Written by Miriam McCarty

Experiment Completed: 3/22/17

Report Completed: 4/12/17

Introduction

Gas chromatography (GC) is used to separate substances from one another

in the gas phase. It has a limited range as to what compounds can be analyzed

with this instrument. Some of these constraints include that the temperature

cannot go below 40 degrees Centigrade or above 400 degrees Centigrade, the

compounds must be volatile and thermodynamically stable. Reactions should not

take place within the column or else this could skew the results. The column is

made up of a mobile phase and a stationary phase. The mobile phase is helium.

This is chosen because most molecules will not interact with the helium in the

column, they will just flow through it. [1]

There are many different columns that can be in a GC instrument. Figure 1

in the appendix displays the different types ranging from packed capillary column,

support coated open tubular column, porous layer open tubular column and the

column in the GC at Capital University is the wall coated open tubular column. To

allow for better separation within a column, one wants the stationary phase to be

similar in composition to the sample being pushed through the column. This way

the stationary phase and the sample interact and the sample stays in phase longer

which allows for better separation. [1]

The temperature is another aspect of the instrument that can have an effect

on the results. If there is too low of a temperature then it will take a long time for
the analytes to move through the column. The sample needs to be vaporized right

away. To secure that this happens the inlet temperature needs to be higher than

the highest boiling point of the samples being run through the instrument. Split

ratio is another aspect of the GC that is able to be manipulated to produce the

best results. The split ratio allows for only a certain amount of your sample to

reach the column for analyzing. An example is a 50:1 split ratio. This means that

only half of your sample is making it through the column. A higher split ratio can

result in narrower peaks. If one chooses to use a split less ratio, then 100% of the

sample will flow through the column. [1]

There are many different types of detectors that a GC instrument can

contain. These include a mass spectrometer, FTIR, flame ionization, electron

capture and thermal conductivity. The GC at Capital University uses the mass

spectrometry along with quadruple mass analyzer. Figure 2 in the appendix

displays the schematic for a quadruple mass analyzer. In this, ions are accelerated

through four rods; two which are positive and two which are negative. The positive

poles work as a high mass pass filter whereas the negative poles work as a low

mass pass filter. Thus, only a narrow range of ions makes it through to the end of

the detector. [1][5]

For the experiment done in class students were asked to change parameters

on the instrument, such as temperature and flow rate, to produce the best results
and narrowest peaks. The samples that were run through the machine were fatty

acid methyl esters (FAMEs), rapeseed oil and nutmeg samples from the

biochemistry classes. The FAMEs and the rapeseed oil were run numerous

amounts of times until the students felt they secured the best results. The FAME

sample should produce 5 peaks and the rapeseed sample should produce 10-11

peaks. The method that was run for the FAME was then used to also run the

nutmeg samples.

Procedure

All samples were prepared for the students before the lab. This procedure

outlines the students methods for changing the parameters on the instrument.

Attached is an instruction sheet for how the students were able to change all the

parameters. This attachment was produced for English-310 as a guide to students

for running samples through the GCMS at Capital University. The students did not

want to go into the experiment blindly and thus papers on gas chromatography of

FAMEs were found and starting methods were derived from these papers. [2][3]

The first samples tested by the students were the FAME. These runs lasted

around 15 minutes. Multiple runs were performed before the final method was

confirmed. Examples of different methods and their results can be seen in the next

section. The method used by the group that resulted in the narrowest peaks

started at 190C for 5 minutes, then ramped at 15C per minute till the temperature
reached 215C. This temperature was then held for one minutes and then ramped

again at 15C per minute until the temperature reached 230C. This temperature

was held for 5 minutes. This method was performed with a split ratio of 50:1. This

will be called Test 1. Another method was performed on the FAME this time with a

split less ratio. This will be called Test 2. The unknown sample the students tested

was the nutmeg. This sample was run twice using the FAME method- one with the

50:1 split ratio and the other with a split less ratio. These will be called Test 3 and

Test 4.

The second samples tested by students were the rapeseed oil samples.

These runs lasted around 55 minutes. The method used by the group that resulted

in the narrowest peaks started at 140C and was held for two minutes. Then the

temperature increased by 2C per minute until the temperature reached 167C.

This temperature was held for 12 minutes. Then a second ramp was performed at

an increase of 2C per minute until the temperature reached 230C. This

temperature was held for 5 minutes. This will be called Test 5.

Results

Figure 4 represents the chromatogram for Test 1. The peaks displayed in order are

tridecanoic acid, methyl tetrdecanoate, pentadecanoic acid, hexadecanoic acid

and heptadecanoc acid.

 Figure 5 represents the chromatogram from Test 3. The peak displayed at 2.9

minutes is methyl tetradecanoate.

Retention time (k) Number of Plates (N) Height of Column (H)

4.1 0.51 20

Table 1 displays the retention time (k), number of plates (N) and height of column

(H) for the peak circled in Figure 5.

Figure 6 represents the chromatogram for Test 2. The peaks displayed in order are

tridecanoic acid, methyl tetrdecanoate, pentadecanoic acid, hexadecanoic acid

and heptadecanoc acid.

Figure 7 displays the chromatogram for Test 4. The peak circled at 5.0 minutes is

hexadecanoic acid.

Retention time (k) Number of Plates (N) Height of Column (H)

1.9 5.8 6

Table 2 displays the retention time (k), number of plates (N) and the height of

column (H) for the peak circled in Figure 7.

Figure 8 displays the chromatogram for Test 5. The peaks in order of appearance

are methyl tetradecanoate, hexadecanoic acid, octadecadienoic acid,

octadecenoic acid, methyl stearate, cis-11-eicosenoic acid, eicosanoic acid, 13-

docosenoic acid, docosanoic acid, and tetracosanoic acid.

Resolution between 13-docosenoic acid and docosanoic acid is 3.

Discussion

Through many trials and plenty of time spent with the GCMS instrument, the

students were able to not only identify substances using the instrument, but also

gain a better understanding of how the GCMS works. At first, parameters were

changed, not knowing exactly what it was going to do to the outcome. After

analyzing the peaks on the chromatograms the students were able to use their

knowledge about how to narrow peaks in order to produce a better chromatogram

for the sample. Using temperature and split ratios the students were able to gather

accurate results.

The FAME samples were first run using isothermal methods. This consisted

of a method at 185C for 15 minutes and then a second run at 220C for 15

minutes. These resulted in either very broad peaks or not even all 5 peaks that

should have been seen for the FAME samples. The group then tried different

methods that included temperature ramps. Most of the first attempts resulted in

heavy amounts of noise at the beginning or the end of the run. This noise was

seen once the temperature was above 220C. The final method did not exceed

215C for this reason. Split ratios of 100:1 and 50:1 were used for these varying

methods. The group decided that a split ratio of 50:1 produced narrower peaks

than the 100:1 split ratio. With the 100:1 split ratio there wasnt enough sample
moving through the column and there was poorer sensitivity- hence all the noise.

[4]

The nutmeg samples prepared by the Biochemistry students were run using

the same method for the FAME samples. Figure 5 displays one peak that was

identified as methyl tetradecanoate. This is one fatty acid that makes up nutmeg.

The second nutmeg test was done using the FAME method with a split less ratio.

This time 3 peaks were produced, but only one matched a fatty acid that was seen

in the FAME. This fatty acid was hexadecanoic acid as seen in Figure 7.

The rapeseed oil samples were tested with many different methods as well.

All of these methods included a ramp. The group achieved 9 peaks after a couple

trials, but wanted to reach at least 10. By looking at the chromatograms the group

was able to distinguish which areas needed ramps and thus the final method was

created and 10 peaks were produced. Earlier methods that were used took a

longer amount of time for the peaks to exit- the group fixed this by increasing the

temperature since they knew that at a higher temperature the sample would exit

the column faster. [5] Earlier methods attempted resulted in lots of noise near the

end of the run as well as less than 7 peaks showing up in the final chromatogram.

These methods started at lower temperatures as low as 85C. The group realized

that the rapeseed oil samples needed to be run at higher temperatures to ensure

all 10 peaks would be separated.

 The number of plates (N) for Test 3 is 0.51 and the N for Test 4 is 5.8. The

higher the N the better separation there is between the fatty acids. [4] Test 3

revealed only one clear peak so it makes sense that the N was a low number. For

test 4, the number was not too high, meaning that the peaks could have been

better separated. The height of column is related to number of plates. The smaller

the N, the higher H is- they are inversely related. This then leads to higher

efficiency [5]. The H for Test 3 was 20 and the H for Test 4 was 6. This makes

sense because Test 3 had the smaller N, thus has the larger H and test 4 has the

larger N and thus the smaller H. The resolution (Rs) was calculated for peaks 8

and 9 of Test 5. The Rs came out to be 3. Any resolution above 1.5 is said to

mean that the peaks are very well separated. [5]

Temperature played a large role in how well the peaks exited the column.

The students learned by trial and error what the best methods were for determining

the fatty acids in their samples. After the trials the students knew what outcome

they wanted to achieve and observing the chromatograms the students were able

to determine at what times the ramps needed to happen and for how long. The

GCMS ended up being a great instrument to separate substances. By just

manipulating one parameter the students were able to achieve interesting and

accurate results.
Appendix

Figure 1 displays the different types of columns that can be found in the GCMS

instrument. Letter A is the column that is in the GCMS instrument at Capital

University. [1]
 Figure 2 represents the quadrupole mass analyzer. [1]

References

1. Granger, R., Yochum, H., Granger, J., & Sienerth, K. (2017). Atomic Absorption
Spectroscopy. In R. Granger, H. Yochum, J. Granger, & K. Sienerth, Instramental Analysis
(pp. 207-242). New York: Oxford University Press.

2. Wilson, Z. (2000). .METHODS AND PROTOCOLS FOR ARABIDOPSIS LIPID ANALYSES. In Z.

Wilson, Arabidopsis: A Practical Approach (pp. 1-27). Oxford Press.

3. Michel, F. (2012). Analysis of FAMEs in Edible Oils with GC. Retrieved from Sigma Aldrich
Analytical: https://www.sigmaaldrich.com/content/dam/sigma-
aldrich/docs/promo_NOT_INDEXED/General_Information/1/09-fame-analysis-pl-sem-
2015.pdf

4. Agilent Technologies. (n.d.). Method Development for Capillary GC Systems. Retrieved from
Agilent Public Seminars: https://www.agilent.com/cs/library/eseminars/Public/GC%20e-
seminar%20meth%20dev.pdf

5. Agilent Technologies, Inc. (2016). Agilent 7820A Gas Chromatograph Operating Guide.
Shanghai.
 Instructions for 7820A Gas Chromatography Mass Spectrometry
1. Turn on the Geer Gas Pressure Sources located near the instrument. Consult your professor
for the settings that the gas should be maintained.
2. Turn on the Gas Chromatograph machine. This is done by clicking the power button on the
instrument as shown below.

3. Once the machine is turned on, it will sit idle until the program is opened on the computer. The
program will be titled Agilent GCMS Open Lab. Double click to open and start the program.
A green light should shine on instrument when it connects to the computer
program.
4. Once the program is opened it is time to create a method that will be used to run a sample.
a. At the top of the page there is a bar with the words File, Edit, Method, etc. Click
on Method and scroll down to Edit Entire Method
b. Once clicked you will have the option to name and save your method in a folder. Be
sure to name the method accurately so if someone else, or yourself, plans to
come back to use this method it is easy to find.
5. Once the new method is opened the program screen will appear as this.

a. This is the screen that you are going to use to help run your unknown samples.
 b. Here we must turn on the oven by clicking the box where it says On. The oven can
be left on whatever default temperature it was last left on.
c. Next, the detector needs to be turned on. Click on the Detectors tab, right next to
the Oven tab and check the box for the front AND back detectors.
6. Once the gas, oven and detector are all on, now you are ready to create a method.

7. Creating a method (this section will go through each of the tabs on the previous
figure, what they do and how you can change the settings on each)
a. Injector determines the amount of your unknown sample that is injected into the
machine for testing. Under this tab you will have the option to wash the needle that
takes up sample. This is recommended so previous samples do not contaminate your
own. In the box titled Solvent Washes put a number between two and five. This is a
good amount of washed to ensure your sample will not be contaminated.

b. Skip Vales and DO NOT change any settings under this tab.
c. Inlet is the part of the column, indicated with a purple line, where the sample is actually
injected into the machine. The temperature of this column should be higher than the
highest boiling point of the sample.

d. Column is where the flow and pressure of the sample can be adjusted. Pressure is not
changed, but flow rate is changed. The amount of your sample moving through the
 instrument will have an effect on the output, or chromatogram. Flow rates need to
run under three milliliters. Too much of a sample can ruin the instrument.
e. Oven is the most important when running samples through the GCMS. Controlling
temperature is your best bet at obtaining the most accurate chromatogram.

The max value you can set any temperature at is 350C. Anything above that
could ruin the instrument. The purple box highlights how you are able to change
temperature. In this example method, the user starts the run at 50C and holds that
temperature for 2 minutes. Then they increase the temperature by 40C per minute
until the temperature rises from 50C to 200C. Once the temperature reaches 200C
it is held constant for 2 minutes. That is how the table is read and how you are able to
set different temperatures for various lengths of time.
f. No other tabs need to be used or changed. You are ready to save your method
8. To save a method follow these steps. In the top left corner, click File, Save Method As, and
remember to name the method accurately so you or another user is able to
understand the method and use it in the future.
9. Now it is time to run the unknown sample through the GCMS.
10. Your sample will already be in the appropriate vial and once it is
 The inlet is a small part on the outside of the instrument. There will be small slots for you to
input the glass vial with your sample in it. If your unknown is the only sample being run, then put
it in slot 1.
11. Once your sample is in the inlet you can return to the program on the computer. To run the
method you have created go to the top left corner and click on Run Control, then select Run
Method. The machine will now wash your sample, if you selected that option in step 7a. Then it
will take part of your sample, however much you indicated in the flow rate in step 7d, and inject
it into the instrument. The more changes in temperature you chose in step 7e the longer it will
take to run a sample. Unknowns can take anywhere from 15 minutes to 90 minutes.
12. Once the sample is fully run a chromatogram will appear on screen such as this one from Figure
1.

The peaks will NOT be labeled. To view the instruments best matches for which compounds
correspond to each peak you will need to double click on a specific peak. Once clicked another
screen will appear and it will give you the best matches in percents- form there the user can
make an educated guess on what compounds make up their unknown sample.
13. Before leaving the instrument, the user must start the cool down method. If no one else will
be using the machine right after you, you must set the cool down method. This keeps
the machine in an idle position. This method will already have been created by the professor and
can be loaded as follows: In the top right corner click File, then Load Method and select the
Cooldown option. Then click on Run Control, click on Run Method and the cooldown
will begin.

References
Agilent Technologies, Inc. Agilent 7820A Gas Chromatograph Operating Guide.
Shanghai, 2016. PDF.

Concordia College. Determination of Caffeine in Beverages using SPME-GC-MS.

n.d. 8 March 2017.

Masclef, Amedee. Brassica napus. 31 January 2001. painting. 8 March 2017.

Rattray, Chris. When GC Injection Issues Arent Syringe Issues. 9 November 2012.
10 March 2017.

The Hebrew University of Jerusalem . Thermo Polaris Q - An Ion Trap GC/MS.

2009. <http://departments.agri.huji.ac.il/zabam/Polaris-Q.html>.