In the Laboratory
An Environmentally Significant Experiment Using GC/MS and GC Retention Indices in an Undergraduate Analytical Laboratory Rebecca Guisto-Norkus, Giv Gounili, Peter Wisniecki, John A. Hubball, S. Ruven Smith, and James D. Stuart* Department of Chemistry, U-60, University of Connecticut, Storrs, CT 06269-4060 A laboratory experiment is described in which a rapid solvent extraction followed by GC/MS is used to separate and analyze a number of plasticizers that have become serious environmental problems. Plastics have numerous industrial applications ranging from food packaging to automobile chassis. Their practical uses, however, generally require the incorporation of various additives such as plasticizers, antioxidants, and slip agents. To fulfill the requirements of our society, plasticcontaining products are manufactured by the hundreds of tons annually. Compounds used as plasticizers or modifiers may leach out of these products. These compounds are generally not biodegradable, and as a result their concentrations continue to increase in the environment, including living tissue (1–3). There is evidence that bis(2-ethylhexyl)adipate, one common plasticizer, is carcinogenic (4) and that dibutyl adipate (DBA) and bis(2ethylhexyl)phthalate (DEHP), two other common plasticizers, affect the reproduction of aquatic organisms (5). In this experiment the additives are extracted from plastic food wrap, Tygon® tubing, and chopped-up plastic soda bottles or any other plastic material, using either methylene chloride or methanol. Tygon® tubing is a low-density polymer that contains a high proportion of plasticizers and guarantees results, and plastic soda bottles and plastic food wrap are “real-world” samples that everyone has encountered. The analyses of three of the above examples were carried out in our laboratory with a Hewlett–Packard GC-MSD (gas chromatograph–mass selective detector). The mixture of additives is separated by temperatureprogrammed gas chromatography. Primary identification of the individual components is accomplished by the computerized library that is part of the mass spectrometer software. Since a number of these esters have very similar mass spectra or are not present in the computer library, manual library searches (6, 7) and chromatographic retention indices are valuable adjuncts in the identification of the plasticizers. After identification, the plasticizer(s) could be quantified in a Selective Ion Monitoring (SIM) mode. Procedure
Sample Preparation Three different plastic samples were used: (i) plas-
*Corresponding author. The present address of RGN is Cytec Inc., P. O. Box 60, 1937 W. Main St., Stamford, CT 06904-0060. The address of JAH is Connecticut State Police Forensic Laboratory, 294 Colony St., Meriden, CT 06450. SRS and JDS are members of the Institute of Materials Science, University of Connecticut.
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tic food-wrap stretch film, (ii) chopped soda bottles from a recycling machine, and (iii) unused Tygon® tubing. Plasticizers were obtained from the samples by extraction with organic solvents: (i) methylene chloride (Fisher D 143-1), and (ii) methanol (Fisher A 452SK-4). Between 5 and 10 g of each sample was placed in a 250mL container and 150 mL of solvent was added. The container was covered with aluminum foil and the sample was allowed to soak in the solvent for 48 h at room temperature. During this period the flask was swirled several times. Methanol Samples A 50-mL aliquot of methanol was removed from the extraction flask and passed through a Millex® -GV 0.22 µm filter to remove particulate material. The volume was reduced to about 3 mL by rotoevaporation and further reduced to about 1 mL with zero grade nitrogen. The volume reduction required about 30 min. Methylene Chloride Samples A 50-mL aliquot of methylene chloride was removed from the extraction flask and passed through a Millipore Millex®-LCR 0.5 µm filter (a modified hydrophilic Teflon® filtering medium compatible with methylene chloride) to remove particulate material. This extract was reduced to 1 mL either by rotoevaporation and subsequent zero grade nitrogen blowdown, by nitrogen blowdown alone, or by allowing the sample to stand in a fume hood overnight.
Gas Chromatographic Standards Two standard solutions were used in this experiment: • A solution (1 mg/mL) of n-alkanes ranging in length from 9 to 30 carbon atoms, C-9 to C-26, C-28, and C-30 (#90967, Absolute Standards, New Haven, CT). The elution temperatures were used to calculate the temperature-programmed retention indices of the unknowns in the extracts from the plastics. (See the appendix for a sample calculation.) • A solution containing 5 mg/mL of the common adipate and phthalate plasticizers dibutyl adipate (Aldrich 30,949-4), bis(2-ethylhexyl) adipate (Aldrich 29,118-8), bis(2-ethylhexyl) phthalate (Aldrich D20,115-4), dibutyl phthalate (Aldrich 24,047-8), and also tributyl O-acetylcitrate (Aldrich 38,837-8) and erucamide (Aldrich 28,057-7). The chromatogram of the standard mix of plasticizers is shown in Figure 1. The mass spectra and retention times obtained from this sample were used to assist in the identification of the unknown esters in the samples.
Gas Chromatography/Mass Spectrometry
Journal of Chemical Education • Vol. 73 No. 12 December 1996
In the Laboratory
Table 1. Elution Times, Elution Temperatures, and Retention Indices of Standard Additives Standard
Elution Time Elution Temp. Retention Index (min) (°C) (Ipr)
Dibutyl adipate
11.151
191.51
Dibutyl phthalate
13.148
211.48
1735 1920
Tributyl o-acetyl citrate
16.067
240.67
2224
Bis(2-ethylhexyl) adipate
17.383
253.83
2374
Bis(2-ethylhexyl) phthalate
18.506
265.05
2511
Erucamide
20.262
282.62
2740
Figure 1. Mass chromatogram of six-component standard solution.
The GC/MS instrumentation used was a HewlettPackard Model HP 5890 gas chromatograph interfaced to an HP 5970 mass selective detector. The GC column was a methyl siloxane bonded capillary column, 12 m long with an 0.20 mm i.d. and a 0.33-µm film thickness (HP-1, the column provided as the checkout column for the MSD system by Hewlett–Packard). Samples were run under the following conditions: Solvent delay 2.0 min Initial temperature 100 °C Rate 10 °C/min Final Temperature 300 °C Run time 22.0 min
ing at 18.51 min matched that of bis(2-ethylhexyl)phthalate and the identification was accepted on the basis of these two facts. The computer library search identified the second peak, which eluted at 16.07 min, as 1,2,3-propane tricarboxylic acid-2-acetyloxy tributyl ester (common name tributyl O-acetylcitrate) with 60% “quality fit”. We confirmed this marginal identification by running an authentic sample of tributyl Oacetylcitrate on our GC/MS. The authentic sample had the same mass spectrum and temperature-programmed retention index as the peak eluting at 16.07 min and the identification was considered confirmed. The presence of tributyl O-citrate was confirmed by the manufacturer.
Results and Treatment of Data
Tygon® Tubing The gas chromatograms of the methanol and methylene chloride extracts from the Tygon® tubing gave only one major peak. On the basis of temperature-programmed retention times and mass spectral comparisons this component was identified as bis(2-ethylhexyl) phthalate.
The elution temperatures of the temperature-programmed runs of the n-alkane standards were used to determine the temperature-programmed retention indices of the phthalate and adipate standards and other additives extracted from the plastics. The retention indices were calculated by the following equation (8). I pr = 100(T x – Tz) / (T z+1 – Tz) + 100z where Ipr Tx Tz Tz+1 z
is the temperature-programmed retention index of the component of interest is the elution temperature of the component of interest is the elution temperature of the alkane eluting just before the component of interest is the elution temperature of the alkane eluting just after the component of interest is the number of C atoms in the n-alkane standard eluting just prior to the compound of interest
The retention times, elution temperatures and temperature programmed retention indices of our standard mix of additives are listed in Table 1. Identification of Plasticizers
Plastic Food Wrap (Saran® Wrap) Both methanol and methylene chloride extracts from Saran® Wrap gave identical chromatograms, which contained two peaks. The mass spectra of these peaks were identical in the two cases. The mass spectrum and temperature-programmed retention index of the peak elut-
Crushed Plastic Soda Bottles The extract with methanol was red and the extract with methylene chloride was yellow. The gas chromatogram of each solution gave only one major peak and several minor peaks. The peak from the methanol extract eluted at 18.51 min and the peak from the methylene chloride eluted at 20.26 min. The peak at 18.51 min was identified as bis(2-ethylhexyl)phthalate on the basis of its mass spectrum and retention time. Initially, no satisfactory identification was made on the peak from the methylene chloride extract at 20.26 min. The mass spectrum of this peak did not contain the peaks characteristic of either adipates (m/e = 129) or phthalates (m/e = 149); rather there was a straight-chain, alkane-like fragmentation pattern between m/e = 55 and m/e = 130 and a single peak at m/e = 337. Computer and manual library searches produced no satisfactory result. The closest match was a computer match with erucamide, but the quality match was only 47%. However, comparison of the mass spectrum and retention time of the peak matched those of an authentic sample of erucamide. Various handbooks of polymeric materials note that erucamide is often added as a slip agent in plastic products.
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In the Laboratory
Conclusions This experiment takes about three 3-hour laboratory periods. One period is for the extraction steps, the second for the GC/MS training and instrument instruction, and the third for making the GC/MS runs. This experiment has given students experience in extracting and analyzing the additives from a number of everyday plastic samples and can be expanded or modified to identify the extracts from students’ favorite “environmental problems”. In earlier work, we had quantitated adipate and phthalate esters from drinking water and groundwater samples by EPA Method 525.1 (9). We obtained positive results by this approach, but an additional two days of work were needed for the extractions. We concluded that the time and amount of work required made this procedure impractical for an undergraduate laboratory. Therefore methanol and methylene chloride were chosen as solvents in a batch extraction procedure. The quantitative approach could be used for a more advanced laboratory or a student project. Particulate matter has been found in the extracts with both solvents. The filtration step is necessary to prevent plugging of the syringes and capillary columns. Whereas almost any filter would be adequate to filter the methanol extracts, special organic-resistant filters (Millex®-LCR or equivalent) with a fluoropore filtering membrane and appropriate housing must be used for the methylene chloride extracts. If there is an aversion to using methylene chloride, the experiment can be performed with methanol as the extraction solvent. The trade-off is the extra time in volume reduction.
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Literature Cited 1. Andelman, J. B.; Wachter, J. K.; Nolle, S.; Beck, J. M. Water Res. 1984, 18, 843. 2. Felder, J. D.; Adams, W. J.; Saeger, V. W. Environ. Toxicol. 1986, 5, 777. 3. Ogino, Y.; Saito, N.; Nagao, M. Okaya ma-ken Kankyo Hoken Senta Nempo 1979, 3, 167. 4. Hsia, M. T. S. NIST Report, 1990, Order No. PB90-21567; U. S. Department of Commerce, Springfield, VA. 5. Sanders, H. O.; Mayer, F. L., Jr.; Walsh, D. F. Environ. Res. 1973, 6, 84. 6. Eight Peak Index of Mass Spectra, 3rd ed.; Mass Spectrometry Data Centre, Royal Society of Chemistry; The University, Nottingham, NG7 2RD, UK, 1983. 7. Heller, S. R.; Milne, G. W. A. EPA/NIH Mass Spectral Data Base; National Standard Reference System, NBS Publication 63, Vol. 1–4; U. S. Government Printing Office: Washington, DC, 1978. 8. Poole, C. F.; Poole, S. K. Chromatography Today; Elsevier: New York, 1991; p 179. 9. Method 525.1 Determination of Organic Compounds in Drinking Water by Liquid–Solid Extraction and Capillary Column Gas Chromatography/Mass Spectrometry; U. S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH, May 1991.
Appendix. Calculation of Elution Temperatures and Retention Indices Example: Dibutyl adipate Elution time from chromatogram: 11.151 min Elution time after onset of temperature program ramp: 11.151 min – 2.000 min = 9.151 min Elution temperature (Tx): 100 °C + 9.151 min × 10 °C/min = 191.51 °C Elution temperature (Tz) of C17 (z) alkane: 187.60 °C Elution temperature (Tz+1) of C18 (z+1) alkane: 198.79 °C (C17 and C18 are the alkanes that elute immediately before and after dibutyl phthalate.) I pr = 100 × (191.51 – 187.60) + (100 × 17) = 1735 (198.79 – 187.60)
Journal of Chemical Education • Vol. 73 No. 12 December 1996
