AMAlYTlCAL TECHNIQUES By Alan Newmon Some new t e c h n i q u e s a r e creeping into EPA’s venerable list of analytical methods for organic compounds in the environment. Speakers at the recent EPA Conference on Analysis of Pollutants i n the Environment, in Norfolk, VA, described experiments with liquid chromatographylmass spectrometry (LCIMS), supercritical fluid extraction (SFE), a n d robotic systems. These state-of-the-art techniques offer ways to measure nonvolatile pollutants, improve laboratory efficiency, and reduce costs. Compared to the number of procedures involving gas chromatography, few EPAmethods currently rely on liquid chromatography. However, says William Budde of EPA’s Environmental Monitoring Systems Laboratory in Cincinnati, “I predict more growth for LC.” Budde lists a host of environmentally important compounds that are too temperature sensitive to survive GC conditions ( 1 3 1 , for instance, pesticides such as carbamates (e.g., carbaryl and alicarb sulfone),thioureas (e.g., diuron and linuron), and rotenone with five temperature-sensitive ether linkages. What makes liquid chromatography particularly attractive is that it is now possible to interface high performance liquid chromatography (HPLC) instruments with mass spectrometers. Mass snectrometrv offers sensitive and generally unequivocal identification of analytes. A number of different designs are currently available for LCIMS interfaces ( 4 , 5 ) . At the EPA meeting Budde described experiments with one configuration, labeled a particle beam interface. As t h e mobile phase elutes off the HPLC column, the solution is sprayed into a heated de-
solvation chamber. Aerosol particles containing the dissolved nonvolatile solute are forced through the chamber and exit via a nozzle or beam colliminator. The resulting particle beam travels through two more chambers in which vacuum pumps strip off solvent vapor and reduce the pressure. Particles and solvent vapor that diffuse out of the beam are removed by
0013-936x191/0925-1363$02.50/0 @ 1991 American Chemical Society
momentum separator skimmers. Those particles with the greatest momentum stay focused in the beam and thus pass into the mass spectrometer. “Momentum carries the particles through the interface to the mass spectrometer,” explains Budde. Using LCIMS, Budde and his colleagues have developed EPA Method 553 for the identification and quantitative analysis of temperature-sensitive benzidines, which are known h u m a n c a r c i n o g e n s . Benzidines may be forming in the environment as a result of anaerobic reduction of azo dyes. “There are millions of pounds of azo dyes being produced, and the waste has to go somewhere. It is out there percolating and we don’t know if or when it will be a serious problem.” To identify benzidines, samples are run on a reverse-phase liquid chromatography column and eluted with a wateracetonitrile gradient solution containing ammonium acetate to improve separations. Lowbleed HPLC columns are employed i n order to minimize the introduction of contaminants that might interfere with the mass spectrum. The solvent and ammonium acetate that pass through the particle beam interface obscure only the low mass region of the spectrum (63 m/e and lower). The higher mass region reveals patterns similar to standard electron impact spectra for each benzidine congener. To further evaluate this method, EPA conducted a study with 13 laboratories that used particle beam interfaces. These laboratories employed five different combinations of commercially available mass spectrometers and HPLCs. With benzidine samples that ranged from 5 to 100 ng/FL, Budde reported that the mean accuEnviron. Sci. Technol., VoI. 25, No. 8, 1991 1363
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Environ. Sci. Technol., Voi. 25, No. 8, 1991
racy was approximately 97% and, with the lOO-ng/yL sample, the mean precision was < 10%. To insure the quality of the LC/ MS spectra, Budde’s group has evaluated decafluorotriphenylphosp h i n e oxide (DFTPPO) as a performance test compound. This compound features a number of strong peaks in the mass spectrum, ranging from an m / e of 77 to the molecular ion at 458. The spectrum of DFTPPO varies with different spectrometer conditions and thus could indicate system problems that would affect the analysis. Another new technique offers an easier method for collecting samples for analysis. SFE, explains Merlin Bicking from Twin City Testing Corporation of St. Paul, MN, requires less material and time than traditional solvent extractions. (Supercritical fluids form when materials are raised to temperatures and pressures at which the liquid and gas phases are indistinguishable.) Furthermore, by varying the pressure and temperature, analysts can “fine-tune” the properties of a supercritical solvent for a particular pollutant. To handle all the variations, Bicking’s group employed a statistical approach to determine the best temperature and pressure for extracting a particular pollutant. This approach, labeled a “star-square” or central composite design, determines the percent recovery under nine different extraction conditions. The data lead to a three-dimensional plot of recovery versus temperature and pressure that pinpoints the best extraction conditions. As an example, Bicking and his colleagues demonstrated the versatility of this approach by adapting it for EPA Method 413.2, which involves the extraction of oil and grease. To simulate this analysis, hexadecane and chlorobenzene were extracted from diatomaceous earth. The current method calls for the soxhlet extraction of a 20-40-g sample with 300 mL of freon. After 48 h, the solution is concentrated and examined by FT-IR in a 100-mm path length cell. Recovery was determined to be 97%. The equivalent SFE approach used just 2-5 g of sample and 6 mL of freon-eliminating 98% of the chlorofluorocarbon solvent. After only an hour the extract was diluted to 1 0 mL and examined by FT-IR using a 10-mm cell. Recovery was about 91% Bicking calculated that the cost of
the analysis dropped from $12.50 for the standard soxhlet extraction to $1.65 for SFE; “For an analysis that you charge $30 for, that is a significant cost reduction.” He also reported that about six SFE analyses could be performed daily, compared to eight by soxhlet. Automating the supercritical extractions would improve that rate, and thus the efficiency of this new approach. Bicking has also looked at introducing SFE for the recovery of dioxins and dibenzofurans (EPA Method 8290). The standard SFE fluid, supercritical CO,, failed to adequately recover these organics even after long extractions. However, Bicking finds that the extraction improves with methanol mixed in with the C0,another indication of the many variations available with SFE. As Bicking mentioned, automation is one way to improve the efficiency of a test. W. A. Michalik described the experience of Shell Oil’s analytical laboratory in Roxanna, IL, with robotic systems for performing biochemical oxygen demand (BOD) analyses on waste water. At the heart of the system is a Zymark robotic arm. Arranged in a circle around the arm are various automated stations that transfer the analyte; add the biochemical seed, phosphate buffers, and water; insert and remove the glass stopper: take pH and oxygen measurements; and even wash the bottle for the next analysis. To prepare 30 samples the robot takes 60-90 min, says Michalik. The precision and accuracy of the automated system match the results of human operators. A technician is needed only to move prepared samples in and out of the incubator and to daily calibrate the peristaltic pumps that transfer solutions. As a result, human involvement in the BOD analyses at Shell has decreased by approximately 75%, yielding another significant savings. References (1) Ho, J, S. et al. Environ. Sci. Techno]. 1990,24,1748. ( 2 ) Beller, T. A , ; B u d d e , W. L. Anal. Chem. 1988,60, 2076. (3) Behymer, T. D.; Bellar, T. A.; Budde, W. L. Anal. Chem. 1990,62, 1686. (4) Covey, T. R. et al. Anal. Chem. 1986, 58,1451A. (5) B u d d e , W. L. et al. 1. A m . Water Works Assoc. 1990,82, 60.
Alan Newman is an associate editor on the Washington editorial staff of ES&T.
