ACKNOWLEDGMENT
(3) J. C. Gage, J. Sci. Instrum., 30, 25 (1953). (4) K. Ashida, F. Yamauchi, M. Katoh, and T. 181-185 (1974).
The author acknowledges the help given by P. Maddison in the construction of the analyzer and in its commissioning in field trials.
Harada, J. Cell. Plast.
10 (4),
RECEIVED for review June 14,1976. Accepted November 9, 1976. This work was partly supported by a grant from the
LITERATURE CITED
Products Research Committee (U.S.A.) in association with the International Isocyanate Institute Inc. of New York.
(1) C. Louden and H. Antrobus, Ana/yst(London),64, 187-189 (1939). (2) A. Chaston Chapman, Analyst (London),35, 470-477 (1910).
Chlorine-Selective Detection for Liquid Chromatography with a Coulson Electrolytic Conductivity Detector John W. Dolan and James N. Seiber* Department of Environmental Toxicology, University of California, Davis, Calif. 956 16
The design and operation of a liquld chromatography/Coulson electrolytic conductivity detector (CECD) analytical system are described. Vydac totally porous reversed phase column effluent (aqueous methanol) was introduced to the CECD quartz combustion tube through a stainless steel inlet. The conductivity response of pyrolysis products formed In the presence of hydrogen was monitored with slight modifications of the standard CECD cell operation. The system showed high selectivity to organochlorine compounds relative to hydrocarbons, with a linear range of lo5 and minlmum detectability of 5-50 ng for lindane. The CECD detectability was better than that of the UVSe4detector and its selectivity greater than that of the UV22. detector, when CI-containing aliphatlc pesticides were examined. This was reflected in the successful CECD analysis of uncleaned extracts of lettuce and river water fortified to sub-ppm levels with aldrin and dieldrin and the corresponding failure of the UV detector at either 254 or 220 nm in the same analyses.
High performance liquid chromatography (HPLC) has become increasingly useful for analysis of low levels of chemicals in biological and environmental samples. For example, HPLC represents a potential alternative to gas-liquid chromatography (GLC) for the residue analysis of pesticides lacking sufficient volatility or thermal stability for GLC determinations ( 1 ) . The utility would likely be extended were there available element-selective detectors for HPLC comparable to those in current use with GLC. Electrochemical (2-5), spray impact (6), alkali-flame ionization (7), electron capture ( 8 ) ,and other experimental detectors offer some selectivity which may be useful in pesticide residue analysis. But among those commercially available, only the ultraviolet (UV) and fluorescence photometric detectors have provided the sensitivity and detectability needed for practical applications in this field (1, 9, 10). Selectivity with the photometric detectors is limited to those compounds or their derivatives having UV absorbance or fluorescence properties not possessed by sample interferences; these requirements are not met by many pesticides and their conversion products of current environmental importance. The Coulson electrolytic conductivity detector (CECD) is an example of an element-selective detector widely used in the GLC determination of pesticides (11,12).In the reductive mode of the CECD, with no pyrolysis catalyst, compounds 326
ANALYTICAL CHEMISTRY, VOL. 49,
NO. 2,
containing C1 or Br respond with little or no interference from chemicals which lack these heteroatoms. This selectivity, which may be extended to S- and N-containing compounds through appropriate choice of pyrolysis gas, catalyst, and effluent scrubber, reduces the need for time-consuming cleanup of environmental samples prior to determination (13). We have adapted the CECD to monitoring the effluent from HPLC. Our system was aimed at providing selective detection of poorly UV-absorbing organochlorine pesticides resolved by reversed phase chromatography. We examined the system’s design and optimization, its response t o some representative pesticide standards, and its applicability to analysis of a few environmental samples fortified with aldrin and dieldrin. A UV detector was used for comparison with the CECD, to aid in evaluating the latter’s performance with HPLC. GLC/ CECD was also used to compare CECD performance with the two modes of chromatography.
EXPERIMENTAL Materials. Pesticides and other chemical standards, generally of 99% or greater purity, were obtained from manufacturers. The following Mallinckrodt solvents were used: Nanograde acetonitrile and hexane, absolute diethyl ether and methanol, and reagent grade isopropanol and petroleum ether. Ion exchange resin (AG 501-X8(D), Bio-Rad Laboratories, Richmond, Calif.) was used to deionize the recirculating CECD water supply. HPLC Components. The HPLC system was assembled in this laboratory from several components. Single piston or dual piston pumps, 5000 psi, with pulse dampers (Laboratory Data Control, Riviera Beach, Fla.) were used at various times with similar results. Injection was through a Valco valve (Laboratory Data Control) fitted with a 12.5- or 30-pl sample loop, or a Rheodyne Model 70-10 valve (Rheodyne, Berkeley, Calif.) fitted with a 20-pl sample loop. A 5 cm X 4 mm i.d. guard column packed with 37-40 pm Vydac reversed phaqe packing (Separations Group, Hesperia, Calif.) was placed between the injector and column during analysis of environmental samples. A 25 cm X 3.2 mm i.d. reversed phase column of 10-wm nominal particle size (Vydac 201 TP reversed phase, Separations Group) was used in all analyses.The mobile phase, approximately80% methanol in deionized water pumped at a flow rate of 0.5 ml/min, was adjusted slightly in composition to effect solute elution within 20 min of injection. A Spectrometer I variable wavelength detector (Laboratory Data Control) was used for UV detection. CECD Interface and Components. A Coulson electrolytic conductivity detector furnace, cell, and deionizer (Tracor, Austin, Texas) were used with the modifications noted below. In the final configuration of the LC/CECD system (Figure l), effluent from the analytical column entered a 1.59-mmo.d., 0.18-mm i.d. No. 304 stainless steel tube (the inlet) extending 6 cm into the furnace.The inlet tube, a 1.59-mm0.d. stainless steel sidearm allowing
FEBRUARY 1977
-LCEFFLUENT lNLEl
TO CE
SWAGELOK FITTINGS
Figure 1. Schematic diagram of LCICECD interface
entry of hydrogen reaction gas, and a 24 cm X 6 mm o.d., 4-mm i.d. quartz combustion tube were held by a modified %- to %-in.Swagelok reducing union with Teflon ferrules. The 20 cm X 1.27 cm i.d. horizontal furnace was held at 900-975 "C unless otherwise noted. The furnace effluent passed into the CECD cell's glass transfer line, heated at 100-150 "C from the combustiontube joint to the gas-liquid contactor with Nichrome resistance wire. The CECD cell was operated as specified by the manufacturer except that a piece of clamp-restricted latex tubing was fitted to the exit of the conductivitysolution overflow to increase the delivery of solution to the gas-liquid contactor, and 50% aqueous methanol was used as the conductivity solution. The cell voltage was maintained at 9 V dc. GLC Conditions. An F&M Model 400 gas chromatograph was equipped with a 1.2 m X 6 mm 0.d. glass column packed with 2% SE 30 on acid-washed, DMCS-treated Chromosorb W, and a Coulson electrolytic conductivity detector. Nitrogen carrier and oxygen combustion gas flows were each 40 ml/min. Column oven and detector furnace temperatures were 230 and 850 "C, respectively. Sample Preparation. Two mature lettuce heads were chopped, thoroughly mixed, and divided into 100-g portions. Three portions served as controls. Two were fortified to 1 ppm, and two were fortified to 0.1 ppm by adding aliquots of 1 mg/ml standards of aldrin and dieldrin in methanol. Samples were extracted with 2 1 hexane-isopropanol by a published method (14). Concentrated extracts freed of isopropanol were made to 2 ml for the controls and 0.1-ppm fortifications, and 25 ml for 1.0-ppm fortifications, in hexane for GLC analysis, or exchanged to methanol for HPLC. River water, collected from the Sacramento River, Sacramento, Calif., was filtered and divided into 1-1. portions. Three portions served as controls, two were fortified to 0.1 ppm, and two were fortified 40 0.01 ppm with aldrin and dieldrin. Samples were extracted with hexane by a published method (14). Concentrated extracts were made to 2 ml in hexane for GLC analysis,or exchanged to 2 ml in methanol for HPLC analysis. R E S U L T S AND DISCUSSION A major consideration in the design of an LC/CECD interface lies in accommodating the massive increase in volume (2300 times for methanol, 5300 times for water) upon conversion of liquid column effluent to the gaseous phase. A too rapid throughput could well have overwhelmed the capacity of the combustion tube of the CECD cell, while any unevenness in the phase transition would have affected baseline stability. Furthermore, carbon deposition in the inlet or combustion tube from incomplete methanol pyrolysis would have contributed noise t o the system a t the very least, and perhaps resulted in plugging under unfavorable circumstances. Removal of a major part of the solvent before solute combustion, as done in the moving wire detector (151, was considered; the expectation that the aqueous mobile phase from reversed phase LC lacked sufficient volatility to allow efficient removal from organic solutes led us to pursue a different approach. The system we adopted, in which the mobile phase and solutes were simultaneously vaporized in the combustion tube, allowed for a very simple interface design and reproducible operation. Inlet. The inlet ( h g u r e 1) served to vaporize column effluent and introduce hydrogen reaction gas to the heated
quartz combustion tube of the CECD. Vaporization took place largely inside the tip of the stainless steel inlet, heated by its alignment near the front edge of the hottest zone of the furnace. Other inlet configurations, including those in which the liquid was introduced to the combustion tube as drops, droplets, or in an atomized form, resulted in uneven vaporization or rapid carbon deposition. The hydrogen reaction gas served t o sweep vapors emanating from the inlet tip into the hot zone of the furnace. The mass of the inlet served as a heat sink, providing for nearly complete vaporization while maintaining its structural integrity over many hours of operation. The inlet did, however, occasionally become plugged with carbon deposits, usually after 4 to 40 h of continuous operation; restoration was accomplished by cutting 2-3 cm from the tip, and repositioning the tip adjacent to the hot zone of the furnace. This shortcoming of the inlet was offset by its simplicity and low cost. Combustion Tube. Studies of CECD optimization for GLC indicated that smaller combustion tube diameters gave an enhanced responss, the optimum being 0.5-mm i.d. for the tubes tested (16).With the LC/CECD system, however, no difference in peak height was observed ( F test, a = 0.05) for combustion tubes of 2.5, 4, 6, and 9-mm i.d. An increase in tube diameter from 2.5 t o 9 mm results in a 3.6-fold increase in surface area, a 13-fold increase in volume, and a corresponding increase in combustion tube residence time for vapors; this situation might favor HCl loss through adsorption and lower the conductivity response. Since this was not observed in LC/CECD, HCl adsorption is apparently not as important a factor with this system as with GLC/CECD, perhaps because of the large excess of water vapor present in the former. Nor did a larger or catalytic surface tend to enhance response, from improving pyrolysis efficiency; packing the combustion with quartz chips or platinum gauze gave no improvement in CECD response when compared with an empty tube. Carbonization in the quartz combustion tube was minor to non-existent, but some deterioration with time was observed. After one to several weeks in the furnace, the tubes appeared etched and cloudy; this gradual process was accompanied by an increase in background noise which could be corrected only by replacing the tube with a new one. Tube deterioration was greatest near the inlet tip, indicating its origin was in thermal stress from constant contact of the red hot tube walls with relatively cool effluent vapor. F u r n a c e Temperature. From investigations of CECD optimization with GLC, both molecular structure and furnace temperature affect response toward organochlorine compounds. In fact, the structure-temperature determinants were predictable for organochlorines varying in the nature of the C-Cl bond (16).These same relationships were observed for LC/CECD (Figure 2); heptachlor, which contains no aromatic or vinylic C1, showed a regular increase in response with fur-
ANALYTICAL CHEMISTRY, VOL. 49, NO. 2, FEBRUARY 1977
*
327
I*
9 r
A
lB
0, = I C
L
0 450
500
1 550
600
650
700
TEMPERATURE
750
1
I
800
850
I 900
0
1
Figure 3. LCICECD chromatograms for lindane standard p,p'-DDE
(A)
nace temperature over the range examined. For p ,p'-DDE, however, in which all the C1 atoms are bound to aromatic or olefinic C, very little response was observed at furnace temperatures below 700 OC, and then response increased disproportionately with temperature in the 700-900 "C range. Thus a high temperature ( X O O "C) was required for nonselective C1 detection, and lower temperatures favored selectivity towards compounds containing C-Cl bonds of lower bond strength. Since the temperature limit of the CECD furnace was approximately 1000 "C, it was routinely operated at 900-975 OC for nonselective detection of organochlorine compounds of different structural classes. Reaction Gas. In the reductive mode of the CECD, all substances entering the combustion tube are theoretically converted to their most reduced states. For conpounds containing C, H, 0, and C1, the products are CH4, Hz, HzO, and HC1. Calculations indicate that a mobile phase of 80% aqueous methanol introduced at 0.3 ml/min would require 160 ml/min of hydrogen for complete reduction; 1kg of lindane as solute would need ml/min of hydrogen, an insignificant amount compared to that used in solvent conversion. For replicate injections of lindane or Kelthane, no difference in response ( F test, (Y = 0.05) was found for hydrogen flow rates in the range 25 to 215 ml/min. Flow rates below 25 ml/min gave only a slight decrease in response. However, complications of practical significance did arise for some flow rates. Very high flows (greater than 150 ml/min) were greater than the CECD cell gas-liquid separator could accommodate, causing gas bubbles to enter the conductivity cell. With no hydrogen flowing, combustion tube vapors condensed on the inlet end. The resulting liquid droplets apparently absorbed HC1, causing a loss in response. Thus, while some gas flow was required as a carrier to sweep the combustion tube, the results did not establish the need for hydrogen as opposed to another gas. In fact, there was no difference in detector response ( F test, 01 = 0.05) when hydrogen and helium were compared a t 23-44 ml/min with replicate injections of lindane or DDT. This raises a question regarding the origin of HC1 during combustion. Dehydrochlorination may account for the release of some HC1 from solutes such as lindane and DDT which are known to undergo such a conversion readily (17). This is supported by the successful use of the CECD in the pyrolytic mode (no hydrogen) for C1 detection by some workers (18), and our own identification of DDE in the pyrolysis effluent during DDT analysis. For solutes such as DDE which do not readily dehydrochlorinate, HC1 release at temperatures above 700 "C may involve reaction with hydrogen produced from dehydrogenation of mobile phase methanol. That such a solvent conversion occurs was indicated by the presence of 328
I
TIME
('C)
Figure 2. LCICECD response to heptachlor ( 0 )and at various combustion temperatures
'
(A) 5 ng at attenuation 4; (B) 10 ng at attenuation 4; (C) 50 ng at attenuation 8; and (D) 100 ng at attenuation 8. I = injection point, S = solvent response, L =
lindane. The time spent for each chromatogram in this and the subsequent Figures was 20 min
formaldehyde in the furnace effluent even when hydrogen was externally introduced. Detector Cell. A standard CECD cell and Wheatstone bridge were used with only minor modifications. The Teflon transfer line insert was removed, and the transfer line was heated to 100-150 "C to prevent condensation of combustion tube effluent. In GLC operation, modifications have been reported which reduce the water flow to the cell and thus increase the proportion of the sample entering the cell (19).In HPLC, however, the large volume of gas from the combustion tube required a higher flow of conductivity solution to the gas-liquid separator, to prevent bubbles from entering the conductivity cell. Addition of a restrictor to the cell pressure head overflow served this purpose. The restrictor decreased the amount of sample entering the conductivity cell, but effectively increased the signal-to-noise ratio by lowering cell background. Use of a conductivity solution of approximately 50% aqueous methanol rather than water significantly reduced background noise and decreased the time needed for system stabilization after replacement of the ion-exchange resin. The use of aqueous methanol may account a t least in part for the need for the overflow restrictor, since its viscosity and flow characteristics are materially different from water. Fortunately the percent methanol in the conductivity solution was not critical; the composition changed during operation from input of unreacted methanol from the combustion tube. Column Flow. HPLC column flow rates were limited to approximately 0.5 ml/min; higher flows resulted in excessive introduction of bubbles to the conductivity cell. A column effluent splitter would be necessary for HPLC operation at higher flow rates. System Performance. M.ultiple-injections of lindane or heptachlor were made a t hydrogen flows ranging from 10-70 ml/min, to determine reproducibility of response under typical operating conditions. The average relative standard deviation (RSD) of peak height, 3.4%for 16 data sets of 6-11 injections each, compared favorably with a 2.2% RSD with the same HSLC system and a UV detector in this laboratory. To determine the system response linearity, replicate injections were made for lindane quantities in the range of 5 ng to 500 yg. The linear range, lo5,had a coefficient of determination of 0.99. The smallest quantity injected, 5 ng, corresponds to the detection limit since the resulting peak was about twice the background noise (Figure 3). From peak quality and precision on replication, however, 50 ng of lindane represents a more practical detection limit.
ANALYTICAL CHEMISTRY, VOL. 49, NO. 2, FEBRUARY 1977
Table I. CECD Response and Relative CECD/UV254 Response for Pesticides and Related Chemical Standards
Chemical Mirex Heptachlor Heptachlor epoxide Hexachlorobenzene Aldrin Dieldrin Lindane Endrin o,p’-DDT p,p’-DDD Pentachloronitrobenzene Pentachloroanisole p,p’-DDT p,p’-DDE Methoxychlor Trifluralin Dursban Kelthane Ronnel Pentachlorophenol Captan Perthane Dichlorobenzophenone Paraoxon Disyston p,p’-DDA 2,4-D Parathion Abate Guthion Phosdrin Meta-Systox-R Azodrin Methyl parathion Omite Methomyl Carbarvl
Retention relative Response to p , p ’ - Hetero- CECD (area/ CECD/ DDT uv254 PW m 2.39 207 26.3 0.94 113 25.0 0.72 105 2.15
101
0.46
1.21 0.84 0.62 0.84 1.08 0.76 1.01
84 77 71 66 56 41 41
6.98 10.4
0.95
40
0.64
1.00
40
1.15 0.73 0.78 0.79 0.81 0.78 0.37
40 40
0.58 0.03
36 36 35 31 30
0.008 1.04 1.16 3.66 0.04
0.53 1.04 0.71
30 20 15
0.51 0.01
0.50 0.63 0.47 0.49 0.59 0.72 0.55 0.49 0.47 0.47 0.53 0.76 0.49 0.50
11 11 10
10 9.9 8.3 7.1 7.1 6.5 5.1 4.1 3.8 2.3 0.08
m
4.06 1.23
1.33 0.24
0.10
2.41
0.16 1.33 0.33 0.73 0.02
0.01 0.02 m m m
0.01 0.65 m
0.004
T o evaluate selectivity, the LC/CECD system response was determined for 37 pesticides and related compounds covering a wide range of chemical classes (Table I). In general, compounds containing C1 or F responded better than nonhalogenated chemicals, and response increased with halogen content among related chemicals. However, as noted previously, the C1 response was somewhat dependent on structure (cf. endrin and hexachlorobenzene), indicating that a combustion temperature even higher than 975 OC would be needed to eliminate inter-halogen selectivity. The nature of other functional groups in organochlorine compounds might also influence response; for example, pentachlorophenol and Kelthane, both of which contain an -OH group, gave a lower response than expected based on C1 content. Among compounds containing heteroatoms other than C1, some gave significant response, others very little. For those containing halogen along with other heteroatoms, the response was apparently due primarily to halogen. N contributed little to response, since carbaryl, which contains only N as heteroatom, gave the lowest response of all the chemicals exam-
ined. This was as expected, since a Ni catalyst is needed to produce ammonia during reductive pyrolysis of N-containing compounds in the CECD (20). Omite, the only compound examined which contained S as the sole heteroatom, gave a small but measurable response; this may have resulted either from reduction to HzS, a weak electrolyte, or from partial pyrolysis to SOB,a strong electrolyte. Some response was obtained for all P-containing compounds examined, probably from the production of conducting phenolic or acidic moieties during pyrolysis. These observations, necessarily qualitative because of the heterogeneity of the compounds examined, indicate that the LC/CECD was selective toward halogencontaining compounds, but less so than GLC/CECD. The LC/CECD system did, however, maintain a high selectivity for halogenated compounds over hydrocarbons, an important consideration in any application to environmental samples. The response of lindane was lo4 that of hexane or pentane, both of which gave a very broad HPLC peak a t quantities needed to produce a measurable conductivity signal. To compare CECD and UV254 response for individual compounds, a factor was determined by normalizing response to conditions giving equivalent baseline noise for the two detectors (0.04 AUFS for UV254, attenuation of 32 a t 9 V for CECD), then dividing CECD response by that for UV2.54 (Table I). A response factor larger than one favored CECD detection, while a factor less than one favored UV254 detection. Among the extremes of the 37 test chemicals, lindane and mirex clearly required CECD detection, while carbaryl required UV254. Kelthane and Dursban represent an intermediate group giving roughly equivalent response to the two detection modes. This comparison was limited to UVz54; the use of a variable wavelength UV detector would undoubtedly influence detector choice, but within the limitations imposed by sample-derived interferences. To investigate the applicability of the LC/CECD system to the analysis of environmental samples, lettuce and water were fortified with aldrin and dieldrin, two organochlorine pesticides having low UV absorbance. Sample extracts were determined without prior cleanup by three methods: GLC/ CECD was used to verify recovery, and to compare response and convenience of an accepted method with the experimental LC/CECD system; the same extracts were then transferred to methanol and analyzed by HPLC using both the CECD and UV detectors, the latter operated a t 254 or 220 nm. Recoveries calculated from analyses with the LC/CECD and GLC/CECD were in good agreement, greater than 69% for both pesticides in all the fortified samples. While aldrin and dieldrin peaks were clearly visible a t the levels examined by both techniques, the GLC system gave superior chromatograms in terms of peak quality and response. Chromatograms from the LC/CECD system were somewhat ragged, and suffered a partial interference for the lower fortification in lettuce (Figure 4). Both systems had a relatively high initial setup and maintenance requirement, but neither was operating at optimum during these particular tests. The same samples, adequately clean for LC/CECD, were unsuitable for HPLC determination by UV at either 254 or 220 nm (Figure 5 ) . At the longer wavelength, neither aldrin nor dieldrin standards gave a measurable response a t the levels needed for the analysis. While the standards responded well to UV a t 220 nm, the water blank produced an interference which completely obscured dieldrin, and the lettuce blank obscured both pesticides. These limited tests point out some advantages and weaknesses of the LC/CECD system. For poorly UV-absorbing organochlorine compounds, the CECD has considerable selectivity and some detectability advantages over the UV detector. This is best reflected in the comparison of lettuce and
ANALYTICAL CHEMISTRY, VOL. 49, NO. 2, FEBRUARY 1977
329
IC
IF
150 ng Sld
Inject
Dld
Ald
Inject
D
150 ng Sld.
Did
Ald
TIME
Figure 4. LC/CECD chromatograms for analysis of lettuce and water extracts (A) lettuce blank extract analyzed at levels corresponding to 0.1-ppm fortification, (B) extract of lettuce fortified at 0.1 ppm with aldrin (Ald) and dieldrin (Dld), and (C) 150 ng each of aldrin and dieldrin. (D)-(F) represent similar chromatograms corresponding to analysis of river water
lnlecl
Dld
Ald
Inject
Old
Ald
TIME
Figure 5. LC/UV chromatograms for analysis of lettuce and water ex-
tracts
river water blanks. Analysis of water samples for organochlorines, perhaps after concentration on the reversed phase column by continuous sampiing, seems to offer a good possibility for application in an area of current environmental concern (21). The LC/CECD system cannot, however, be considered in its present form to rival GLC and C1-selective detection for residue analysis of those organochlorine compounds which can be readily gas chromatographed. While the principle of conductivity detection of LC effluent has been established in the present work, improvements in selectivity, detectability, and operational quality are needed to make the LC/CECD system competitive with GLC/CECD. Some improvements can be expected from further refinements in inlet-combustion-cell operation in the present system, similar to what has occurred with the GLC/CECD system (16,18,19).But perhaps the best possibility for a major advance might entail the use of a concentric electrode cell design as described recently for the microelectrolytic conductivity detector (22). When a commercial version of this detector cell was attached to our inlet-combustion tube, inefficient and irregular gas-liquid separation in the cell, apparently resulting from too high gas flow to the cell, prevented successful operation. Reducing the gas flow entering the cell by an effluent split, or a change in cell electrode spacing, might overcome this problem such that the advantageous detectability of the microelectrolytic conductivity detector could be transferred to HPLC. Our system was evaluated with only a single chromatographic mode (reversed phase partition) and a single solvent (aqueous methanol); while these conditions are suitable for a large group of pesticides (23), more choice in chromatographic parameters would give the flexibility needed to optimize conditions in specific analyses. Since the CECD has Sand N-selective detection capabilities in addition to halogen, 330
(A) 150 ng of aldrin and dieldrin monitored at 254 nm; (B) 150 ng of aldrin and dieldrin monitored at 220 nm; (C) and (D) water and lettuce blanks analyzed at levels corresponding to 0.1-ppm fortifications at 220 nm
adaptation of the appropriate modes to HPLC would further increase the potential applications of this system, and perhaps make it competitive with gas chromatographic techniques for particular applications.
LITERATURE CITED (1) H. A. Moye, J. Chromatogr. Sci.. 13, 268 (1975). (2) J. G. Koen, J. F. K. Huber, H. Poppe, and G. Den Boef, J. Chromatogr. Sci,, 8, 192 (1970). (3) P. T. Kissinger, C. Refshauge, R. Dreiling, and R. N. Adams. Anal. Lett., 8, 465 (1973). (4) B. Fleet and C. J. Little, J. Chromatogr. Sci, 12, 747 (1974). (5) R. C. Buchta and L. J. Papa, J. Chromatogr. Sci., 14, 213 (1976). (6) R. A. Mowery, Jr., and R. S. Juvet, Jr., J. Chromatogr. Sci., 12, 687 (1974). (7) K. Slais and M. Krejbi, J. Chromatogr., 91, 181 (1974). (8) F. W. Willmott and R. J. Dolphin, J. Chromatogr. Sci., 12, 695 (1974). (9) F. Eisenbeiss and H. Sieper, J. Chromatogr., 83, 439 (1973). (10) D. F. Horgan, Jr., in "Analytical Methods for Pesticides and Plant Growth Regulators", Vol. 7, J. Sherma and G. Zweig, Ed., Academic Press, New York, N.Y., 1973. (11) D. M. Coulson, J. Gas. Chromatogr., 3, 134(1965). (12) D. F. S. Natusch and T. M. Thorpe, Anal. Chem., 45, 1184A (1973). (13) D. M. Coulson, Adv. Chromatogr., 3, 197 (1966). (14) G. Zweig and J. Sherma, "Analytical Methods for Pesticides and Plant Growth Regulators", Vol. 6, Academic Press, New York, N.Y., 1972, pp. 275-7. (15) R. J. Maygs, Chromatographia, 1, 43 (1968). (16) J. W. Dolan and R . C. Hall, Anal. Chem., 45, 2198 (1973). (17) N. N. Melnikov, "Chemistry of Pesticides", Voi. 36 of Residbfe Rev., Springer-Verlag, New York, N.Y., 1971. (18) W. P. Cochrane, B.P. Wilson, and R. Greenhalgh, J. Chromatogr., 75, 207 (1973). (19) J. F. Lawrence and N. P. Sen, Anal. Chem., 47, 367 (1975) (20) D. M. Coulson, J. Gas Chromatogr., 4, 285 (1966).
ANALYTICAL CHEMISTRY, VOL. 49, NO. 2, FEBRUARY 1977
E. Dowty, D. Cartisle, J. L. Laseter, and J. Storer, Science, 187, 75 (1975). (22)R. C. Hall, J. Cbromatogr. Sci., 12, 152 (1974). (23) J. N. Seiber, J. Cbromatogr., 94, 151 (1974). (21)
RECEIVEDfor review September 1, 1976. Accepted November 8, 1976. This work was supported in part by NIH Training Grant ES 00125 and NIH Grant ES 00054.
Analysis of Cigarette Smoke by Fourier Transform Infrared Spectrometry Wayne L. Maddox* Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830
Gleb Mamantov Department of Chemistry, University of Tennessee, Knoxville, Tenn. 379 16
The application of Fourier transform infrared spectrometry (FT-IR) to the quantitative determination of several components in the gas phase of whole, dilute tobacco smoke was demonstrated. The 18-cm absorptlon cell was part of a cigarette smoking system similar to the intermittent inhalation exposure devices used in smoking and health research with rodents. Concentrations were measured for carbon monoxide, carbon dioxide, methane, ethylene, and methanol in 7 to 22% smoke. The precision of a measurement in 22% smoke ranged from 3 % for carbon dioxide to 34 % for ethylene. Absorbances measured for isoprene and hydrogen cyanide followed expected concentrations in different cigarette smokes. It was shown that the concentrations of these components remain constant during a 30-s hold-up following each puff on the cigarettes.
Investigation of the relationship between smoking and health has resulted in an increasing emphasis on inhalation studies wherein animals are confined in an exposure device and exposed to mechanically generated smoke. Placing these experiments on an improved quantitative basis requires means of chemically and physically characterizing the environment within the exposure chamber during the exposure. Chemical characterization of tobacco smoke in an exposure device containing animals is a challenging analytical problem, since the smoke is neither a simple nor a stable mixture, and is furthermore being disturbed by the animals’ respiration. Tobacco smoke is a complex mixture which consists of a particulate phase that has lo8 to 1010particles/cm3 (diameters of most of the particles fall in the range of 0.1 to 1.0 hm) surrounded by a gas phase (GP) containing air and the volatile products of tobacco combustion (1). The particulate matter is conveniently removed from the gas phase by filtration with a glass-fiber Cambridge filter ( 2 ) which removes 99.9% of particles greater than 0.3 pm in diameter; this is the customary basis for an arbitrary definition of the two phases. The analytical task involves determining rapidly as many smoke components as possible, preferably without affecting either the animal subjects or the atmosphere to which they are exposed. At present, chemical analysis of smoke in exposure chambers is accomplished by withdrawing a portion of the smoke from the chamber and determining selected comPresent address, ORGDP, P.O. Box P, Bldg. K-l004B, Mail stop 449, Oak Ridge, Tenn. 37830.
ponents in that sample by gas chromatography (GC) ( 3 ) .Up to now, dosimetry data have been available after the fact, e.g., by addition of radioactive tracers to the tobacco and sacrifice of the animals immediately after a short exposure to the smoke ( 4 ) .An instrument that could frequently observe in situ some of the smoke constituents would enable the acquisition of data on chemical composition and hence dosimetry immediately. Rapid-scan Fourier transform infrared (FT-IR) spectrometry is characterized by speed and sensitivity which may make it an attractive approach for such on-line analysis of smoke in exposure chambers. Lephardt and Vilcins (5, 6) described the application of FT-IR to the study of kinetics in undiluted gas-phase smoke. A review of the work by Vilcins and Lephardt (7) has appeared since the submission of this paper. We are grateful to one of our reviewers for calling our attention to this reference. We have applied FT-IR to the determination of certain gas-phase components in whole smoke under conditions (path length, cell volume, dilution, exposure time) which might be encountered in an animal exposure experiment. Strong absorption bands due to carbon dioxide, carbon monoxide, methane, water, and hydrogen cyanide are readily observed in the mid-IR spectrum of dilute whole smoke. Other components such as isoprene, methanol, and ethylene, yield weaker yet analytically useful bands.
EXPERIMENTAL Materials. Cigarettes. T h e University of Kentucky Standard Reference Cigarette-lR1, and other special cigarettes, manufactured to National Cancer Institute (NCI) specifications (8),are supplied by the NCI to the Oak Ridge National Laboratory and other NCI contractors for research in smoking and health. Codes 1, 6, 13, and 16 from the first experimental series were used. Code 1 is the Kentucky Reference cigarette and the higher-numbered codes are variants produced by various manufacturing techniques from a “Standard Experimental Blend” of tobaccos and other ingredients. When smoked, these variants produce smokes which differ in chemical composition and consequently in biological effects. Apparatus. Spectrometer. The spectrometer used was the Digilab Model FTS-20 (Digilab, Inc., Cambridge, Mass.), a high-resolution, rapid-scanning, computer-controlled instrument with a range capability of 10 to 10 000 cm-l and having nominal resolutions of 16, 8, 4, 2, 1, 0.5,0.25, and 0.125 cm-’. Absorption cells or accessories up to 20 cm in overall length may be utilized in the sample compartment. The mid-IR source is a heated nichrome filament and the detector a TGS (triglycine sulfate) pyroelectric bolometer. The beamsplitter is a film of germanium deposited on a potassium bromide substrate. Data acquisition times during each scan with this instrument are 0.4 s at 8 cm-l resolution, 1.6 s a t 2 cm-’ resolution, and 3.2 s a t 1 cm-’ resolution. Running times are longer; with the software employed, ANALYTICAL CHEMISTRY, VOL. 49, NO. 2 , FEBRUARY 1977
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