (applying scintillator admixtures in the sample). For most of the particle-detection procedures, the samples are left undisturbed and free of contaminants (such as scintillator materials) after the radiation measurement. In many investigations, where the activity measurements are to be followed by chemical analyses, this is a strong requirement. Before discussing the available methods of P-particle detection further, some remarks will be made on the film registration techniques. Film Registration Techniques. From densitometric evaluation of X-ray film blackening due to spots of 3Hglucose on Whatman paper No. 1 and 3, we have found (T. Kristiansen and S. Prydz, unpublished results, 1965) a (13) L. G. Car0 and M . Schnos, Science, 149, 60 (1965). (14) F. Pochiari and C. Rossi, J. Chromatogr., 5 , 377 (1961). (15) M. Wenzel and P.-E. Schulze. "Tritium-Markierung." W. de Gruyter, Berlin, 1962. (16) "Symposium on Radioisotope Sample Measurement Techniques in Medicine and Biology," International Atomic Energy Agency, Vienna, 1965. (17) A. W. Rogers, "Techniques of Autoradiography," Elsevier, Amsterdam/London/New York, 1967. (18) "Advances in Tracer Methodology," S. Rotchild, Ed., Vol. 1 (1963). 2 (1965), 3 (1966), 4 (1968), Plenum Press, NewYork, N.Y. (19) F. L. Snyder, lsotop. Radiat. Techno/., 6, 381 (1969). (20) K . Randerath, Anal. Biochem., 34, 188 (1970).
A N A L Y T I C A L CHEMISTRY, VOL. 45, NO. 14, DECEMBER 1973
2317
~~
~
Table I. Efficiency of a Standard Operated Windowless Gas-Flow GM Tube Given as 100 cpm/dprn in the Spot Typical overall efficiency (cpm/dpm) %
Radionuclide
Tritium, 3H
Radiocarbon, 14C Radiosulfur, 35S Radioiodine, l 3 l I Radiophosphor. 3zP
Half-life
12.3 years 5730 years
87 days 8days 14 days
E,,
keV
18.5 159 167
250-810 1710
Thinlayer Paper ( 2 ~ ) ( 4 ~ )
1.5 8
8 10 15
3.5 15 15 20 25
reduction factor of about 2 for the exposure time required when applying a moderate vacuum. This clearly shows that the blackening is due to @-particlesof which many are absorbed in the thin air gap between the sample and the film under normal conditions. On the other hand, with scintillator admixtures the use of a photographic emulsion sensitive in the visible range is much more efficient. Thus when scintillators are added, it is fluorography (optically produced film blackening obtained through 0-excitation of the scintillator) which is to a much greater extent responsible for the 3H detection rather than the direct 0-particle exposure. Exposure at low temperature is advantageous (20-25). This rather unexpected temperature dependence of the photographic sensitivity was first observed by Webb already in 1934 and thoroughly investigated with the techniques available at that time (21). A method applying this enhancement of the photoemulsion sensitivity at low temperatures to radiochromatic detection on film has been studied, and its usefulness evaluated. Luthi and Waser (22) added anthracene as a scintillator to their radiochromatograms and detected the luminescence on photographic film emulsions. Upon lowering the temperature to that of dry ice, they found a great sensitivity enhancement for the method, which they attributed to an enhancement in the scintillation efficiency of anthracene. We were able to show (24) that an increased light sensitivity of the photographic emulsion at the low temperature was the reason for the increased overall sensitivity. This is in fact the opposite of the normal behavior of the film materials at higher light intensities. At low photon fluxes, the stabilization of nucleation centers in the alkali halide crystals of the emulsion becomes a limiting factor for the buildup of the exposure. The lifetime of the precursor of a nucleation center is temperature-dependent and gives the relationship found. If one compares a faint mono-photon source of light with a @-activatedscintillator of the same average intensity, the latter gives off scintillations which, during their lifetime (about 2 x sec for anthracene) maintain very much higher (shortterm) intensities. Correspondingly, the temperature effect was larger for the weak light source than for the tritiuminduced scintillations. For radiocarbon, the effect is less than for tritium. This might be due to the fact that the short-time scintillation intensity is larger for the radiocarbon-induced scintillations. Additionally, however, radiocarbon gives an electron exposure of the film which is much more prominent than for tritium and which is not J. A. Webb, J. Opt. SOC.Amer., 25, 4 (1935). U. Luthi and P. G. Waser, Nature (London), 205, 1190 (1965) K. Randerath, Ana/ Chem., 41, 991 (1969). J. F. Koren, T. B. Melo. and S. Prydz, J. Chromatogr., 46, 129 (1970). (25) S. Prydz, T. B. Melo, and J. F. Koren. Anal. Chem., 45, 2106 (1973).
(21) (22) (23) (24)
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thought to show a similar temperature effect. The lowtemperature method seems to be the most sensitive yet available for photographic recording. The lowest limit of detection known for any other method of photographic detection, except for the methods employing spark chambers or image intensifiers, is that of Chamberlain et al. (26), which uses the impregnation of the chromatogram with an X-ray emulsion, namely 33 nCi.day/cmZ. However, his treatment precludes the subsequent recovery of the spot material for further analyses. Randerath (20, 23) uses the term “sensitivity” with the dimension nC/crnZ/day to classify the various detection methods. Obviously he means to give their low level limit of radionuclide detection which should have the dimensions nCi.day/cmZ. Assuming this to be the case, he is claiming (20) a low level limit of 3 nCi.day/cm2 for tritium and 0.06 nCi.day/ cm2 for radiocarbon when using the PPO scintillator and photographic film detection a t low temperature, which represent great improvements over the earlier low level limits. A product containing an organic scintillator in a solvent forming an aerosol is offered by New England Nuclear Corp. for spraying of chromatograms which are to be recorded subsequently by a photographic film at dry ice temperature. In Europe the product is available from NEN Chemicals GmbH, Siemensstrasse 3, 6072 Dreieichenhain, bei Frankfurt am Main, Germany. The physicochemical mechanism responsible for the low-temperature enhancement of the sensitivity of the photographic emulsion has recently been investigated and the use of the effect in scintillation fluorography of radiochromatograms has also been reviewed (25). Beta-Particle Detection Methods. Leaving now the film registration methods, we shall discuss a t some length the various methods and detectors suitable for the counting of single active spots or, primarily, for the one-dimensional scanning of radiochromatograms. For the @-particledetectors available, we can state as a general requirement that the detector aperture should be mounted as close to the scanned sample surface as possible, consistent with there being no risk of contaminating the detector. In this way, the excessive absorption of lowenergy electrons before they reach the detector is avoided. This applies to all types of Geiger-Muller (GM) detectors. The channel electron multiplieE detectors that will be discussed can be operated only in a vacuum which is, nevertheless, the best possible condition of measurement according to the above mentioned requirement. It sometimes happens, however, that the chromatographed components are damaged by excessive drying and, of course, they must not be too volatile if detection by this method is to be possible. One can say in general that if the detector is operated in a vacuum one can expect an increased detection efficiency for low energy particles. For windowless gas-flow GM tubes, various geometries have been produced and tested as shown by the literature. Flat ionization chambers (27) now seem to be paramount, and with these one obtains a relatively large collection efficiency by increasing the acceptable angular variation for the paths of the electrons to be collected. Nowadays nearly all radiochromatogram scanners use windowless gasflow GM tubes. The sensitivity of this detector to various radionuclides may be judged from Table I. The sensitivity is seen to be quite energy dependent and very low for tritium electrons. For detection on paper (26) J. Chamberlain, A. Hughes, A . W. Rogers, and G. H. Thomas, Nature (London), 201, 774 (1964). (27) P. -E. Schulze and M. Wenzel, AngeN. Chem., 74, 777 (1962).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 14, DECEMBER 1973
By applying simultaneously two detectarS instead 01 one. one can change from 2 r to 4r detection. covering the full solid angle. around the sample. ~ i g - : "re la shows the Packard 7201 instrument. Figure lb shows the Tracerlab SC525B type. Figure 1c Shows the Baird Atomic 1-363version. All models are made tor Scanning Paper strips but can alternatively Count single 5 X 20-cm thin-layer strips
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b
Figure 38 shows a 2-week film exposure by the paper chromatogram. Figure 36 shows the dot print resulting from an overnight run of the
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principle of a channel electron multiplier .~ , . . . . An eiectron cascaae IS prwucea DY seconoary e m m m rrom me lnmrnal wall of t h e tube 8s a result of a primary electron entering the low potential end after having been released from t h e sample. For each cascade. a pulse may be counted. T h e figure is reproduced from a recent Work 1288) Figure 4. Wcirking
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In Figure 3 is shown a two-dimensional scan obtained by a scanner of the type shown in Figure 2 in combination with a dot printer. Part 3a is a %week exposure on photographic film and part 36 an overnight recording hy the Berthold scanner. In this case, a 14C distribution has been used as an example and a 0.4 mg/cm2 window has been applied in front of the counter. Solid state particle detectors are known to he noisy a t low energies and, even when cwled to liquid nitrogen temperature, the noise increases rapidly for particle energies below 20 keV. Therefore, while they are probably useful for '4C electrons they are not so well suited to 3H electrons. However, the possibility of the future appearance of improved solid state detectors cannot be overnuled. Their great advantage is their small size, which makes possible
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Figure 5. Block diagram of the channeltron p-detector equipment The movement of the step motors in both directions is controlled by a puke generator. The scanner is contained in a stainless steel housing and the detector requires that the gas pressure be kept below 5 X lo-' Torr. The figure is reproduced lrom a recent work (28)
h Figure 6. The electron defector. claseiy coupled to its preamplifier, may b e adjusted in height above the chromatogram which is seen fixed to the three-point support table of the vacuum x-y scanner. The figure is reproduced from a recent work (28) chromatograms i t is possible to use two detectors, one a t each side of the chromatogram, and to add their signals to get an increased sensitivity (4r-detection). In Figure 1 are shown three different paper strip scanners for the recording of one-dimensional radiochromatograms. In all cases, gas-flow windowless GM detectors are employed in pairs so as to cover a full 471 solid angle. Figure 2 shows a scanner for thin-layer preparations, equipped with a single windowless gas-flow GM tube, covering a maximum solid angle of 2r. 2320
-
a simultaneous detection with many detectors placed in an array or a matrix to cover the chromatogram area. For nuclear physics experiments, such a "checker board" counter has been built. The most direct method of multichannel detection in any system is to use a series of detectors, each of which is followed by a complete set of the necessary additional electronics (e.g., transducer, counter, memory) for recording the signal. In a few types of measurement, this multidetector system is a possible approach, but in practice, economic considerations strongly limit its application. We have attempted to use different versions of an experimental channel electron multiplier, obtainable from Mullard Research Laboratories, Redhill, Surrey, England, for electron detection (28). The working principle of the detector is demonstrated in Figure 4. A vacuum better than 5 X 10-4 Torr was required to operate this detector and therefore the whole chromatogram scanner was put into a vacuum chamber together with the detector. The setup is shown in Figures 5 and 6. As a consequence we got rid of any absorption due to air between the sample and the detector. This kind of detector has a further great advantage: It requires no cooling and yet its noise level is extremely low, of the order of one count in 10 minutes under the best of circumstances. The three featuressmall space requirement, vacuum operation, and very low noise-make this type of detector seem very promising for further developments and applications to nonvolatile systems. Figure I demonstrates the possibility of applying computerized data processing to obtain iso-activity contours (281 S. Prydz. (19711.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 14, DECEMBER 1973
T.
B. Melo. and J. F . Koren. J. Chromafogr., 59, 99
for the various spots. Part n shows the results of a 10-day film exposure of a two-dimensional 14C distribution. Part b shows the result of a corresponding 12 hour scan by the above channel electron detector system. Scintillation detectors have already been employed in nuclear physics for a long time. Such a detector comprises a high-efficiency scintillator crystal mounted in front of a photomultiplier (PM), and the pulses from the latter due to y or particle-induced crystal scintillations are counted by standard techniques. A recently released stepped scanner for radiochromatograms applying an ordinary scintillation detection technique is the "Low energy double labelling chromatoanalyzer" ATS-1003, obtainable from Nuclear Supplies Inc., P.O. Box 312, Encino, Calif. In this version, two PM's are connected, each by a separate lucite light pipe to a common anthracene crystal embedded in the front of a lucite prism and equipped with a slit aperture. Below the slit the radiochromatogram strip is driven a t a selected (stepped) speed. Double-channel recording is possible (e.g. 14C and 3H concurrently) and a coincidence circuit should effectively reject all single-detector pulses such as the noise pulses produced by the spontaneous release of photocathode electrons without any scintillator photon being absorbed. However, some noise counts will result from background radiation traversing the anthracene crystal. It should he borne in mind, however, that with the exception of the fluorography method, all the detection techniques so far mentioned suffer from the predominant absorption of the &particles within the samples themselves. Therefore, their use is to a great extent dependent upon the method of sample preparation applied. For most types of chromatogram, it is probable that there is a drying effect: When the preparations are drying by evaporation, the activities may to some extent be concentrated in the surface layers of the chromatogram. This could probably he utilized as a very effective means of raising the detection sensitivity, at least for tritium, hut would certainly introduce some degree of uncertainty into the measurements. Luminescence Detection Methods. The other main group of detection techniques makes use of the luminescence induced by the @-particlesin a scintillator dispersed within the sample itself. Of the photons produced in this 8-radioluminescence (b-RL) process only a few are absorbed in the sample, leaving a sufficient flux out of the sample to enable detection on photographic film or by high sensitivity PM detectors. Since the light absorption in the sample is much smaller than the absorption of the 8-particles for tritium and probably also tor other radionuclides, the p-RL sensitivity seems to appreciably override the low sensitivity of the GM detectors. The scintillator may he a liquid solution, a gel, or a solid. The first and the last (20, 22-24) alternatives have been combined with detection on photographic film materials. All three alternatives have been combined with methods using photodetector reading of either the intensity of the luminescence, or the number of counts during a certain integration time, as a measure of the activity in the sample. When using liquid scintillation counting, one usually puts the sample and the scintillator solution into a flat chamber or vial between two cooled PM detectors with head-on photocathodes, and coincidence techniques are used to get rid of PM noise counts. Numerous hiochemical applications of this method have been reported in the literature. In some cases the sample material can he dissolved in the scintillator solution, but otherwise the material is sus-
a b Figure 7 . Figure 7a shows an autoradiogram of a two-dimensional radiochromatogram of an ethanolic extract of Papaver Somniferum. The chromatography was performed first in the a.and second in the b-direction. Figure 76 shows how iso-activity contours may be constructed, in principle from any type of scanned detector, by Computer processing of recorded count data (53).This figure is reproduced from an earlier work (28)
pended in the solution. This may he facilitated, and quenching to some degree avoided, hy means of thixotropic additives. Liauid scintillation detection is an exnensive but rather common procedure nowadays. It is very sensitive, but in most cases it involves a rather drastic treatment of the sample. In some instances the sample material cannot be recovered, which excludes this method of detection if the sample is required for further analyses or experiments. Normally the spots must be localized on the chromatogram by another method prior to the liquid scintillation measurement, which is applied for quantitation purposes. References to some textbooks on liquid scintillation detection may he given (29-31). Since the various liquid scintillation techniques are both relatively well known, and will probably not undergo any appreciable further development, they will not he discussed any further. They are nevertheless of great practical value in various versions for detection of 3H as well as of 14C and other radiolahels. Further, the technique allows the simultaneous determination of different radionuclides in the sample by means of pulse-height analysis. Some special techniques for strip counting have been developed by Snyder et nl. (32-37). The main idea is to use an, automated scraper (designed by Snyder and others and commercially available) to take out section by section of the radiochromatogram for liquid scintillation counting (sections as narrow as 1 mm are required) and in this way establish a histogram profile of the activity distribution along the travelling direction in the TLRC sample. This method of "zonal scanning" is exact, but time-consuming and expensive. There is only one scanning detector system based on scintillator admixture to the sample available, namely the French strip scanner DMSL-3 of S.A.I.P., 38 Rue Gabriel Crie Malakoff, Paris, France, which uses a water-cooled
(29) E. Schram and R. Lombart, "Organic Scintillation Detectors." Elsevier, Amsterdam, 1963.
(30)J. B. Birks. "The Theory and Pracfice 01 Scintillation Counting." Pergamon Press. New York, N.Y..1964. (31) "Organic Scintillator$ and Liquid Scintillation Counting,'' D. L. HOCrochs and Chin-Tzu Peng, Ed., Academic Press. New York and London. 1971. (32) F. Snyder and N. Stephens. Anal. Biachem.. 4,128 (1962). (33) F. Snyder. Anal. Biochem., 9. 183 (1964). (34) F. Snyder and H. Kimble. Anal. Biachem., 11,510 (1965). (35) F. Snyder, Separ. Sci., 1,655 (1966) (36) F. Snyder and D. Smith. Separ. Sci., 1 . 709 (1966). (37) F. Snyder and E. A. Cress. Clin. Chem., 14, 529 (1968).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 14, DECEMBER 1973 * 2321
l
.-.-. A-A-A
"C 3H
10 T L R C spot aclivity
1800V /*'
10'
1
103
IO'
i n nCi
Figure 8. Spot detection by @-radioluminescence( 4 4 ) Sensitivity curves for both tritium and radiocarbon are drawn, for three different photomultiplier voltages, on the basis of roughly 1 cm2 spots on 0.2-mm thin layers in the range from 1 to l o 4 nCi. 75% w/w of the scintillator 2nZSiOa:Mn was added to the Merck Silica Gel 7736
PM detector and a thixotropic organic scintillator additive (38-40). PM detection of radionuclides in chromatograms by means of solid scintillators admixed to the preparation has as yet only been carried out by Seliger and Agranoff (41) and in our own laboratory during some of our work on methodology (42-45). For the p-scintillation method of detecting tracers in radiochromatograms with added solid scintillators, we found a strict linearity between the signal intensity and the tracer activity in the measured spot. A more or less linear increase in the efficiency of detection was also found with increased scintillator addition. A long-term deterioration in the efficiency of anthracene as a scintillator additive was found and given a tentative explanation. Most of the scintillators tested were somewhat sensitive to humidity, although in most cases they recovered when stored in dry air. These solid scintillators showed very minute changes in efficiency upon cooling to liquid nitrogen temperatures. Figure 8 shows the PM detector responses to spots of labeled glucose on thin-layers with added solid inorganic scintillator of a chemically inert type. The whole circular spot areas were viewed one at a time by the detector. (38) J.-C. Roucayrol and E. Oberhausen, Nafurwissenschatten, 42, 41 1 (1955). (39) J.-C. Roucayrol and P. Taillaidier. Cornp. Rend., 256, 4653 (1963). (40) J.-C. Roucayrol. J.-A. Berger, G. Meyniel, and P. Taillandier, in "Radioactive Isotope in Klinik und Forschung," Vol. 6. K. Fellinger and R. Hofer, Ed., Urban 8 Schwarzenberg, Munchen/Berlin, 1965, pp 474-479. (41) H. H. Seliger and B. W. Agranoff, Anal. Chern., 31, 1607 (1959). (42) S. Prydz, C. Petersen, and J. F. Koren, Phys. Norvegica, 2 ( 4 ) , 343 (1967), (43) S. Prydzand K. S. Skammelsrud, J. Chrornatogr., 32, 732 (1968). (44) S. Prydz, T. 6. Melo, E. L. Eriksen, and J. F. Koren, J. Chrornatogr., 47, 157 (1970).
2322
It may be mentioned that an estimate was made ( 4 4 ) of the efficiency of the @-radioluminescencedetection method for tritium. As scintillator additive, 75% w/w of ZnzSiOc: Mn in Silica Gel (Merck 7736H) was used. At a detector high voltage of 1800 V, an efficiency greater than 60% (cpm/dpm) was found. This is extremely high as compared to that obtained for gas-flow windowless GM tubes and makes a quantitation of a 1nCi tritium spot with an accuracy of 10% possible in about 100 seconds (stationary counting of the whole spot). These data were obtained with a light collection efficiency of only about 8%. With a good sample-detector geometry (a large solid angle covered by the detector window), a collection efficiency of about 40% should be obtainable. These relationships seem to warrant a very high sensitivity for a solid-scintillation photodetector scanner for radiochromatograms. A series of experiments (46) demonstrated that heavy loads of scintillators may be incorporated in chromatographic thin-layers without causing interference effects to a degree which would preclude the practical use of the suggested method of solid-scintillation detection for radionuclides. Randerath holds the view (23) that scintillator addition to the chromatoplates prior to carrying out the separation may cause separation problem (artifacts, inadequate resolution, etc.). However, Luthi and Waser (22) have shown, as is evident 'also from our own results, that for many chromatographic systems such problems do not occur. On the other hand, even if scintillator addition prior to the separation does disturb the separation procedure, the method is still useful with scintillator impregnation after a completed separation. Such methods for scintillator impregnation have been given by Randerath (20, 23) among others. In the solid scintillation detection of tracer activities in radiochromatography, different radionuclides can be distinguished by their characteristic @-energies.These are reflected in the photon content in the scintillations and thus in the largeness of the PM scintillation pulses recorded. If now a pulse-height discriminator is used, the pulses b e - , longing to, for example, radiocarbon and tritium may be distinguished and counted separately even if both nuclides are present in the same spot on the radiochromatogram. This possibility is not unexpected as it exists and is well known for liquid scintillation counting. In a recent work ( 4 5 ) , it was shown that it was also possible when counting directly on the chromatogram, using anthracene as solid scintillator additive. This is demonstrated in Figure 9. No scanner has yet been developed to take advantage of the established procedure except for the ATS-1003 version, which is based on direct electron detection by the aforementioned scintillation detector system. It should further be mentioned that PM detectors can now be produced as miniaturized solid state devices with high sensitivity. However, the best signal-to-noise ratio is .still found for the conventional types. As a future possibility, one could perhaps use a series of microdetectors mounted so as to trace the luminescence along the whole chromatogram strip at one and the same time, which would make the measurement much faster. In the near future, various types of image intensifiers might also be applied to the simultaneous detection over the whole of a 20 cm x 20 cm radiochromatogram area. This group of electron-optical systems comprises a variety of new instrument types. In many applications, their performances are steadily being improved. These instruments (45) T. B. Melo and S. Prydz, Anal. Chern., 42, 1093 (1970). (46) L. H. Landmark, A. K . Hognestad, and S. Prydz, J. Chrornatogr., 46, 267 (1 970).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 14, DECEMBER 1 9 7 3
PHOlOHULllPLlER VOLTAGE
Figure 9. A demonstration of the possibility of the COnCL counting of tritium and radiocarbon. based on t h e pulse-t discrimination between PM pulses from either tritium or I carbon In the anthracene impregnated sample. electrons emitted from Carbon release larger scintillalionr. and thus produce greater PM F than do t h e tritium electrons. The PM noise pulses. again. are 5 than the tritium pulses. T h e curves in the figure Show, for each I pulse, t h e relative number of pulses passing a discriminator stag< function Of t h e PM voltage (a measure lor the internal amplifica the detector). For B lower discriminator level, corresponding here volts, nearly all “C pulses will be counted and virtually none 01 the In a channel corresponding to the voltage range 900-1100 V. aboL of t h e ’H pulses will be counted with an addition of less than 10% “C pulses and 5% of the noise pulses. The figure is reproduced I recent work (451
are aimed at low level detection, partly of particle fluxes and partly of light. The following types: electronographic “cameras” (for film exposure by multiplied and/or accelerated electrons), secondary electron image intensifiers, and cascade image intensifiers give, by electron amplification, enhanced pictures of the investigated flux distributions which may then be photographed. Further, the above types can be applied in combination with TV camera-tube principles to enable immediate readout of the picture information as an electronic signal. Instrument types such as the “vidicon” and the “image orthicon” may he mentioned. The detection of 6-emitting radinuclides by means of either the bremsstrahlung or the Cerenkov-radiation produced is possible in principle although it has not yet been applied t o radiochromatography. For 3zPan efficiency for Cerenkov-radiation detection of 25% is claimed (47) and for 8eRb and 36Cl in biological materials, efficiencies of respectively 60% and 13% are claimed (48). However, for the Cerenkov-radiation detection to have a useful sensitivity, the 8-energies should not he less than 0.5-1 MeV, which precludes the use of this method for tritium detection. Other Methods. The two main groups of detection methods were those using @-particle detection directly and those using scintillation detection. There are some additional methods which belong to these groups hut which are yet of little importance because they are either inconvenient, inferior to other methods, too expensive, or simply too recently conceived to have gained any acceptance. The use of frozen benzene scintillation detection of radio-labels in TLRC has recently been suggested (49).At liquid nitrogen temperatures, benzene behaves like a high-sensitivity solid scintillator, and after the measurement has taken place the benzene may he made to evaporate completely, thus leaving the sample undamaged and free from contamination. (47) R. H. Eirick and R. P. Parker. lnf. J. Appl. Radial. Isatop.. 19, 263 (1968). (48) A. Lauchli, Inf. J. Appl. Radiat. isolop., 20. 265 (1969). (49) S. Prydr, T. 8. Melo. J. F. Koren. and E. L. Eriksen. Anal. Chem.. 42, 156 (1970).
Figure IO. The
Bawd-Atomic Beta camera
The 3H and 14C contained in organic materials will after combustion be found in the form of 3HzO, l4C02, and other active gases. These may be counted in ionization chambers. However, this metbod, although well established, does not yet seem to have been applied in combination with TLRC. On the other hand, ionization chambers have been employed to measure the activity of spots scissored from paper chromatograms. This method may equally well be applied to the spot material scraped off from the TLRC preparations. However, the methods of Snyder (32-37) would be preferable if a liquid scintillator detector were available. Finally the recent construction and application of “spark chambers’’ or “beta cameras” should be mentioned. Of the two versions which are now available commercially, the one is a cheap spark chamber type, available from Panax Equipment Ltd., Holmethorpe Industrial Estate, Redhill; Surrey, England, while the other, available from Baird-Atomic, 125 Middlesex, Turnpike, Bedford, Mass. 07131, is a very expensive instrument employing the beta camera principle. The spark chamber from Panax (50) has two horizontal planes separated by ‘/e inch in the vertical direction, and each of the planes contains a set of parallel wires where the wires in the one plane run in a direction perpendicular to the wires in the other plane. Thus, viewed from above the unit appears as a grid. The two sets of wires are maintained a t a potential difference of around 3 kV, and surrounded by an ionizing gas. The 20 cm x 20 cm radiochromatogram is placed beneath the wire sets. Radiation entering the wire structure from below will result in ionized particles in the gas, which in turn will cause a discharge between the two wires forming the nearest crossing point in the grid. The discharges may he recorded on film using a standard Polaroid Camera. The spark pattern observed will indicate the position of the active spots in the radiochromatogram. The great advantages of the spark camera system are that the whole spot distribution is viewed simultaneously and (50) Leaflet from Panax Equipment Ltd , Holmethorpe Industrial Estate, Redhill. Surrey. England, 1968
ANALYTICAL CHEMISTRY, VOL. 45, NO. 14. DECEMBER 1 9 7 3
-
2323
Table II. Buyers Guide to Radiochromatogram Scanners (Spring 1972) Efficiencv com/dDm f % ) Producer
Model
Detector type: GM windowless gas flow 1 . Baird Atomic Inc. 11363 33 University Road Cambridge, Mass. 2. Berthold/Frieseke G m b H 75 Karlsruhe-Durlach, BF 300 Bergwaldstrasse 30 BF 200 Postfach 76 3. PhilipsfBerthold PW4009/01 PW4007f8 4. Packard Instr. Co. 7201' 220 Warrenville Rd. Downers Grove, Ill. 5. Nuclear-Chicago Actigraph 333 E. Howard Ave. 1002 with Des Plaines. 111. thin-layer adaptor 1006' 6. Panax Equipm. Ltd. E.0111 /P.7900A Holmethorpe Ind. Estate Redhill, Surrey 7. ICN Tracerlab SC-525B Antwerpse Steenweg 277 2800 Mechelen SC-527 Belgium 8. Nuclear Supplies, Inc. ATS-1001 P.O. Box 312, ATS-1002 Encino, Calif. Detector type: Anthracene scintillation crystal detector 9. Nuclear Supplies, Inc. ATS-1003
Detector type: photomultiplier,with scintillator in the layers 10. SAlP Rue Gabriel-Crie Malakoff, Seine, Paris, France Detector type: Spark Counters and @-Cameras 1 1 . Panax Equipment Ltd. RCIS-1
12. Baird-Atomic Inc
Type of detection
Beta Camera Mod. 6000.
14c
35s
...
32p
...
4 a paper 2a thin-layer
3
35
7Q
30
40
4a paper 2a thin-layer 4a paper 2a thin-layer
as above (approx.) as above (approx.) 2 30 30 1 15 15
80 45
4 a paper 2n thin-layer
6.5 3
30 15
2a thin-layer
2
15
paperb
3.5
15
15
25
4a paper0 2a-thin layer
3.5 1.5
15
15
25
8
8
15
44
50 50
4a
2
paper & thin-layers paperb
30 25
...
paper, simultaneous detection of 3H and 14C is possible
Polaroid film detection of two-dimensional spark distribution over chromatogram. 2n detection on paper and thin-layers. Method of detection as described in text. Paper thin-layer
Prices given in currencies other than Norwegian Kroners are F.O.B. prices. Peak integration facilities with printer are available.
that the record may be integrated on the film until sufficient. After a fast and good localization with this equipment, the spots may be quantitated by using either a strip scanner or the method of liquid scintillation counting. A somewhat similar spark chamber, which achieved a spatial resolution of 0.5 cm, has been constructed and tested by Pullan et al. (51). Spots of 0.5-cm diameter with a radiocarbon activity of 0.03 nCi or with a tritium activity of 0.3 nCi became visible on the film after a 10minute exposure. In addition Pullan et al. could deduce the spark coordinates electronically and record these on paper tape for subsequent computer analysis. (61) B. P. Pullan, R. Howard, I . Kaye, and H. Lowe. Biochem. J., 102, 6P-7P (1967).
2324
3H
...
5 7
44 44
44
Dot printer for scanning of two-dimensional chromatograms is available.
The Baird-Atomic Beta Camera has recently been described elsewhere (19, 5 2 ) . It is constructed as a combination of 1622 miniature gas-flow GM tubes in a matrix covering an area of 20 cm X 20 cm and an imaging cathode-ray tube. The instrument is shown in Figure 10. The whole spot distribution may either be viewed on a scintillating screen or photographed, and digital quantitation of selected spots is also possible. The method is claimed to be roughly lo4 times faster than ordinary film detection (without scintillator enhancement techniques). Figure 11 demonstrates the high sensitivity of the method. The av(52) The Beta Camera Modei 6000, Description and preliminary operators manual from Baird-Atomic, 125 Middlesex, Turnpike, Bedford, Mass 01730
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1 4 , DECEMBER 1973
Low level of detection
3H
Noise, cDrn
’4C
...
Pricea U.S.$3295
300 dpmb 600 dpmb
20
30 dpmb 50 dpmb
D M 16190 D M 17265 N.Kr. 18000 N . K r . 27600 N . K r . 28300
0.5-1 nCi
0.05 nCi 10-1 7
5
U.S.5 3515
...
(with adaptor $ 3800)
f
3
500 dpm
100 dpmC
12
200 dpm
3 0 dprn
U.S.$3300
12 15
200 dpm 350 dpm
3 0 dprn 50 dpm
U.S.$3800
1628
U.S.$4800 U.S.$8975
...
...
, . .
U.S.$ 15000
f 1295
I
.
.
...
...
U.S.$20895
erage background count rate per detector unit is about 0.5 cpm. For example, a spot which covered an area of ten detectors would possess a background count of 5 cpm and would probably be seen if it gave twice the noise count rate. However, the dpm corresponding to this count rate would be expected to be quite sensitive to the type of chromatogram preparation. By using an easily installable window screen in front of the detector, a discrimination between two different labels is achievable. A movable collimator frame may be used so as to increase the spatial resolution from that given by 1622 picture elements contained in the 8-inch square area to that containing 12976 picture elements within the same area. The exposure time may be varied in second steps from 1 second up to 166 minutes. According to Snyder
(19) the quantitation, resolution, and sensitivity of the beta camera are not so good as those attainable from his liquid-scintillation-scraping techniques, but the “betagrams” are obtained in a period of time very short compared to the time required to carry through the procedures in the method of Snyder, especially when applied to two-dimensional chromatograms. Tentative Comparison of Sensitivities. This presentation of radionuclide detection methods of widely differing natures is meant partly to be a very short introduction to the common practice in this field and to some possible principles of detection which are not at all obvious or already in common use, and partly to give an insight into the interrelationships between the various methods. It might be useful now to sum up very briefly what has been said: A major difficulty is a weak @-radiation from tritium, a most important radio-label, which renders the detection somewhat more difficult for this particular label than for the other ones. Most of the presently available methods for radionuclide detection can be grouped into one of the four categories A-D. We have A) film detection of electrons (autoradiography); B) film detection of scintillations (fluorography or scintography); C ) detector measurement of emitted electrons; and finally D) detector measurement of electron-induced luminescence. (When this principle is used with a scanned detector one could perhaps use the term “scintiscanning” as has been done in Figure 12.) Here A and B are useful mainly for localization purposes and B is better than A. C and D are useful also for tracer quantitation and D can normally be assumed to be better than C. A tentative comparison of the sensitivities of the various methods which have been discussed in the previous sections is given diagrammatically in Figure 12. Film detection of @-particles(autoradiography) has been used as a reference and given a value of 1 on a relative sensitivity scale. To be able to cover the very great range necessary to pin-point the various techniques, a logarithmic scale has been chosen. Photographic techniques will be found equally sensitive for both alternatives, one- and two-dimensional recordings, while scanners will of course operate a t a reduced speed in two dimensions, the reduction factor being equal to the number of parallel scans necessary to cover the area which is to be detected and give a reasonable resolution. Snyder’s method of applying the scraping off of narrow zones from the chromatogram and successive liquid scintillation counting, is said (19) to be even more sensitive than the use of the beta camera, but of course is a very time-consuming technique. For the other methods in the diagram, there is a correspondence between the sensitivity and the speed of detection. For the low-temperature scintillation fluorography, the sensitivity enhancement due to the low temperature is somewhat smaller for radiocarbon than for tritium ( 2 5 ) . There is one important point which has not so far been discussed, even if its practical relevance is quite obvious: There is a possibility that the detection signals from spots lying close to each other are smeared together under the activity recording even if they have been satisfactorily separated in the chromatogram. For scanned detectors, this problem can be partly treated as one regarding the steepness of sensitivity increase across the border of the detector aperture (28, 53, 5 3 ) . Quite naturally, such effects would be most prominent for measurements aimed a t the detection of faint activity spots lying close to spots (53) S. Prydz and T.’Seim, J. Chrornatogr., 73,183 ( 1 9 7 2 ) . (54) M . J. Johnsen, J. Chromatogr., 20, 100 (1965).
A N A L Y T I C A L CHEMISTRY, VOL. 45,
NO.
14, DECEMBER 1973
2325
50,000
' it
OP M
OPM
A. Automdiogmm (Time: 17 &yl)
8.
k t o g m m (Time:
5 minutes)
~.
Figure 11. A demonstration of the achievements of the Baird-Atomic Beta Camera Part A Shows a 17-day exposure by Conventional autoradiography Of two "C Spots. Part B Shows the corresponding betagram with a 5-min exposule time. The sample is a 8 in. x 8 in. Eastman Chromagram
1
A
1
10000
Two dimcnsionr-
Figure 12. Approximate comparison of t h e sensitivities of the various detection methods The values are taken as the inverse of the respective iow level limits of detection and are given relative to that Of the Conventional film autoradiography. Note the different sensitivities Occurring in one and two dimensions for the method3 based on the scanning of a detector. These differences are consistent with the successi~eapplication of 100 parallel scans in the two-dimensional case. The method of Snyder, where the Spots ere scraped off for liquid scintillation counting. is more sensitive than the beta Camera method. However. the practical working time required to perform the measurements would always be much in excess of that required for a single Camera exoosure
of much larger activity. Regarding this point, the method of Snyder, employing scraping and individual counting with a liquid scintillation detector, is by far the most accurate one. Here the interspot activity interference has been maximally reduced to become of exactly the same order as the degree to which the chromatographic separation has left the components unresolved. Finally, one should perhaps mention that a gain in the signal-to-noise ratio, and hence in the sensitivity, can be achieved for the methods applying scanning of a single detector, by computer techniques (53). This is obtained most easily in an off-line mode after storing of the digitized activity readings (e.g., on paper tape) by the application of a mathematical low-pass filtering procedure, reducing the statistical fluctuations in the countings suhstantially. When the smoothing is applied to two-dimensional recordings, the smoothing procedure is operative also across the direction of the parallel scans. One should hear in mind that the computer facilities make such possibilities as writing out the activity distributions on various scales and intergrating the various spots available.
ACKNOWLEDGMENT I would like to thank K. Randerath for drawing my attention to the problem of the spatial resolution in the various methods of activity detection-a point of ohviously great practical importance-and which has perhaps not been treated in sufficient detail in the present work.
APPENDIX A Buyers Guide to radiochromatogram scanners, where data on two spark chambers have been added as well, is included (Table 11). The principle of detection in each case is stated and the counting efficiency is given for 3H, *4C, 35S, and 32P. when these numbers have been obtained from either literature statements or directly from the producer. Similarly, the noise count rate and the low level of detection are added in some cases. Mostly, prices are given in the currency of the producer's country.
Received for review January 23, 1973. Accepted June 22, 1973.
2326 * ANALYTICAL CHEMISTRY, VOC. 45. NO, 14. DECEMBER 1973
