HPLC: high performance liquid chromatography One of the fastest growing segments of analytical instrumentation finds increasingly popular and versatile applications f o r organic pollutants in environmental samples
Michael Lynch Ed Weiner Waters Associates Milford, Mass. 01 757
High Performance Liquid Chromatography (HPLC) is a fast, precise, and economical technique for detecting organic pollutants. Consequently, H P L C has become one of the most popular approaches to environmental surveillance. This article presents an overview of high performance liquid chromatography intended to inform the environmental scientist of what H P L C is, how it works, and specifically, how it can be practically applied to the field of environmental surveillance. High performance liquid chromatography is a technique for making precision separations of complex chemical mixtures into their individual compounds. It is based on the phenomenon that under the same conditions, each component in a solution ordinarily interacts with its environment differently from all other components. T o the environmental scientist, therefore, H P L C is a technique for separating organic pollutants from complex mixtures occurring in air, water, or soil. H P L C can help determine the number of pollutants in a mixture, how much of each pollutant is present, and the purity of the sample. When employed in conjunction with other analytical techniques, H P L C can assist environmental scientists in specifically identifying individual components, by separating individual pollutants in pure form for subsequent identification. A basic high performance liquid chromatography system is illustrated. The H P L C process involves the introduction of a small amount of sample into a stream of solvent (the mobile phase) that flows through a column 666
Environmental Science & Technology
containing a bed of packing material (the stationary phase). The sample is then carried through the column by a continuous flow of the mobile phase which is delivered by a precise pumping system. With selection of the proper solvent and column packing material, some components of the sample mixture will travel through the column more slowly than others. Ideally the components are separated by the time they emerge “or elute” from the column end. The underlying principle of separation is based upon the affinity of the sample mixture components for the packing material. Sample components strongly attracted to the packing material will move slowly through the column. Sample components with a weak attraction for the packing material will move rapidly through the column. Thus a separation is achieved. As the separated components leave the liquid chromatography column, they are sensed by an appropriate detector. The detector then transmits a signal to a recorder and the separation is documented as a chart in the form of a sequence of peaks called a chromatogram. Each discrete peak may represent a particular compound. Practical environmental applications of high performance liquid chromatography are numerous and growing rapidly. Some of the more important of these applications are described; each illustration provides a brief background on the history and importance of the application, the specific methodology used to achieve the separation and detection, and the benefits H P L C provides.
Carcinogens in workplace air Benzo[a]pyrene (B[a]P) is a carcinogenic polynuclear aromatic hydrocarbon ( P N A ) which occurs in steel coking operations, asphalt production, automobile exhaust, aluminum production and cigarette smoke. In a 1977 study conducted for N I O S H , Jack
Holt of the Utah Biomedical Test Laboratories in Salt Lake City, developed a high performance liquid chromatography method for determination of B[a]P and other PNAs in the workplace air of steel coking facility. Workplace air samples are collected and passed through a filter at a fixed rate. When sampling is complete, the filter is extracted three times with benzene. The benzene-soluble extract is then injected directly (without any derivatization or further cleanup) into the liquid chromatograph for analysis. In liquid chromatography the distance from the injection point to the midpoint of a specific peak on the chromatogram (elution volume) is a convenient parameter used for peak identification. The UV absorbance ratio at two different wavelengths, however, is a molecular property, and therefore is a more precise indicator of peak identity. Holt identified peaks by use of both parameters. H e obtained an absorbance ratio and elution volume for each P N A peak, in a single analysis, by monitoring the separation simultaneously a t two different wavelengths. The L C procedure, as shown in Figures 1 and 2 , consists of two simple analyses. First, an analysis of P N A standards is made to determine the characteristic elution volume and absorbance ratio for each P N A peak. An aliquot of air filter extract is then injected directly into the chromatograph, and analyzed under the same chromatographic conditions used in the standard analysis. Elution volumes and absorbance ratios are then calculated for each peak appearing in the sample chromatogram. A comparison of data obtained from the standard and sample analyses is shown in Figure 1. Benzo(a)pyrene, fluoranthene, and pyrene were determined to present in the coke oven sample. In H P L C , peak area is proportional to compound mass. Holt
0013-936X/79/0913-0666$01.00/0
@ 1979 American Chemical Society
HPLC: the basic system
-*--.--
High pressure Iniecrwr
Liquid chromatography column
High pressure pumping system
Sample mixture containing compounds A B & C starts through the column
\h
"a Little Later" the separate compounds elute or pass from the column end.
Reservoir of
solvent
Chart record (Chromatogram)
FIGURE 1
FIGURE 2
Standard analysis
Workplace air sample analysis
I
280nm - \r
*'i
280 nm
t
c
0
.-
S
I
I .
365nm
Shown above is an analysis of six PNA standards. This analysis is made to determine the characteristic elution volumes and absorbance ratios for each PNA peak.
365 nm This actual sample analysis took only 27 minutes to complete. Three PNAs, Fluoranthene, Pyrene, and Benzo[a]Pyrene, were identified in the sample by comparing absorbance ratios and elution volumes to the standard values obtained in the standard analysis.
Volume 13, Number 6, June 1979
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FIGURE 3
ppb residues of isocyanates accurately determined in 16 minutes *T
1 .
1 , NitroReagent
MDI
I
I 0
1 4
I
I
J
8
12
16
Time (min) Figure reprinted with permission from Analytical Chemistry, 48 (1976),497499,K. L. Dunlap, R. L. Sandridge and Jurgen Keller, “Determination of Isocyanates in Working Atmospheres by High Speed Liquid Chromatography”. Copyright, 1976 by the American Chemical Society.
quantitated B(a)P in air samples by measuring the area of the B(a)P peak a t either wavelength (365 nm or 280 nm) used to monitor the separation. Reference to the simple calibration curve enabled him to calculate a concentration value. Wherever a manufacturing process produces or involves the use of toxic organic compounds, H P L C may provide quick, sensitive, reproducible determinations to help ensure compliance with industrial hygiene regulations and more important, the safety and health of manufacturing personnel. Atmospheric monitoring with liquid chromatography is not limited to determination of PNAs.
Isocyanates in industrial atmospheres Another example of how atmospheric pollutants can be monitored by H P L C is the analysis of isocyanates. Isocyanates are a highly versatile class of compounds used in the manufacture of polyurethane foams, coatings and elastomers. Their use in chemical manufacturing operations must be monitored to avoid potential health hazard to personnel and to ensure compliance with industrial hygiene regulations. T o ensure the safety and health of chemical manufacturing personnel who work closely with isocyanates, a team of chemists from the Mobay Chemical Corporation in New Martinsville, W. Va. and from Farbenfabriken Bayer A G in Leverkusen, Germany, has developed a routine method for monitoring airborne isocyanates i n the working atmosphere. Their task was difficult because the 668 Environmental Science & Technology
analytical method for isocyanate determinations must be highly sensitive since the threshold limit for toluene diisocyanate (TDI) is only 0.14 mg/m’ (0.02 ppm) for an 8-hour workday and 40-hour workweek. In 1971, scientists a t Farbenfabriken Bayer AG developed thin-layer chromatographic (TLC) methods for determination of isocyanates. Research colleagues a t Mobay adapted the methods for use, and extended their application. A t that time, T L C seemed the best method to use since it could measure either aliphatic or aromatic isocyanates without interference from amines. However, analysis by T L C required several tedious steps. Quantitative results depended entirely on the subjective judgment of the analyst to make accurate visual comparison of the standard and sample spots. A Mobay research team headed by Dr. R . L. Sandridge investigated liquid chromatography as an improved means for measuring isocyanates in air. The result was a new method incorporating liquid chromatographic instrumentation which relied on the proven “nitro reagent” chemistry from the earlier T L C work. HPLC was able to avoid the additional steps of reduction diazotization and coupling needed to visualize the T L C spots. The H P L C analysis is simple, quick, and straightforward. Sandridge and his colleagues used a high performance liquid chromatograph with a pellicular silica column for their method. The resulting chromatogram, Figure 3, shows determination of the four urea derivatives of isocyanates. If necessary,
the detection limit of this liquid chromatography method can be lowered to the parts per trillion range: by concentrating the sample directly onto the column before analysis by increasing the size of the sample directly onto the column before analysis by increasing the size of the sample injected from 9 0 ,uL to 300 ,uL allowed the Mobay team to extend the detection limit of TDI to 0.2 ppb. This represented a detection limit 100 times lower than required. Wherever a manufacturing process involves the use of toxic organic compounds, H P L C will provide quick, sensitive, reproducible determinations to help ensure compliance with industrial hygiene regulations and more important, the safety and health of chemical manufacturing personnel.
Wastewater surveillance Pentachlorophenol (PCP) is a n acutely toxic, highly persistent chemical often present in the effluent of facilities which manufacture pesticides, wood pulp and paper, wood preservatives, and many other products. In order to meet discharge requirements, a manufacturer required a n analytical method that was specific and accurate for P C P a t the 1 ppm level. They asked Waters environmental chemists to develop a method to be cost effective in a routine wastewater monitoring program. Described below is the development of an H P L C method that meets these criteria, and turns a potential problem analysis into a routine test. The first step was to determine if H P L C could separate and detect P C P in water. T o d o this, water was spiked with I O ppm P C P and IO0p L injected directly into the chromatograph. This analysis was successful and also permitted the determination of the characteristic elution volume (distance from the injection point on the chromatogram) for P C P in water. Next, a standard mixture of P C P and other phenols which were expected to be in the wastewater as co-contaminants h e r e chromatographed. This analysis was made to ensure that the H P L C method was specific for PCP. Upon completion of these two tests, the L.C method was tested on the ma n u facture r ‘s w a s t ewa t e r sample. A 150 p L aliquot was injected directly into the chromatograph. The chromatogram shows a very small peak eluting a t the characteristic elution volume for PCP. The relative concentration of P C P in the wastehater can be determined by injecting a small amount of sample
FIGURE 4
Wastewater sample analysis and recording the liquid chromatographic separation. Then inject another aliquot of the same sample spiked with 4 ppm P C P into a liquid chromatograph. A comparison of these two chromatographs (see Figure 4) will show the relative level of P C P in the sample wastewater.
Sample preparation T h e previous applications have not used any special sample concentration or collection techniques. However, many environmental applications require special sample handling. To meet this demand a unique collection and concentration technique has been developed by environmental chemists at W a t e r s Associates. This sample preparation method is called trace enrichment, and is a major advantage in simplifying the analysis of environmental pollutants. T r a c e enrichment permits the quantitative analysis of pollutants a t the ppb level with speed, accuracy and efficiency. Trace enrichment achieves this by eliminating the need for liquid/liquid extraction on large volumes of water samples, and by permitting direct 1000:1 concentration of organics from water. T h e trace-enrichment procedure uses a 5-cm-long economical sample preparation and collection cartridge called S E P - P A K and involves two major steps: concentration and selective elution. First the sample is passed through a S E P - P A K cartridge and the organic pollutants are retained on the L C separating material. Then those concentrated organics are selectively removed from the SEP-PAK cartridge by pumping one mL of an appropriate organic solvent through the cartridge. T h e collected cartridge eluent sample is then injected into a liquid chromatograph for a precise analysis. Using the trace enrichment process, environmental chemists can accelerate sample preparation to streamline analyses, permit detection of trace components, facilitate on-site sampling, and reduce consumption of costly solvents and reagents. The following environmental H P L C applications use trace enrichment as an integral part of the procedure.
A. Wastewater Sample
8. Wastewater Spiked to 4 ppm PCP
/,
i
I
Shown above is an analysis of the wastewater sample, and wastewater spiked to 4 ppm PCP. As shown, PCP is present in the wastewater sample at less than 1 ppm.
wastewater samples for trace levels of organics and simultaneously collect fractions for subsequent testing. This H P L C method overcomes traditional drawbacks such as tedious extraction routines, multiple analyses and poor recoveries. H P L C was used to monitor highly complex wastewater from a specialty chemicals manufacturer. The manufacturer was interested in determining the organic content of the influent water (before use in a manufacturing process) and comparing this to the organic content of the plant effluent (after use) and the effluent after a treatment process. T h e information used for this comparison was obtained by simply checking the chromato-
graphic profiles of the different water samples. Figure 5 shows the characteristic profile of the influent water. The trace level organics, which are normally difficult to isolate, were concentrated by trace enrichment. Figure 6 is the chromatogram of the organics present in the plant effluent after use. The chromatographic profile was arbitrarily divided into four sections, based on the polarity of the organics, to simplify determinations of the efficiency of the treatment process. A comparison (Figure 7 vs. 6) of the effluent profile from the treatment plant indicates that the majority of the organic content was removed by treatment, as shown in Figure 6. However, there is a relatively large
Monitoring industrial wastewater H P L C offers sufficient capability to screen a water sample for total organic components or to allow isolation and collection of a single component for subsequent identification or toxicological testing. Environmental applications chemists at Waters Associates, Inc. have developed a rapid, multiresidue method to screen industrial Volume 13, Number 6, June 1979
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FIGURE 5
Effluent from a manufacturing process (influent to a waste treatment plant)
increase in the amount of polar organics (polarity section 1) present in the effluent from the treatment plant compared to the influent. This is possibly due to the treatment process. A 2 mL plant effluent sample of the collected fraction (Figure 7) was then rechromatographed under different chromatographic conditions to separate a number of individual components. Any of these could be easily collected for further toxicity testing or identification by G C / M S .
w
u
.-alC
II
I
Ill
IV
This trace-enriched sample shows the organics in the water prior to the treatment process. The polar organics elute first followed by organics of continuously decreasing polarity.
FIGURE 6
Effluent from a waste treatment plant
1
0
.-alC
UV at 0.32 AUFS
I
A 1 ml trace-enriched sample was injected into the liquid chromatograph. Comparison of the influent and effluent shows that most of the organics were removed during the treatment process. However, polar organics still remain even after treatment. (Section I)
FIGURE 7
Collection of a plant effluent fraction
Frac-
tion
0.32 AUFS
2.0 ml of another trace-enriched plant effluent sample was injected. Then a specific fraction was collected for subsequent analysis.
Agricultural run-off Run-off water from pesticidetreated agricultural land is a source of pollution. In some areas it is the primary factor in the determination of water quality. Standards for waterquality planning must be established. Therefore, it is very important that all of the organics (pesticide and nonpesticide, polar and non-polar) in the sample are collected, detected, and quantitated. Often this cannot be done with commonly used solvent extraction procedures. Frequently, extraction procedures fail to extract all of the organics. Clean-up procedures frequently result in low recoveries. Multi-residue, multi-class analysis of organics in water is common in agricultural screening. T o help meet the demands of this screening, Waters Associates e nv i ro n menta I c he m is t s have developed a liquid chromatography procedure which: allows easy on-site collection and concentration of organics in the water sample using the trace-enrichment technique eliminates time-consuming extraction, clean-up and derivatization procedures screens multi-class organic residues in a water sample
-
I
4
0
.-a) -c
12 9,8
5' 1 6
19,20
10
7 254 nm
I
1 '?\ 14
allows easj collection of sample fractions for subsequent analysis. The method developed by Waters' chemists allows the collection and concentration of virtually all but the most water-soluble organics via the trace-enrichment process. Thermally labile compounds are easily analyzed without fear o f decomposition because LC is an ambient temperature technique. Because of the ability to vary solvent strength over a wide range, both polar and non-polar organics can be analjzed in the same run. Figure 8 illustrates a chromatogram resulting from the H P L C method for a sample of agricultural run-off water. The sample was spiked with 20 ppb for each of 18 pesticides. These pesticides were o f four different classes: organophosphates, triazine herbicides, carbamates and chlorphenoxy acids (and esters). After spiking, 100 mL of the sample was trace enriched on a SEP-PAK cartridge and the pesticides
18
I
11 12 13 14 15 16
Propham Propazine 2,4,5, T Silvex 2,4, D Methyl ester Parathion/ Methyl Parathion 17 Malathion 18 Methoxychlor
eluted with 1 mL of acetonitrile (100 x concentration factor). H P L C can be effectively used to monitor water samples for organic pollutants from both point and nonpoint agricultural run-off sources. H P L C offers a rapid, trace-level method which supports federal goals for establishment of water quality criteria. The H PLC technique provides the unique capability to collect a fraction, or individual components. for subsequent toxicological testing or mass spectral identification. H P L C is a technique which has grown in popularity among environmental scientists because of its versatility, sensitivity, speed, accuracy and economy. Its practicality and ease of use will continue its growth in the future.
International activity: earthNatching GEMS, Canadian and Wexican monitoring global atrnospheric monitoring and more
U.S. activity: NPDES permits air monitoring equivalency regulations oil spill control environmental labs and air pollution models
Business: associations and organizations Air: instrumentation Philips SO2 monitor continuous stack monitoring, automotive emissions testing 3rganic vapors environmental stress plume opacity air quality at power plants burning solid wastes and more Water: instrumentation carbon analyzers chemical-sensing electrodes, sampling standards GC and MS pyrographic analysis wastewater treatment water purity aerial photos to monitor algae and more Miscellaneous: minicomputers. pesticide residues, and listings of 43 related books and 38 news leads 197 pages (1976) LC 76-54966 ISBN 0-8412-0346-6 Hardback $13 50 ISBN 0-8412-0295-8 Paperback $8 50
Michael Lynch ( I ) i.c the Technical Enrironniental Writer re.spon.sible for w i r i n g coniprehensice technical support literature for custotwrs and the Waters sales force. H e is also responsible f o r collecting and comniunic'ating practicnl information ahorrt specific enrironmrnral and industrial health applications.
Ed Weiner ( r ) is the leader of the En1.ironniental Group at Waters Associates and has o w r n l l responsibility f o r the derelopnient of H P L C f o r encironntetital and industrial h,rgiene applications.
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