Seattle Central Community College Science and Math Division. Chemistry 211 Analysis of Flavor Component of Gum and Candy by GCMS

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1 Seattle Central Community College Science and Math Division Chemistry 211 Analysis of Flavor Component of Gum and Candy by GCMS Introduction Fruity, juicy, aromatic or creamy the joys of flavor are sensuous. The work of flavor chemistry is a marriage of art and science, created from a diverse palette of tastes. Budding artists often start out by imitating other artists. The same is true with flavor chemists. Flavors are original works of art; they are entirely created by the chemist's own hand. Flavorists create various flavors from a palette of natural and artificial ingredients to fit food manufacturers needs. Sometimes, 'Mother Nature' does not always guarantee a consistent high quality product. Foods are affected by various factors, such as time of year and heat-processing of a product. Flavors enhance enjoyment and provide stability and improved quality to foods that are found on supermarket shelves. Previously bland, artificially sweetened and fat-free foods are made more enjoyable with the addition of flavors. Flavors also are used in pharmaceuticals. Cough syrups and tablet-form medications are made more palatable to those who find medication difficult to ingest. The addition of citral imparts a lemon flavor to food products and is often used in confections and juices. The characteristic aroma and taste of a solution, tonality may be described in the following terms: jammy, juicy, fruity, buttery, green, brown, sweet, ripe, creamy, sour and berry. Some of the simple chemicals: allyl caproate a pineapple aroma; benzaldehyde a pleasant cherry and almond; and ethyl butyrate fruity, strawberry and bubble-gum-like notes provide the first layer of flavor, the aroma. Aroma is considered before taste, when learning about flavors. The creation of a flavor is personal. Each flavorist has his own interpretation of what a strawberry, apple, orange or any fruit or vegetable tastes like. For instance, five flavorists may create five varying orange flavors, depending on one's background, personal experiences and where one lived as a child. A flavorist who lived in Germany would remember that an orange had sour tonalities and therefore would create an orange flavor that contains more sour notes. A flavorist in the United States may create an orange flavor with sweeter and fresher tonalities. Flavors are created in the laboratory by combining and measuring varying amounts of chemical or natural solutions to produce the desired flavor. When a food company requests a kiwi flavor, the flavorist selects from a palette of various tonalities, those compounds that when added to a food product will produce a kiwi flavor. On the lab bench, she combined several parts of chemicals with strawberry, banana and green tonalities, since she recognized that a natural kiwi was a combination of these flavors. Current flavor trends include exotic flavors. Many food companies are requesting papaya and mango flavors to be used in juice and other beverage products. Coffee and tea flavors are also popular. To produce a mango flavor, chemicals with peach tonalities are used. Adding a sulfur compound to the flavor base helped produce the overripe exotic character of the fruit. Flavor chemistry has evolved as a complex discipline which measures and quantifies sensory-significant compounds in foods. Typically, the flavor constituents, which are most influential in food flavors, are present at trace levels, which present analytical challenges for their identification and assessment. Significant advances have been made over the last several decades in the separation and identification of critical compounds in flavors via GC/MS techniques. The Gas Chromatograph

2 Gas chromatography is often used as a tool to separate, identify and quantify components of a mixture. The sample is injected through a heated injection port. It vaporizes and is carried through the heated column by the carrier gas, commonly helium. The carrier gas is the mobile phase. The components are separated through a column that contains an adsorbent that is coated with a liquid. The liquid coated adsorbent is the stationary phase. The sample is passed through the column and separates. The separation occurs because the components are attracted to mobile and stationary phases differently. As the components separate, they go through the detector at different times. A signal is sent to the detector and peaks are generated as each component goes through the detector. The distance from injection mark to the peak, multiplied by the chart speed is called retention time (RT). Given there is no change in the flow rate of carrier gas and temperature, the retention time for the given compound will always be the same. The retention times of the components are compared to the retention time of the known standards to identify the components. The areas of the peaks are proportional to amount of the components. To determine the percent component, the areas of the peaks are measured. Each of the component areas is divided by the total area and multiplied by 100. In the past, the areas of the peaks were calculated by triangulation of the peak, height (at ½ width) x width. For this experiment, the integrator will measure the areas for you. A computer prints out with the areas and % areas will accompany your chromatogram. The areas of the peaks are proportional to amount of the components. To determine the percent component, the areas of the peaks are measured. Each of the component areas is divided by the total area and multiplied by 100. In the past, the areas of the peaks were calculated by triangulation of the peak, height (at ½ width) x width. For this experiment, the integrator will measure the areas for you. A computer prints out with the areas and % areas will accompany your chromatogram. Mass Spectroscopy

3 Mass spectrometry is a powerful analytical technique that is used to identify unknown compounds and quantify known compounds. Mass spectrometry is a sensitive technique that can be used with separation techniques, such as chromatography, to analyze complex mixtures. GC/MS is a technique that combines Gas Chromatography (GC), a powerful analytical separation technique, with the powerful detection technique of mass spectrometers (MS). A small amount of sample is introduced into the mass spectrometer where it is bombarded by a stream of high-energy electrons. When high-energy electrons strikes an organic compound, it dislodges a valence electron from the molecule producing a cation radical - cation because the molecule has lost a negatively charged electron and radical because the molecule now has an odd number of electrons. In the ion source of the MS, the electron bombardment transfers such a large amount of energy to the sample molecules that the cation radical fragments after ionization. The fragments fly apart into numerous smaller pieces, some of which retain a positive charge (ions) and some are neutral. The ions then pass through a mass filter where they are separated by mass. The ions are counted by the detector which records them as peaks at the proper m/z ratio. The lines in the mass spectrum represent the mass-to-charge ratio (m/z) of the molecular ion and the mass-to-charge ratio of fragment ions produced. Since the number of charges, z, is usually 1, the peaks of ratio, m/z, are simply, m, the masses of the ions. The mass spectrum of a compound is usually presented as a bar graph with unit masses (m/z values) on the x-axis and the abundance (number of ions of a given m/z striking the detector) on the y-axis. The tallest peak is called the base peak is assigned a relative abundance of 100. Below is a mass spectrum of methane (Figure 1). Figure 1 Mass spectra of methane, CH 4 The spectrum is relatively simple, since few fragmentations are possible. The spectra show the base peak has m/z =16 which corresponds to the unfragmented methane cation radical. It is the parent peak or the molecular ion (M + ). The mass spectrum also show ions at m/z =15 and 14 fragments.

4 Mass spectral interpretation is the process of determining the identity of a molecule by deducing information from its mass spectrum. Interpretation is done by an 'expert' or by a program that mimics an 'expert' by finding a matching spectrum from a data library. Interpretation methods can be used to solve questions of identity when the mass spectrum of the molecule does not presently exist in a database. Interpretation of Mass Spectral Fragmentation Patterns Mass spectroscopy would be useful even if molecular weight and formula were the only information that could be obtained. In fact, though, we can get much more. The mass spectrum of a compound serves as a kind of "molecular fingerprint." Each organic molecule fragments in a unique way depending on its structure, and the chance that two compounds will have identical mass spectra is small. Thus, it's sometimes possible to identify an unknown by computer-based matching of its mass spectrum to one of the more than 130,000 mass spectra recorded in a reference library. It's also possible to derive structural information about a molecule by looking at its fragmentation pattern. Not surprisingly, the positive charge usually remains with the fragment that is better able to stabilize it. In other words, the more stable carbocation is usually formed during fragmentation. For example, propane tends to fragment in such a way that the positive charge remains with the ethyl group rather than with the methyl, because the ethyl carbocation is more stable than the methyl carbocation. M mlz = 29 Neutral;not observed Propane therefore has a base peak at m/z = 29 and a barely detectable peak at m/z = 15. (Figure 2.) Figure 2. Spectra of Propanol Since mass-spectral fragmentation patterns are usually complex, it's often difficult to assign definite structures to fragment ions. Most hydrocarbons fragment in many ways, as the mass spectrum of hexane, a typical alkane, demonstrates (Figure 3). The hexane spectrum shows a moderately abundant molecular ion at mlz = 86 and fragment ions at mlz = 71, 57, 43, and 29. Since all of the carbon-carbon bonds of hexane are electronically similar, all of them break to a similar extent, giving rise to the observed ions.

5 Figure 3. Mass spetra of hexane, C 6 H 14 ; molecular weight 86. The base peak is at m/z -57 Figure 4 shows how the hexane fragments might arise. The loss of a methyl radical from the hexane cation radical (M + * = 86) gives rise to a fragment of mass 71; the loss of an ethyl radical accounts for a fragment of mass 57; the loss of a propyl radical accounts for a fragment of mass 43; and the loss of a butyl radical accounts for a fragment of mass 29. With skill and practice, chemists can learn to analyze the fragmentation patterns of unknown compounds and work backward to a structure that's compatible with the available data. Figure 4. Fragmentation of hexane in a mass spectrometer. An example of how information from fragmentation patterns can be used to solve structural problems is given in Figure 5. Assume that we have two unlabeled bottles, A and B. One contains methylcyclohexane,

6 the other contains ethylcyclopentane, and we need to distinguish between them. The mass spectra of both samples show molecular ions at M + = 98, corresponding to C 7 H 14, but the two spectra differ considerably in their fragmentation patterns. Sample B has its base peak at m/z = 83, corresponding to the loss of a - CH 3 group (15 mass units) from the molecular ion, but sample A has only a small peak at m/z = 83. Conversely, A has its base peak at m/z = 69, corresponding to the loss of a CH 2 CH 3 group (29 mass units), but B has a rather small peak at m/z = 69. We can therefore be reasonably certain that B is methylcyclohexane and A is ethylcyclopentane. Figure 5. Mass spectra of unknown samples A and B. This example is a simple one, but the principles used are broadly applicable for organic structure determination by mass spectroscopy. Specific functional-group families such as alcohols, ketones, aldehydes, and amines often show specific kinds of mass spectral fragmentations that can be interpreted to provide structural information. Spectra Samples of Compounds listed by Functional Groups The examples below demonstrate the patterns which can be seen in mass spectra of compound ionized by electron impact ionization.

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11 In this experiment, the flavor components of candy and gum will be identified. Background information on the flavor components of candy and gum can be found in Page 211, Box 20-3 of your lab text. A small portion of the sample is added to acetone to dissolve the flavor components. The sample will be analyzed in the GCMS. The signals produced by the flavor components will be isolated to generate its mass spectrum. To identify the compound, the mass spectra are compared to a library and the computer provides the best match. A percent report will also be generated by the computer to give the relative percent concentration of the components. Procedure 1. Place a lifesaver candy in between a folded weigh paper. Use a pestle and gently crush the candy. If your sample is a gum, tear a 0.5 mm strip and shred finely. 2. Cover the bottom of the sample vial with a thin layer of the candy or gum sample 3. Add acetone to the 1mL mark. 4. Place your sample vial on the next available slot. Note the slot number. 5. From the MSD System Window, the "Sample Name" button using the mouse. This is the button with the green arrow. 6. A sample form will pop up. Fill the necessary information. Your data file will be your "name".d. Enter the slot number as the vial number. When completed press "Start run." The analysis will commence. 7. To perform a data analysis while still running the analysis, maximize the Enhance Data Analysis window and click on "Take a snapshot." 8. To perform a data analysis after a run is completed, maximize the Enhanced Data window. Click load and find your file and click. 9. Your total ion count (TIC) signal window will pop open. 10. Right double click on the signal. The MS window will pop up with your spectra. 11. Right double click on the spectra. The matching spectra will be listed. Pick the spectrum that you feel is the best match and print. 12. The percent report will automatically print after the completion of the run.