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Analysis of a Commercial Oil by Gas Chromatography / Mass Spectrometry 1. Introduction

This experiment differs from most others in this course in three ways. First, its focus is qualitative analysis and so only the identity of the unknown must be determined instead of the concentration. Second, it is a union of two techniques, gas chromatography (GC) and mass spectrometry (MS). This combination creates a 3-dimensional data analysis the gives us additional useful information. Lastly, the unknown is a real-life sample, cooking oil. As is the case with many “real-life” samples, the oil is not ready for analysis and must be processed prior to injection into the instrument. The purpose of this laboratory is to prepare the sample, analyze it with the GC/MS instrument, and identify the oil and its fatty acid components.

GC/MS has numerous applications to chemical identification and analysis. It is the “gold standard” in the field of forensics and has been used for drug detection, fire investigation, environmental analysis, and explosives testing. GC/MS is also used by airport security to detect substances in luggage or on human beings. Additionally, it can identify trace elements in materials that were previously thought to have disintegrated beyond identification.

The GC/MS is composed of two major components: the gas chromatograph and the mass spectrometer. The GC column separates analytes based on their relative boiling points and affinity for the stationary phase. The molecules travel through the GC column at different rates and therefore exit the column at different times. These “retention times” are the first dimension of the GC/MS data on the x-axis. The analytes remain separated as they enter the mass spectrometer, where they are ionized and the ions separated based on their mass to charge (m/z) ratios. The mass spectrum of a species (ion abundance versus m/z) represents the second and third dimensions of the analysis. See Figure 1 below for an example of the three dimensional data.1

Figure 1: GC/MS 3-D Data Plot1

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Much research has been performed developing ionization methods for MS. The ionization method determines the type of samples that can be analyzed by mass spectrometry. Electron impact (EI) ionization and chemical ionization are used for gases and vapors. The instrument in this laboratory uses electron impact (EI) ionization.

In an EI source, electrons are produced by heating a wire filament through which electric current is run. The electrons are accelerated through the ionization space towards an anode; in the ionization space, they bombard analyte molecules (M) in the gas phase and knock off a valence electron. This creates a radical cation with an odd number of electrons:

M(g) + e- → M+● + 2e- (1) The molecular ion (M+●) can also fragment into smaller ions which are present at smaller m/z in the mass spectrum. Each molecule has a specific fragment spectrum known as its “fingerprint”.

In this lab, your unknown is a cooking oil sample. Fats and oils belong to a class of biomolecules called triglycerides. Triglycerides are derived from a glycerol core, with three fatty acid chains attached via an ester linkage. A triglyceride has the shape of an “E” as shown in Figure 2 below. The fatty acid chains can be identical, or, as is more common, are different. Because fats and oils have such low volatilities, they cannot be analyzed directly by gas chromatography. They must be broken down into more volatile components. It is possible to decompose triglycerides into glycerol and the conjugate bases of their constituent fatty acids by heating them with a strong base, such as NaOH. This hydrolysis process which breaks the ester linkage is called saponification. The general reaction is shown below.

C

C

C

H

H O

H O

H O

HC

C

C

O

O

O

R

R'

R"

C

C

C

H

H O

H O

H O

HH

H

H

+NaOH

CH3OH

C R

O

ONa

C R'O

ONa

C R"

O

ONa

Triglyceride glycerol conjugate base

Figure 2. Saponification of a triglyceride.

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It is common to analyze fats and oils by performing this saponification reaction then replacing the sodium with methanol to form three methyl esters of the constituent fatty acids (Figure 3).

C R

O

ONaBF3

CH3OHC R

O

OH3C

Conjugate base fatty acid methyl ester (FAME)

Figure 3. Esterification reaction. These esters then can be extracted and injected onto the GC/MS instrument and the original structure of the fat or oil determined. This type of analysis is called a fatty acid methyl ester (FAME) determination.

2. Safety Protective eyewear must be worn at all times. Perform all steps of the extraction in the fume hood. Boron trifluoride is toxic, so handle with caution; the boron trifluoride/methanol solution is flammable. Many internal parts of the GC/MS are very hot or carry dangerous voltages. Do not open the cover of the GC. Notify the instructor if there is a problem with the instrument. Discard all hazardous waste as directed in this procedure. For more general safety in the laboratory, please refer the appendix.

3. Preparation of the unknown:

Obtain two large test tubes containing your unknown oil from your lab instructor. The test tubes contain a few drops of identical unknown oil. Verify that the unknown ID numbers are the same and document in your notebook. Retain the corks for each test tube, as you will use them later.

Saponification and Esterification of the unknown oil. Add 3 mL of methanolic NaOH solution to each test tube. Be careful not to use too much methanolic NaOH! Place the test tubes in a beaker with hot water until all the oil dissolves into the solution. You may need to remove the test tube from the water bath and gently mix the contents by shaking. The methanol in the test tube should not be boiling vigorously. If it is, then, you may consider removing the beaker off the hot plate, and possibly removing the test tube from the hot water bath. Make sure to watch the test tube and not the beaker. Because the boiling point of methanol is less than that of water; do not let the beaker of water come to a boil – this is too hot. Do not let your sample dry out.

Obtain the boron trifluoride/methanol solution (which is stored in the refrigerator) from your instructor. Add 5 mL of boron trifluoride solution to each test tube. Gently boil the test tubes in the hot water bath for 3 minutes. The boron trifluoride/methanol solution must be kept refrigerated when not in use. While you are esterifying the fatty acid, return the bottle

Instrumental Analysis Laboratory Safety Rules A. Instructions: Carry out all manipulations in accordance with instructions and the

safety rules and procedures given herein. B. Eye Protection: All students and staff working in the laboratory must wear safety

glasses at all times. If a student needs to be reminded more than three times to wear goggles, she/he will

be dismissed from lab for the remainder of the day, and will not be given an opportunity to make up the work.

C. Apparel: The clothes you wear in lab are an important part of your “safety

equipment,” and should offer protection from splashes/spills. Closed toed shoes (sneakers are fine), Full-length pants or a full-length skirt, and A shirt that completely covers your torso (i.e. at minimum, a t-shirt).

In other words, you must NOT wear shorts to lab. You must NOT wear flip-flops, sandals, or crocs. You must NOT wear tank tops, halter tops, spaghetti-strap tops, or low cut jeans to lab. Exposed abdomens, hips, and backs are not safe in the lab.

D. Gloves: Gloves are an important part of personal protection. Gloves will be available

at all times in the laboratory. Your instructor will require their use when appropriate. E. Food: Food, drinks, and gum are not allowed in lab. None at all, not even water

bottles. F. Sanitation Issues: Be sure to wash your hands before leaving lab, before you eat

anything outside of lab, and before you answer your cell phone. G. Music: Individual headphones are not allowed. Your may choose to play music for

the entire class. H. Cell Phones and Other Electronic Devices: Cellular phones and other electronic

devices that you do not need to perform your laboratory work should be put away. I. Other: All students are explicitly prohibited from:

1. conducting any unauthorized experiments. 2. removing chemicals or apparatus from the laboratory for any reason. 3. working in the lab alone, or at other than regularly scheduled lab periods. 4. smoking in the laboratory or within 20 feet of any doorway. 5. impeding movement in aisles or through doorways with bags, skateboards, etc.

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to your instructor immediately. After 3 minutes of gentle boiling, remove the test tubes from the hot water bath, and allow them to cool to room temperature. If your sample dries out, it is okay to add a few mL’s of methanol to it. You may then have to briefly heat the test tube to dissolve the sample back in to solution. Extraction of fatty acid methyl ester (FAME). After the test tubes have cooled, add 5 mL of saturated sodium chloride solution and 10 mL of cyclohexane to each test tube. Cork each test tube and shake well. If the aqueous solution appears cloudy, add a few milliliters of nanopure water and shake again. The organic and aqueous layers will separate upon standing.

4. Operation of GC/MS

Note: The screenshots in Figures 4 – 6 are shown to give you a general idea of what the instrument controller (aka “gameboy”) screen should look like. The specific parameters on the controller should be verified versus what is written in the text of this procedure, not versus the picture. Load the proper method on the GC. Use the control module on the right side of the GC. All operations on the controller module begin at the Status screen shown below. If the screen does not appear like shown in Figure 4, press the ESC button until you see the Status screen.

Figure 4. Sample Status Screen on GC/MS

Note: this image is here to give you and idea. It may look somewhat different from the real screen

At the Status screen, select the "Method Files" (F3 button), then select GC Methods (F5). Several different methods will be listed. Press the up or down button to select the method called "INSTRUMENTAL_LAB", then select Load (F8). If the correct method has already been loaded, the control module will indicate this. If the method parameters have been changed, the control module will ask if you want to save the changes. If the control module prompts you to save the method before loading it, seek immediate help from your instructor. Once the proper method has been loaded, press Esc button until you are back to the Status screen.

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At this point, the GC is set up to run. While you should not have to change any parameters for the GC, you should verify the settings of the GC. You can do so by selecting Settings (F1) from the Status screen.

Figure 5. Sample Oven Settings Note: this image is here to give you and idea.

It may look somewhat different from the real screen The oven (Figure 5) settings should be:

Initial temperature 190°C Initial time 5 min Rate 2°C/min Final temperature 200°C Final temp time 5 min

Once the oven settings are verified, select the Inlet (Figure 6) settings (F1). They should be set to the following values:

Inlet temperature 250°C Split ratio 1:50 Flow 1.20 mL/min

Figure 6. Sample Inlet Settings screen Note: this image is here to give you and idea.

It may look somewhat different from the real screen

Finally, check the Detector settings (F4). There is only one value for the Detector setting: Detector temperature = 280°C

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At the computer start the ChemStation software by double-clicking the "Instrument #1" icon on the desktop (Figure 7). Do not click the one it says “Instrument #1 Data Analysis”, as it will not help you collecting data. Once ChemStation starts up, two windows (Instrument Control and Top) are visible on the computer monitor. The Instrument Control window will primarily be used.

Figure 7. ChemStation Software

Load the proper method file. Go to "Method" and select "Load". Select "C:\MSDChem\1\Method\Instrumental_Analysis.M" as the method file. Before injecting samples on the GC/MS system, you must evaluate the tune used by the mass spectrometer for that analysis. In the Instrument Control window, go to the "View" menu, select "Tune and Vacuum Control…" and wait a few seconds.

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Once the Instrument Control window has entered the Tune and Vacuum control mode, go to the "Tune" menu, and select "Tune Evaluation". Evaluation of the mass spectrometer tune will start. The instrument will make a clicking sound, and the computer monitor will display a series of plots. When the instrument is finished performing the Tune Evaluation, the results will print automatically. Next, check the results. All the checks should read "OK". If any do not read "OK", talk to your instructor immediately. Retain the tune evaluation along with your results.

Once tuning is finished, go to the "View" menu and select "Instrument Control". At this point, a dialog box will read "Be sure tune file is saved. Switch view now?" Click "yes" to continue.

Once the method has been loaded, go to "Method" and select "Run". A new window will pop up (Figure 8). Fill the following in each box:

Data Path: D:\Instrumental_analysis\ Data File Name: Enter your name Operator Name: Enter your name

Figure 8. Start Run Window When all the information is entered, click the "Run Method" button (not "OK" button). If you click "OK", you will have to go to "Method" and select "Run" again. You have just setup the computer to collect data upon request. You will have to start from this part every time when you run the instrument.

Next, go to the GC, press the "Prep Run" button and wait until the "Not Ready" LED is off (Figure 9).

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Figure 9. GC Front Panel Fill the syringe (Figure 10) with the top organic layer which contains the analyte, and expel excess liquid, bringing the volume to 1 µL. If you need a demonstration of proper injection into a GC, ask your instructor.

Figure 10. GC Syringe for Injection of Sample

Carefully insert the needle into the center of the injector (Figure 11) so that the hub of the needle butts up against the injector. The needle will penetrate into a rubber septum (inside the injector).

Figure 11. GC Injection Port

Inject the sample in one swift, quick motion, then quickly withdraw the needle from the injection port. Immediately press the "Start" button located on the front of the instrument (Figure 8). It is important not to keep the needle in the injector for a long time. Once it has penetrated the septum, the sample must be injected as soon as possible and as smooth as possible.

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Once the run has started, you should see a message (Figure 12) on the computer saying "Acquisition Override Solvent Delay (3.00 minutes)?" DO NOT CLICK ON ANYTHING! SIMPLY IGNORE! This is normal.

Figure 12. Message Window upon Starting Run - ignore

Each run lasts 15 minutes. While waiting for the run to complete, clean the syringe with methanol and work on any cleaning or laboratory tasks. Repeat the previous procedure for your second sample. Two pages will print out per injection: one page with the peak area percent report and the other with the chromatogram. Repeat the above procedure for each sample. When the analysis is complete, DO NOT TURN ANYTHING OFF! THE GC/MS MUST BE KEPT RUNNING AT ALL TIMES! Dispose all of your sample in the test into the large “Recovered cyclohexane” flask in the middle fume hood. Discard the empty test tubes and corks.

5. Data analysis

In order to analyze the results, switch to the Data Analysis module of ChemStation. Go to "View" in menu, select "Data Analysis (offline)" and wait. The Data Analysis module should start at this point. Once Data Analysis has started, load your data file to analyze the results. Go to "File" and select "Load Data File…" from the menu. A new dialog box will ask for the data to be selected. Select the data you have just collected. If you don't see your data, ensure that the directory path is set correctly. It should be D:\Instrumental_Analysis\. If not, select the correct path by clicking the "Path" button shown on the window. Once you have located your data, click "OK" to load. Next, print out the mass spectrum of each primary component of the chromatogram; there should be three. Move the cursor to the apex of the chromatogram peak and RIGHT DOUBLE-CLICK. The mass spectrum for that particular peak should be displayed on the lower half of the screen in a new window. Print the mass spectrum. To do so, go to “File”, select “Print”. When asked what to print, select “Selected Window”, and select “1”. This will print your mass spectrum shown on the lower half of the screen.

Determination of the cooking oil identity using GC data. You can determine the identity of your unknown cooking oil using the GC chromatograms. Compare the retention times and

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relative peak area percentages of your sample to the sample oil data in Table 1 in the appendix. You may have to use the smaller peak areas and percentages to accurately determine the oil identity. Make sure that you discuss in detail how you determined your oil’s identity (i.e. your reasoning) in your lab notebook. Determination of fatty acid identities using MS data. Next you will use the mass spectrum of each peak in the chromatogram to elucidate the identity of the fatty acid components of the cooking oil. Remember, each mass spectrum you collected is of a methyl ester derivative of the fatty acid. For example, the structure of methyl myristate, a derivative of myristic acid (tetradecanoic acid), is shown below in Figure 13.

CH3 CH3

O

O

Figure 13. Chemical Structure of Methyl Myristate You can think of your unknown as having the general structure of H3COOC-(CH2)n-CH3. For example, methyl myristate, a 14 carbon fatty acid derivative, would have n = 12 by this notation. Also, note that the 14 carbon chain possesses no double bonds. The shorthand notation for this fatty acid is C14:0. You can see from this structure that each fatty acid methyl ester (FAME) has the same basic structural functional (end) groups (shown in bold here): H3COOC-(CH2)n-CH3. You must determine two things: the value of n and the number of double bonds in the chain (if any). The highest m/z peak in a mass spectrum is called the molecular ion (M+) peak. This peak is for the unfragmented FAME; it has not lost any atoms, only an electron. Remember, mass spectrometry discriminates between isotopes, so we can use the nominal masses of carbon (12 amu), hydrogen (1 amu) and oxygen (16 amu) for determining mass. For example, for methyl myristate2, the molecular ion is the peak at 242 in Figure 14 below. This peak is composed of the end groups (H3COOC- & -CH3, which contribute to 74 of the 242. The remaining 168 (242-74) is attributed to the 12 CH2 groups (12 x 14 = 168). When the carbon chain contains no double bonds, as in this example, the mass of the chain (i.e. 168) is a multiple of 14 (the mass of CH2). If the chain contained any double bonds we would expect the mass to be smaller by 2 amu per double bond (i.e. 166 if it contained 1 double bond).

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Figure 14. Mass Spectrum of Methyl Myristate2

Use this information about interpretation of mass spectra to determine the identities of the fatty acid chains in your unknown. The first step is to determine the molecular ion peak in each mass spectra. Next, determine the n value in your FAME (H3COOC-(CH2)n-CH3) and the number of any double bonds, if present. Remember that although we know that a double bond is present, we do not know the location of this bond. Don’t forget: to accurately determine the identity (original chain length) of the fatty acid, you must also account for the two carbons that are present on each end of the chain. For this experiment you are graded not only on your correct determination of the unknown oil and the formula of its three largest fatty acid components, but also how well you demonstrate your reasoning and construct your argument. We are looking for you to present your data and logic as if you are presenting to your boss or PI in a lab. Please present your data in tables with brief and succinct explanations. We are not looking for a multipage paper of prose. We are looking for clear, effective and efficient data presentations and accompanying descriptions. First, you are given an Excel sheet (posted on BlueLine and/or the web page where you downloaded this instruction) that contains standard oil data. On the Excel sheet, insert a row for your oil. You will ultimately enter your oil adjacent to the standard with the best match. You may be able to rule out some oils right away. Show in your notebook how you did this. Don't write a long treatise on this, present the data in an efficient format (e.g. a table) and briefly describe it. Or you can use Excel and some of its cool features to show which ones you can rule out. You may narrow your potential oils to a few. At this point, you should use the spreadsheet to manipulate the numbers to mathematically show which is likely your oil. Print out your spreadsheet and make sure that your math is clear and labeled. Give a brief description and show a sample calculation in your notebook.

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Generally, after reading through the mass spectral analysis in the procedure, most students know what to do. Please document this as if you are explaining to a fellow student who doesn't understand. Again, data tables make it visually easy for a reader to process multiple numbers and manipulations.

Reporting Requirements Once your data analysis is complete, report the following: 5.1. The identity of your unknown oil (determined by retention time and area percent

comparison with standard oils using the spreadsheet from Blueline). 5.2. The identity of each fatty acid in your oil (determined from FAME mass spectra

identification). (If double bonds are present, report the chain length and number of double bonds). Report in the format of C16:0, as was described for myristic acid.

You can either print out the sheet on the following page or pick up a copy in the lab. Report your data on this sheet rather than the cards. Make sure that your reasoning and mass spectrum interpretation is clearly documented in the results and discussion section of your notebook for each FAME. Your laboratory grade depends on both of these items. It is important to clearly describe your reasoning. Your grade will be determined out of 100% as follows: 70% Oil identification (50%) and explanation (20%) 30% fatty acid formula and explanation (10% for each)

6. References

1. http://www.spectroscopynow.com/FCKeditor/UserFiles/Image/specNOW_advertorials/1105_MS_Thermo/Thermo_msadvertorial_nov05_fig1a_large.jpg

2. http://www.lipidlibrary.co.uk/ms/arch_me/me_sat/M0014.gif