Measurement of Tritium in the Effluent of a Gas Chromatography

Measurement of Tritium in the Effluent of a Gas Chromatog r a phy Column

Downloaded by UNIV OF SUSSEX on August 31, 2015 | http://pubs.acs.org Publication Date: April 1, 1963 | doi: 10.1021/ac60197a050

ARTHUR KARMEN, IRMGARDE McCAFFREY, JAMES W. WINKELMAN, and ROBERT L. B O W M A N laboratory of Technical Development, National Heart Institute, Bethesda 7 4, Md.

b Two methods for assaying tritium in compounds analyzed by gas chromatography are described. When the tritium in each component i s sufficient for accurate assay in less than 10 seconds, the assay is performed during the analysis. The column effluent is passed through a combustion train consisting of a heated tube containing copper oxide in which organic materials are converted to carbon dioxide and water; a tube containing heated iron, maintained in the reduced state by a stream of hydrogen gas, in which the water reacts to liberate tritium-labeled hydrogen gas; a magnesium perchlorate water trap; and an ionization chamber or a transparent tube filled with anthracene crystals for scintillation counting. When the samples contain insufficient tritium, the effluent is passed through cartridges containing p-terphenyl crystals coated with silicone oil. High boiling materials are trapped and retained. Each cartridge i s transferred to a vial containing diphenyloxazole-toluene for radioassay by liquid scintillation counting for the time required for statistically accurate results.

W

described several sensitive methods for assaying radioactivity in compounds analyzed by gas chromatography. iilthough we found these methods useful for assaying carbon-14 in samples of biological origin, tritium assays were less satisfactory. Because of the availability of many tritium-labeled compounds of biological importance and their usefulness as metabolic tracers, the specific problems of tritium assay were studied more intensively and modified procedures were developed. Compounds analyzed by gas chromatography can be assayed for radioactivity either by placing a radioactivity detector in the effluent gas stream and monitoring the gas as i t leaves the column (1, 4, 6, 9 , 11, Id, 14, 17-19) or by condensing the materials out of the effluent for subsequent radioassay (3, 6, 8, 10, 13). When there is sufficient radioactivity in the sample t o permit accurate measurement in a few 536

E H ~ V Erecently

ANALYTICAL CHEMISTRY

seconds, the first approach is more convenient. When the levels of radioactivity are too low for this, the second approach offers additional sensitivity, because the sample can be assayed as long as required for statistically accurate results. Since the amount of radioactivity in our experimental samples may vary, we have used both approaches. Condensation of the materials out of the effluent for subsequent radioassay must be quantitative. This may require more than merely delivering the effluent gas into a cooled volume, since many materials form aerosols on condensing and condensable material may be carried along for a considerable distance as an aerosol even at temperatures far below its boiling point. Several procedures have been reported (3, 5 , 6 , 10, 13). We have found that a short section of gas-liquid chromatography column a t room temperature is a convenient and quantitative trapping device for high boiling materials in the effluent (IO), and developed an automatic device for fractionating the effluent of a GLC column based on passing the effluent through a succession of sections of GLC column (8). By constructing the sections of glass, and substituting anthracene crystals for the usual solid support, carbon-14 radioactivity in each fraction was measured by scintillation counting without further transfer of the material. I n this report a modified procedure is described which makes it useful for assay of tritium as well as earbon-14. TTe have previously described experiences with two methods for monitoring tritium and carbon-14. I n both methods, the effluent is delivered to a combustion train, in which the organic materials are continuously converted to carbon dioxide and water, and the water is then reduced to hydrogen. The carbon dioxide and hydrogen are delivered to flow-through ionization chamber a t room temperature (18), or to a flox-through transparent detector cell containing anthracene crystals at room temperature or below, which is monitored by scintillation counting (9). When either procedure was used, after several hours of operation, the elution of unlabeled material from the column caused the evolution of detectable

amounts of tritium from the combustioii train. To enable these methods to be used with samples containing small amounts of radioactivity, the source of this effect was traced and a modified water reduction procedure was developed to eliminate it. EXPERIMENTAL

Method 1. Tritium Assay by Fractionating Eflluent for Subsequent Radioassay. REAGENTSAND XPPARATUS. p-Terphenyl (Packard Instrument Co., Inc.) is recrystallized from hot toluene to reduce the amount of very fine particles. It is then coated n-ith lOy0 by weight of Dow Corning DC 550 silicone oil by adding a n appropriate amount of the oil dissolved in acetone and evaporating the acetone under vacuum. -4series of glass cartridges identical to those described for use with anthracene (1.75 inches long, 7-mnl. i d . , 9-mm. 0.d. borosilicate glass tubes) are then packed with these crystals by placing a cellulose filter a t one end of the cartridge, applying suction a t this end, and inserting the other end into the terphenyl. The automatic fraction collector, it< attachment to the gas chromatographic equipment, and the details of operation in our laboratory have been described in detail (8). PROCEDURE 1. The changing niechanism of the fraction collector is triggered by an automatic timer a t regular intervals, so that the effluent of the column is fractioned into equal parts. For a given analysis the frequency with which fractions are collected ia chosen on the basis of the resolution required. .It the completion of the analpi., each cartridge is removed from the fraction collector, inverted, and placed in its entirety in a glass liquid scintillation counting vial. The cellulose filter is removed, and 15 ml. of diphenyloxazole (DPO) in toluene ( 5 grams per liter) is added. The vial is then stoppered and inverted several times. The terphenyl and contained radioactivity promptly flon- out of the caitridge, and the excess terphenyl settles to the bottom of the counting vial in equilibrium with the terphenyl in solution. The vial and its contents are then counted in a liquid scintillation counter (Tricarb liquid scintillation spectrometer, Packard Instrument CO., Inc.) a t photomultiplier voltage and amplifier settings determined to be optimal for counting tritium, carbon-14,

+

I

coDDer ox # n e

I

'

I

I

I

stee: w o o l

'17(I

i

I1 I1

COMBUSTiON FURNACE

WATER

*

REDUcTiON'

FURNACE

H2


Figure 1.

Schematic diagram of combustion train

or tritium and carbon-14 for the particular instrument being used. The counting time is determined by the levels of radioactivity present and the accuracy desired. EXPERIMENTAL 1. A series of cartridges was prepared containing coated terphenyl and another series containing Chromosorb W (Johns-Manville Co., Inc.) coated with 10% by weight of silicone oil (DC 550). Samples of a solution of tritium-labeled methyl palmitate in iso-octane were then pipetted into cartridges containing coated terphenyl, cartridges containing coated Chromosorb W, and empty counting vials. The cellulose filter was removed from each cartridge and the contents of each cartridge were expressed with the aid of a glass rod and toluene rinsing into a vial for liquid scintillation counting. Fifteen milliliters of diphenyloxazole in toluene waj then added t o each, and the counting rates were compared a t two photomultiplier voltage settings. At the higher setting the counting rate of tritium in the 10to 100-volt window was maximal for the solution containing DPO-toluene. A series of cartridges containing coated terphenyl tvas then installed in an automatic fraction collector in the effluent of a 5-foot glass column containing ethylene glycol adipate (5%) on Chromosorb W (95%). Appro=mately of the effluent 1%-asdiverted to a mass detector. Aliquots containing approximately 28,000 c.p.m. (110,000 disintegrations per minute counted in DPO-toluene) of H3-labeled methyl palmitate and a rnixture of unlabeled esters were injected into the column. The effluent of the column, from the time of injection until the methyl stearate in each sample had emerged (methyl stearate has approximately tlvice the retention volume of methyl palmitate), was collected in a single cartridge, which was transferred to DPO-toluene solution and counted. Method 2. Tritium Assay by Monitoring Effluent during Analysis. =IPP A R A T C S A N D A I A T E R I A L S . The combustion train (Figure 1) consists of a n 18-inch length of Vycor brand glass tube fitted with a side arm a t its midpoint. -411 8-inch-long electric furnace (Catalog Yo. 5682-A, Arthur H. Thomas Co., Philadelphia, Pa.)is placed over each arm. One arm is filled with copper oxide (15-ire form), and the second Lvith steel wool, coarse grade. The side arm between the tubes is filled with broken sections of Vycor tubing, n-hich also separate the steel wool from the copper oxide. -4mater trap consisting of a 3-inch length of 5-mm.4.d. glass tubing con-

taining magnesium perchlorate is placed in the effluent of the combustion train following the tube containing the heated iron. The effluent of the water trap is delivered to the radioactivity detector cell. In these experiments the detector was a flow-through, transparent detector cell containing anthracene crystals and monitored by scintillation counting. The construction of the detector, and its sensitivity to carbon-14 and tritium have been described in detail (9). PROCEDURE 2. The contents of the combustion-water reduction tube and the water trap are replaced daily. The tube is replaced in the furnaces, the column effluent is connected to its inlet, and power is supplied. Sufficient time is permitted for the furnaces to come to operating temperature (750' C.). Then approximately 5 cc. of hydrogen gas per minute is bled cautiously into the side arm. The gas flowing out of the water trap is directed to the inlet of the scintillation detector cell. The gas from the detector cell is delivered to a fume hood through a carbon dioxide trap (Ascarite). EXPERIMENTAL 2. To calibrate the system for sensitivity to tritium, a heated glass T connection fitted with silicone rubber seals was used in some preliminary experiments instead of a column to introduce radioactive specimens into the combustion train. Aliquots of a solution of tritium-labeled methyl palmitate were counted by liquid scintillation counting with comparison to a known standard and then injected into the heated T connection. The number of distintegrations detected during the passage of the peak was counted. dfter subtraction of background, the efficiency of the detector a t a given voltage and window setting was determined from the relationship : Quantity of radioactivity injected, d.p.m. = counts recorded X detector volume (cc.) X efficiency flow rate (cc./min.)

Table 1.

When optimal conditions for tritium counting had been determined, the output of the ratemeter was recorded on a strip chart. Samples of tritium-labeled methyl palmitate were injected and several curves obtained of counting rate with respect to time. Following the injection of many samples, the sensitivity setting of the ratemeter was increased so that fluctuations in background counting rate could be clearly seen. AIicroliter quantities of unlabeled methyl palmitate were then injected to determine if their passage through the combustion train caused evolution of tritium from the train or from the pseudo-column that preceded it. Folloning this, the output of a column containing Chromosorb W coated with Carbowax 400 (Union Carbide Chemicals Co.) (10% by weight of Chromosorb a t 70" C.) was connected to the train, replacing the heated glass T. Through the use of a stream-splittei, approximately of the effluent 11as directed to a hydrogen flame ionization detector. Tritium-labeled acetic anhydride was esterified with a mixture of methanol, ethanol, and propanol, n ith the alcohols in excess. To a small aliquot of the resulting mixture, a mixture of unlabeled acetates n aadded and aliquots n ere chromatographed. For combined carbon-14 and tritium determinations, carbon-14-hbeled butyl acetate was prepared from carbon-14-labeled acetic anhydride and excesr butanol, and unlabeled butyl acetate was added. Mixtures of the CI4-labeled butyl acetate, C14-labcled methanol, and tritium-labeled mixture of acetates were then injected into the column. RESULTS

When tritium labeled methyl palmitate was pipetted directly into DPOtoluene, or into cartridges containing coated terphenyl or Chromosorb which were then placed in DPO-toluene, the counting rates in the DPO-toluene solution and the DPO-toluene solution containing terphenyl were not significantly different; both were higher than those obtained in the vials containing Chromosorb (Table I). That the counting rates in each windom- setting and a t each voltage did not differ showed that there n as no significant difference in the size of the light pulse resulting from the average tritium disintegration in each mixture and no loss in transfer.

Counting Rates of Cartridges Containing H3 Methyl Palmitate

(C.p.m. X 10-3) Injected into: DPO-toluene directly Coated terphenyl; counted in DPO-toluene Coated Chromosorb; counted in DPO-toluene

Sample 1 2 3 4 5 6

10-100Tap 610-0, 32.5 32.7 31.2 31.3 31.9 32.0 32.2 32.3 28.0 28.1 27.5 27.6

VOL. 35,

NO. 4,

10-100 Tap 44.7 42.7 43.0 43.4 40.5 39.4

APRIL 1963

81 0 - m 53.0 50.9 51.3 52.1 47.2 46.3

537


--

I

i

Unlabeled Compound

il '\ Figure 2.

Analysis of mixture of H3-labeled methyl, ethyl, and propyl acetates followed by injection of unlabeled iso-octane

Record of hydrogen flame ionization detector Is superimposed on record of scintillation counter ratemeter.

Table II. Effect of Adding Chromosorb or p-Terphenyl to Vial Containing H3 Palmitate and DPO-Toluene

DPO-toluene p-terphenyl Chromosorb

+ +

Table 111.

2

538

Sample 4 5 6 c.p.m. (10 volts to infinity)

1

2

3

37.6 37.6

37.7 37.7

34.3 36.6

. ..

...

...

34.4 36.7

...

7

8

33.9

34.0

35.1

35.2

30.7

30.8

32.3

32.5

...

...

...

...

Recoveries of Ha Methyl Palmitate from Column Effluent

Injected directlv into DPO-toluene, c.p.m. X 10-3 1

I

Split

Count rate, c.P.m.

x

lo-'

Recovery,

fraction

Expected

Found

70

27.9 27.3

7/s /8

24.4 24.0

24.4 24.6

100 102

25.1 25.2

7/s /8

21.9 22.1

21.5 21.2

98 96


Full scale for ratemeter record is 1000 c.p.m

Expressing the contents of cartridges containing Chromosorb into vials containing DPO-toluene and tritium-labeled methyl palmitate which had already been counted showed that the addition of the Chromosorb reduced the counting rate to 90% of its previous value (Table 11). Recovery of tritium radioactivity from the effluent of the column using cartridges filled with coated terphenyl was quantitative (Table 111). Results 2. The eficiency of t h e 7.5-cc. 1-cm.-diameter U-shaped detector used was appro\imately 11% for tritium a t optimum conditions of counting, confirming previous results with this detector (9). Figure 2 shows the results of an analysis of the mixture of tritiumlabeled acetates recorded on a two-pen recorder which wperimposed the record of the output of the hydrogen flame ionization detector on the record of the ratemeter output. The analysis was performed folloning the elution of many other samples of tritium-labeled acetates. When 3 ~ 1 .of unlabeled iso-

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Figure 3.

Simultaneous scintillation counting of carbon-1 4 and tritium in effluent of column Lower. Pulses between 10 and 100 volts Upper. Pulses larger than 100 volts Samples injected 1. Mixture of H3-labeled acetates 2. C14-lobeled methanol 3. Mixture of 1 and 2

octane was injected into the column, a large mass peak was recorded but there was no detectable response of the radioactivity detector. In Figure 3, the loiver graph is a record of the light pulses in the spectrometer window (10 to 100 volts), and the upper graph of pulses that exceed the window or produce an amplified electrical pulse larger than 100 volts. At the photomultipler voltage setting chosen all of the tritium pulses were less than 100 volts and were, therefore, recorded only on the lower record; many of the carbon-14 pulses were larger than 100 volts, while many were smaller. When a mixture of carbon-14 methanol and tritium-labeled acetates was chromatographed, propyl acetate and methanol were superimposed. The population of pulses greater than 100 volts was a measure of the carbon-14, while the ratio of number of pulses less than 100 volts to the number greater than 100 volts was greater than expected from carbon-14 alone, and indicated the presence of tritium as well as carbon-14. An analysis of a mixture of tritiumlabeled methyl, ethyl, and propyl acetates and carbon-14-labeled butyl acetate is shown in Figure 4.

H3 d ! -

Figure 4. Analysis of mixture of Ha-labeled methyl, ethyl, and propyl acetates and C14-labeled butyl acetate Lower.

Upper.

Pulses from 10 to 100 volts Pulses larger than 100 volts

DISCUSSION

I n the work reported here, as well as in the studies reported previously, i t has been our aim to develop methods for measuring radioactivity in the effluents of gas chromatography columns that

fulfill two primary requirements: permit compounds bearing the radioactive label to be identified with the same certainty as unlabeled peaks on the same columns, or with minimal loss in the

resolving power of the analysis; and permit reasonably accurate measurement of the small quantities of radioactivity present in some samples. If small quantities of radioactivity in VOL. 35,

NO. 4,

APRll 1963

539


the effluent of a gas chromatograph are to be measured while the analysis progresses, the time available for making the measurement must be extended as much as possible. When a detector cell is used so that the radioactivity is measured during its passage through the detector, the time available for counting is determined by the ratio of the detector volume to the gas flow rate. The maximum time is limited by the requirement to maintain the resolving power of the analysis. I n many packed column chromatographic analyses. we have noted no significant loss of resolution if the time is extended to 10 seconds, but more apparent losses if the time is increased. (This response time may be considered distinct from the electrical time constant of the ratemeter or electrometer circuitrp, which only forms part of it. K e have generally chosen the electrical time constant of the equipment to be less than that of the whole system, and h a r e noted no appreciable change in the amount of real information available from the analysis, when the electrical time conrtant was varied.) I n analyzing samples with less radioactivity, the time available for counting can be extended appreciably without significant decrease in reqolution only by fractionating the effluent for subsequent radioassay; then the time for counting and the resolving power of the radioasray, determined by the number of fractions taken, are independent. Many methods have been proposed for collecting materials from the effluent of a gas chromatograph for characterization by infrared spectrometry. mass spectrometry, and chemical procedures, as well as measurement of radioactivity. Fractionation for measurement of radioactiritv requires a conceptually different approach, primarily because the methods for detecting radioactivity are very sensitive and are not influenced by the presence of unlabeled material. A. a result. the record of radioactivity eluted from a gas chromatography column need not bear any relationship either qualitatively or quantitatirely to the record of the analyqis s h o m by any mas. detector such as the hydrogen flame ionization detector. Therefore, a method for condenqing materials from the effluent for radioassay should be independent of the output of the mass detector and should make i t feasible and conrenient to fractionate the effluent. rather than to “collect peaks” as they emerge from the column. It is easier to interpret the results of an analysis if the effluent is fractionated into equal parts. It became apparent that the ability to handle a large number of fractions conveniently and automatically was a primary requirement. The method of collecting components in short sections of gas chromatography

540


column was developed to facilitate the use of an automatic fraction collector. T h a t these devices permit quantitative trapping of relatively high boiling materials from the effluent has been documented (8, 9) and repeatedly confirmed. Few studies have been carried out with materials of volatility greater than that of methyl laurate, and appreciably more volatile materials may require that the cartridges be maintained a t less than room temperature. I n most of our previous work, F e used coated anthracene crystals as the solid support in the cartridges used for trapping. Anthracene is an efficient scintillator and permitted us to assay accurately for carbon-14 by direct scintillation counting with no further transfer of the material. R e considered this important, 4nce we expected to collect a large number of fractions during each analysis. During earlier experiments we tried p-terphenyl crystals in pIace of the anthracene for C14 and found only minimal differences in scintillation efficiency. However, we chose to use anthracene crystals, despite their higher cost, because of the greater experience with anthracene crystals for measurement of radioactivity in aqueous qolution (16). We n-ere not able to evolve a satisfactory procedure for tritium using direct scintillation counting on the crystals. The presence of liquid phase on the surface of the crystals diminished the efficiency of counting tritium, but not of countjnq carbon-14. A similar diminution in efficiency u-as noted when appreciable quantities of unlabeled carrier were collected with the tritiumlabeled material. Absorption of tritium beta particles in the liquid phase and self-abwrption in unlabeled material, rather than lack of efficiency of the anthracene as a scintillator, prevented counting of the tritium directly on the cartridges. Crvqtalline anthracene is efficient as a scintillator for tritium as well as for carbon-14. To avoid the effects of absorption and self-absorption from the small amounts of material involved, we used liquid scintillation counting. Since transfer of the materials appeared to he required, our aim waq t o develop a procedure for transfering the material from a large number of fractions quantitatively with minimal inconvenience. The ease with which the terphenyl flows out into the toluene as well as the fact that the scintillation counting efficiency of the liquid scintillation solution was not changed by the addition of the terphenyl made it ideal for this purpose. Anthracene crystals cannot be substituted for terphenyl in this procedure. Not only is anthracene an inefficient scintillator in toluene solution, but in solution i t absorbs strongly at wave-

lengths from 300 to 400 mp, and addition of even small quantities will markedly lower the counting efficiency of DPO-toluene solutions. The counting efficiency of terphenyltoluene for tritium is not appreciably lower than that of DPO-terphenyltoluene, so that including D P O in the toluene solution is not essential. Since the solubility of the terphenyl in the toluene is limited and decreases significantly as the temperature is decreased, i t was felt that addition of the DPO was warranted, if only to ensure greater reproducibility and to avoid having to consider the temperature of the freezer housing the multiplier phototubes and the length of time the samples had been kept a t that temperature. The possibility of using coated white Chromosorb to trap volatiles from the effluent, followed by transfer of the Chromosorb to the liquid scintillator, was investigated. N o t only was transfer of the Chromosorb into the solution somewhat more difficult, but the counting efficiency was less. Extracting of the radioactivity off the Chromosorb r a s , of course, possible, This approach is also open to error because of possible failure to extract quantitatively. Transfer of the sample by the procedure described requires that the outside surface of the collecting cartridge be free of radioactivity. Since the fraction collector may become contaminated after repeated analyses, it is advisable to monitor the background counting rate frequently. When the sample contains sufficient radioactivity, i t is almost invariably more convenient to measure radioactivity in the effluent as it emerges from the column. A number of reports have described methods for using ionization chambers or proportional counters at ambient temperature, elevated temperatures, or ambient temperature following combustion of the sample. K e have explored the use of ionization chambers and scintillation counters in come detail and have chosen the approach described on the basis of the folloR-ing observations and reasoning. Ionization chambers respond to the passage of many unlabeled or nonradioactive materials through them. This responsiveness rather than fluctuation in base line current limits the sensitivity of the system, since increasing the electrical amplification to the degree necessary to detect small quantities of radioactivity results in detection of unlabeled materials. We noted this effect with several commercial chambers as m-ell as several constructed in our laboratory. When Cacace et al. ( 2 ) described use of a large (275-cc.) ionization chamber, in conjunction with a brisk flow (more than 1000 cc. per minute) of diluting gas, for gas chromatography, we were interested to see if dilution sufficiently


i .. .

Figure 5. Sensitivity of flow through ionization chamber to nonradioactive compounds Full scale, 3 X amp. 1-PI. liquid samples introduced as vapor into gas tlowing into ionization chamber. Record shows characteristic response to vapors of (bottom to top) petroleum ether, ethyl acetate, chloroform, methanol, acetone, and iso-octane

reduced the sensitivity of the chamber to unlabeled compounds. The effect of injecting 1-pl. samples of several commonly used solvents into the gas stream well ahead of the large chamber is shown in Figure 5 . We had no satisfactory explanation for this effect and tentatively attributed it to a change in the dielectric constant of the gas or in surface potential of the electrodes. The responses produced by injecting different materials were different. Although n-e considered exploring this phenomenon further with the thought that it might lead to the development of a qualitative detector for gas chromatography, we have not done so because of the small size of the signals produced. We also attributed the fact that no reports of this phenomenon had appeared in the literature to the relatively small signals produced, which presumably appeared insignificant in the presence of highly radioactive compounds. Since in many experiments we find it necessary to detect radioactivity in compounds with low specific radioactivity in which relatively large quantities of unlabeled material must be analyzed, we explored the possibility of

eliminating this effect by burning the sample in a copper oxide-iron metal combustion train such as that described by James and Piper (6) and used by prior to delivering i t Cacace et al. ( I , i?), to the ionization chamber. Combustion of the sample followed by an effective water trap almost completely eliminated the sensitivity of the chamber to unlabeled compound, if the combustion oven was sufficiently hot to burn the samples completely (I8). When we attempted to extend the use of either this ionization chamber method or the scintillation counting method developed subsequently t o routine assay for tritium, after several hours of operation and after several analyses, the emergence of unlabeled material from the column was accompanied by the evolution of small but detectable amounts of tritium from the system. Since this effect was observed using the scintillation detector, which had never been noted to be sensitive t o changes in gas composition, and because of the height distribution of the pulses produced, we were forced to conclude that the counts produced were attributable to tritium. When we replaced the

contents of the combustion train with new materials, we noted for the first time that precisely the same effect could be obtained by injecting repeated samples of carbon-14-labeled material. Since this effect had not been noted previously, me were led to suspect the water-reduction segment of the combustion train as the source of radioactivity, and thir suspicion was confirmed. We attributed this effect to reversible adsorption of labeled water or carbon dioxide on the oxidized surface of the steel wool or iron. The effect became more and more pronounced the longer the combustion oven was in operation. During this time the steel wool became noticeably blue over its entire length. Experiments with a d.c. discharge detector ( 7 ) following a similar combustion train showed that not only is gaseous oxygen released from the copper oxide a t the temperature necessary to effect complete combustion of organic materials but hot steel wool can remove it from the gas stream. The accessory stream of hydrogen added to keep the steel wool reduced and thus to test this hypothesis completely eliminated the release of tritium by unlabeled solvent. Since the hydrogen keeps the iron in its original, reduced state, the over-all reaction may be considered to be : H:O

+ Hz % Hz0 + H;

with heated iron acting only as a catalyst. If complete equilibrium is reached the ratio of gaseous hydrogen to water a t the outlet will be the same as a t the inlet, and if the gaseous hydrogen is added in excess, and isotope effects are comparatively small, the ratio of tritium as gaseous hydrogen to tritium in the water in the effluent will likewise be high. Although the effect of adding the hydrogen did not prove that the oxidized iron had reversibly adsorbed the radioactivity, it represented a working solution to the problem of retention of tritium in the furnace. Tolbert and Siri (I6) have described several other procedures for preparing hydrogen from water for tritium assay. ilmong reactions which appear applicable to GLC are the reactions of magnesium with water a t 625’ C. and of zinc n-ith water a t 420’ C. Both are described as having “memory” effects, which presumably are similar to the effects described here. The addition of hydrogen gas as described here may prove useful under some circumstances with these methods as well. LITERATURE CITED

(1) Cacace, F., Guarino, A., Inam-U1-Haq, Ann. Chim. (Rome) 50, 919 (1960). (2) Cacace, F., Inam-U1-Haq, Science 131, 732 (1960). VOL. 35, NO. 4, APRIL 1963

541

( 3 ) Dutton, H. J., Pittsburgh Conference on Analytical Chemistry and Applied

Spectroscopy, Feb. 28, 1961.

(4) Evans, J. B., Quinlan, J. E., Willard, J. E., I n d . Eng. Chem. 50, 192 (1958). (5) Hajra, A. K., Radin, N.S., J . L i p i d Res. 3, 131 (1962). (6) James, A. T., Piper, E. A., J . Chro-


matog. 5 , 265 (1961). (7) Karmen, A., Bowman, R. L., in “Gas Chromatography,” N. Brenner, ed., p. 189, Academic Press, N.Y. (1962). (8) Karmen, .4.,Giuffrida, L., Bowman, R. L., J. L i p i d Res. 3 , 44 (1962). (9) Karmen, A., McCaflrey, I., Bowman, R. L., Ibid., 3, 372 (1962).

(10) Karmen, A., Tritch, H. R . , Nature 186, 150 (1960). (11) Lee, J. K., Lee, E. K. C., Musgrave, B., Tang, Y. X., Root, J. W.,Rowland, F. S., ANAL.CHEM.34, 741 (1962). (12) Mason, L. H., Dutton, €1. J., Bair, L. It., J . Chromatog. 2, 322 (1959). (13) Meinertz, H., Dole, V. P., J . L i p i d Res. 3, 140 (1962). (14) Popjak, G., Lowe, A. E., Moore, D., Brown, L., Smith, I?. A,, Ibid., 1, 29 (1959). (15) Steinberg, D., Nature 183, 1253 (1959). (16) Tolbert, B. &I., Siri, W. E., “Determination of Radioactivity” in “Phys-

ical Methods of Organic Chemistry,” rirnold Weissberger, ed., p. 3335, Interscience, h-ev Pork, 1960. (17) Wilzbach, K. F., Riesz, P., Science 126, 748 (1957). (18) Winkelman, J. W., Karmen, A., ANAL.CHEM.34, 1067 (1962). (19) Wolfgang, IL, Roviland, F. S., Ibid., 30, 903 (1958). RECEIVED for review Xcvember 19, 1962. Accepted February 8, 1963. Presented at the International Symposium OII Advances in Gas Chromatography, Universit’y of Houston, Houston, Texas! Janusry 21-24, 1963.

Determination of Methyl-Ethyl Lead Alkyls in Gasoline by Gas Chromatography with an Electron Capture Detector HAROLD J. DAWSON, Jr. Research and Development Deparfmenf, American Oil Co., Whifing, Ind.

b Tetraethyllead, tetramethyllead, and the three methyl-ethyl lead alkyls are determined in gasoline directly b y temperature-programmed gas chromatography with an electron capture detector. The lead scavengers, ethylene dichloride and ethylene dibromide, are removed in the gas chromatography column to avoid interference. Satisfactory conditions have been found for operation of the detector with a temperature-programmed column. Standard deviations of the 25-minute analyses vary from hO.01 to h0.08 grams of lead per gallon for the individual lead alkyls; accuracy is approximately the same.

T

ETRAETHYLLEAD (TEL) has been used as an anti-knock agent in gasoline for 40 years. Tetramethyllead (TRIL) and the mixed lead alkylstrimethylethyllead (MesEtPb), dimethyldiethyllead (RlezEtzPb), and methyltriethyllead (RIeEtsPb)-are currently being evaluated by petroleum refiners. The lower-boiling alkyls are reportedly advantageous in gasolines high in aromatics and low in sulfur (15). If only one lead alkyl is present, determination of total lead in gasoline (1, 3, 6) is adequate. If only T M L and T E L are present, the sample can be separated into fractions by distillation and total lead in each fraction can be determined (2). The only published method for determining individual mixed lead alkyls (13) separates them by gas chromatography, absorbs them in iodine, and determines the lead in

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the fractions by a dithizone spectrophotometric procedure. Both latter methods are slow and laborious. A rapid and convenient method for determining the individual lead alkyls in gasoline directly is needed for research purposes and, ultimately, for control of refinery blending operations. Lovelock and Zlatkis (11) reported the use of an electron capture detector to determine TEL in gasoline and suggested it could be used for determining the individual lead alkyls. The response of electron capture detectors for lead alkyls and organic halides is several thousand times that for hydrocarbons (7, 10, 11). Consequently, the lead alkyls and organic halides can be detected with substantially no interference from hydrocarbons. Development of a gas chromatographic method for determining the individual lead alkyls in gasoline presents three major problems: preventing interference by the halide scavengers, preventing interchange of methyl and ethyl radicals, and establishing satisfactory operating conditions for the electron capture detector. lF7ith a nonselective column packing, ethylene dibromide eluted with MesEtP b and interfered with its determination. Separating the scavengers from the lead alkyls chromatographically was not attempted; instead, the scavengers were removed with a chemically active stationary phase-silver nitrate dissolved in Carbowax 400. Interchange of methyl and ethyl radicals between T M L and T E L oc-

curred on a column of 5% SC-30 Glicone rubber on acid-viashed Chroniosorb W. Calingaert found that the alkyl radicals of organo-lead compounds interchanged readily on slightly acidic catalysts; he called this the redistribution reaction (4). Coating the Chromosorb with sodium hydroxide before the stationary liquid phase is applied reduces interchange of radicals to a nondetectable level. Slight interchange takes place when the silver nitrate packing is located at the column inlet. Vhen this packing is located at the column exit, the lead alkyls are separated before contact with the silver nitrate and interchange is avoided. The electron capture detector is prone to instability; several variables had to be investigated to establish satisfactory operating conditions. Detector stability is known to be affected by water ( 9 ) , and the level and stability of the applied voltage ( 5 ) . The detector is temperature sensitive so the increase in temperature of gas entering the detector that accompanies temperature programming causes instability. A stainless steel block regulates the temperature of gas entering the detector. EXPERIMENTAL

Apparatus. An upstream flow control valve maintains a constant flow through a packed column. Liquid samples (1 J.)are charged with an Osage linear slide valve. A Jarrell-Ash 26-755 electron capture detector is thermostatted in a 26-750 oven. Drycell batteries deliver a constant d.c. voltage to the detector. X Gyra

Measurement of Tritium in the Effluent of a Gas Chromatography

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