1) Loss of the analyte may be caused by the interferent during the preatomization cycle. This is exemplified by the interference of CaClz and MgCl2 on Mn. The volatility of the analyte depends on the solution composition. Therefore, it is not recommended to measure the analyte vs. pure aqueous standard solutions because the behavior of the analyte depends strongly on the matrix composition. Standard addition methods or chemical extraction methods are preferable. 2) The introduction of a programmed ashing cycle, i.e., the increase of the ashing time, results in the elimination of the matrix compounds MgC12 and CaClz so that the interference is reduced or nullified in those cases. Of course, the ashing temperature must be selected so that no losses of the analyte occur (see conclusion 1.) 3) The integration method of evaluating absorption peaks instead of measuring peak height reduces the positive interferences but does not affect negative interferences. We are aware that the experiments described in this article do not explain the reason for the occurrence of interferences in flameless AAS and that the mechanisms proposed
are, a t best, plausible hypotheses. However, one conclusion is quite clear: most articles about this subject state that interference effects in AAS are complex. These results show that they are even more so than generally accepted. Therefore, more systematic investigation is needed. I t was the purpose of this work to contribute toward fulfilling this need.
LITERATURE CITED (1) L. Ebdon, G. F . Kirkbright, and T. S. West, Anal. Cbim. Acta, 58, 39 (1972). G. Baudin, M. Chaput, and L. F e w , Spectrocbim. Acta, 26, 425 (1971). D. A. Segar and J. G. Gonzalez, Anal. Cbim. Acta, 58, 3 (1972). R. B. Gruz and J. C. Van Loon, Anal. Chim. Acta, 72, 231 (1974). W . C. Campbell and J. M. Onoway, Talanta, 21, 837 (1974). C. W . Fuller, Ana/yst(London), 00, 739 (1974). E . L. Henn, Anal. Cbem., 47, 428 (1975). C. W. Fuiier, Anal. Cbim. Acta, 82, 442 (1972). A. Syty, CRC Crit. Rev. Anal. Cbem., 4, (2). 155 (1974). Y. Michotte, unpublished results. J. Smeyers-Verbeke, unpublished results. F . J. H. J. Maessen and F . D. Posma, Anal. Cbem., 48, 1439 (1974).
(2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12)
RECEIVEDfor review July 11, 1975. Accepted September 29, 1975. Presented in part a t Euroanalysis 11, Budapest, August 1975.
Isotopic and Quantitative Analysis of the Major Carbon Fractions in Natural Water Samples Larry M. Games and J. M. H a y e s * Deparlments of Chemistry a n d Geology, Indiana University, Bloomington, lnd. 4 740 7
Carbon present in natural water samples is quantitatively transformed into COP for quantitation and carbon isotope ratio measurement. Fractions representing inorganic C02, volatile organic carbon, invoiatiie organic carbon, and CH4, and CO are obtained separately with detection limits of 5, 5, 50, 1, and 5 ppb, respectively. Separation of the water from the invoiatiie organic carbon, which Is released by uv photooxidation of an O2-satura1ed solution, Is not required. The results of extensive control experiments validating the procedures and analytical conditions are presented.
The task of determining the physical and biological processes that govern the movement and degradation of carbon compounds in ground water has only begun recently ( I ) , mainly because of difficulties in following the movement of ground water and the uncertainties involved in assessing the interactions of the carbon compounds with the sedimentary environment. Investigations have generally been limited to phenomenological studies, such as measuring the extent of ground-water pollution after it has occurred (2-5). Work in this laboratory on the processes controlling the transport of carbon into ground water from a sanitary landfill has involved using variations in the natural abundance of carbon isotopes as a tracing technique, and, in order to provide a detailed picture, has included development of procedures to separate quantitatively the total carbon present in ground water into four fractions. Variations of the l3C/I2Cratio have been used previously to verify the presence or absence of pollutant carbon in natural aqueous environments (6, 7 ) and the technical re130
ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
quirements for this type of investigation are stringent. All procedures must result in the quantitative removal of all carbon compounds, and should facilitate the conversion of the separated material to COS, the gas universally used to measure 13C/12C ratios. Quantitative removal is important because partial removal of any individual carbon compound can have an isotope effect that precludes use of natural abundance isotope ratio variations as a tracing technique. The methods must be relatively simple to allow routine analysis, but provide sufficient subdivision of the carbon present to furnish useful information. The methods must also be adaptable to wide ranges of concentration, since even in unpolluted ground water the organic carbon concentrations vary from 0.1 to 15 mg C/1. (8-10) while the amount of inorganic carbon varies over an even broader range ( 7 , I I ) . Here, analytical methods are described that provide for the determination of the total carbon present in water in four discrete fractions which are, to a certain extent, unique in terms of the processes leading to their formation (12).The fractions and their primary constituents are: ino r g a n i c c a r b o n , free carbon dioxide and acid-labile carbon released as carbon dioxide [usually bicarbonate (13)l;volat i l e o r g a n i c c a r b o n , all organic carbon that can be stripped from a water sample a t room temperature ( m e t h a n e and c a r b o n m o n o x i d e are isolated as subfractions); and nonvola t i l e o r g a n i c c a r b o n , all remaining organic carbon. Inorganic carbon normally arises in ground water from three sources, namely CO2 from the atmosphere dissolved in rainfall (a minor component), COZ from the microbial metabolism of organic compounds, and C02 from the dissolution of carbonate rock (14). Organic carbon is present in
natural waters in many types of well-defined compounds, polar and nonpolar, volatile and nonvolatile ( 1 5 ) , and also in less well-defined species called humic acids (16). Methane is produced by methane bacteria during the anaerobic degradation of organic compounds (17), and, although carbon monoxide has never been shown to be present in ground water, its formation has been demonstrated in living and decomposing plant material (18, 19). Numerous techniques have been developed to isolate the various types of carbon compounds from natural water samples for isotopic analysis, and some of these techniques, with suitable modifications, have been adapted here. Inorganic carbon in water has generally been removed by acidification with 95-100% HsP04 (11).This process shifts the equilibrium among the various inorganic carbon species, forming free COn which can easily be removed from the water. Two basic techniques have been utilized previously for the isolation of organic compounds for isotopic analysis, both of which result in the conversion of the compounds to COn prior to removal from water. The two methods, photooxidation of the organic material (20-22), and chemical oxidation by persulfate ( 2 3 ) give comparable results, although the photooxidation technique recovers somewhat more organic matter ( 2 4 ) . Both techniques as previously described result in the unavoidable loss of the volatile organic carbon during sample preparation, and, in the present work, the volatile organic matter has been analyzed separately prior to use of the photooxidation process. Techniques for the analysis of methane and carbon monoxide have been described previously (25, 26) and the modification reported here has been to provide for the analysis of both gases simultaneously.
EXPERIMENTAL
YSampIe
0 - ring compression
seal
TC gouge m
(1)
where R = (13C/'2C), and PDB represents the carbon isotopic standard (29,30). Inorganic Fraction. This fraction of carbon is collected by acidification of the water sample with 100% H3P04 [prepared by the method of Urey et al. (3011 and vacuum stripping of the resul-
TC gauge m
n
,.
1I 1 9 , m
-
To vacuum
ond samDle
Figure 1. Vacuum system used for extraction of inorganic acid-labile CO2
The sample bottle is closed by a vacuum valve prior to drainage into the H3P04 flask
a
\Ve Stripping Chamber
__
Handling
Sample Collection a n d Handling. All volatiles present in the water a t the time of sampling are retained by collecting the water samples in evacuated glass cylinders. These cylinders are closed by a break-seal up to the time of sampling (the seal is broken under water), and by a vacuum valve after sampling. Samples not to be analyzed for volatile constituents are filtered through Whatman GF/B glass fiber pads and 0.45-fi Miliipore filters, then stored (one week maximum), a t 4 "C until analysis. The glass fiber filter pads are pre-cleaned by rinsing in distilled water followed by ignition a t 500 "C, and the Millipore filters are rinsed in distilled water immediately prior to use. Apparatus. Water samples for all fractions are processed on one multipurpose vacuum line. The section of the line used for sample handling (distillation, measurement of amounts, and withdrawal of aliquots for mass spectrometric analysis) is evacuated by a 25 1./ min mechanical pump backing a three-stage all glass mercury diffusion pump; and the sample collection section of the vacuum line is pumped by an identical mechanical pump with no diffusion pump. All stopcocks are lubricated by a light application of halofluorocarbon grease, Kel-F No. 90 (3M Co.). Oxygen used as a stripping gas is purified by passage through a 20 mm X 280 mm tube filled with cupric oxide and heated to 850 "C to oxidize all organic contaminants to C o p , through a 10- by 350-mm bed of molecular sieves (Linde 5A) to remove water, and through a bed of soda asbestos of the same size to remove the COz produced during combustion of the contaminant organic matter. Carbon dioxide resulting from the combustion of each carbon fraction is distilled a t -120 "C to remove water of combustion and other impurities before measurement of the amount and carbon isotope ratio. The sample COz amount is measured in a device analogous to the "microvol" designed by Sanderson (27). Isotopic Analysis. Isotope ratios are measured mass spectroscopically (281, and are reported as c?'~CPDB,defined by: 6 1 3 C ~ ~ ~=([% ( R~s a) r n p i e / R~ ~11~io3 )
9)
I1
-196°C
uu
Flgure 2. Gas-handling system used for analysis of the volatile organic fraction
Traps A and C incorporate fritted glass disks in order to eliminate transmission of COP mists tant free Con. The analysis system shown in Figure 1 is evacuated with the filled water sample vessel and phosphoric acid in place and is then isolated from the pump by closing stopcock A. The -196 "C and -78 "CJraps are cooled to trap COz and H20,respectively, and the water sample is allowed to enter the acidification chamber with the leak valve closed. The leak valve is slowly opened, the COz evolving from the water trapped, and the leak valve closed after a short time. A large amount of noncondensable gas comes out of the water with the COz at this point; and after waiting to ensure complete trapping of the COz present, stopcock A is opened to remove these gases. The whole process, opening and closing the leak valve and removing noncondensable gases, is then repeated until no more COz is evolved, as determined by monitoring the thermocouple gauges. During this time, the emptied sample vessel remains open to the acidification chamber to avoid loss of any COz that might escape into the sample vessel when the sample is first added. The COz is then distilled, measured, and removed for isotopic analysis. (Note that in unusual cases some potentially interfering gases such as N p 0 and SO2 might be released from the water sample and collected in this step. When this is judged to be a possibility, some modifications may be required.) Volatile Organic Carbon. The volatile organic fraction is comprised of organic material which can be stripped from the water a t room temperature by purging with a stream of purified oxygen. Practically, its precise composition is determined by the conditions of analysis, and the analysis train is shown in Figure 2. A maximum oxygen flow rate of 12-15 ml/min is used. A total purging time of 3 hr has been adopted (see Results and Discussion section). After leaving the oxygen purification system, the gas is bubbled through the sample, passed through a -78 OC cold trap (A) for water vapor removal (some organic material is retained but this material is later recovered), through a soda asbestos trap (Ascarite, 8-12 mesh, 25 X 350 mm) to remove inorganic Cop, and then
ANALYTICAL CHEMISTRY, VOL. 48, NO. 1. JANUARY 1976
131
Lamp
Leads
\\SampleI I
Leak
TC gzuge I1
d,
Figure 3. Analytical system for nonvolatile organic carbon
The quartz reaction vessel is cylindrical in shape and is shown here in crosssection
..
.
D
through an oven (10 X 70 mm, Pt catalyst) maintained a t 1000 "C in order to oxidize the volatile organic material. The combustion oven is followed by an oven (10 X 70 mm) maintained a t 500 "C and filled with Korbl's catalyst ( 3 1 ) in order to remove sulfur oxides and halogens from the gas stream prior to the -196 O C trap. The -196 "C trap (C) is maintained a t low pressure by use of a leak valve (see Figure 2), and collects the H20 and COZ comhustion products derived from the volatile organic material. After 3-hr purging, the oxygen stream leaving the stripping chamber is switched to bypass the soda asbestos trap. Trap A is warmed to room temperature to release any organic material condensed there, while the second -78 "C trap (B) is cooled to remove water previously condensed in Trap A. After 20 min, the oxygen stream is stopped, and the leak valve is slowly opened completely to evacuate the whole line, thus ensuring that all of the CO2 from the combustion of the previously trapped organic material is collected in the -196 "C cold trap (C). Nonvolatile Organic Fraction. An ultraviolet photooxidation technique, utilizing a 1200-Watt Hanovia (189A) mercury arc lamp converts the remaining nonvolatile organic carbon to COZ ( 2 1 ) . The reaction vessel, shown with the analysis train in Figure 3, has two inner cylinders (-5.0 cm and -7.0 cm 0.d.) that are made of clear fused quartz to minimize absorption problems (21, 32, 33). The outer cylinder (9.0-cm 0.d.) contains the water sample and is composed of Vycor as is the frit a t the bottom where stripping gas enters. This one-piece design eliminates the need for any type of joints that can cause leakage problems, and the use of Vycor (since it can be sealed directly to quartz) eliminates the need for large, fragile quartz-to-Pyrex graded seals. The vessel is 60 cm long and designed to hold 800-ml water samples. After placing the prefiltered sample in the reaction vessel, 100% phosphoric acid is added to lower the pH to 1, and the volatile organic and inorganic carbon are purged from the sample in 3 hr with purified oxygen. The reaction vessel is then sealed, the sample photooxidized for 5 hr, and the COn resulting from the oxidation of the nonvolatile organic carbon stripped from the water by a stream of oxygen for 3 hr (see Results and Discussion for explanation of time sequence). Carbon Monoxide and Methane. Individual carbon dioxide samples representing each of these compounds are obtained by a modification of the volatile organic fraction procedure, as shown in Figure 4. After removal of water and inorganic carbon from the 0 2 purge stream, a leak valve reduces the pressure to 1.2 Torr, and the purge gas stream is led through a -196 "C trap to remove most organic compounds less volatile than methane (see Results and Discussion section for comments on the detailed composition of the methane fraction). The gas stream is then led through the bed of 1 2 0 5 (10 X 380 mm, see Figure 4) in order to selectively oxidize CO ( 2 5 ) ,through a second -196 "C trap (B in Figure 4) to collect COn derived from CO, through an oven (1000 "C, 10 X 70 mm, Pt catalyst) to oxidize the methane, and through a third -196 "C trap (D) to collect methane-derived COz. Alternate Methane Analysis. Because some organic compounds other than methane may escape the first -196 O C trap in Figure 4, the methane was also separated by the method of Swinnerton and Linnenbom ( 2 6 ) .In this technique, all hydrocarbons other than methane are trapped on a bed of activated alumina cooled to -78 "C.The alumina trap (15 X 250 mm) is placed after trap B (Figure 4), so that any volatile hydrocarbons escaping the first -196 "C trap are removed before the methane is combusted. The precise nature of the samples which may require use of this alumina trap is discussed later. 132
ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
-7VC C
Figure 4. Gas-handling system used in the analysis of the CO and CH4 fraction
Traps A, B, and D incorporate fritted glass disks in order to eliminate transmission of COP mists
RESULTS AND DISCUSSION The optimum analysis time for each procedure, with the exception of the inorganic fraction in which the time required is roughly dependent on sample size and the amount of noncondensable gas present, has been established by testing with standard compounds, and the rationale for each of the times chosen will be considered in some detail for each fraction. Volatile Organic Carbon Analysis. The 3-hr stripping time used for this fraction was chosen after experiments with two compounds, heptane and acetone. These standards represent large differences in polarity and volatility, and the results give a general indication of the importance of these two factors in determining the extent of removal. The more polar molecule, acetone, has a vapor pressure of 190 Torr a t room temperature, while heptane, a hydrophobic molecule, has a vapor pressure of 40 Torr. Both have vapor pressures significantly lower than 1 Torr a t -80 "C, so that if they are removed from the water, a large fraction of each should be trapped in the first Dry Ice/acetone trap (see volatile organic analysis, Experimental section) and only be oxidized when this trap is warmed. Table I shows the results of tests measuring the removal rate of acetone and heptane from water in the analysis system, and indicates that while heptane recoveries are complete, only a fraction of the input acetone appears as COa in the analysis procedure. T o demonstrate that the acetone was being retained in the water and that its low recovery was not due to incomplete combustion or loss in the analysis train, the acetone was also added to the sample vessel in Figure 2 without water. Complete recovery was obtained, as shown a t the bottom of Table I. Table I. Removal of Acetone and Heptane from Water by Oxygen Stripping Percentage of input removed Compound, initial concn
Acetone, Acetone, Acetone, Heptane, Heptane, Acetone, Acetone,
2.4 mg/l. 3.1 mg/l. 2.3 mg/l. 4.3 mg/l. 3.1 mg/l. 2.1 mg, pure 2.6 mg, pure
Hour 1
Hour 2 Hour 3
1.6 1.2 0.5 1.2
5.6 5.4 4.7 103 105
0.9
...
...
...
...
0.7 0.9
... .. . .. . ... ...
Total 7.9 7.5 5.2 104.1 105.9 99.6 100.3
______
~
Table 11. Time Required for Complete Photooxidation of Mannitol and Resorcinol Recovery
Table 111. Amounts of Various Gases Which Escape Trapping at -196 C in the Normal Methane and Carbon Monoxide Analysis Estimated vapor pressure at
for
Added,
Recovered as CO,,
mg C
mg C
Mannitol 1)First 2 hr Third hr 2 ) First 2 hr Second 2 hr 3 ) 5 hr 4 ) 5 hr Resorcinol 1) 5 hr 2 ) 5 hr 3 ) 5 hr _________-~__
each step, %
Total recovery,
%
0.760
1.94 0.17 0.665 0.067
3.70 5.37
3.74 5.40
101.0
3.81 2.34 4.62
3.73 2.37 4.53
97.9 101.3
2.15
90.2 8.0
98.2
87.5 8.8
96.3 100.6
98.0
______~__-
These tests indicate that removal of many nonpolar volatile organic compounds should be complete within 3 hr, but that some polar substances may be only partially removed. The portion of any compound not completely removed will then be photooxidized as nonvolatile organic carbon in a later procedure so that no carbon is lost. However, partial removal may have associated with it an isotope effect ( 3 4 ) , and this problem will be discussed later. If the COz trap in the volatile organic procedure failed, erroneously high results would be obtained. Therefore, the effectiveness of soda asbestos in removing inorganic COS was also tested. A water sample was sparged free of any volatile organic carbon and a large quantity (50 mg as carbon) of NaHC03 was added. The water was then acidified and analyzed for volatile organic carbon. Since no volatile organic carbon was actually present, the amount of COz found was a good estimate of the extent to which COS can move through the soda asbestos trap without being removed from the gas stream. Triplicate analyses gave 0.0004, 0.0005, and 0.0008 mg C , indicating that the soda asbestos is extremely effective in removing CO2 from the moving gas stream under the conditions of this analysis. Nonvolatile Organic Carbon. The nonvolatile organic carbon analysis can be divided into three steps including 1) acidification and removal of the inorganic and volatile organic carbon from the water, 2) photolysis of the nonvolatile organic carbon, and 3) stripping and isolation of the resulting COz. Step 1 is continued for 3 hr, and tests were conducted to make certain that inorganic carbon (usually removed by vacuum-degassing) was completely removed during this time interval. In these tests, the oxygen purging gas was led directly into a -196 OC trap without passing through the combustion ovens, so that only inorganic carbon was condensed as COS. The amount of COS collected during the second three hours of purging was measured, and triplicate analyses gave 0.018, 0.021, and 0.004 mg C. This indicates that the first 3 hr of purging was sufficient to remove the inorganic carbon, since these values are near or below the blank associated with this procedure (see Table IV). The time required for photolysis to complete the conversion of the organic carbon to C 0 2 (step 2), has been studied in detail by Armstrong et al., (21), who demonstrated that the photooxidation technique results in complete oxidation of the total range of organic compounds, including humic acids, that would be expected to occur in a natural water sample. In their work on sea water, complete oxidation was observed within 1 hr. The rate of oxidation found in studies of pure compounds was such that essentially complete oxidation occurred within 2 to 3 hr except for urea, in which
-196OC,
Gas
Tor@
Methane 9.7 Carbon monoxide 333 Ethylene 3.1 x 10-3 Ethane 4.6 x 10-4 Acetylene 7.0 X Propane 2.0 X Propene 1.6 X Butane 3.1 x 10-9
Maximum contribution to apparent methane content, mg ~b
17,200 removed b y I,05/liq. N, 5.49 0.81
0.012 0.003
0.003 5 x 10-6
a Based on data in the “Handbook of Chemistry and Physics” 53rd ed., 1972-73, The Chemical Rubber Co., Cleveland, Ohio. b The amount of carbon which would be swept through the -196 C trap at standard conditions: oxygen flow rate = 1 2 ml/min, trap pressure = 1.2 Torr, trap temperature = 77 K, and time of flow = 3 hr.
less than 50% oxidation occurred. In the present work, mannitol was used as a standard to monitor the reliability of the photooxidation method. The results of these measurements, shown in Table 11, indicate that 5 hr is sufficient to ensure complete recovery of the input mannitol as COz. An aromatic compound, resorcinol, was also tested and 100 f 2% recovery was observed in each of three 5-hr trials. The third step, removal of the COS resulting from the photooxidation of the organic material, is accomplished in 3 hr, the same length of time required for the initial removal of the inorganic carbon. Methane and Carbon Monoxide Analysis. The normal method of methane analysis (without alumina) does not preclude the inclusion of some other volatile hydrocarbons. Given the vapor pressure of these hydrocarbons a t - 196 OC, the flow rate of oxygen carrier gas, the pressure a t which trapping takes place, and the time of analysis, the maximum amount of these hydrocarbons that could escape trap B in Figure 4 and be analyzed as “methane” can be estimated, as shown in Table 111. The calculations indicate that a significant quantity of the Ca gases, a t least, could escape the -196 OC trap, and if they are present in amounts significant compared to the methane present, they would affect the results. These calculations are conservative in that the vapor pressures are for a bulk phase, and trapping efficiency in a thin film on a cold surface would undoubtedly be much better. For example, when 3.34 mg carbon as ethane is added to the purge gas stream to test these calculations, only 0.307 mg C appears in trap C (Figure 4) as COz resulting from untrapped ethane. While this is less than half of the 0.81 mg C calculated as a worst case, enough ethane does escape trapping to interfere with the analysis for methane. However, because the abundance of ethane and other potentially interfering gases is low in normal ground water samples, both types of analysis (with and without alumina) usually give equivalent results, and both can be used in most cases. The efficiency of the 1 2 0 5 [Schutze’s reagent ( 3 5 ) ]in oxidizing CO and its inability to oxidize methane have been shown recently ( 2 5 ) .To test the reagent prepared and used in analyses performed here, individual samples of each gas and mixtures of the two were analyzed after being placed in water. The tests indicate that a maximum of 2 X of either gas introduced appears as a cross-contaminant in the CO2 produced from the other gas. Blanks of Procedures. The blanks for the various proANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
.
133
Table IV. Procedural Blanks for Each of the Analytical Procedures Amount
Isotope ratio 95% Conf.
limits0
0.100
;0:004 i 0.05
-38.4 -25.2
+1.2 t1.2
0.002 0.017
+0.0006 t0.004
-33.6
il.l i1.2
mg C/1.
0.005 0.017
CH,
6I3c
(a,) ...
Fraction
Inorganic Volatile organic Nonvolatile organic CO and CH,
co
95%Conf. limits0
-38.4
...
0 95% confidence limits = it,.,,,,^ S, where t is Student’s t, 0.025 refers to the probability of error, and @ is the number of degrees of freedom available for the calculation of s, the standard deviation of the population of single observations of the blank.
cedures were determined by removing all of the carbon present from a quantity of distilled water, and then carrying out the respective analyses as for a natural sample. The amount and isotope ratio of the blanks for each fraction are shown in Table IV. Sample requirements are determined by these blanks, and the size of the water sample needed varies. For uncontaminated ground water which we have analyzed in southern Indiana, an amount of carbon ten times that of background is present in approximately 800 ml of water for the nonvolatile organic fraction, 600-700 ml for the volatile organic fraction, and 2 ml for the inorganic carbon. Particular care is required in establishing the blank for the inorganic carbon procedure, which involves acidifying the sample with 100%H3P04 prepared from P205 and commercial 85% H3P04 (30). Remarkably, this preparation is always contaminated with NzO, a gas with vapor pressure vs. temperature characteristics very similar to those of COz, and even having the same molecular weight (14N2160 = l2C16Oz = 44). These coincidences mean first that ordinary COZ-purification procedures do not remove NzO, and, second, that mass spectrometric isotope ratio measurements can be seriously in error. Because 15N is much less abundant than 13C, carbon dioxide samples contaminated with N20 often appear to be sensationally “light,” perhaps yielding S13C = -200%0. In the present instance, errors of this type are avoided by adding the water sample to preevacuated 100% H3PO4 (exposure to vacuum for -30 min is sufficient to remove all N20 from the acid). Isotope Ratio Measurements of Standards. The reproducibility of isotope ratio measurements of standards analyzed in the same manner as natural samples is shown in Table V. All of the organic compounds and gases have been analyzed isotopically by standard combustion techniques not involving their removal from water, and, as noted in Table V, excellent agreement between the two methods of analysis was obtained.
The results for the volatile organic carbon standards require some further explanation. As stated earlier, acetone was not quantitatively removed from water in the 3-hr purging time, although its recovery was complete if no water was added. The isotope ratio results in Table V indicate that an isotopic shift of -2.4 f 1.0% occurs because of this incomplete removal. The effect is small and normal, since the lighter isotope is preferentially removed by stripping, and if the magnitude of the effect is similar in other partially stripped compounds, it should have little influence on the results for natural samples. A similar effect, also involving solvent-solute and solute-solute interactions, has been observed for the vapor pressures of isotopic species of amines diluted with nonpolar hydrocarbon solvents ( 3 4 ) . Methane was analyzed in three separate ways to test for the occurrence of any significant isotope effect which may result from the loss of small amounts in the analysis train. The methane was analyzed as a pure gas passing directly into the combustion ovens, as a pure gas passing through the soda asbestos into the combustion ovens, and as a dissolved gas stripped from water and passing through soda asbestos into the combustion ovens. The results for all three analyses agreed within the limits of the mass spectrometric measurement ( f l % ~ ) . N a t u r a l Samples. The system has also been tested with natural samples to determine reproducibility of the measurements of amounts and isotope ratios, and Table VI illustrates the results of these tests. The rather poor reproducibility in the measurement of the amount of inorganic carbon is due to the variable inclusion of carbonate debris in the sample collection tubes, and the degree of variation observed is dependent upon the particular locale and type of well studied. The uncertainties of the isotope ratio measurements in each fraction listed in Table VI are based on duplicates of three separate samples. Determination of 95% confidence limits in such a case requires the use of the coefficient t with only three degrees of freedom, and the broadening of the confidence limits caused by this is fairly large. It seems likely, in fact, that substantially higher precision is furnished by this technique. APPLICATION The techniques described permit the monitoring of all of the carbon present in ground water, and provide subfractions of the total carbon that can be useful in delineating the processes leading to the precise composition of the water. Specifically, the proportion of volatile organic to nonvolatile organic carbon would be different in aerobic vs. anaerobic degradation (12), and the isotope ratio of the inorganic carbon has been shown to be useful in studies of the hydrologic cycle (7, 11). It is frequently important to trace the pathway and chemical history of pollutant carbon in the sedimentary environment. To a substantial degree, natural isotopic con-
Table V. Isotopic Composition of Standards as Determined by Direct Combustion and by the Present Techniques Present techniques No. of
Fraction
Compound
detns
Volatile organic Nonvolatile organic Inorganic Carbon monoxide and methane Volatile organic Volatile organic Volatile organic
Methane Mannitol Sodium Carbon monoxide Acetone, pure Acetone, in H,O Heptane
8
134
ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
6 6 3 6 5 2
6
”C,
(%O)
-36.1 -10.2 - 5.5 -40.9
-27.8 -29.5 -28.4
95% Conf. limits
i0.03 t0.06 i0.03 il.l t0.06 tl.0
i0.5
Direct combustion 6
I’C.(%O)
-36.3 - 9.8 - 5.5
-40.7 -27.1 -27.1 -28.1
Table VI. Overall Reproducibility of Analysis for Natural Samples 95%
Fraction
Nonvolatile organic Volatile organic Inorganic
Amount, mg Cjl.
Confidence limits Isotope ratio, %o
*0.17 i0.02
*3
t2.2
i0.5
k3
tents, which are less easily modified than chemical structures, can serve as "tracers" for various carbon inputs, even when the input is mixed with indigenous carbon. When two carbon pools are mixed, the relationship between the mixing ratio and isotopic compositions can be summarized by an isotopic mass balance equation:
In this expression, the 6 terms represent the 6 1 3 C p values ~~ for the mixture (m) and two contributing pools (a and b ) . The x terms represent the fractional abundance of a and b in the mixture. The first demonstration of this approach was provided by Calder and Parker (6), who were able to determine the contributions of natural and pollutant carbon to the organic matter in a marine bay system. A more detailed and informative investigation can be carried out when subfractions of the carbon are investigated in the mixture and in the two precursor pools, a and b. If two pools, each comprised of four distinct carbon fractions, are combined, calculations based on Equation 2 will also be useful in evaluating the extent to which the fractions are simply mixed vs. the extent to which not only mixing but, in addition, transfer of carbon between fractions takes place. If transfer of carbon between fractions occurs, the isotopic mass balance (Equation 2) will fail on a fraction by fraction basis. If not all the input carbon is retained in the aqueous phase, not even an overall isotopic mass balance based on the sum of the four fractions in the two inputs will succeed. This type of effect, in which the isotopic mass balance equation fails, might easily occur in ground water in which interactions between the carbon compounds and the sedimentary environment can take place. An example of the application of these techniques and of the tracing concept is furnished by our study of the interaction between normal ground water and the leachate from a sanitary landfill (36). It was, in this instance, possible to show by an application of the techniques reported here that a well thought to be contaminated by the landfill was being polluted instead by another source.
ACKNOWLEDGMENT We appreciate the skillful technical assistance of S. A. Studley and the generous cooperation of our co-workers D. A. Schoeller and D. H. Hauber.
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RECEIVEDfor review June 27, 1975. Accepted September 22, 1975. Our laboratory is supported by grants from the National Aeronautics and Space Administration (NGR 15003-118) and the National Institutes of Health (GM18979). LMG acknowledges with thanks a fellowship from the Lubrizol Foundation and the receipt of an NIH biochemistry traineeship, Grant. No. GM-1046-12.
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