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

GC/MS Page 1 12/31/2017

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

H

C

C

C

O

O

O

R

R'

R"

C

C

C

H

H O

H O

H O

H

H

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.

GC/MS Page 2 12/31/2017

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

ONa

BF3

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 derivatization and extraction steps

in the fume hood. Boron trifluoride is toxic and so handle with caution; the boron

trifluoride/methanol solution is flammable. Notify the instructor if there is a problem with

the GC-MS. 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 caps for each test tube, as you will use

them later.

Saponification and Esterification of the unknown oil. Add approximately 3-mL of

methanolic NaOH solution to each test tube. 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. If the solution starts boiling vigorously,

consider removing the beaker from the hot plate, and possibly removing the test tube from

the hot water bath. 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 refrigerated boron trifluoride/methanol solution 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. 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, add a few

milliliters of methanol to it. You may then have to briefly heat the test tube to dissolve the

sample back into solution.

GC/MS Page 3 12/31/2017

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. Cap each

test tube and swirl vigorously to mix the layers together, but do not invert the test tube as

the cap is not leak-proof. 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, with the FAMES in the upper hexane layer.

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 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 an idea of the screen.

It may look somewhat different from the actual 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.

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 and document the settings of the GC in your notebook. You

can do so by selecting Settings (F1) from the Status screen.

GC/MS Page 4 12/31/2017

Figure 5. Sample Oven SettingsNote: this image is here to give you an idea of the screen.

It may look somewhat different from the actual 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 screenNote: this image is here to give you an idea of the screen.

It may look somewhat different from the actual screen

Finally, check the Detector settings (F4). There is only one value for the Detector setting:

Detector temperature =280°C

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.

GC/MS Page 5 12/31/2017

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.

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.

GC/MS Page 6 12/31/2017

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.

Next, go to the GC, press the "Prep Run" button and wait until the "Not Ready" LED is off

(Figure 9).

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.

GC/MS Page 7 12/31/2017

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.

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

GC/MS Page 8 12/31/2017

Each run lasts 15 minutes. While waiting for the run to complete, clean the syringe withmethanol 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 replicate sample. When the analysis is complete, DO

NOT TURN ANYTHING OFF. The GC-MS is designed to remain operational at all times.

Dispose all of your sample in the test into the large “Recovered cyclohexane” flask in the

fume hood. Discard the empty test tubes and caps.

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

relative peak area percentages of your sample to standard oil data as discussed below.

For the identification of unknown oil, download the Excel spreadsheet for GC/MS analysis.

It is at the same site where the laboratory procedures are posted

(http://chemistry.creighton.edu/~jiro/CHM466/) You must use this spreadsheet. You should

compare the retention times and the signals (as measured by area percentages) of your

sample numerically to the standard oil values listed on the spreadsheet. Prepare a figure

(data table or graph) that demonstrates how you numerically determined your unknown’s

identity. This figure should be appropriately labeled as if you are using it in a presentation or

manuscript. In addition, type up a brief description of this figure. The quality and clarity of

the figure and description will be used to determine part of your grade for this experiment.

Attach this printout to your lab notebook pages.

GC/MS Page 9 12/31/2017

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 fattyacid. For example, the structure of methyl myristate, a derivative of myristic acid

(tetradecanoic acid), is shown below in Figure 13.

CH3CH3

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

structural pattern of: H3COOC-(CH2)n-CH3 (the end groups are shown in bold here) You

must determine two things: the value of n and the number of double bonds in the chain(if any). Then you will report the formula of each original fatty acid in the shorthandnotation.

The highest m/z peak in a mass spectrum (Figure 14) is called the molecular ion (M+) peak.

The molecular ion is 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 amu in Figure 14.

This peak is composed of the end groups (H3COOC- & -CH3, which contribute to 74 of

the 242. The remaining 168 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).

GC/MS Page 10 12/31/2017

Figure 14. Mass Spectrum of Methyl Myristate2

Use this information about interpretation of mass spectra to determine the identities of the

original fatty acid chains in your unknown. 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. You will be graded on both the

accuracy of your logic along with the clarity of your work and reasoning for your results, as

described in your notebook. Record your reasoning as if you are teaching someone about

interpreting the mass spectrum.

Reporting Requirements

Once your data analysis is complete, report the following on the reporting sheet:

• The identity of your unknown oil (determined by retention time and area percent

comparison with standard oils using the spreadsheet from Blueline).

• 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.

Your grade will be determined out of 100% as follows:

70% Oil identification (20%) and explanation (50%)

30% fatty acid formula and explanation (10% for each)

6. References

1. http://www.spectroscopynow.com/FCKeditor/UserFiles/Image/specNOW_advertorial

s/1105_MS_Thermo/Thermo_msadvertorial_nov05_fig1a_large.jpg

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

GC/MS Page 11 12/31/2017

GC/MS Fatty Acid Reporting

Name: _________________ Lab Section: _____ Unknown Number: ____________

Date: _________________ Unknown Oil Identity: ___________________________

Grading:

Possible score Your score

Oil identification 20

Oil ID explanation 50

Fatty acid 1 ID and explanation 10

Fatty acid 2 ID and explanation 10

Fatty acid 3 ID and explanation 10

TOTAL 100

GC/MS Page 12 12/31/2017

Peak#

RT (min)M+●

m/zFatty acid formula

(in the format of C24:2)

1

2

3

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.