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Liquid Extraction of Caffeine and Identification Using Mass Spectrometry and Gas
Chromatography
Abstract
Caffeine is a widely used simulant to give people energy. The stimulant is found in many
energy drinks, tea, and coffee. This experiment aims to successfully extract and identify a caffeine
sample from coffee. This is done by liquid-liquid extraction to separate the caffeine from coffee then
gas chromatography (GC) would separate the caffeine. After that it goes through mass spectrometry
(MS) to identify the molecule as caffeine. These methods are very precise and give an exceptionally
good idea of what a molecule is and how much of it was extracted. Using these methods, caffeine
was successfully extracted and identified from the sample of coffee. The fragmentation from mass
spectrometry was not as accurate as predicted, hinting at some error in the extraction process.
Overall, the methods were highly successful at completing the goals.
Introduction
The goal of this lab was to take a caffeinated sample (coffee) and use various methods (MS
and GC) to extract all of the caffeine from the liquid and correctly identify it as such. Caffeine can be
extracted from coffee using a liquid-liquid extraction with ethyl acetate. This separates the caffeine
from the water. Using the difference in densities, a separatory funnel can be used to create two
distinct layers of ethyl acetate and caffeine on the bottom and everything else above it. After
filtering the liquids, the ethyl acetate must be evaporated to leave only caffeine. After weighing the
sample, ethyl acetate could be added to the beaker. At this point, the sample can be put into a gas
chromatograph instrument to be fully separated. The GC works by injecting the sample into a
column that is microns thin using a hypodermic needle. The interior of the column is coated in a
non-polar, silicon based, material that allows the sample to travel through it without sticking. The
column is placed into an oven where the sample is vaporized. The vaporized sample is pushed
through the column using an inert gas. The sample vaporizes into two separate substances
depending on its properties and as it is pushed through the exit, a signal will be output to show the
abundance of the substance. The time it takes to get through the column will identify the substance.
The substances will then be immediately put through the mass spectrometer where they are hit
with high energy electrons, ionizing them so that it is able to measure the mass to charge ratio of
the ion. Once the entering particles have been ionized, they flow into a magnetic field that directs
them through an arc into a detector where a computer reads it m/z ratio1.This fragmentation is
then read to identify the parent ion and which ions it broke in to. The mass spectrometer will then
return a graph with peaks of the fragments which can be traced to their parent ion.
Methods
The experiment started by measuring 100 mL of coffee into a graduated cylinder which was
then poured in an Erlenmeyer flask. Then, 2 g of sodium carbonate was added and dissolved into
the flask. After the sodium carbonate had dissolved, the ethyl acetate was added and gently stirred
for 10 minutes by swirling the flask. Once the time had passed and the contents were mixed, it was
poured into a separatory funnel. After the ethyl acetate separated in 10 minutes and floated on top,
it was collected into a pre-weighed 50 mL beaker. Once completely collected, the beaker was placed
on a hot plate under the fume hood where it could be evaporated to leave only the caffeine in the
beaker. Then the beaker was weighed to find the total mass of caffeine collected. Finally, ethyl
acetate was added back to the beaker to dissolve the caffeine and a small amount of the sample was
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put into a vial to be injected into the GC. The full, in-depth lab instructions can be found in the
Chemistry 110 lab manual on pages 11 and 122.
Results
Table 1: Quantitative analysis
Identity of Sample
Volume of Sample Used
Mass of Empty Beaker
Mass of Beaker and Caffeine
Mass of Caffeine per serving
Coffee (caffeine)
100 mL
27.370 g
28.840 g
14.7 mg / mL
Table 2: Gas Chromatograph peaks with corresponding retention time and identity of molecules
Peak
1
2
3
Retention Time (min)
.803.
.844
1.497
Identity of Molecule
Diethyl Ether
Diethyl Ether
Caffeine
Table 3: Mass spectrum of caffeine and assigned fragment ions
Peak
A
B
C
D
E
F
G
m/z value
194.05
167.12
134.09
130.06
124.12
91.11
77.10
Identified Fragments
C8H10N4O2
C7H9N3O2
C7H8N3
C7H4N3
C6H8N2O
C6H5N
C6H5
Table 4: Mass spectrum of ethyl acetate and assigned fragment ions
Peak
A
B
C
D
m/z value
88.04
86.00
83.96
51.00
Identified Fragments
C4H8O2
C4H6O2
C4H4O2
C4H3
Discussion
The first step in the extraction process involves liquid-liquid extraction with immiscible
liquids. Liquid-liquid extraction is driven by the difference in chemical potential between the polar
and non-polar liquids3. The solution wants to be at a lower energy state which is what prompts the
transfer of solute. Diethyl ether was used as an organic solvent. Diethyl ether can form stronger
bonds and a more stable state in the non-polar solvent. Ethyl acetate is immiscible in water, so it
will form its own layer beneath the water after extracting the caffeine. Once the transfer of caffeine
is completed, the liquids are transferred to a separatory funnel to complete the process. Gas
chromatography is a powerful method that can separate mixtures and identify them based on
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retention times in the column. Solution is injected into the microscopic column and heated in an
oven where the molecules separate based on boiling points. The column is coated in a non-polar
silicon-based compound which ensures all the molecules injected will come out and not stick to the
inside. Diethyl ether has a lower boiling point and will exit the column first which was seen in the
first peak with a retention time of .844 minutes. The second and largest peak was caffeine based on
its 1.497-minute retention time. Immediately after chromatography, the sample was bombarded
with high energy electrons in the mass spectrometer. The multiple peaks show the possible
fragments of caffeine. Each smaller peak can be identified as a fragment of caffeine (as shown above
in table 3). The expected m/z value of caffeine is 194.05. The data collected had the highest m/z
value of 167.12 which is a fragment of caffeine without CHN. Since this is still an expected peak
resulting from caffeine, it can be concluded caffeine was identified, but in a small enough
concentration where only fragments were identified.
Conclusion
In conclusion, liquid-liquid extraction, gas chromatography, and mass spectrometry are
effective combinations for the separation and identification of a sample. The goals of the
experiment were met in identifying and separating caffeine. There was some variability in the mass
spectrometry of the experimental and expected values, this could be due to the lack of
concentration of the molecule. There were 7 peaks identified in the mass spectrometer. While
separating in the separatory funnel, some of the water remained with the diethyl ether due to
human error. This could have caused variability in the mass spectrometry as well as diluting the
sample making it less pure. Most of the fragments obtained are smaller than expected, which is due
to an amount of diethyl ether being present lowering the mass charge ratio. The diethyl ether
fragments are much smaller than caffeine which is what promotes the idea that it was present
because of smaller caffeine fragments than expected. This lab successfully verified gas
chromatography and mass spectrometry as methods to separate molecules from a substance. This
can be useful in many fields and industries needing separation. For example, the coffee industry
where caffeine needs to be extracted to produce decaffeinated coffee beans.
Supporting Information
Figure 1: Chromatogram experimental values
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Figure 2: Spectra experimental values
E
D
F
G
B
C
A
Figure 3: Spectra diethyl ether standard
B
D
C
A
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References
1. Mass Spectrometry
https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/spectrpy/massspec/masspec1.h
tm (accessed Mar 23, 2023).
2. Kautz, J.; Periago, J. Taking things apart…Putting things together In 110 Chemistry; Hayden McNeil
Lab
Solutions: Department of Chemistry, University of Nebraska-Lincoln. 17 November 2021.
3. Chaugule, A.; Patil, H.; Pagariya, S. International Journal of Advanced Research in Chemical
Science 2019, 6 (9).