may require from 50 to 150 seconds for processing. A spectrum containing 200 lines will usually require about 3 to 7 minutes for processing. The program HIRES2 usually requires 10 to 60 seconds for identifying the standard masses and computing the masses of all the lines. The computing time required by HIRES3 depends on the number of atoms to be considered and on the size of the constants which define the maximum number of each atom permitted in a given formula. About 40 to 60 seconds is typical
for computing all possible combinations of C, C13, H, 0,and N for a spectrum containing 200 lines when one C13 and 2 to 4 0 and N atoms are permitted in each ion. ACKNOWLEDGMENT The authors acknowledge the valuable advice of A. C. Jones and J. H. Schachtschneider in some of the mathematical aspects of these programs. RECEIVED for review June 10, 1968. Accepted July 19, 1968.
Automatic Apparatus for Sampling and Preparing Gases for Mass Spectral Analysis in Studies of Carbon Isotope Fractionation during Methane Metabolism Melvin P. Silverman and Vance 1. Oyama Exobiology Division, Ames Research Center, NASA, Moffett Field, Calif. 94035 An automatic apparatus is described which samples the gas phase above a microbial methane metabolizing system every two hours, separates and measures the individual gas components (H2, 02,Nz, C02, CH,) by dual-column gas chromatography, combusts methane quantitatively to CO,, and collects separately the metabolic CO, and COz derived from methane combustion for subsequent mass spectral analysis of carbon isotope ratios. With this apparatus, it was found that the aerobic methane oxidizing bacterium Methanomonas methanooxidans p refe rentia IIy utiI izes the Iig hter isotope of carbon, thus leaving the residual methane enriched in *3C. The microflora in Bower’s clay soil also preferentially utilize 1zC during anaerobic methane production from hydrogen and carbon dioxide; residual carbon dioxide becomes enriched in 13C, and biogenic methane becomes progressively more enriched in l3C.
FRACTIONATION of the stable isotopes of carbon by biological systems has been well established (I-5),and it may be possible to expioit this phenomenon as an indicator of life processes. Microorganisms that metabolize methane, either oxidizing it to COZin the presence of oxygen or producing it anaerobically from COz or other organic compounds, are widely distributed on Earth. Methane producing bacteria are responsible for some of the highest carbon isotope fractionations known (6,7) but no such information is available for the methane oxidizing bacteria. The possibility that certain extraterrestrial bodies may harbor similar microorganisms led us to study carbon isotope fractionation during methane metabolism in order to evaluate this process as the basis for the design of a life detection tool. Standard methods of preparing samples for mass spectrometric analysis of 1“c/ lZCratios demand considerable attention to detail ( I , 8). The samples must be converted completely to COZ, freed of water, and transferred to a suitable (1) H. Craig, Geochim. Cosmochim. Acta, 3, 53 (1953). (2) R. Park and S. Epstein, ibid., 21, 110 (1960). (3) P. H. Abelson and T. C. Hoering, Proc. Nut. Acad. Sci. U.S., 47, 623 (1961). (4) P. L. Parker, Geochim. Cosmochim. Acta, 28, 1155 (1964). (5) E. S. Cheney and M. L. Jensen, ibid., 29, 1331 (1965). (6) W. D. Rosenfeld and S. R. Silverman, Science, 130,1658 (1959).
(7) N. Nakai, “Geochemical Studies on the Formation of Natural
Gas,” Ph.D. diss., Nagoya Univ., Nagoya, Japan (1961); cited
in (5). (8) N. Nakai, J. Earth Sci., Nagoya Uniu., 8, 174 (1960).
vessel before they enter the mass spectrometer. These procedures, which require the use of leak-proof combustion and vacuum systems with their attendant problems, involve time consuming and tedious manual operations, and limit the number of samples that can be processed in a reasonable time. It was necessary for our studies of carbon isotope fractionation during microbial methane metabolism to develop an automatic apparatus that would periodically sample the gas phase above a methane metabolizing culture, separate and measure 0 2 , Nz, COZ,CHd), comthe individual gas components (Hz, bust methane quantitatively to COZ,and collect separately metabolic COSand the COzderived from methane combustion for subsequent mass spectrometric analysis. This paper describes such an apparatus capable of processing a gas sample every 2 hours. Preliminary results are given for experiments on carbon isotope fractionation by an aerobic methane oxidizing system and an anaerobic methane producing system. EXPERIMENTAL Automatic Apparatus. The apparatus is essentially a dualcolumn gas chromatograph connected in series with a combustion system and collection tubes. It is mobile and selfcontained except for a source of ac power. Table I lists the construction features and operational modes that were found to provide satisfactory performance. A flow diagram for the apparatus is shown in Figure 1. The programmed sequence of operations and events is illustrated in Figure 2. The water-jacketed fermentation unit, fitted with bacteriological filters at the gas inlets and outlets, is sterilized by autoclaving. Sterile medium is added to the main body of the fermentor and inoculum to the side arm. With stopcock 1 open, the unit is evacuated and then filled with the desired gas mixture. The inoculum is added at zero time through stopcock 2, and stopcocks 1 and 2 are closed. The culture is stirred with the Teflon-coated magnetic stirring bar. The temperature of the culture is controlled by pumping water from a water bath through the water jacket of the fermentor. Gas enclosed in the fermentation unit is circulated through the sample loop by a diaphragm pump to provide thorough mixing of the gases. At the time of sampling, the gases in the sample loop are injected into the helium carrier gas and pass through the Poropak T column where a composite peak (H,, 02,N,, CHI) is separated from a subsequent COz peak. Both peaks are sensed by a detector and the composite peak passes through valve V1 into the molecular sieve column. VOL. 40, NO. 12, OCTOBER 1968
1833
Figure 1. Gas flow diagram of automatic apparatus
CO2
COLLECTION TRAPS Valves V1 and V2 are actuated, diverting the C02 peak to the liquid nitrogen trap previously flushed with helium. Valve V2 is then deactuated. Valve V1 remains actuated until the water vapor injected with the other gases in the sample is diverted to exhaust. Then valve V1 is deactuated. The composite peak in the molecular sieve column is separated into individual peaks in the sequence HB, 0 2 , NP, CHa and measured by the other detector. These gases then enter the CuO furnace (850 "C)where Hz is oxidized to water, O2 oxidizes any reduced copper, and CHI is converted to C 0 2 and water. The water of combustion is trapped in the Drierite trap and the C02 derived from methane is captured in the preflushed liquid nitrogen trap when valve V3 is actuated. When the carrier gas is dehydrated by appropriate means, regeneration of the molecular sieve column is not necessary because water or COznever enters the column. Because very little water of combustion is produced, the Drierite trap can function for long periods before it must be renewed. The ability of the apparatus to detect and measure 02 and N2serves as a check for leaks in the closed cycle fermentation unit. A small quantity of NZis routinely added to the ferTable I. Construction Features and Operational Modes Sampler: Loenco sample valve, model L208-6, with LVO-200Aair cylinder operator Sample loop: 2.09-ml capacity Valves V1, V2, V3: Chromatronix, model CAV 4031, 4-way, 0.03 inch only Columns: Poropak T, 60/80 mesh, 12 ft X inch o.d., stainless steel. Molecular sieve 5A, 40/50 mesh, 8 ft X '/a inch o.d., stainless steel. Capillary restrictors: 500 ft X 0.02 inch i.d., stainless steel Tubing: Copper or stainless steel, inch 0.d. Combustion tube: Stainless steel, 9 inch X ' 1 2 inch, o.d., filled with CuO Detectors: Loenco, thermal conductivity, Model D-204-TWH Detector circuits: Loenco, Model 40 Temperature programmer: Loenco, Model 63A Oven: Loenco, Model 90, held at 80 "C Recorder: Texas Instruments, Model F4L Carrier gas: Helium, 90 psig, 30-37 ml/min Sequence programmer: Actan (Sealectro Corp.), l/l~o rpm, 2 4 1 - 1 resolution COz traps: U-tubes, 8 mm 0.d. borosilicate glass, ca. 14 inches long with 10/30 inner joints and 2-mm oblique bore hollow stopper vacuum stopcocks
1834
a
ANALYTICAL CHEMISTRY
PUMP
COMBUSTION FURNAC CHART DRIVE SAMPLE VALVE
v-I H2O v-2 co2
v-3 CH4 0
10 20 30 40 50 60 70 80 90 100 110 120 TIME, minutes
Figure 2. Programmed sequence of operations and events mentor at zero time. Any anomalous change in the concentration of Nz in aerobic systems, or Nzand OZin anaerobic systems, is taken as evidence of a leak. However, all operations to date with the apparatus have been leak-free. The entire sequence of operations is automatic from sampling to the point of trapping the COZwhereupon some manual operations are necessary. These involve attaching the COz traps to the instrument, flushing them with helium, opening
r
Figure 3. Calibration curves for 02,COZ, Nz,and CH4
2.5
2.0
>
E
+* 1.5 I (3 W
I Y
3 a
1.0
.5
100
200
32
B
HYDROGEN
p'
ZONE
0
&
36
300
400
HYDROGEN,
500
600
mm
700
Figure 4. Calibration curve for H2 and closing their vacuum stopcocks, and removing them from the apparatus whenever collection of a sample for mass spectral analysis is desired. Further development should make it possible to pass the COSdirectly into the inlet of a mass spectrometer. The apparatus can also be switched from automatic to manual operation of the sampling valve, valves V1, V2, V3, the diaphragm pump, the combustion oven, and the recorder chart drive. This feature is essential for calibrating the apparatus, for determining the proper timing of sequential events for automatic operation, and for making measurements at times other than those set on the Actan programmer. Calibration. The apparatus was calibrated for the gases of interest by measuring peak height in millivolts as a function of partial pressure. Calibration curves were nonlinear as illustrated in Figure 3 for oxygen, nitrogen, carbon dioxide, and methane. Although inversion occurs for hydrogen, a calibration curve could be obtained (Figure 4) that was satisfactory for quantitative estimations except in the hydrogen inflection zone. Mass Spectral Measurements. Isotope ratios were measured on an Atlas M86 180", 65-mm radius, dual collector isotope ratio mass spectrometer equipped with a dual inlet system. The ion current was 60 PA. The instrument was not operated in the ratio mode; instead, each l3C/lZCratio of a sample or standard was calculated as the mean of 10 consecutive scans of the mass 44 and 45 peaks. Every sample analyzed was preceded by an analysis of the reference standard (tank CO,). The background at the mass 44 and 45 peaks was less than 0.25% of the sample and reference signals. The results were calculated as per mil (%,J deviation from the reference standard according to the formula 813C(%c)= [(l3C/'*C ~ a m p l e / l ~ C standard) /~~C - 11 X loa
No corrections were made for the small contribution of 1 7 0 to the mass 45 peak (9). Student's t test at the 1 % level of confidence was used to test the significance of differences between the 13C/12Cratios of samples and standard. RESULTS
For accurate measurements of isotope fractionation during methane metabolism, none of the operations performed by the apparatus should cause a change in the carbon isotope ratio of a gas sample. Altered isotope ratios could result from incomplete combustion of CH4 to C o n ,incomplete diversion of all the C 0 2 to the collection trap, or incomplete trapping of COSin the liquid nitrogen trap. (9) H . Craig, Geochim. Cosmochim.Acta, 12, 133 (1957).
20 40 60 80 100 120 140 TIME, hr Figure 5. Gas changes during methane oxidation wand groth of Methanomonas rnethanooxidans at 30 OC. Arrows indicate samples analyzed in mass spectrometer 0
Efficiency of Methane Combustion. To test the efficiency of methane combustion, the apparatus was modified by placing the CuO furnace in line between the sampling valve and the Poropak T column. Known mixtures of CO2:N2 or CHd:Nz were flushed through the sampling valve at 1 atm pressure and injected into the flowing carrier gas. The efficiency of converting CH4 to C 0 2 was estimated by comparing the peak heights of C 0 2 from methane combustion with the peak heights of COz from the known COz:Nzmixtures. At a furnace temperature of 850 "C, the mean efficiency of combustion of CHI to COz for 3 samples was 101.4% =t3. Complete Diversion of COS to Traps. The sequence of operations and events (Figure 2) was programmed to provide 6 minutes for collecting both carbon dioxide and the carbon dioxide derived from methane. Collection of the gas responsible for a peak was initiated just prior to the appearance of the peak, and base line was reestablished well within 3 minutes. For a peak with a gaussian distribution, four standard deviations will encompass 99.99 of the material in the peak. Because the shape of the peaks was skewed owing to slight tailing which increased the base width slightly beyond that of a normal gaussian distribution, an estimated six standard deviations were used for collection. Thus, even if isotope separation were possible with the relatively low number of theoretical plates (200) estimated for the columns, the quantity of material that escaped diversion to the liquid nitrogen traps was negligible. Efficiency of Trapping, The efficiency of liquid nitrogen trapping of COZ or COZderived from methane combustion was tested with the aid of a second gas chromatograph fitted with an all glass valving system. This analyzing gas chromatograph was calibrated for COz with a standard mixture of 25 % C 0 ~ : 7 5 %H2. Known quantities of COz or CHI were then injected into the carrier gas stream of the automatic apparatus and the chromatographically separated peaks were collected in the liquid nitrogen traps. The trapped COzwas transferred Table 11. COz Trapping EAiciency Trapped
coz
Added, mmoles coz 0.0528 CH4 0.0358 Mean of 4 replicates.
derived from a
Found, mmoles 0.0524 0.0347
Recovery,
z
99.2 =k 3. 96.9 i 3=
VOL. 40, NO. 12, OCTOBER 1968
1835
Table III. Carbon Isotope Fractionation by Methanomonas methanooxidans during Growth and Methane Oxidation at 30 "C 0 Time 135 Hours Net change mmoles 6 '3C mmoles 6 lac moles 6 '*C CH4 COZ
34.8 0.8 21.4
0 2
-9.5 +1.2"
21.6 5.7 2.4
...
Biomass ... Value statistically insignificant.
...
...
+7.8 +1.5O
-13.2 +4.9 -19.0
*..
$17.3 +O. 3"
... *..
.-_ _._
...
-12.0
0
Table IV. Carbon Isotope Fractionation at 23 "C during Anaerobic Methane Production from Hz and COz by Microorganisms in Bower's Clay Soil 45 HoursG 66 Hours 0 Time Net change, 0-45 hr Net change, 45-66 hr mmoles 6 W mmoles 613C mmoles 613C mmoles 613C moles 6'3C Hz C02
22.10 7.40 0
...
+O.F
CH4 ... Pooled 44-and 46-hr samples. * Value statistically insignificant.
11.20 3.40 0.37
...
-10.90
.*.
+19.8 +23.1
-4.00
+19.1
+o. 37
5.70 1.95 0.56
...
...
+18.5 +26.4
...
-5.50 -1.45 +0.19
-1.3 +3.3
5
to the analyzing chromatograph and measured, taking into account the efficiency of transfer of COz from trap to analyzing chromatograph. The results (Table 11) show that, within the limits of accuracy of our detection methods, essentially all the carbon dioxide was trapped. Because the gas samples went through the entire sequence of automatic operations, the data in Table I1 also are a measure of the overall efficiency of operation of the automatic apparatus. Carbon Isotope Fractionation in an Aerobic Methane Oxidizing System. Fifty milliliters of a stationary phase culture of Methanomonas methanooxidans was the inoculum for 450 ml of Pope and Skerman's mineral salts medium (IO) modified to contain KNO, (1 .O gram/liter) as nitrogen source. The gas phase in the fermentor was 1612 ml of a mixture of methane, oxygen, and carbon dioxide. The initial pH was 7.0 and the temperature was maintained at 30 "C. The gas phase above the culture was sampled automatically every two hours beginning with the fifteenth hour. Figure 5 shows that after a lag phase of approximately 60 hours, rapid consumption of methane and oxygen occurred accompanied by carbon dioxide production. The arrows indicate those samples that were collected and analyzed for their 13C/12C ratios. The pH at the end of the experiment was 6.6. The biomass was collected, washed, dried, and converted to C 0 2 in a CuO furnace at 850 "C in an oxygen atmosphere. The 13c/lzC ratios of gas samples and biomass are listed in Table 111. It is evident that the lighter carbon isotope is preferentially metabolized during bacterial growth on methane because the residual methane became enriched in '3c and the biomass enriched in W. There was no significant change in the isotopic ratio of the carbon dioxide in the gas phase although no effort was made to recover the C 0 2in solution at pH 6.6. According to the energy yielding reaction CH4
+ 202 = COz + 2Hz0
complete oxidation of the methane consumed (13.2 mmoles) should have resulted in the consumption of 26.4 mmoles of oxygen and the production of 13.2 mmoles of carbon dioxide. This clearly was not the case. But since the principal source of carbon as well as energy for the growing culture was methane, a considerable quantity undoubtedly was diverted by (10) V. B. D. Skerrnan, "A Guide to the Identification of the Genera of Bacteria," Williams and Wilkins, Baltimore, 1959, pp 143-4.
1836
ANALYTICAL CHEMISTRY
22 20 18 In
$j 16
E 14
HYDROGEN INFLECTION ZONE
v) Q)
.6z E .4 E . 2 I" 0
n u I O 20 30 40 50 60 7f TIME, hr
Figure 6. Gas changes during methane production at 23 "C from H2and COS by anaerobic microflora in Bower's clay soil. Arrows indicate samples analyzed in mass spectrometer the bacteria into the synthesis of cellular material and was not oxidized completely to COS. Carbon Isotope Fractionation in an Anaerobic Methane Producing System. Bowers clay soil (100 grams) was enriched for anaerobic methane producing bacteria by the addition of 150 mg acetic acid, 90 mg glucose, 50 mg ammonium sulfate, 60 ml water, and incubation under 3 :1 (v/v) helium-nitrogen at room temperature. After several weeks of methane production, the system was evacuated and filled with 3 :1 (v/v) hydrogen-carbon dioxide. After vigorous production of methane with concomitant consumption of hydrogen and carbon dioxide, the system was finally evacuated and refilled with the hydrogen-carbon dioxide mixture and incubated at room temperature (23 "C). The volume of the gas phase over the soil was 734 ml. Figure 6 shows that there was immediate hydrogen and carbon dioxide consumption and methane production which continued at a constant rate for the duration of the experiment. The l3C/I2Cratios of samples taken at the time indicated by the arrows are listed in Table IV. After 45 hours, the carbon dioxide was enriched in 13c with little further change after 66 hours. The biogenic methane measured at 45 hours was enriched in l3c and became further enriched after 66 hours. These data indicate that the microflora in Bowers clay preferentially consumed the light carbon dioxide, and as the residual
carbon dioxide became heavier, the biogenic methane derived from carbon dioxide became heavier as well. According to the reaction 4Hz
+ C02 = CHI + 2H20
the consumption of 5.45 mmoles of COZin 66 hours should have resulted in an equivalent production of methane, but only about 10 of the required methane was produced. Evidently large quantities of C 0 2were converted into cellular material or other nongaseous species. DISCUSSION Earlier studies of carbon isotope fractionation during microbial methane metabolism were limited to anaerobic methane producing systems. It has been demonstrated that with a finite supply of methanol (6, 7) or acetic acid (7), the methane producing bacteria preferentially utilized the 12Cof these substrates. As a consequence the biogenic methane and carbon dioxide became heavier as the reactions proceeded. Our preliminary data (Table IV) now show that this also occurs during microbial methane production from hydrogen and carbon dioxide. Residual carbon dioxide and biogenic methane both became heavier with time because of preferential utilization of 12c. To our knowledge, carbon isotope fractionation during
microbial methane oxidation has never been reported. Our data (Table 111)indicate that the methane oxidizing bacterium M . methanooxidans preferentially consumed 12CH4so that the residual methane became enriched in I3C. Thus, from the limited studies to date, a general picture seems to be emerging in which the processes involved in microbial methane metabolism-i.e., both methane production and methane oxidation, result in methane becoming progressively enriched in 13C. Although the apparatus described in this paper was designed specifically for studying carbon isotope fractionation during microbial methane metabolism, it should be useful also for studying the kinetics of isotope fractionation in any reaction where one or more carbonaceous gas species are present. With suitably designed reaction vessels and sampling systems, it should be possible to study the kinetics and extent of carbon and/or oxygen isotope exchange reactions between CHb and COz,COzand its dissolved species or solid carbonates, etc., as well as the influence of environmental variables and the intervention of biological systems on these processes. ACKNOWLEDGMENT We are grateful to H. Taube, Stanford University, for the use of the Atlas M86 mass spectrometer. RECEIVED for review April 25,1968. Accepted July 29,1968.
Identification and Estimation of Choline Derivatives by Mass Spectrometry G . A. R. Johnston Department of Physiology, John Curtin School of Medical Research, Australian National UniFersity, Canberra
A. C. K . Triffett and J. A. Wunderlich Division of Applied Chemistry, C.S.I.R.O., Chemical Research Laboratories, Melbourne The halide salts of choline and related derivatives including acetylcholine, when heated under vacuum, dissociate by N-demethylation to the methyl halide and to the tertiary amine, the latter giving characteristic mass spectra under electron impact. Some fragment ions are common to all or most of the compounds studied; however, those arising from the loss of (CH3)2N. are specific to each compound. These characteristic ions and the less abundant molecular ions permit identification of suitable choline derivatives at the 1-10 nmole level. The lower limit of detection and estimation of total cholines in a mixture is approximately 0.1 nmole, while the estimation of individual choline derivatives requires approximately 10 nmole of each derivative.
TRACEAMOUNTS OF CHOLINE derivatives, acetylcholine in particular, in biological extracts place considerable demands on current methods of estimation and identification. Bioassay procedures permit the estimation of minute traces of physiological activity but parallel bioassay techniques represent only “a valid first approach to the identification of cholinomimetic substances” ( I ) . Recently, two gas chromatographic procedures have been developed for the assay of choline esters but both require chemical pretreatment of the involatile salt to the (1) C. Hebb and D. Morris, Nature, 214, 284 (1967).
tertiary amine by N-demethylation in one case (2) and by borohydride reduction of the ester group to the alcohol in the other (3). These approaches allow the estimation of very low levels of acetylcholine and related compounds, but the problem of identification other than by parallel bioassay techniques and the various chromatographic comparisons remains. Under favorable circumstances, mass spectrometric methods can provide an absolute identification of trace amounts of suitable substances ( 4 , 5 ) and their application to the estimation and characterization of choline derivatives forms the basis of this paper. EXPERIMENTAL Materials. The following were purchased and used without purification: choline chloride (BDH); acetylcholine chloride (Merck); acetylcholine bromide and propionylcholine iodide (2) D. J. Jenden, I. Hanin, and S. I. Lamb, ANAL.CHEM., 40, 125 (1968). (3) W. D. Stavinoha and L. C. Ryan, J . Pliarmncol. Exptl. Therap., 150, 231 (1965). (4) J. H. Beynon, “Mass Spectrometry and Its Applications to Organic Chemistry,” Elsevier Publishing Co., Amsterdam, 1960, p 178. (5) D. J. Parker and W. S. Ruliffson, Anal. Biochem., 19, 418
(1967). VOL. 40, NO. 12, OCTOBER 1968
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