Michael L. Gross, Virgil K. Olsen, and R. Ken For& University o f Nebraska-Lincoln Lincoln, Nebraska 68508
Mustrating Gas Chromatography and Mass Spectrometry A n undergraduate experiment
The combined technique of gas chromatography and mass spectrometry (GCIMS) has in recent years provided one of the most powerful analytical techniques for rapid separation and identification of complex mixtures. A recent review with over 700 references shows the current level of active research in this a1ea.l In view of the role the method is playing now in such fields as petroleum, forensics, environment, and medicine, we have developed a pseudo GC/MS experiment for use in our undergraduate junior and senior analytical instrumentation courses. It is our primary objective t o illustrate both the separate POWers of GC (for separation) and M S (for identification) and the complement& nature and tremendous potential of the coupled techniques. At the same time the students gain experience in GC operation and sample collection, and are introduced to some basic principles of M S instrumentation and interpretation. Most institutions, like ourselves, will have separate instruments available for instructional use (an interfaced GC/MS being the exception rather than the rule). In our case, the dual aspects of the experiment are conducted se~aratelvwith Dart of the MS experiment conducted as a grbup dekonstrkion. However, the availability of relatively inex~ensiveMS instrumentation such as the Varian EM-~~OOwill permit the preferred approach of "hands on" M S data collection by the students. Whatever the instrumentation availahle, the basic fundamentals of GC/MS can he illustrated and appreciated.2 The compounds used (isomeric ketones containing six or seven carbons) are ideally suited for this experiment in that they (1) are readily available, (2) dramatically illustrate the capability for separating a group of closely related compounds (a situation commonly encountered in actual GC/MS applications), (3) are readily separated on a "typical" GC column (SE-30 on Chromosorb), (4) permit flexibility and ease in preparing a variety of unknown mixtures for student use, (5) are volatile (permitting MS
operation a t ambient temperatures), (6) produce mass spectra which are both unique and amenable to a logical (yet simple) interpretation scheme. Reference spectra are availahle for many of the ketones allowing a "dry-lab" approach in the M S section if a n instrument is not available. The reference files can he used in a "fingerprint" method of compound identification. In addition t o these advantages, the experimental approach itself is highly flexible and can he conveniently adjusted to meet both the pedagogical goals of the instructor and the needs of the students. The approach described here divides the experiment into three parts requiring two laboratory periods. One lab period (part 1) is used to separate and collect the components of a ketone mixture. A second lab period (part II) is used to explain and demonstrate the MS instrumentation and the interpretation of ketone spectra. In part III, spectra of the separated ketones are analyzed by the individual students. GC Separation of Ketones (Part I )
Of the C B H ~ Z ketone O isomers, all are availahle commercially, and we have found reference spectra3-%n all six isomers (given in part in Table 1).For C1H140 iso1 Junk, G.
A,, Int. J.Mass Speetmm. IonPhys., 8,1, (1972). 2For detailed reviews on GC/MS and computer applications
see: (a) McFadden, W. H., "Techniques of Combined Gas Chmmatography/Mass Spectrometry; Applications in Organic Analysis," Wiley-Interscience, New York, 1973. (b) Williams, D. H., and Howe, I., "Principles of Organic Mass Spectrometry," McGraw-Hill, New York, 1972, pp. 208-242. (c) Roboz, J., "Intmduction to Mass Spectrometry; Instrumentation and Techniques," Wiley-Interscience,New York, 19fi8, pp. 354-373. Stenhagen, E., Abrahamson, S., and McLafferty, F. W., (Editors), "Atlas of Mass Spectral Data," Wiley-Interscience, New York. American Society for Testing and Materials, "Index of Mass Spectral Data," 1919 Race St., Philadelphia, Pa. 19103.
Volume 52. Number 8, August 1975 / 535
Table 1. Partial Mass Spectra of CsH,10and C,H,,O Ketonese No.
m / e = 114
100
99
mers, 12 are available commercially and we have found reference spectra for seven of the 14 isomer^.^-^ Baseline separation is obtained for any combination of two of the six isomeric CB-ketones using a % in. X 5-ft metal column packed with SE-30 on Chromosorh (20% load) a t an operating temperature of approximately 100'C with a helium flow rate of 60-100 mlls. On occasion. mixtures of Cs- and C7-ketones are issued, and, of course, the separation is accorndished more easilv in this event. The separations Dortion-of the experiment can he conducted rabidly hecause the retention times of CB-ketones are on the order of 2-3 min. Thus, the tedium of repeated injections is minimized. As the unknowns are meoared and distributed, each student may he informed o i the number of components. S a m ~ l einjections are made to observe and maximize the sepa;ationand to establish the points a t which collection of each eluted component should he started and ended. At this point, i t is convenient to point out and discuss with each student their role as the GC/MS "interface." The student collects the components by repeatedly injecting several microliters of the mixture and condensing the eluted gaseous components in glass collecting tuhes . ~ find that as much as 20-25 cooled in liquid n i t r ~ g e nWe can be injected without overloading the column. Injection and condensation is repeated until a drop of each component is collected. The ends of the glass tuhes are then sealed in a burner flame and stored for use in part 111. MS Instrumentation and Spectra Interpretation (Part II) Following the GC separation of the ketone mixture, the students are introduced to the M S instrumentation and the interpretation of ketone spectra is explained. As i t hecomes appropriate during the demonstration and data collection, we reinforce both the individual powers of GC and M S and the complementary nature of the techniques. The design of a n actual interface (molecular separator) and *American Petroleum Institute Research Project 44, "Selected Mass Spectral Data (Standard)," Thermodynamics Research Center, Dept. of Chemistry, Texas A & M University, College Station, Texas. Wudents can prepare their awn collection tubes from 2-mm i.d. glass tubing. Leaving 3-5 em side arms, make right angle bends to produce a "U" shape 5-7 cm long and 2-4 cm wide. Students should be reminded to carefully label the tubes to prevent accidental remixing of the isomers during collection. It is preferable to use liquid nitrogen rather than various Dry Ice slushes, because of the hieh of condensine " orobabilitv . " slush vaoor in the sample tube. Acetone, because of its abundant m l e 58 peak, should be avoided. 7 For monofunctional ketones, the molecular mass is sufficient for determining the molecular formula. The formula can be caleulated easily from M + 1and M + 2 abundances. 8McLafferty, Fred W. "Interpretation of Mass Spectra," 2nd Ed., W. A. Benjamin, Inc., New York, pp. 49,120-123. 9Reference(81,p p 5659, 120,123. 536 1 Journal of Chemical Education
85
72
58
71
57
43
29
Ref.b
the con\.enience and sensitivity of the combination are discussed. We feel this experiment is an ideal lead-in for a discussion on the role o i small laboratory computers in chemistry. As the mass spectra become available, the students see the data-rich character of this analytical tool and can easily appreciate the role of on-line computer for data acquisition and reduction especially for complex mixtures. More specialized computer techniques such as specific ion detection, file searching, etc., can he discussed if time and interest permit.' The logical procedure for interpreting mass spectra of ketones can be described either in lab lecture or following the instrument demonstration. Often, i t is helpful if the instructor interprets one actual spectrum. Three features of ketone spectra are applied. The Molecular Ion The parent ion a t either rnle 100 or 114 is used to identify the molecular mass and the molecular formula of each ketone component.' M d M.+ em l e = 100 if M = CsH,?O m l e = 114 if M = CjH,,O
+
Simple Cleavage Reactions Species with odd rnle values can result from a variety of primary cleavages, and i t should he emphasized that odd rnle ions found a t the hieh mass wrtion come from " simple cleavage reactions. Fragmentations which are "carhonvl directed" (ems. (1)-(2)) ~ r o d u c ethe lareer relative . . ahukdances including the base peak.8 ~ h i s ' i m ~ o r t a n t to fraementation involves the loss of alkvl m o u ~ attached s th'carhonyl with the loss of the larger &kyi favored a t 70 eV. I t is obvious from Table 1 that the loss of methyl mouos (M-15) from the methvl ketone isomers (1. . , 3., 4., 6., 7, and 10) result in relatively small abundances when c o m ~ a r e dto the sienificant abundances resultine from the loss bf ethyl groups (M-29) from the ethyl ketones (2, 5, 8, and 12) or loss of propyl groups (M-43) from the C7H140 propyl isomers (9, 11, and 13). As the first step in interpreting the ketone structure, the students compare the relative abundances of the M-15, M-29, and M-43 peaks (the M-43 peak being considered only for C,HlrO isomers). Generally, this will provide sufficient information to identify the composition of the groups attached to the carhonyl since an ethyl ketone or propyl ketone will give a much larger M-29 or M-43 than M-15. The students are also instructed to keep in mind that the base peak must he a result of a carhonyl directed cleavage reaction analogous to either eqn. (1) or (2).
-
. ,
m l e observed
P
R-C-R'
43 if R 57 if R
0 +
R-C+
1I
+
R'
= =
CH, C,H, (1)
Table 2. Mass SDectrum of Student Collected Ketonea
P
R-C-R'
-
0
R+
+
II
C-R'
"Spectrum obtained on a Hitaehi RMU-6D Double Focvssing Mass Spstmmeter at 200-C with an ionizing energy of 70 eV. Isotopic peaks are deleted for simplicity.
Rearrangements To complete the identification, the actual structure of the various alkyl groups is sought. Of course, there is no problem with methyl and ethyl groups. Often the identity of a propyl, hutyl, or pentyl group can be established by using the results of the McLafferty rearrangement (eqn. (3)).9 Fragment ions resulting from this chemistry will be found a t even mass numbers.
m l e observed 58 if R = CH, and R' 72 if R = CH, and R' 72 if R = C2H, and R'
= = =
Thus, 2-hexanone (R = CH3 and R' = H) will give a McLafferty peak a t m l e 58 while the isomer 3-methyl2-pentanone (R = R' = CHs) will yield a m l e 72 fragment. Also, the lack of abundant even mass numbered fragments a t higher m/e values is informative since, for example, 2-methyl-3-pentanone gives no McLafferty rearrangement, and this negative information supports the assignment of this isomer. It is seen from Tahle 1 that isomers expected to give an m l e 72 fragment (2, 3, 8, and 12) show relative ahundances of 4.5, 17.1, 19.9, and .I%, respectively, while all other isomers produce ahundances 53%. Isomers expected to give an m l e 58 fragment (1, 4, 7, 9, and 10) show relative ahundances of 42.2, 32.3, 50.5, 6.5, and 35.4%, respectively, while all other isomers produce ahundances 54.9%. The relatively small abundance of m l e 58 for 4-heptanone is because this isomer must undergo a double McLafferty rearrangementg to produce this fragment (eqn. (4)).
Certain of the C7-ketones resist complete identification using the above information. For example, the spectra of 3-heptanone and 5-methyl-3-hexanone are very similar: Both give the same simple cleavages and a McLafferty rearrangement a t m l e 72. We give full credit to the stu-
dent for either assignment and i t is pointed out that a final distinction can he made on the basis of reference spectra. There are several additional odd m l e fragments ( m l e 27, 39, and 41) which frequently occur in the lower mass portion with large relative ahundances as a result of secondary fragmentations. These abundances are helpful in makine com~arisonswith reference sDectra. As an example of spectra interpretation, consider the oartial soectrum (Tahle 2) obtained from a student coliected ketone for which no reference spectrum is available. The molecular ion (mle = 114) confirms the ketone is a C7Hl40 isomer. Consideration of M-15 (0.6%), M-29 (2.0%), and M-43 (11.9%) might lead initially to an erroneous conclusion that a propyl group is attached to the carbonyl. This error becomes obvious, however, since none of the propyl isomers can undergo a McLafferty rearrangement to produce the abundant m l e 72 fragment. The only other choice that can produce the m l e 43 base peak fragment by means of a carhonyl directed cleavage is a methyl ketone. Either 3-methyl-2-hexanone or 3,4-dimethyl-2-oentanone are exnected to undereo a McLaffertv rearraigement to produce the m l e 72 c38.5%) fragment (the 3-methyl-2-hexanone being the true identity). Student Analysis of MS Data (Part I l l ) Once the characteristics of ketone mass spectra have been explained and the sample spectra analyzed, experimental data are ohtained on all student collected components and distributed to the students for individual analysis. The students submit a bar graph (relative abundance versus rnle) along with a tabulated list of relative ahundances observed for all m l e fragments and conclusions regarding the identity of each isomer. The students are asked to support their conclusions by submitting a detailed set of cleavage (carhonyl directed) or rearrangement (McLafferty) reactions showing the likely mechanisms for anv- m ,l e 99.. 85.. 72.. 71.. 58.. 57.. or 43 fragments observed with significant relative ahundances. The student's conclusions are evaluated and returned with corrections and any reference spectra needed to illustrate the final distinctions between two possible choices. In conclusion, we have found that students enjoy the GC separation and are eager to try identifying their components once the logic of interpretating mass spectra is explained. In our view, the aliphatic CsHlzO and C7H140 ketones are ideally suited for illustrating the separate powers of GC and MS as well as the potential of the combination. The comoounds are inexpensive and readilv available commerci~lly.Mixtures ard easily separated on commerciallv available GC columns. The mass spectra are characteristcc and readily ohtained on either student or analytical spectrometers. If a mass spectrometer is not availahle, the experiment still can he used by handing out copies of reference spectra such as those presented in Tahle 1. In either event. the soectral interpretation is straightforward and relatively unambiguous. he experiment has been used successfully for over five years a t the University of Nebraska. Volume 52, Number 8, August 1975 / 537
