In the Laboratory
Identification of Volatile Flavor Components by Headspace Analysis: A Quick and Easy Experiment for Introducing GC/MS1 Richard A. Kjonaas,* Jean L. Soller, and Leslee A. McCoy Department of Chemistry, Indiana State University, Terre Haute, IN 47809
Tabletop gas chromatography/mass spectrometry (GC/ MS) has become a common feature in many sophomore organic chemistry laboratories. Several experiments employing this instrumentation have appeared in this Journal (1– 5), and two of these have specifically discussed the advantages of using headspace techniques when adapting an experiment to a large group of students (4, 5). During the past several years we have been introducing GC/MS and headspace analysis to our students by way of an especially simple and interesting experiment. The student places a crushed piece of hard candy or part of a stick of chewing gum into a vial. A 5-µL sample of the headspace is withdrawn and injected into the GC/MS. The mass spectrum of the major volatile component is obtained and identification is made. In some cases, there are minor components that can also be identified. An example is shown in Figure 1. Table 1 lists the samples and the volatile flavoring agents that we have identified. The retention times are given in Table 2. Identification of the compounds can, of course, be facilitated by having the instrument compare each mass spectrum with those in the NIST library. However, we feel that it is a better learning experience for students if they are not allowed to use this feature. To assist them, we hand out a list of the names and structural formulas of several likely candidates. So that cinnamaldehyde will not be a “giveaway,” we list it as 3-phenyl-2-propenal. The contents of the list depends on the choice of samples for that particular group of students, and it usually contains a few compounds that are not found in any of the samples. The terpenes in Table I have very similar mass spectra; because of this, we usually avoid mint-flavored samples and have the students identify major components only. This narrows the list of correct responses to isoamyl acetate, limonene, benzaldehyde, benzyl alcohol, ethyl butyrate, and cinnamaldehyde. These compounds can usually be identified by students who have just a very rudimentary understanding of mass spectral analysis. We have even had several groups of high school students do this experiment and, in each case, a brief prelab lecture on mass spectrometry has been sufficient to enable them to make positive identification of the non-mint major components. Although this experiment seldom presents problems, there are a few details that should be mentioned. The candy samples are crushed by placing them in a piece of weighing paper folded in half and then hitting with a hammer until a powder is obtained. This is placed into a 4-mL vial. Although it is best to use a vial cap that has a septum, excellent results are obtained even when using an ordinary cap; the student simply lifts the cap just enough to insert the needle into the headspace. The syringe (10 µL) does not need to be of any special type. We have noticed, however, that if the syringe is not cleaned after each injection, there can be a considerable amount of contamination from the *Corresponding author.
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previous injection. We routinely clean the syringe by using a vacuum to pull a small amount of methylene chloride through the barrel and needle. It is important to use a vacuum or the chromatogram will be overwhelmed by a large methylene chloride peak. We normally set the lower limit of the mass range at 34. This avoids the detection of N2 and O2 but allows CO2 and argon to be detected. If one does not see a CO2/argon peak, the syringe needle is probably plugged. All retention times in Table 2 are listed at 100 °C. This is done to provide a clear indication of the relative retention times of all components. For many samples, however,
Table 1. Easily Detected Volatile Components of Candy and Chewing Gum Samplesa Sample
Major Components of Headspace
Minor Components of Headspaceb
Banana Runtsc
isoamyl acetate
–
Lime Runtsc
limonene
p -cymene
Cherry Runtsc
benzaldehyde
ethyl acetate isobutyl acetate limonene
Orange Runtsc
limonene
ethyl butyrate
Lime Life Saversd
limonene
β-pinene p-cymene γ-terpinene
Lemon Life Saversd
limonene
α-pinene β-pinene γ-terpinene
Orange Life Saversd
limonene
α-pinene myrcene
Wild Cherry Life Saversd
benzaldehyde benzyl alcohol
ethyl acetate
Pineapple Life Saversd
ethyl butyrate
benzyl alcohol
Butter Rum Life Saversd
ethyl butyrate
ethyl acetate
Pep-O-Mint Life Saversd
menthone menthol
–
Wrigley's Spearmint Gume
carvone limonene
cineole menthone menthol
Wrigley's Big Red Gume
cinnamaldehyde
–
Wrigley's Doublemint Gume
cineole menthone menthol
–
Wrigley's Juicy Fruit Gume
ethyl butyrate isoamyl acetate limonene
–
a
All identifications were confirmed by injecting authentic material.
b
In some of these samples, there are minor or trace components that were detected but not identified. Also, the relative amount of each component in the headspace can vary considerably; this is probably due to variations in sample freshness and handling. c Registered
trademark of SPN.
dRegistered
trademark of Nabisco Foods Group.
eRegistered
trademark of Wm. Wrigley Jr. Co.
Journal of Chemical Education • Vol. 74 No. 9 September 1997
In the Laboratory Table 2. Retention Times (t ) of CO2, CH2Cl2, and Volatile Components
A. carbon dioxide/argon B. methylene chloride C. α-pinene D. myrcene E. limonene
Component
Figure 1. Chromatogram of the headspace of a crushed piece of Life Savers brand orange candy (4.5-min run). A small amount of CH2 Cl2 is present because it was used to clean the syringe.
we sometimes use higher temperatures to decrease the time required for each run. The exact temperature chosen by an instructor will depend on a variety of factors, including the column length and the goal of the experiment (identification of major components only or identification of minor components also). We chose to have the injector in the split mode rather than splitless simply because it is the mode most frequently used in our department; we wanted to avoid having to make frequent changes of the glass insert. The splitless mode would certainly work as well or better. Some experimental conditions are as follows: System: Varian 3400 GC with Saturn MS Column: DB-5,2 30 m × 0.25 mm i.d., 0.25 µm film Carrier gas: helium at 33 cm/s (~1 mL/min) Oven: isothermal 100 °C (or higher for shorter analysis times) Injector: split 1:5, 230 °C Sample: 5 µL of room-temperature headspace from a 4mL vial containing one piece of crushed candy or 1/4 to 1/2 stick of chewing gum torn into several pieces.
Students are often amazed at the fact that even the odor of the candy is enough material for the analysis. Some of the students enjoy the opportunity to confirm a tentative identification by smelling a pure sample of authentic material. We have written to two of the three manufacturers whose products are in Table I, asking for a list of the flavoring agents contained in their products. Both of them told us that this was proprietary information. Students are surprised at how easy it is to obtain information that the manufacturers do not want them to have! It is important, however, to remind the students that this experiment de-
t at 100 °C (min)
Carbon dioxide/argon
1.48
Methylene chloride
1.59
Ethyl acetate
1.62
Isobutyl acetate
1.92
Ethyl butyrate
2.00
Isoamyl acetate
2.39
α-Pinene
2.92
Benzaldehyde
3.17
β-Pinene
3.42
Myrcene
3.44
p -Cymene
3.98
Benzyl alcohol
4.07
Limonene
4.10
Cineole
4.16
γ-Terpinene
4.61
Menthone
7.14
Menthol
7.82
Carvone
11.4
Cinnamaldehyde
13.1
tects only volatile components. Also, because of different volatilities, the relative amounts of the components in the head space may be very different from the relative amounts in the piece of gum or candy. Notes 1. Presented in part before the Division of Chemical Education at the 210th meeting of the American Chemical Society, Chicago, IL, August 20–24, 1995. 2. A nonpolar column similar to SPB-5, AT-5, CP-Sil 8CB, HP-5, Ultra-2, 007-2, RTx -5, BP-5, SE-54, SE-52, and OV-73.
Literature Cited 1. Kostecka, K. S.; Rabah, A.; Palmer, C. F., Jr. J. Chem. Educ. 1995, 72, 853–854. 2. Illies, A.; Shevlin, P. B.; Childers, G.; Peschke, M.; Tsai, J. J. Chem. Educ. 1995, 72, 717 and references therein. 3. Annis, D. A.; Collard, D. M.; Bottomley, L. A. J. Chem. Educ. 1995, 72, 460–462 and references therein. 4. Corkill, J. A.; Raymond, K. W. J. Chem. Educ. 1994, 71, A202–A203 and references therein. 5. Lawrence, S. S. J. Chem. Educ. 1994, 71, 530.
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