Automatic Triparametric Recording in Fluorometry of Polynuclear Hydrocarbons Myron M. Schachter and Edward 0.Haenni, Division of Food, Food and Drug Administration, U. S. Department of Health, Education, and Welfare, Washington, D. C. 2 0 2 0 4
LUORESCENCE spectrometry is a useF F u l technique for the identification of polynuclear hydrocarbons (4, 5) because the characteristic intense and complex fluorescence spectra lead to specific and sensitive determinations. These are extremely important factors in this field, for the analyst is ordinarily dealing with trace quantities of these hydrocarbons and must specifically distinguish the relatively few carcinogens from the great iiumber of closely related noncarcinogenic hydrocarbons. The latter often differ structurally from the former only in the position or the chain length of an alkyl substituent in one or more rings. In currently available spectrophotofluorometers, the observation or recording of a complete series of excitation and fluorescence spectra is a tedious task with complex spectra, for in either mode one wavelength at a time must be fixed while the spectral range in the other mode is scanned. Although it may be ’considered unnecessary in the usual fluorescence analysis to make such complete spectral examinations, the Subtle distinctions required in the identification of po1:ynuclear hydrocarbons involve careful examination of all details of the spectral characteristics. The new oscillogr,aphic technique described in this report permits rapid simultaneous recording of any three variable inputs. We have utilized the
technique to record the excitation and emission wavelengths and the emission intensity. a consequence, a series of fluorographs is obtained a t selected levels of emission intensity. This series may be assembled into a three dimensional model, or stereofluorograph which comprises an integration of all details of the three spectral parameters (Figure 1). EXPERIMENTAL
The basic apparatus conqists of a n hrninco- Bowman spectrophotofluorometer with a Hewlett-Packard Model 13013 oscilloscope equipped with a Beattie-Coleman Oscillotron and periscope camera adapted to use Polaroid film, This equipment is modified by incorporation of a device that triggers the cathode ray beam trace off and on by imposing selected input potentials which are derived from the photometer output and hence are direct functions of the emission intensity. Traces are photographed using Polascope 410 film with f/16 aperture a t time exposure. A schematic diagram of the asbembly incorporating the compatibility device and circuit diagrams of the voltage sensrr and chopper (1-3) are shown in Figure 2 (a, b, and c). The device triggers an on-off signal of 20 volts pulsating (3 kc. per second) d.c. to the cathode ray tube grid. The AmincoBowman photometer derives its output from a cathode follower variable by the “zero adjust” control from 1.1 volt and 25 pa. to 2.2 volts and 130 pa.
D
Figure 1 . Composite of stereofluorograph
three
fluorographs
yields
a
A, B, and C, a r e planes through the peaks scanned by the conventional D, E, and F are the triparaprocedure with a spectrophotofluorometer. metric scans of fluorographs
-
Procedure. T h e spectrophotofluorometer motor switches are turned on full so t h a t both monochromators will rotate simultaneously a t maximum rate (15 seconds per scan). T h e excitation and emission servo outputs are connected, respectively, to the vert’ical a n d horizontal deflection inputs of the oscilloscope. Vernier oscilloscope controls are adjusted for 0.167 volt per em. to give 6-cm. scans on each axis for the 200 to 800 mp spectral range. All instrument components are turned on to stabilize for ‘12 hour. With the photometer “meter multiplier” set at’ “zero adjust,” the “zero adjust” and “dark current” controls are turned until the oscilloscope trace spot is just extinguished. The photometer deflection is noted a t this setting, which is the triggering on-off position of the compatibility device. This critical deflection is used to preselect the gain setting required to produce a fluorograph a t the desired proportionate level of the maximum peak intensity. This gain setting and the emission intensity then determine the attainment of the requisite photomultiplier output to cut off the cathode beam ttace. A solution of the sample at a concentration below that exhibiting selfquenching is placed in the sample cell, and the excitation and emission servos are adjusted manually as usual to produce the maximum emission intensity. The gain is then adjusted by means of the photometer meter-multiplier switch and sensitivity control SO that the peak emission intensity produces the crit’ical deflection of the photometer. If gain is then increased to produce a deflection X% greater than t’he critical deflection, the ultimat’e fluorograph will exhibit a contour pattern of the fluorescence characteristics corresponding to the (100 X)% level of the peak intensity.
LA.-
Figure 29. G-Aminco-Bowman spectrophotofluorometer; H-”meter-multips,er” photometer; I-circuitry of Figure 2 b and 2c (voliage sensor, oscillator, and amplifier); Jcathode ray oscilloscope VOL. 36,
NO. IO, SEPTEMBER
1964
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I
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Dry-cel I battery
+ 25 pf
90 v o l t s
-b Figure 2b.
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800 k
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Voltage sensing circuit
Next the scan motors are turned on full speed, and one is adjusted to put its rotation slightly out of phase with the other. The camera shutter is opened and the exposure continued for at least 50 scans. The finished print is a fluorograph constituting a contour pattern a t a plane passing through the envelope of fluorescence spectral peaks (and others due to normal and Raman scattering) at’ the pre-selected (100 X)% level of the emission maximum. A three-plane composite is obtained by making stereofluoropaph contour patterns a t lo%, 50%, and 90% of the emission maximum. For a four-plane stereofluoroaraph the recommended levels are 20%, 40%) SO%, and 80%. To enlarge the peak cross-section contour patterns, the oscilloscope deflection controls are adjusted to center the contour patt.ern and to expand the scales to read in whole multiples such as 50 mp per cm. For a 25-fold scale expansion the aperture of the camera
lens is changed from 1/16 to fill for Polascope 410 film t o maintain the same contrast. Such enlarged fluorograljhs for chrysene and 3-methJ’lcholanthrene are shown in Figure 3. RESULTS A N D DISCUSSION
Preliminary application to five polynuclear hydrocarbon types [phenanthrene, benzanthracene, dibenz(a,h)anthracene, chrysene, and 3-methylcholanthrene] in n-hexane solution indicates that by using the current technique, they can be identified a t levels of 0.005 p g . per ml (amethyl-
380
cholanthrene) to 0.05 pg. per ml. (phenanthrene, chrysene) and they can be determined a t twice these concentrations with a precision of approximately &lo%,. It should be pointed out that these results by no means represent the full potentialities of this technique. At this time the instrument is being used at about one thousandth of its potential amplification. Moreover, the relatively poor resolution (about 5 mp) of presently available instrumentation is inadequate to fully resolve the closely spaced maxima characteristic of polynuclear hydrocarbons; consequently,
-------380
420 420 Emission ni u Figure 3. Upper: fluorograph for chrysene, 1.1 4 pg./ml. Lowec fluorograph for 3-methylcholanthrene, 1.1 0 pg./ml.
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ANALYTICAL CHEMISTRY
the enlarged fluorographs do not exhibit sharl)ly defined contours. Even a t its present state of development, however, it, is evident that by integrating all the fluorescence spectral parameters into a stereoenvelope, the technique yields a "stereofingerprint" of pure fluorescent compounds. hccordingly, it should permit unique characterization of even very closely related structures. The major contour pattern of the polynuclear hydrocarboni5 appears to be characteristic of the basic skeletal ring structure. By combining the method with such isolation t,echniques as gas chromatography, paper and thin layer chromatography, electrophoresis, etc., it should be possible to make positive identification and determination of highly fluorescent substances at levels far below those required for other techniques of characterization. For this purpose, when the pure compound is
available for comparison, the spectra need not be corrected for the distortions inherent in the instrumental parameters. It is recommended, however, that publication of data, corrected or uncorrected, be referenced to a convenient standard, such as quinine, so that the reader can a t least orient the data in terms of the response of his own instrument. Continuing development is directed toward increasing scanning speed, improving sensitivity and resolution, and extending the technique to recording of phosphorescence phenomena. The last involves a fourth parameter, decay time, which may well be integrated into a stereographic representation. The improved technique will be applied to some thirty polynuclear hydrocarbons now a t hand and to as many others as we can obtain. It is believed that when sufficient data,
corrected for instrumental distortions, have been acquired, not only will specific identification of these hydrocarbons be possible, but also fluorescence characteristics can be correlated with detailed structural features to aid in structure determination of new fluorescent compounds. LITERATURE CITED
(1) General Electric Co., Semiconductor
Products
Department,
Schenectady,
N.Y., Transistor Manual, 6th edition,
p. 194, 1962. (2) Ibid., p. 167. ( 3 ) Ibid.. D . 196. (4)Saw&, E., Hauser, T. R., Stanley, T. W., Intern. J . .4ir Pollution 2, 253 (1960). ( 5 ) Van Duuren, 3. L., J . S a t . Cancer
Inst. 21, 1 (1958).
Pithburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 7 , 1963, Pittsburgh, Pa.
Improved Techniques for Routinely Counting l o w levels of Tritium and Krypton-85 Mary L. Curtis and H. L. Rook, Monsanto Research Corp., Mound Laboratory, Miarnisburg, Ohio
beta emitters R in the gas phase, ofcounted in the ADIOACTIVE ASSAY
Geiger or proportional region as part of the count,er filling, is a well-known analytical method (1, 3 ) . The application of this method to routine counting, using new techniques for precisely and rapidly analyzing tritium in helium-3, and krypt,on-85 in xenon, will be described. An analytical method for krypton-85 by gamma counting will also be presented. The tritium analyses are made in conjunction wit'h Mound Laboratory sales of highly purified He3. The Kr85 in xenon analysis was developed to evaluate the effectiveness of methods of purifying xenon intended for use in scintillation counters. Numerous samples varying from 1 0 - 2 to lo-'* mole yo are analyzed daily by these techniques. Small amounts of nonradioactive impurities, such as water vapor, osygen, nitrogen, argon, and carbon dioxide, are also present in these gases
added in parallel with the existing 47-pf. capacitor to eliminate double pulsing. For proportional counting, the output of the counting tube was connected to the input of the pre-amplifier of the counter. A schematic of the vacuum system (used for gas transfer) is shown in Figure 2. A 2- X 2-inch XaI(T1) well crystal and a Model 39-12 Radiation Instrument Development Laboratory 400-channel analyzer were used for the gamma analyses of Kr*5. Tritium Analysis. GEIGERCOUNTI K G OF TRITIUM I N HELIUM-3. For counting tritium, two counting tubes L A n o d e Terminal
Cathode Terminal T
b
;
y
r
lnlernol Silver Mirror
EXPERIMENTAL
Apparatus. T h e counting tubes (Figure 1 ) were made of 2-inch 0.d. borosilicat,e glass tubing. T h e y were silvered internally by the Brashear process (2) to the same length as the 2mil diameter tungsten anode. For Geiger counting, shielded probes were used to connect the output of the counting tubes to the Geiper input of a Xuclear AIeasurements Corp. Model PC-313 proportional counter. h 105-ohm resistor was added in series with the esiRting 68,000-ohm resistor in the Geiger input circuit to attenuate the pulses, and a 56-pf. capacitor was
Figure 1 . tube
U Borosilicate glass counting
were evacuat'ed and filled with a mixture of 98y0 helium and 2% isobutane to a pressure of 340 torr. After the background was determined, the tubes were placed over the standard volumes on the sample introduction system, and evacuated with stopcock J closed, which separates the cold from the hot side of the vacuum system (Figure 2). Stopcocks B and C (over the standard volumes) and K (leading to the vacuum rack) were closed, and t,he sample was allowed to expand into the standard volumes from the gas sample container through A . The pressure indicated by the micromanometer as read by a cathetometer was measured, stopcocks D and B were closed, and B and C were opened, allowing the sample to espand into the counting tubes. The amount of gas in t'he tubes a t atmospheric pressure and room temperature (20' C.) was computed from the pressure and comparative volumes. The counting tubes were transferred to tapered joints JT and A' on the cold side of the vacuum rack, and the helium isobutane mixture was added to a pressure of 340 torr by: opening stopcock H and the valve controlling the counting gas inlet; opening the stopcock on one of the counting tubes and allowing the counting gas to enter until the mercury reached a predetermined point on the manometer; and closing the stopcock on the counting tube and the valve on the counting gas inlet. Counting gas was introduced into the second tube containing a duplicate sample in a similar manner. A voltage plateau was determined for each tube filling since plateaus vary slightly from sample to sample. The tritium concentration is given by: VOL. 36, NO. 10, SEPTEMBER 1964
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