In the Laboratory
The Separation and Identification of Some Brominated and Chlorinated Compounds by GC/MS An Advanced Undergraduate Laboratory Rebecca M. O’Malley* and Hsiao C. Lin Chemistry Department, University of South Florida, Tampa, FL 33620-5250
The coupling of gas chromatography (GC) and mass spectrometry (MS) has resulted in the extremely powerful technique of GG/MS, which is used extensively in research laboratories, particularly in synthetic organic chemistry and the development of pharmaceuticals. It is also used for the identification and quantification of trace amounts of environmental contaminants. A GC/MS method is used for the urinalysis screening carried out on Olympic and other athletes to check for drug use. For many years GC/MS techniques were too expensive and complicated to be used by undergraduates, except perhaps for individual research projects. However, with the development of reasonable-cost, computer-controlled GC/MS systems it has become feasible to introduce GC/MS methodology into the undergraduate curriculum. A number of articles published in this Journal describe GC/MS experiments designed for use in undergraduate teaching laboratories (1–14). These include experiments in which GC/MS is used to identify individual substances, mixtures, or reaction products (1–9), or to follow the kinetics of certain reactions (10–12). One experiment allows the identification of amino acids in a small peptide (13) and another describes the extraction and identification of plasticizers from a number of different polymers (14). This article describes a laboratory experiment that was developed for advanced undergraduate students. In it the students are provided with a mixture of unknown brominated and chlorinated compounds. They separate the compounds and obtain mass spectra of the individual components in the mixture using a GC/MS method. Armed with the information that each of the unknowns contains at least one and possibly as many as four chlorine or bromine atoms, the students interpret the mass spectral data to obtain the identity of the unknowns. The interpretation of the mass spectra (identification of molecular ions and major fragment ions) is simplified by the presence of the varying numbers of chlorine and bromine atoms, which produce patterns of peaks in the mass spectra. The relative intensities of the peaks within the patterns are characteristic of the number of chlorine or bromine atoms. Experimental Section
Chemicals The unknown compounds used in the experiment are listed in the table. Tetrachloroethylene was obtained from NMR Specialties.1 The solvent was GC Resolv grade hexane and was obtained from Fisher.2 The rest of the chemicals were obtained from Aldrich.3 CAUTION: All procedures with the chemicals—making up solutions, dilutions, etc.—should be carried out in an efficient fume hood. Students should wear protective lab coats, goggles, and gloves.
NOTE: We have used α,α ,2-trichloro-6-fluorotoluene, which is listed by Aldrich as a lachrymator. In this experiment the solutions used by the students are prepared by the instructor, and only one or two drops of each substance are required. The resulting solution is then diluted 1000 times, so we have felt quite safe using the α , α ,2-trichloro-6fluorotoluene and have had no problems. However, as the unknowns given to the students contain a random sampling of five of the eight unknowns listed, this compound could easily be omitted without detriment to the experiment.
Instrumental Conditions There are many bench-top GC/MS instruments that could be used for this experiment. All the GC and MS data reported here were acquired using a Finnigan4 GCQ, which has a NIST mass spectral database (library) stored in the computer. The GC column in this instrument is a DB-5 coated capillary column 30 m long, 0.25 mm i.d., 0.25 µm film.5 GC Method For all of these experiments the following GC program was used: 40 to 70 °C at 10°/min, 70 to 100 °C at 5°/min, and 100 to 180 °C at 10°/min. GC run time was 18 min. The transfer line temperature between the GC and MS was set at 270 °C, the MS source temperature was set at 170 °C, and the splitless injection mode was used. MS Method The instrument was set to operate in the positive ion mode with electron impact as the ionization method. The mass range was scanned from 50 to 250 daltons (Da) with a 3.00-min solvent delay. Experimental Procedure The students are given an unknown solution, which contains four or five of the eight compounds listed in the table. A suitable concentration is obtained by using a dropper to add one or two drops of each compound (approximately 50– Unknowns Used in the Experiment Compound
bp/°C
MW/ (g mol {1)
Tetrachloroethylene
121
165.83
1,2-Dibromoethane
131
187.87
1,1,3-Trichloropropene
131
145.42
1,1,1,2-Tetrachloroethane
138
167.85
1,3-Dibromopropane
167
201.9
1,2,3-Tribromopropane
220
280.80
1,2-Dibromobenzene
224
235.93
α,α,2-Trichloro-6-fluorotoluene
228
213.47
JChemEd.chem.wisc.edu • Vol. 76 No. 11 November 1999 • Journal of Chemical Education
1547
In the Laboratory
100 µ L) to 10 mL of hexane and then diluting the obtained solution 1:1000 with hexane. The dilution is achieved by adding 1.0 µ L of the solution to 1.0 mL of hexane. (The 1.0 µL is measured using a 10-µ L syringe). One microliter of the final solution is injected into the GC/MS system. (NOTE: To prevent cross-contamination of samples the students are told to rinse the 10-µL syringe five times with solvent both before and after sample injection). The students obtain a chromatogram that shows the separation of the unknowns and they print out a mass spectrum for each of the unknown compounds in their mixture.
Results Separation and Identification of Mixture Components Figure 1 shows a total ion current chromatogram in which all eight of the chlorinated and brominated compounds are separated. The compounds elute from the GC column roughly in order of increasing boiling point. The students obtain and print out a mass spectrum corresponding to each of the peaks in their particular chromatogram. Figure 2 shows the mass spectra of two representative compounds, tetrachloroethylene and 1,2-dibromobenzene.
Interpretation of Mass Spectral Data
Calculation of Expected Relative Intensities in the Ion Patterns
The students are asked to calculate the expected relative intensities for the peaks within patterns due to ions containing (i) between one and four chlorine atoms and (ii) between one and four bromine atoms. Comparison of these relative intensities with their experimentally obtained data, coupled with the information that the unknowns contain only either all chlorine atoms or all bromine atoms, makes identification of the ions in the spectra relatively simple. After the ions are identified the students are asked to write down the molecular ion and the most important fragmentation reactions. From this information they can draw conclusions regarding the identity of the unknowns in most cases. If the GC/MS equipment has a mass spectral database (library) associated with it they can use the library for confirmation of their proposed structures. For three of the unknowns, as explained below, structure identification is not quite so simple because the unknowns do not show molecular ions in the mass spectra. In these cases access to a mass spectral database is most helpful and allows simple identification of the compounds.
Chlorine occurs naturally as 35Cl (75.77%) and 37Cl (24.23%), while bromine occurs naturally as 79Br (50.69%) and 81Br (49.31%) (15). If an ion contains one chlorine or bromine atom, this results in the appearance of two peaks in the mass spectrum, two mass units apart. In the case of chlorine the relative intensities of the two peaks will be approximately 3:1; in the case of bromine the relative intensities will be approximately 1:1. If more than one chlorine or bromine atom is contained in an ion then more than two peaks result in the mass spectrum and calculating the relative intensities of the resulting peaks is not quite so intuitive. This is actually a probability– combination problem. So in the situation where there are two Cl atoms in an ion we have to start thinking in terms of the statistical probability of selecting two 35Cl atoms from a large number of atoms of which 75.77% are 35Cl and 24.23% are 37Cl. This probability has to be compared with the relative probability of selecting one 35Cl atom in combination with
a
b
Figure 1. Total ion chromatogram of all eight compounds: (1) tetrachloroethylene; (2) 1,2-dibromoethane; (3) 1,1,1,2-tetrachloroethane; (4) 1,1,3-trichloropropene; (5) 1,3-dibromopropane; (6) 1,2,3-tribromopropane; (7) 1,2-dibromobenzene; (8) α,α,2-trichloro-6fluorotoluene.
1548
Figure 2. (a) Mass spectrum of tetrachloroethylene; (b) mass spectrum of 1, 2-dibromobenzene.
Journal of Chemical Education • Vol. 76 No. 11 November 1999 • JChemEd.chem.wisc.edu
In the Laboratory
one 37Cl atom, and the relative probability of selecting two 37 Cl atoms, all from the same atom population. The expected intensities of the peaks arising from the possible combinations of isotopes can be calculated using the binomial expansion (16 ): (a + b)n = an + na(n–1)b + n(n – 1)a(n–2)b 2/2! + n(n – 1)(n – 2)a(n–3)b3/3! + … where a and b are the fractional abundances of the two isotopes and n is the number of isotopic atoms in a particular ion. A simple way to deal with this equation is to take the intensity of the first isotope to be equal to 1.00 (i.e., for Cl take 35Cl/ 37Cl to be 1.00/x and for bromine take 79Br/ 81Br to be 1.00/x). Then the relative intensities of the peaks in the clusters are: 1:x (ions containing 1 Cl or 1 Br atom) 1:2x:x2 (ions containing 2 Cl or 2 Br atoms) 1:3x:3x2:x3 (ions containing 3 Cl or 3 Br atoms) 1:4x:6x2:4x3:x4 (ions containing 4 Cl or 4 Br atoms) The expected relative intensities for clusters arising for ions containing 1, 2, 3, and 4 chlorine or bromine atoms are plotted in Figure 3. These intensities were calculated by using the values 1.00:0.32 for Cl and 1.00:0.97 for Br and substituting in the above expressions.
Interpretation of Mass Spectra The interpretation of the mass spectra of this type of compound is relatively simple (17). Initially a molecular ion is formed by electron impact: M + e{ → M +? + 2e{ This molecular ion can decompose by a simple bond-cleavage reaction to produce a fragment ion, F1+, and a radical R? (which will be Cl? or Br?): M+? → F1+ + R? Sometimes fragmentation reactions occur in which even-
electron species are eliminated, for example M+? → F2+? + HCl (or HBr) Fragment ions are capable of decomposing to produce further fragment ions in similar reactions: F2+? → F3+ + R? where R? is Cl? or Br?. In some compounds fragmentation reactions proceed so rapidly that they dominate the reaction scheme and no molecular ion is observable. Comparing the relative intensities of the peaks centered at m/z = 166 in the spectrum in Figure 2a with the possibilities shown in Figure 3, we can deduce that this cluster is due to an ion containing four Cl atoms. Likewise, the cluster centered at m/z = 129 is due to an ion containing three Cl atoms and that centered at m/z = 94 is due to an ion containing two Cl atoms. By calculating the mass of four 35Cl atoms (140 Da) and subtracting this from 164 Da (the mass of the ion containing all 35Cl atoms) we get 24 Da, which corresponds to two C atoms. Assuming that this cluster of ions is due to the molecular ion, the formula of the unknown is C2Cl4 (tetrachloroethylene). The following reactions can be written to represent the formation of the molecular ion plus the fragment ions for tetrachloroethylene. Molecular Ion Formation. Loss of an electron: C2Cl4 + e { → C2Cl4+? + 2e { where C2Cl4+? can be C235Cl4+? (m/z = 164) or C235Cl337Cl+? (m/z = 166) or C235Cl237Cl2+? (m/z = 168) or C235Cl37Cl3+? (m/z = 170) or C237Cl4+? (m/z = 172). Fragmentation. Simple bond cleavage involving loss of one Cl atom: C2Cl4+? → C2Cl3+ + Cl? where C2Cl3+ can be C235Cl3+ (m/z = 129) or C235Cl237Cl+ (m/z = 131) or C235Cl37Cl2+ (m/z = 133) or C237Cl3+ (m/z = 135). The small cluster of ions with the most intense peak at m/z = 94 due to an ion containing two Cl atoms is also produced in a simple cleavage reaction: C235Cl3+ → C2Cl 2+? + Cl? where C2Cl2+? can be C235Cl2+? (m/z = 94) or C 235Cl37Cl+? (m/z = 96) or C237Cl2+? (m/z = 98). Looking at Figure 2b and comparing the intensities of the clusters of peaks centered at m/z = 236 and m/z = 157 with the cluster intensities shown in Figure 3, it is apparent that these two clusters correspond to ions containing two and one bromine atoms, respectively. Subtracting the mass of two 79 Br atoms (158 Da) from 234 Da (the mass of the ion containing the two 79Br atoms) leaves 76 Da, which corresponds to C6H4, giving a molecular formula of C6H 4Br2. Again molecular ion formation and fragmentation reactions can be written. Molecular Ion Formation: C6H4Br2 + e { → C6H4Br2+? + 2e{ where C6H 4Br2+? can be C6H 479Br79Br +? (m/z = 234) or C6H479Br81Br+? (m/z = 236) or C6H481Br81Br+? (m/z = 238). Fragmentation: C6H4Br2+? → C6H4Br+ + Br?
Figure 3. Calculated relative intensities for peaks in ion clusters containing one, two, three, and four Cl or Br atoms.
where C6H4Br+ can be C6H479Br+ (m/z = 155) or C6H481Br+ (m/z = 157).
JChemEd.chem.wisc.edu • Vol. 76 No. 11 November 1999 • Journal of Chemical Education
1549
In the Laboratory
The interpretation of the mass spectra of 1,3-dibromopropane, 1,1,3-trichloropropene, and α ,α ,2-trichloro-6fluorotoluene is similarly straightforward, all spectra showing molecular ions and simple fragmentation reactions. For the remaining three compounds, 1,2-dibromoethane, 1,2,3-tribromopropane, and 1,1,1,2-tetrachloroethane, interpretation is more complex because no molecular ions are observed. In electron impact mass spectrometry of all three of these compounds the neutral molecule/molecular ion undergoes a rapid decomposition involving the loss of a Br atom or a Cl atom. For 1,2-dibromoethane: Fragmentation: C2H4Br2 + e{ → C2H 4Br++ Br? + 2e{ where C2H 4Br+ can be C2H479B+ (m/z = 107) or C2H481Br+ (m/z = 109). For 1,2,3-tribromopropane: Fragmentation: C3H 5Br3 + e{ → C3H5Br2+ + Br? + 2e{ where C 3H 5Br 2+ can be C 3H 579Br79Br+ (m/z = 199) or C3H579Br81Br+ (m/z = 201) or C3H581Br81Br+ (m/z = 203), and C3H 5Br2+ → C3H4Br+ + HBr where C3H4Br+ can be C3H479Br+ (m/z = 119) or C3H479Br+ (m/z = 121). For 1,1,1,2-tetrachloroethane: Fragmentation: C2H2Cl4 + e{ → C 2H 2Cl3+ + Cl? + 2e{ where C 2 H 2 Cl 3 + can be C 2H 2 35 Cl 3+ (m/z = 131) or C2H235Cl237Cl+ (m/z = 133) or C2H 235Cl37Cl2+ (m/z = 135) or C2H 237Cl3+ (m/z = 137), and C2H2Cl4 + e{ → CCl3+ + CH2Cl? + 2e{ where CCl 3+ can be C35Cl 3+ (m/z = 117) or C35Cl 237Cl + (m/z = 119) or C35Cl37Cl2+ (m/z = 121) or C37Cl3+ (m/z = 123), and C2H2Cl3+ → C2HCl 2+ + HCl where C2HCl2+ can be C2H35Cl2+ (m/z = 95) or C2H35Cl37Cl+ (m/z = 97) or C2H37Cl2+ (m/z = 99). Most students can make good guesses at the identity of these compounds. However, access to a mass spectral library is most helpful at this stage in the experiment. Comparison of experimentally obtained data with a database is in fact how an investigation of this sort would be carried out in an industrial setting. It should be noted that it is not possible from mass spectral data alone to draw conclusions concerning the exact isomeric structure of the unknowns. For example, in the case of dibromobenzene the students are not able, on the basis of their experimental data, to decide that the unknown is 1,2-dibromobenzene as opposed to another dibromobenzene. Again a mass spectral database can be helpful in this situation and the students can investigate how alike or different the mass spectra of the different possible isomers are. In some situations they find this is useful. However, they will probably find that use of the database has some limitations. For example, the mass spectra for 1,1,3-trichloropropene, 1,2,3-trichloro-
1550
propene, and 1,1,2-trichloropropene are so similar that it is not possible to distinguish between these compounds on the basis of mass spectrometry. Conclusions Students are able to complete this experiment in two 3-hour lab sessions. In separating the components of the mixture and identifying them, the students get experience in the technique of GC/MS. Mass spectrometry is one of the few analytical techniques that are sensitive to different isotopes of elements, so these compounds, which all contain more than one atom of an isotopic element, give the students an appreciation of the fact that isotopes of an element really do differ in mass. The interpretation of the mass spectra in terms of identification of the molecular ion and the fragment ions gives them an understanding of the processes that take place in a mass spectrometer and lead to the recorded mass spectrum. Use of a mass spectral database allows them to discover the uses and limitations of this tool. Acknowledgments We wish to thank the National Science Foundation ILI program (Award number 9550877) and the University of South Florida (Chemistry Department, College of Arts and Sciences, and the Division of Sponsored Research) for providing funds for the purchase of the GC/MS system. We also wish to thank Finnigan Corporation for technical help and support. Notes 1. NMR Specialties, 1410 Greensburgh Road, New Kensington, PA 15058. 2. Fisher Scientific Co., 711 Forbes Avenue, Pittsburgh, PA 15219-1785. 3. Aldrich Chemical Co., 1001, West Saint Paul Avenue, Milwaukee, WI 53233. 4. Finnigan is a subsidiary of Thermoquest Corporation, 450 Franklin Road, Suite 130, Marietta, GA 30067. 5. J&W Scientific Inc., 91 Blue Ravine Road, Folsom, CA 956304714.
Literature Cited 1. McGoran, E. C.; Melton, C.; Taitch, D. J. Chem. Educ. 1996, 73, 88. 2. Amenta, D. S.; DeVore, T. C.; Gallaher, T. N.; Zook, C. M. J. Chem. Educ. 1996, 73, 572. 3. Kostecka, K. S.; Lerman, Z. M.; Angelos, S. A. J. Chem. Educ. 1996, 73, 565. 4. Rowland, A. T. J. Chem. Educ. 1995, 72, A160. 5. Kostecka, K. S.; Rabal, A.; Palmer, C. F. J. Chem. Educ. 1995, 72, 853. 6. Novack, M.; Heinrich, J.; Martin, K. A.; Green, J.; Lytle, S. J. Chem. Educ. 1993, 70, A103 7. Novack, M.; Heinrich, J. J. Chem. Educ. 1993, 70, A150. 8. Asleson, G.; Doig, M. T.; Heldrich, F. J. J. Chem. Educ. 1993, 70, A150. 9. Holdsworth, D.; Ching, G. S.; Hamid, M. J. J. Chem. Educ, 1992, 69, 856. 10. Annis, D. A.; Collard, D. M.; Bottomly, L. A. J. Chem. Educ. 1995, 72, 460. 11. Bishop, R. D. J. Chem. Educ. 1995, 72, 743.
Journal of Chemical Education • Vol. 76 No. 11 November 1999 • JChemEd.chem.wisc.edu
In the Laboratory 12. Brush, R. C.; Rice, G. W. J. Chem. Educ. 1994, 72, A293. 13. Hamann, C. S.; Myers, D. P.; Rittle, K. J.; Wirth, E. F.; Moe, O. A. Jr. J. Chem. Educ. 1991, 68, 438. 14. Guisto-Norkus, R.; Gounili, G.; Wisnieki, P.; Hubball. J. A.; Smith, R. S.; Stuart, J. D. J. Chem. Educ. 1996, 73, 1176.
15. CRC Handbook of Chemistry and Physics, 74th ed.; Lide, D. R., Ed.; CRC: Boca Raton, FL, 1993–94; pp 1-10–1-11. 16. McLafferty, F. W.; Turecek, F. Interpretation of Mass Spectra, 4th ed.; University Science Books: Mill Valley, CA, 1993. 17. O’Malley, R. M. J. Chem. Educ. 1982, 59, 1073.
JChemEd.chem.wisc.edu • Vol. 76 No. 11 November 1999 • Journal of Chemical Education
1551
