An Instrument for the Measurement of Fluorescence of Paper Chromatographic Spots Glen F. Bailey, Western Regional Research .Laboratory, Western Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture, Albany, Calif.
the estrogen content of Iforages, a onconvenient method of analyN WORK
sis for a few tenths of a microgram of coumestrol was required. Other work at this laboratory ( I , 4) had shown that coumestrol absorbs radiation strongly in the near ultraviolet, fluoresces strongly, and can be isolated on paper chromatograms or silicic acid chromatostrips. This note describes a relatively simple instrument for the measurement of materials which produce fluorescent spots on paper chromatograms. The instrument is similar in principle to that described by Semm and Fried (6), modified to provide improved sensitivity and stability. Other instruments which have been used for this purpose (2,5) were considered needlessly sensitive or complex. The design of the instrument provides for the insertion of paper chromatograms between a radiation sourcefilter combination and a detector-filter combination. The source filter is selected for maximum transmittance of the fluorescence-exciting radiation and maximum absorbance of other wave lengths. The detector filter is selected to absorb the exciting radiation while transmitting the fluorescence-emitted radiation. The source, detector, filters, and paper chromatogram constitute a series of essentially horizontal planar elements designated respectively by H,Pc, F, and P in the schematic diagram (Figure l). These components are specified below for an instrument suitable for determining coumestrol by the procedure described in the accompanying paper (3). Any strongly fluorescent paper chromatographic spots can be measured by this method if a suitable combination of source, detector, and optical filters can be found. In the following brief description, component symbols refer to Figure 1. The lamphouse, H , contains three cylindrical fluorescent lamps (simplified drawing shows only L1, S1, and 2'1) with their axes parallel in a horizontal plane. Maximum uniformity of source brightness is achieved by minimizing the space between adjacent lamps by offsetting the middle lamp axially to make room for the lamp sockets. The upper half of the lamps is covered with aluminum foil becured with pressuresensitive cellulose tape to improve the luminosity in the downward direction. A 4.5-cm.-square aperture under the lamps is covered by the 5-cm.-square ultraviolet-transmitting filter, F1, which supplements the filter envelope of the
1726
ANALYTICAL CHEMISTRY
lamp to give more complete suppression of the visible mercury emission lines and the weak visible emission from the internal phosphor. The paper chromatographic spot, P, to be measured is centered in the irradiation beam. Under the lamp house in the center of the horizontal 45 by 60 cm. surface of a box 8 cm. deep is positioned the ultraviolet-absorbing filter, F2, covering the photocell, Pc. The lamp house is hinged to the back of the base box with the source aperture centered over the photocell. An adjustable limit stop
I
I
\ \ \
Figure 1. Schematic diagram of photometer for measuring fluorescence of paper chromatographic spots B = Burgess 4 F H 1.5-volt dry cell battery F1 = Corning No. 5 9 7 0 filter, 5 by 5 cm. F2 = Wratten No. 8 (K2) lacquered gelatin filter, mounted in photocell housing G = Spotlight galvanometer, Rubicon No. 341 4, 0,001 5 po./mm., R = 6 5 0 ohms,critical damping resistance, external = 10,000 ohms, period = 3 seconds H = Lamp house, aluminum chassis 3.8 X 10 X 20 cm. 11, 12, 13 = Sylvania 4 W blacklight blue fluorescent lamps; 12 and 13 ore omitted from tbe diagram P = Paper chromatogram Pc = Weston photronic cell No. 594, 58 mnn diameter, 38 rnm. clear aperture, is mounted with housing flush with top surface of basebox R 1 = 2000-ohm Helipot R2 = 150,000 ohms R3 = May not be required; select for optimum galvonometer damping R4 = Ayrtron Shunt, Rubicon No. 1242 51, 52, 53 = Momentary contact starting switches; 5 2 and 53 ore omitted from diagram 54 = DPSTswitch T1, T 2 , T3 = Ballast G.E. Cat. No. 8 9 6 5 2 5 for 4T5-F lamps, 1 1 8 volts, 60 c.P.s., 0.1 25-amp.; T Z and T 3 are omitted from diagram T4 = Constant voltage transformer, Sola type 30806, 1 20-volt-amp. 1 15-volt output. T5 = Line cord spotlight transformer, Rubicon No. 3 4 4 0
supports the lamp house high enough above the base to permit free horizontal movement of the paper chromatogram between the source and photocell. A sensitive spotlight box galvanometer, G, with Ayrton shunt, R4, is used to measure the photocurrent. Background fluorescence of the chromatographic paper and of the detector filter, F2, is cancelled electrically by a simple network, B, S4b, R1, R2. Variation of excitation intensity caused by linevoltage fluctuations is satisfactorily eliminated by use of a constant-voltage transformer, T4. The lamp ballast, TI, and cancellation network can be located inside the box with the controls accessible. Operation of the device is simple and direct. The switch, S4, turns on power, the lamps are started by their individual switches, 81, and the instrument is permitted to reach thermal equilibrium (about 1 hour). At that time the paper blank is centered over the detector and R1 adjusted to zero the galvanometer. Reference samples are read to establish the scale factor, and unknowns may then be estimated, with occasional checking of the zero point by the reference blank. Since in many paper chromatographic systems a rather high level of background fluorescence proceeds with the solvent front, it is advisable for the reference blank to be measured at a point on the paper a t the migration distance of the sample. This can be conveniently 'measured between spots spaced at 5 c m . intervals. Subdued illumination (not more than 5 foot candles) permits easier visual location of the fluorescent spots. Visible light from sources near the plane of the paper can be scattered into the detector to cause a n erratic blank, and should be avoided. Since the aperture diameter of the photocell specified in Figure 1 is 38 mm., measurements can be made on fluorescent spots not exceeding this dimension with maximum collection efficiency. Masks can be used to reduce the effective photocell aperture to minimize effects of background fluorescence and interference from incompletely separated fluorescent substances. They were used in the development of the analytical method for coumestrol ( 3 ) . Calibration data for. conversion of galvanometer readings to amount of fluorescing substance yielded a standard deviation of about 7% of the quantity being measured in the case of coumestrol ( 3 ) . This variability includes photometric reading errors, paper variability, and nonreproducibility of aliquots applied to the chromatogram. An operator can normally expect to measure about 60 chromatographic spots per hour, including checks of the paper blank.
ACKNOWLEDGMENT
The authors are grateful to E. M. Bickoff and A. L. Livingston for calling attention t o the need for this instrument. LITERATURE CITED
(5) Mavrodineanu, R., Sanford, W. W., Hitchcock, A. E., Coniribs. Boyce Thompson Insl. 18, 167 (1955). (6) Semm, K., Fried, R., Nuturwissenschaften 39,326 (1952).
C. R., DeEds, F., Snence 126, 969 (1957). (2) Brown, J. A., Marsh, M. M., ANAL. CHEM.25,1865 (1953). (3) Livingston, A. L., Bickoff, E. hf., Guggoh, J., Thompson, C. R., Zbid., 32, 1620 (1960). 14) , , Lvman. R. L.. Bickoff. E. hl.. Booth. A. N., Lkngstdn, A. L.,'Arch. Biochem: Biophys. 80, 61 (1957).
MENTIONof specific products does not constitute endorsement by the U. S. Department of Agriculture over similar products not specifically named.
~
(1) Bickoff, E. M., Booth, A. N., Lyman,
R. L., Livingston, A. L., Thompson,
Ammonium Bisulfate Fusion. Other Techniques
Application to Trace Analysis by Spectrochemical and
Cyrus Feldman, Oak Ridge National Laboratory, Oak Ridge, Tenn. P E ~ O C H E Y I S T Soften
dissolve sam-
S ples in order to prepare them for analysis. If the sample is a corrosion scale, refractory oxide, or insoluble fluoride, a potassium pyrosulfate fusion is usually indicated. However, the gram quantities of pyrosulfate involved are often a great inconvenience in performing the subsequent spectrographic, colorimetric, or electrochemical determination. There is no satisfactory way of removing the pyrosulfate alone, so instead the elements of interest are usually removed from an aqueous solution of the fusion cake by precipitation or extraction. This can be done satisfactorily for some groups of elements, but is difficult and uncertain with others--e.g., alkaline earths-and essentially impossible for the alkalies. I n any case, the difficulties of collection increase rapidly as the quantity of material collected decreases. In order to make the fusion technique generally useful for trace analysis, a way is needed to remove the fusion agent after use without losing any of the constituents of the sample. A second disadvantage of potassium pyrosulfate fusion is the tendency of the melt to solidify as SO8 is evaporated. If the sample does not dissolve quickly, it may be frozen in place and lost. A third difficulty arises in connection with the determination of impurities in insoluble fluorides. If trace elements are to be determined in an insoluble fluoride and the impurities must be chemically concentrated, a potassium pyrosulfate fusion would render the sample soluble. However, an additional contamination danger now appears in that molten fluoride attacks most nonmetallic crucible materials, and potassium pyrosulfate attacks platinum a t fusion temperatures (>350' C.). A fusion agent was sought which would be effective as a solvent, completely volatile a t hot plate temperatures, noninjurious to platinum or other common crucible materials, and available in sufficient purity.
ATTACK OF PLATINUM. Since N H r HS04 melts a t a comparatively low temperature, most chemical reactions which require the use of platinum can be accomplished without damage to platinum crucibles. If prolonged treatment a t high temperatures is then necessary to dissolve other constituents of the sample, the operation can be transferred to a quartz container. PURITY. NHJlS04 is available as an analytical reagent. Spectrochemical analysis of the residue from the evaporal tion of 46 grams of reagent grade NHJISO4 showed these impurities (Table I). No other metals were detected. Limits of detection for elements not detected ranged from 2 pg. per gram downward. Submilligram amounts of a carbonaceous contaminant were oxidized during the evaporation. Since NHIHSO~is readily vaporized, a blank determination is easily run for all impurities which would be present in the sample preparation.
ADVANTAGES O F AMMONIUM BISULFATE
Each requirement is satisfied to a large extent by ammonium bisulfate, NHBSO4. (The normal sulfate spatters, and should not be used.) EFFECTIVENESS. NH+HSO4 can perform most of the fusions for which K&O7 is usually used, although the ammonium salt does not attack some refractories if they have been fired a t very high temperatures (TanOs, ALOa, BeO). Some of these can be dissolved by prolonged (1 to 2 hours) fusion with ammonium fluoride in a covered platinum crucible. The normal fluoride, "9, is more effective than the bifluoride, NHJIF2. The bisulfate has both acid and oxidizing effects; it can dissolve many metals directly, including some, such as Zr and Nb and their alloys, which ordinarily require the acid HF for dissolution. ELIMINATION FROM SYSTEM.This compound melts to a clear, colorless liquid a t 146.9' C.; it can be vaporized smoothly and completely a t hot plate temperatures ( 2 O O O t o 300' C.). LIQUIDITY. Under a tight-fitting watch glass, NHJISO, can be kept molten indefinitely a t temperatures up to -475' C. I t boils quietly a t higher temperatures, but does not solidify. A stubborn sample can thus be treated as long as necessary. Once the sample has been dissolved, the beaker can be fitted with a ribbed watch glass and the fusion agent eliminated with no loss of metallic constituents. Volatile acidic oxides which are normally lost from pyrosulfate fusions (B203, AszO8) would be lost in this case also. The less volatile oxides, such as Moo3 and W03, are retained.
Table I.
APPLICATIONS
The following are examples of analyses in which the ammonium bisulfate's felicitous combination of properties has been of decisive importance in preparing samples for spectrographic analysis. PLUTONIUMANALYSIS. Two milligrams of an impure plutonium oxide residue were submitted to our high radiation level analytical facility for quantitative determination of all detectable impurities. The sample was fused with NHJlSO4 in a small quartz beaker, the bisulfate evaporated, and the sulfate residue dissolved. Then the
Impurities in NHlHSOd
(pg. metal per gram of NH4HS04)
AI
Be
Ca
Cr
Cu
Fe
0.36
0.02
1.7
0.16
0.08
0.96
Mg 0.30
Mo
Ka
0.23
8.7
Ni 0.36
VOL. 32, NO. 12, NOVEMBER 1960
Sr 0.5
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