v-xEPA United States Environmental Protection Agency Industrial Environmental Research Laboratory Research Triangle Park NC 27711 Research and Development EPA-600/S7-82-033 August 1982 Project Summary Interpretation of Low Resolution Mass Spectra for Level 1 Analysis of Environmental Mixtures James L. Stauffer This report is a set of guidelines for interpreting the low resolution mass spectra (LRMS) of complex chemical mixtures, within the context of EPA's Level 1 Environmental Assessment Program1. It discusses principles underlying direct mass spectrometric analysis of complex mixtures, tech- niques for optimizing the analyses, and interpreting and evaluating the results. A chapter presents some interpretive aids for LRMS analysis of environmental mixtures. The guide- lines are illustrated by a step-by-step detailed analysis of the mass spectra of four representative samples. The final chapter gives direction for reporting the results in the EPA Level 1 LRMS report format. LRMS plays an important role in determining the chemical composi- tion of environmental mixtures. The other components of the Level 1 organic analysis scheme, liquid chromatography (LC) fractionation and infrared analysis (IR), also con- tribute significantly to the overall analysis. The LC and IR procedures are described elsewhere1. This report is limited to a detailed discussion of the LRMS component of the Level 1 scheme. This Project Summary was devel- oped by EPA's Industrial Environ- mental Research Laboratory, Research Triangle Park, NC, to announce key findings of the research project that is fully documented in a separate report of the same title (see Project Report ordering information at back). Summary Requirements for mass spectrometric analysis of the environmental samples obtained in the EPA Level 1 program are for identification of chemical class, and order of magnitude quantitation. These standards are met through the com- bination of complementary analysis methods: (1) a liquid chromatographic (LC) fractionation of the environmental mixture, followed by (2) mass spec- trometric and infrared spectrometric (IR) analysis of each LC fraction. The LC fractionation narrows the range of chemical classes that can be present in any of the individual fractions to a level which is manageable by LRMS; the mass spectrometric analysis of each LC fraction establishes the molecular weight range of the fraction, and the chemical classes present. To eliminate the chemical discrimination effects that are likely to be encountered when gas chromatography is used for LRMS sample introduction, the samples in this program are introduced in the LRMS via the direct probe. This provides the most representative aliquot of the sample to the LRMS source, but also yields spectra that, typically, contain the superimposed spectra of several chem- ical components. A batch inlet is used ------- for samples that are too volatile for successful introduction via the probe inlet. Complementary sources of informa- tion available in the EPA Level 1 program that may be useful in interpret- ing the mass spectra include the IR spectra that are obtained on all of the LC fractions. These support (confirm) the assignments obtained via LRMS, and signal the presence of chemical func- tional groups that may not be recogniz- able solely on the basis of the mass spectra. The LC f ractionation scheme provides an initial polarity separation of the mixture into seven fractions that range from non-polar (fraction 1) to most polar (fraction 7). Table 1 shows the types of separations that are obtained. The analysis results obtained by LRMS are reported primarily as chemi- cal classes and molecular weight ranges of those classes, with subcate- gory or specific compound or composi- tion designation whenever possible. The categories defined for reporting LRMS data are shown in Table 2. Direct Analysis of Mixtures by LRMS The detail that may be obtained from the mass spectrum of multi-component mixtures depends both on the complexity of the spectrum itself, and on the amount of supplemental information that is available. The precision of the identification that may be obtained will vary accordingly, ranging from specific compound or composition assignments for all of the spectrum, to simply an indication of the chemical classes that are present. The task confronting the analyst of the mass spectra of multi- component mixtures is to discover the correct combination of individual spectra that will adequately account for the experimentally observed spectrum. The additive nature of superimposed mass spectra ensures that this is possible, and the multi-peak nature of electron impact mass spectra makes it practical in most cases. The combina- tion of the two aspects ensures that, if the observed mass spectrum is fully accounted for by the combined indi- vidual assignments, then those assign- ments are an accurate indication of the chemical class makeup of the sample. Two principal techniques provide clues to the analyst for tentative individual chemical class or compound Table 1. Representative Checmical class Distribution by LC Fraction* LC Fraction 1. aliphatic hydrocar- bons, sulfur 2. aromatic hydrocar- bons 3. PAH species 4. carbazoles 5. 6. 7. halogenated aliphatics halogenated aromatics aromatic hydrocarbons nitroaromatics carbolic acids phenols sulfonic acids heterocyclic sulfur, PAH heterocyclic N heterocyclic O ketones esters *This list represents the type of partitioning that may be expected to occur, but it should not be taken as definitive. Many conditions could cause compound class distribution to vary from that shown here. The illustrated overlap between fractions similarly is representative, rather than definitive. assignments. The first and most im- portant Off these is the fractional distillation'of the sample that occurs as the direct insertion probe is slowly taken through its complete temperature cycle from cool to hot. The second is the use of both high (70 eV) and low (10 - 2O eV) electron impacts or chemical ionization modes, at or near the same probe temperature. The thermal disti Na- tion provides a separation into suc- cessive molecular weight ranges, and the change of ionization mode differen- tiates between parent and fragment ions. All of the data, taken in combina- tion, provide enough information for overall spectral interpretation. Tentative assignments made on the basis of the above information are confirmed or modified in the confirma- tion phase of the analysis. In the first phase of confirmation, standard spectra obtained either from the literature2"3 or from reference compounds are used to evaluate how completely the experi- mentally observed mass spectrum is accounted for by the combined tentative individual assignments. The second phase is to make a similar comparison against the data obtained in the IR analysis, to see if all fractional groups and IR spectral features have been recognized and accounted for in the mass spectral analysis. An accurate set of assignments will account for all spectral features of both the MS and the IR data. Several interpretation aids can be helpful in analyzing the LRMS data. The first of these is a table of mass numbers and associated Z values, where the Z value is given by the relationship: mW = CnH(2n + Zl A Z value for any ion in the spectrum can be correlated with a limited range of possible chemical classes, and a very limited range of possible chemical compositions. Mass values for PAH species can be correlated to specific chemical compositions and numbers of rings, although not to specific isomers. In most cases, similarily specific chemi- cal composition assignments can be made to individual mass values for aza- arenes, and for oxygen- or sulfur- containing polycyclic species. Several step-by-step examples of analyses are included in the report. The samples used for examples are: (1) A synthetic mixture of polar materials (LC fractions 5, 6, 7). (2) A simple PAH mixture (LC fraction 3). (3) A complex hydrocarbon mixture (without LC chromatographic separation). (4) A set of potentially confusing spectra, obtained from cigarette tar (LC fractions 3 and 4). ------- Table 2. Categories for Reporting LRMS Data Category (Subcategory) Most probable LC fraction* Category (Subcategory} Most probable LC fraction* Aliphatic hydrocarbons (Alkanes) (Alkenes) (Alkynes) Halogenated aliphatics (Saturated) (Unsaturated) Aromatic hydrocarbons (Benzenes} Halogenated aromatic hydrocarbons Nitro aromatic hydrocarbons Fused alternate, nonalternate hydrocarbons MW<216 (methyl pyrene) MW<216 Ethers (Halogenated ethers) Epoxides Aldehydes Heterocyclic oxygen compounds Nitrites (Aliphatic) (Aromatic) Alcohols (Primary, secondary, tertiary) (Glycols) 1 1 1 1 1,2 1,2 1,2 2,3 2,3 2,3 4,5 2.3 2,3 2,3 4 4 4 4 3.4 4 4 4 6 6 6 Phenols (Alkyl, etc.) (Halogenated phenols) (Nitrophenols) Esters (Phthalates) Ketones Amines (Primary, secondary, tertiary) (Hydrazines, azo compounds) (Nitrosoamines) Heterocyclic nitrogen compounds (Indoles, carbazoles) (Quinolines, acridines) Alkyl sulfur compounds (Mercaptans) (Sulfides. disulfides) Heterocyclic sulfur compounds (Benzothiophenes) Sulfonic acids, sulfoxides Amides Carboxylic acids Silicones Phosphates 6 6 6 6 6 6 6 6 6 6 6 4 6 6 6 6 7 6 6,7 2,3,4 5.6.7 *Possible assignments. Fractions 4-5, 5-6, 6-7 generally overlap to a considerable extent. Also, additional components of a particular molecule may cause it to elute in an LC fraction other than that expected. For example, a short-chain ester would probably elute in LC Fraction 5 or 6, whereas a long-chain ester would elute in Fraction 3 or 4. The several examples are designed to illustrate: (1) The establishment of mass spectral peak associations. (2) The use of reference spectra for identification assignments in a complex spectral matrix. (3) The principle of total spectrum accounting. (4) Utilization of the LC fractionation scheme for interpreting LRMS spectra. (5) Chemical class assignments in a complex spectral matrix. The spectra utilized in the analysis examples are then used as reporting examples to illustrate the use of the standard EPA Level 1 Environmental Assessment Program LRMS report form. References 1. Lentzen, D.E., Wagoner, D.E., Estes, E.D., and Gutknecht, W.F., "IERL- RTP Procedures Manual: Level 1 Environmental Assessment (2nd edition)," EPA-600/7-78-201, NTIS PB 293-795.(1978). 2. "Eight Peak Index of Mass Spectra," 4 volumes, 2nd edition, published by Mass Spectrometry Data Centre, AWRE, Aldermaston, Reading, RG7 4PR, United Kingdom, 1974. 3. Heller, S.R., and Milne, G.W.A., "EPA/NIH Mass Spectral Data Base," 5 volumes, U.S. Department of Commerce/National Bureau of Standards, NSRDS-NBS 63 (1978). * U.8.30VEHNMEWTPHIKT1NQOFFICE:HtS-559-017/0787 ------- James L. Stauffer is with Arthur D. Little, Inc.. Cambridge, MA 02140. Larry D. Johnson is the EPA Project Officer (see below). The complete report, entitled "Interpretation of Low Resolution Mass Spectra for Level 1 Analysis of Environmental Mixtures, "(Order No. PB 82-232 455; Cost: $15.00, subject to change) will be available only from: National Technical Information Service 5285 Port Royal Road Springfield, VA 22161 Telephone: 703-487-4650 The EPA Project Officer can be contacted at: Industrial Environmental Research Laboratory U.S. Environmental Protection Agency Research Triangle Park, NC 27711 United States Environmental Protection Agency Center for Environmental Research Information Cincinnati OH 45268 Postage and Fees Paid Environmental Protection Agency EPA 335 Official Business Penalty for Private Use $300 CHICAGO ------- |