Direct Recording of High Resolution Mass Spectra of Gas

Volatile fatty acids and methane production in relation to anaerobic carbohydrate fermentation in Oryctes nasicornis larvae (Coleoptera: Scarabaeidae)...
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water-peak (D). This is because peak (C) includes tritiated acetone as well as tritiated water. The actual amount of water carried in the acetone peak was estimated from the area of the negative radioactivitypeak. A typical result found is that a 10-11. sample of acetone removes from the column 0.004 11. of water resulting in 400 p.p.m. water in the acetone peak. This value was derived from the

area of the negative radioactivity-peak calibrated by using arem of the positive radioactivity peaks obtained from injection of known amounts of tritiated water. LITERATURE CITED

(1) Halford, J. O., Anderson, L. C., Bates, J. R., Swisher, R. D., J . Am. Chem. SOC.57, 1663 (1935). (2) Howard, K. S., Pike, F. P., J . Phys. Chem. 63, 311 (1959).

(3) Seita, R. O., Central Research Laboratories, Air Reduction Co., Murray Hill, N. J. 07971, private communication, 1964. (4)Weinstein, A., ANAL. CHEM.32, 288 (1960); 33, 18 (1961). (5) Zhukhovitakii, A. A., Turkeltaub, N. M., Gaier, M., Lagashkina, M. N., Malyasova, L. A., Shlepuzhnikova, G. P., Zavodsk. Lab. 29, 8 (1963). RECEIVED for review November 18, 1964. Accepted April 5, 1965.

Direct Recording of High Resolution Mass Spectra of Gas Chromatographic Effluents J. THROCK WATSON and KLAUS BIEMANN Department o f Chemistry, Massachusetts Institute of Technology, Cambridge, Mass.

b A technique for the recording of complete high resolution mass spectra (resolving power: 1 part in 10,000 or better) of compounds emerging from a gas chromatograph i s discussed. The pressure reduction system used for the connection of chromatographic column and mass spectrometer is short, simple in design, and made entirely of glass which make it suitable for the study of complex, polar, and high-boiling compounds. The quality and magnitude of mass spectral data obtainable by this technique demonstrate the value it will have in the identification or determination of the structure of compounds separated by gas chromatography.

B

gas chromatography andalthough less widely availablemass spectrometry have become valuable techniques in organic chemistry. While the former is, strictly speaking, a separation technique and the latter provides unique characterization of organic molecules, two of their common characteristics, namely high sensitivity and the requirement of a vaporized sample, make a combination of these two techniques a desirable method more powerful than either one considered individually. Some of the immediately obvious advantages are: an increase in the speed of analysis, elimination of the necessity for isolation of minute quantities of pure sample, elimination of sample alteration when coming in contact with the atmosphere (air oxidation, hydrolysis) , and greater certainty in identification of the eluted component than that achieved by retention time alone. A number of laboratories have successfully operated a gas chromatograph in combination with a conventional single focusing mass spectrom844

OTH

ANALYTICAL CHEMISTRY

eter. The earlier work ranges in implementation from the simple monitoring of a single mass (10, 11) to the fast scanning of a preselected part (12) of the spectrum, or all of it, using oscillograph recorders (5, 7 , IS, 14, 17) or oscilloscope photography (5, 10). Inline trapping of fractions in a manifold system located between gas chromatograph and mass spectrometer has also been used (8, 16). Coupling of gas chromatography and mass spectrometry is extremely useful in the study of natural products where one may encounter complex mixtures containing trace components that are difficult to isolate and identify. I n a study of neutral fecal steroids, impressive results have been obtained with a combination of a gas chromatograph and a fast scanning, conventional (low resolution) mass spectrometer (18). Data obtained with a mass spectrometer of much higher resolving power provide, however, unique information not derivable from conventional spectra and this becomes very important in the work on complex molecules of unknown structure (2). Careful measurement of the molecular and fragment ions permits determination of their elemental composition (1) which enables one to make a less ambiguous interpretation of the spectrum than when examining a conventional low resolution mass spectrum in which case a number of different elemental compositions have to be considered for each nominal mass. A resolution of 1 : 10,000 is generally sufficient to permit mass determination with the accuracy required for work with compounds of molecular weight up to 500, although 1 :20,000 or more is sometimes required. We have, therefore, explored the feasibility of obtaining complete high resolution mass spectra of compounds such as amino

esters, alkaloids, carbohydrates, and steroids while they emerge from the gas chromatograph directly into a double focusing mass spectrometer. We are using a CEC 21-110 mass spectrometer which is based on the Mattauch-Herzog design and thus focuses all ions in a focal plane. A photographic plate placed in this focal plane is used for the recording of the high resolution mass spectra. This technique eliminates many of the problems inherent in the direct combination of gas chromatograph and mass spectrometer, First, the disturbing effect of the change in sample concentration associated with the emergence of gas chromatographic fractions is eliminated due to the integrating effect of the photographic emulsion which is bombarded by all the ions all the time. Second, since the entire high resolution mass spectrum is simultaneously recorded , the necessity of rapid scanning during the time of emergence of the gas chromatographic fraction is eliminated. A point concerning the relative sensitivities of a us. electron photographic plate multiplier should be made. When using magnetic or electric scanning at high resolving power the electron multiplier is exposed to each peak in the spectrum only for a very small fraction of the time taken for the total scan. When the lifetime of the sample in the ion source is so short that one must scan the spectrum in 1 to 10 seconds, the signal recorded for each peak in the spectrum becomes diminishingly small. On the other hand, the photographic plate is exposed to all the ions simultaneously and thus achieves greater over-all sensitivity. Based on the sensitivity of photographic emulsions [io5 ions/mm.2 necessary to produce visible blackening (IS)] one can conclude that a measurable line (1 mm. long, 10 mic-

the residence time of the sample in the ion source. I n addition, the high resolving power of a double focusing mass spectrometer makes it possible to feed continuously a calibration compound into the ion source to provide mass standards for the mass determinations. The calibration compound is carefully chosen so that there is no overlap with compounds being analyzed-Le., a t any given nominal mass the Calibration line is well resolved from the compound lines. This contribution of added calibration compound is later automatically removed during the computation process (6). PRESSURE REDUCTION SYSTEM

The most critical part in any such combination of gas chromatograph and mass spectrometer is the pressure reduction system connecting t h e two instruments. T o minimize memory effects (tailing after the column) and ___r_l ___-__, ..._ catalytic or thermal decomnn*it.ion the connection has to be as sf 101%as possible (particularly in the re:gion of high pressure), easily heatabl e, and free of metal. The pressure reductic)n system we are using serves two purl:boses. First, it provides a pressure drop from 1 atm. to 10-6 mm. Hg and, secoiid, it leads to concentration of the saniple in the gas stream entering the ion source of the mass spectrometer. Figure 1 shows a sckiematic of an improved modification of our earlier system (19). I t involveri a fritted glass tube instead of the metal valve generally used (8, 9, 11, 14, 16) t(> pump off the The ultrafine excess carrier gas. porosity of the tube has the additional effect of permitting heliiim, the carrier gas, to he removed at a higher rate (due to preferential effu.sion) than the eluate (sample), the molecular weight of which is, of course, muc:h higher . . than . that of helium. This results m the relative enrichment of the sample (with

Figure 2.

Pressure reduction system, dirosrembled VOL. 37, NO. 7, JUNE 1965

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Figure 3. Schematic of spectrometer with gas chromatograph attached to modified ion source housing

respect to carrier gas) which passes through the second capillary, constriction into the ion source of the spectrometer. This enrichment was found to be, for example, 50-fold for diethyl ether, by introducing a mixture of helium and diethyl ether both through the pressure reduction system as well as the conventional reservoir into a Bendix Timeof-Flight mass spectrometer and measuring the abundance ratio of mass 31 us. mass 4 in each case. The enrichment factor is, of course, related to the molecular weight of the sample-Le., better for higher molecular weight compounds. For the purpose of this experiment the gas chromatographic column was removed. From this enrichment factor and an estimate of the total amount of gas entering the ion source one can roughly estimate that

Figure 4.

846

at least loyo,but probably not more than 50%, of the sample injected into the gas chromatographic column reaches the ion source, the rest being lost through the walls of the fritted tube. The fritted tube is held by means of two silicone rubber O-rings a t either end of a small copper pipe which constitutes a vacuum chamber when connected to a mechanical pump. The conditions of effusion (20) must be satisfied inside the fritted glass tube for the pressure reduction system to provide an enrichment of the sample with respect to the carrier gas. To maintain molecular flow (effusion) the mean free path of the gas must be 10 times the diameter of the pores through which it passes. Since the pores in the fritted tube are approximately 1 micron wide, the pressure inside the tube should be of the order of a few millimeters of

mercury. Thus the diameter of the entrance constriction is important in effecting a pressure drop sufficient to satisfy conditions for effusion and in establishing the flow rate into the pressure reduction system. The exit constriction diameter controls the sensitivity of the apparatus limited only by the maximum pressure that can be tolerated in the ion source of the mass spectrometer. The constrictions are prepared by collapsing the capillary walls by a process of trial and error. A diameter of approximately 0.2 mm. for the entrance orifice and 0.1 mm. for the exit orifice were found to be best. The apparatus is shown disassembled in Figure 2 . The gas chromatographic column is made of 3-mm. i.d. borosilicate glass (Pyrex) tubing (6 feet effective length) wound into a helix (2-inch o.d., 8 inches long). The injection port consists of a serum cap of silicone rubber. Helium is supplied a t a slight over pressure (0.5 p.s.i.) to the injection port via the borosilicate glass (Pyrex) tube intersecting the column just below the serum cap. The first 2 inches of the column below the injection point are filled with glass wool and wrapped with a heating tape to form the vaporizer. The column is connected directly to the capillary containing the entrance constriction to the pressure reduction system via a short piece of silicone rubber tubing, The porous glass tube (8-mm. o.d., 2-mm. wall thickness) is 8 inches long and of ultrafine porosity (average pore diameter 1 micron, obtainable from Corning Glassware, Corning, N. y.). Beyond the exit constriction a short piece of glass tubing (4-mm. 0.d.) extends through the walls of the ion source just into the ionization

"Beam monitor chromatogram" of a series of esters from methyl caprate to methyl arachidate

ANALYTICAL CHEMISTRY

chamber. Thus the effluent from the gas chromatograph contacts only glass and ,is channelled directly into the electron beam of the ion source, no more than ,I4 inches from the end of the column packing. The evacuation chamber .consists of a &//.-inch copper pipe fitted on both ends with "Quick-Connect" fittings which provide the means for vacuumtight O-ring seals (silicone rubber) around the glass capillary. The small pipe a t right angles to the evacuation chamber leads to a mechanical pump. The evacuation chamber is connected to the mass spectrometer ion source housing by means OE a stainless steel flange and is wrapped with heating wireabove the flange; theentirechamber is kept at a reasonably uniform temperature because of the high thermal conductivity of copper. Because of its low thermal conductivity, stainless steel was used for the flange to avoid heat loss to the ion source housing. The large cylinder a t the top of Figure 2 provides both a support as well as an oven for the gas chromatographic column when bolted to the circumference of the flange. The lower slotted portion is of stainless steel and the solid walled upper portion is of brass which provides even distribution of heat when wrapped with heating wire. All of the components have separate heaters and their temperatures are monitored by appropriately placed thermocouples. Practical limits of the temperature of the components are as follows: injector, 350" C.; column oven, 300" C.; pressure reduction system, 275'-300° C.; and ion source 225'300' C. Since we use the high resolution mass spectrometer as a highly sophisticated fraction detector and are-at least at present-more interested in qualitative work rather than quantitative aspects, it was possible to greatly simplify t,he gas chromatographic section of the system. I t has been reduced to the only essential part, a heated column. Thus it represents more a modified inlet system for the ma= spectrometer rather than a separate unit. The pressure reduction system remains permanently attached to the ion source housing and, with the helium flow clamped off and the mechanical pump running, does not interfere with the normal operation of the instrument using individual. samples. The column is attached to the pressure reduction system by a simple silicone rubber connection which facilitates interchange of various columns without completely dismantling the apparatus. For a display of the chromatogram in the conventional sense the variation in the intensity of total (unresolved) ion beam is recorded using the beam monitor of the mass spectrometer. As shown in Figure 3, it intercepts and

PP P3 24 PI

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Figure 5.

69

74

Section of photo plate containing rpectto of the esters shown in Figun5 4

measures part of the ion beam after leaving the electric sector and before entering the magnetic field. In the absence of a chromatographic fraction entering the ion source, the beam is mainly due to the calibration campound (perfluorokerosene). Helium seems to contribute little since all the parameters are set for maximum intensity a t high masses (300-500). Mass discrimination is thus greater for very low masses preventing most of the helium ions from passing through the first slit of the electric sector. When a fraction emerges from the chromatograph, the total ion intensity increases and this change is displayed on the pen-and-ink recorder connected to the beam monitor. Only if a very large amount of organic material-e.g., the solvent-emerges do the potentials in the source get sufficiently disturbed &s to lead to a temporary loss of intensity by "defocusing" the source and thus to a dip in the signal or even to a negative peak.

EXPERIMENTAL In operation the pressure in the ion source is about 1-2 X mm. Hg when a good mechanical pump-i.e.,

Duo-Seal 1400B-is

connected to the pressure reduction system. 1:he entrance of the gas chromatograp hic column is kept at a slight overpressure (about 0 5 p s i ) of helium. 'Un,der these conditions a flow rate of 20 nlI./ minute (into the column) is established. Although'the beam monitor was Inot specifically designed to function a:; a detector for a gas chropatogFaph, I t behaves quite well as shown in FigU l t 4. Since thm trace represents the Ion beam1.e.. sample concentration in the ion source us. time-it also demonstra tes that there are no prohibitive memq3rY effects due to adsorption or conden ation in the pressure reduction system and ion source. Figure 4 is a chromatogram of'l pI . of a solution containing 2-4 pg. each of 11 fatty esters (3% JXR on Gaschrom P. injector: 260°/eolumn temp.: pcogrammed, start a t I6Oo/vacuum jacket: 24Oo/glass extension into ion source: 185'). The vertical slots in the chromatogram trace indicate the sh ort periods of time'when the ion beamI is deflected.off the plate,(and thus off the beam monitor) which IS necessary wlien changing the position of the pha,tographic plate, a process that takes ab,out 2-3 seconds with our present arran gement. This pen deflection provide!3 a useful indication of the exact portitons of the chromatogram being reconied VOL. 37, NO. 7, JUNE 1965

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P l a t e 237 chromatogram by ion beam monitor

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Figure 7. Magnification of 0 P O rtion gram represented by Figure 6

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07

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293

28 I Figure 8.

Densitometer trace of region of plate shown in Figure 7

with the mass spectrometer and aids in the rorrelation of the spectra on the plate with the chromat,ogram. The photographic plate provides space for up t o 29 usable spect,ra and it is thus possible t,o analyze reasonably complex mixtures by this technique. The numbers along the baseline between the vertical slots correspond to the position of the photographic plate exposed for that length of time. Figure 5 shows the full width and approximately one tenth the length of the plate where many of the masses of the fatty esters are discernible (the magnetic field was set to focus the region from mass 12-360 on the plate. The column of numbers a t the left in Figure 5 refers to the exposure positions on the photographic platme. Those lines which are of similar density in all exposures are due to the calibration compound-e.g., m/e 69. Others, as for example, portions of the multiplets at m/e 74 and 87 appear and disappear; these are fragments which are characteristic of methyl esters of fatty acids. Multiplets are discernible a t several nominal masses even under this quite low magnification, not only rather widely spaced ones, due t'o ions of the perhalogenat'ed mass

standard and the ester, but also narrow doublets of the C , H us. C,H,O type from the ester-e.g., a t m/e 83-85. Exposures 5 and 7 are overexposed as far as masses 74 and 87 are concerned, which could still be measured on exposure 8 as it cont,ains the tail of the fraction represented by exposure 7. X very short exposure (1 second) during the emergence of such an intense peak would still be better. Figure 6 shows a chromatogram of a fraction of alkaloids extracted from the bark of Aspidosperma quebracho blanco. In an attempt to improve the information obtainable from the poorly-resolved gas-chromatographic fraction, the doublet was recorded in three separate parts (19, 20, and 21 in Figure 6). X closeup view of the photographic plate (Figure 7 ) shows a definite trend in the appearance and disappearance of two different compounds in consecutive exposures during the emergence of the incompletely resolved fraction. Figure 7 is of that region of the plate which corresponds to m/e 280 through m / e 293. The numbers a t the left correlate the plate positions esposed to the various segments of the chromatogram (Figure 6)-e.g., positions 17 and

22 are background. Koniinal mass 281 is always a doublet; one peak, C6Fll,is from perfluorokerosene, the other, C7H2102,8Si4, from the bleeding of silicone compounds from the gas chromatograph. The spectrum a t position 18 was obtained during the emergence of the first chromatographic peak which contained a compound having a molecular ion a t mass 280. Positions 19, 20, and 21 show the trend in the appearance first of a compound that has a fragment a t mass 284. h mixture appears in position 20 and finally in position 21 mass 284 virtually disappeared and the compound with molecular ion a t mass 282 clearly dominates the spectrum. The lines a t 279 and 294 in exposure 23 illustrate that even one half of a very weak chromatographic peak gives well-discernible spectrographic lines. The two major fragments of the calibration compound (perfluorokerosene) in this region are C6Fl,and CiFll (see Figure i). The exact masses of these fragments are used to calculate the masses of all the other lines in the region by carefully measuring their distances relative to the 1)ositions of the two calibration fragments. This formidVOL. 37, NO. 7,JUNE 1965

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Figure 9 . Magnificotion of photo plate in region of molecular ions of androstane-3,17-dione and AP-andrortene3.1 7-dione able task is accomplished using a semiautomatic comparator-densitometer (4) for the distance measurements, followed by feeding the resulting data into a digital computer (InM 7094) for further conversion t o masses and elemental composition (6) or further treatment (2, 3). The masses determined for a few lines are shown in Figure 7; the difference (in millimass units) between the mass calculated from the lines on the plate and the theoretical mass of the combination of the elements indicated is given in parentheses. It can he seen that the mass measurement is sufficiently accurate to assign unambiguously a definite elemental composition to each of the ions observed. While it may be difficult for the untrained eye to see the subtle differences in intensity on the plate, they are readily distinguished in densitometer scans of the same area of the plate as shown in Figure 8. The bottom trace shows only the calibration compound with nominal masses at 269, 281, and 293. The third trace shows the appearance of mass 284 from deacetylaspidospermine (molecular weight 312) while the fifth trace shows the virtual disappearance of mass 284 as quehrachamine clearly dominates the spectrum with its molecular ion at mass 282. I t should he noted that the recording densitometer used for this scan (which is shown only as a pictorial representation of the relative densities but is not used for any actual measurements) was not designed to measure lines as sharp as those of a high resolution mass 850

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spectrum. The slit on this densitometer is much wider than the line width, hence the resolution in the doublet at m/e 281 appears to he quite poor in this trace in contrast t o the appearance of the lines on the plate where they are completely separated (see Figure 7). The baseline appears to be quite noisy; this is due to the slight grain in the photographic emulsion, an effect exaggerated in the densitometer scan because of the logarithmic representation of the output. In a similar experiment a mixture containing androstane, androstane-17one, androstane-3,17dione, and Atandrostene-3,17dione was injected into the column (3% JXR on Gas-Chrom P, injector: 280" C./column: 260' C./ pressure reduction system: 230' C.) and the chromatogram indicated incomplete resolution of the last two components under these conditions. T o obtain spectra of each pure component, three exposures (15, 16, 17) were taken during the emergence of this chromatographic doublet. Figure 9 is that section (in the region of m/e 281-293) of the photoplate which contains the molecular ions of the saturated and unsaturated diketones. A definite trend can he seen starting with exposure 15 which contains the molecular ion of androstaue-3,17dione a t mass 288. The next spectrum is that of a mixture because A4-androstene-3,17dione (mass 286) begins to emerge, which finally dominates in the third spectrum (exposure 17) while the line at m/e 288 virtually disappears. Some of the determined masses and

corresponding elemental compositions derived from measurement of line positions are incorporated in Figures 7 and 9. These values demonstrate that the molecular composition of even a rather complex organic molecule such as a difunctional steroid or an indole alkaloid, can be determined regardless whether emerging as a single fraction or as an incompletely resolved mixture. These values shown are hut a few of the many mass measurements (35WfW performed on each spectrum using the techniques discussed elsewhere (4). The completeness and accuracy of these measurements, even though obtained on a minute, transient sample such as a gas chromatographic fraction, are sufficient for data processing and conversion to complete high resolution mass spectra in the form of "element maps," the principle and interpretation of which has been discussed elsewhere (2, 3). Since the computer can automatically subtract the lines produced by the simultaneously recorded calibration compound as well as those of the column bleeding, spectra free of this hackground and representing only the emerging fraction are thus produced automatically. LITERATURE CITED

(1) Beynon, J.

H.,"Advances in Mass Spectrometry," J. D. Waldron, ed., 1, pp. 328-54, Pergamon Press, London,

1959. (2) Biemmn, K., J. Pure A p p l . Chem. 9 , 95 (1964).

(3) Biemann, K., Bommer, P., Desiderio, D. >I., Tetrahedron Letters, Yo. 26, 1725 (1964). (4) Bommer, P., Mchlurray, W.J., Biemann, K., Twelfth Annual Cons. Mass Spectrometry and Allied Topics, Montreal, June 1964. (5) Brunnee, C., Jenckel, L., Kronenberger, K., 2. Anal. Chem. 189, 50 (1962); 197, 42 (1963). (6) Desiderio, 11. M., Biemann, K., Twelfth Annual Conf. Mass Spectrometry and Allied Topics, Montreal, June

1964. (7) Dorsey, J. A., Hunt, R. H., O'Neal, M. J., ANAL.CHEM.35, 511 (1963).

(8) Ebert, A. A,, ANAL.CHEM.33, 1865 (1961). (9) Gohlke, R. S., Ibad., 31, 535 (1959). (10) Gohlke, R. S., Ibid., 34, 1332 (1962). (11) Henneberg, D., 2. Anal. Chem. 183, 12 (1961). (12) Holmes, J. C., Morrell, F. A,, A p p l . Spectry. 11, 86 (1957). (13) Lindeman, L. P., Annis, J. L., ANAL. CHEM.32, 1742 (1960). (14) McFadden, UT.H., Teranishi, R., Black,'D. R., Day, J. C., J . Food Science 28, 316 (1963). (15) Miller, D. O., ANAL.CHEM.35, 2033 (1963). (16) Owens, E. B., Zbid., 35, 1172 (1963).

(17) Ryhage, R., Zbid., 36, 759 (1964). (18) Ryhage, R., J . Lipid Res. 9, 245 (19641. (19) Watson, J. T., Biemann, K., ANAL. CHEM.36, 1135 (1964). (20) Zemanv, P. D., J. A m ..l i e d Phvsics 23, 924 (i952). RBCEIVEDfor review January 26, 1965. Accepted March 24, 1965. Eastern Analytical Symposium, New York, N. Y., Kovember 11, 1964. Work supported by research grants from the National Science Foundation (G-21037) and the Kational Institutes of Health, Public Health Service (RG-9352).

Radiochemical Determination of Lead-21 0 after Solvent Extraction as Iodide and Dithizonate N. A. TALVlTlE and WILLIAM J. GARCIA Colorado River Basin Water Quality Control Project, Public Health Service, U . S. Department o f Health, Education, and Welfare, 1750 South Redwood Road, Salt lake City, Utah 8 4 1 04 Lead-210 is determined radiochemically in environmental samples. Iron, zinc, tin, and similar heavy metals are removed by extraction of the thiocyanates with methyl isopropyl ketone. Lead-2 10 and stable lead are isolated by successive extractions as the iodide and dithizonate. The activity of the lead-2 1 0 is determined by direct counting of its beta radiation or by counting the beta and alpha radiation resulting from ingrowth of bismuth-2 10 and polonium-2 1 0. The recovery determined with standard lead-2 10 is 95% with a relative standard deviation of 6.470 when polonium-210 ingrowth is measured.

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~ ~ ~ - 2 1as0 ,a member of the uranium-238 chain, appears in the waste products of uranium ore processing. Because of its assimilable nature and because of the length of its half-life relative to human longevity, its presence in culinary and irrigational waters is of concern. A method for the determination of lead-210 was needed which would be applicable to source samples such as uranium mill tailings and effluents as well as to river waters, sediments, and biological specimens. Separation techniques applicable to radiometric determination have been reviewed by Gibson (3) and Millard (6). R e s t and Carlton ( 7 ) have shown that lead in hydrochloric acid solution can be separated from a large number of metals by a preliminary extraction of metal thiocyanates with methyl isopropyl ketone followed by the extraction of lead as the iodide with the same solvent. hlcCord and Zemp ( 5 ) separate lead from partially ashed urine by extraction of

lead iodide with methyl isopropyl ketone and determine the lead spectrophotometrically as the dithizonate. Frank ( 2 ) ,in a modification of the hlcCord and Zemp procedure, extracts lead iodide with a 6: 3 mixture of methyl isobutyl ketone and methyl ethyl ketone. I n the following procedure, iron and other heavy metals are removed by extraction of their thiocyanates with methyl isopropyl ketone after which radiolead and stable lead are separated by extraction as iodides. Following wet-ashing of the extract and a waiting period to allow for decay of short-lived lead isotopes, final purification of the lead and its separation from bismuth210 and polonium-210 are accomplished by extractions as the dithizonate. The lead-210 is determined radiometrically by direct count of its beta radiation or by counts of beta and alpha radiation resulting from the subsequent ingrowth of bismuth-210 and polonium-210. EXPERIMENTAL

Apparatus. Counts were obtained with Suclear Measurements Corp., Model PC-3A, internal proportional counters and with a n Isotopes, Inc., Model CLL-4-C, low-level beta counter. Reagents. Methyl isopropyl ketone (3-methyl 2-butanone), redistilled. Immediately before use, shake with 10% of its volume of 5y0 (0.6A7) hydrochloric acid. Weak and strong dithizone solutions. 25 and 500 mg. of diphenylthiocarbazone per liter of chloroform. Buffer solution. Adjust 0.05.11 sodium formate to pH 3.4 with 90% formic acid. Ammonia-cyanide solution. Mix 2 liters of 1 : 1 ammonium hydroxide with 400 ml. of a 1% solution of anhydrous

sodium sulfite and 400 ml. of a 2% sodium cyanide solution. Procedure. THIOCYAKATE EXTRACTION. Transfer a 50-ml. aliquot of the prepared sample solution in 5oj, (0.6S) hydrochloric acid to a 250-ml. separatory funnel. Add 10 ml of 20.11 ammonium thiocyanate and 25 ml. of methyl isopropyl ketone and shake for one minute. Drain the aqueous phase into a second separatory funnel and discard the organic phase. Repeat the extraction with additional 25-1111. portions of the ketone until the aqueous phase returns to a nearly colorless state. IODIDE EXTRACTION. Add 10 ml. of 9 X sodium iodide and 70 ml. of methyl isopropyl ketone to the aqueous phase from the thiocyanate extraction and shake for 2 minutes. Drain the aqueous phase into a separatory funnel and the organic phase into a 25O-ml. beaker. Repeat the extraction of the aqueous phase with an additional 70-ml. portion of the ketone and evaporate the combined extracts to dryness. ASHING OF RESIDUE. Cover the beaker containing the dried extract and cautiously add a few drops of concentrated nitric acid. When the foaming subsides, continue a d d q nitric acid in small increments until 20 ml. have been added. Digest just below the boiling point on a hot plate until a clear solution results. Inasmuch as the decomposition of the organic matter proceeds slowly and time should be alloued for the decay of short-lived lead isotopes, it is advantageous to continue the digestion overnight or longer. Raise the temperature of the hot plate to cause moderate boiling of the acid. When the volume has been reduced to about 5 ml., add 2 ml.of 70y0 perchloric acid. Continue to heat, while covered, to fumes of perchloric acid and then evaporate to dryness. Ilissolve the residue with 4 ml. of concentrated nitric acid and dilute with 10 ml. of water. VOL. 37, NO. 7 , JUNE 1965

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