Machado - REPORT GC-MS Biofuels

Machado et al 1
© 2015 Volume 1 Issue 2 – MJ0IAC. For course purposes only.
THE MOCK JOURNAL OF Article/Mini-Review
INSTRUMENTAL ANALYTICAL CHEMISTRY – CHEM 4123.11
Gas Chromatography and Mass Spectrometry (GC-MS)
Analysis of Synthesized Biofuels
John-Hanson Machado†**~, Grégoire Romano†~, & Susan Gillmor†^^
† The George Washington University Department of Chemistry
Received: October 16, 2015
Abstract: In this lab, biodiesel was synthesized from corn oil and methanol using two
separate conditions: 1:6/1 wt% and 1:6/3 wt% (Oil:Methanol, wt%= wt. KOH/wt. oil).
Using GC-MS matching spectra to a compound database, we found glycerin and fatty
acid methyl ester predominance in both reaction conditions as expected based on the
general chemical reaction for the production of biodiesel. Furthermore, we found that
the reaction condition differences were capable of synthesizing unique compounds
and even constitutional isomers.
Introduction
Gas chromatography (GC) is a technique used mainly for mixture separation but
can also be used for mixture identification.1 Since this lab’s sample is a liquid, gas-liquid
chromatography (GLC) will be reviewed, though it should be noted that gas-solid
chromatography exists and sometimes used in limited applications.1
There are two phases in GLC:
1. Stationary Phase
2. Mobile Phase
When the sample is injected into the GLC, the liquid is vaporized and is considered
to be in the mobile phase.2 Once heated to a gas, the sample is moved through a column
by an “inert” carrier gas. Typical carrier gases are noble gases such as Ar and He, though
some reactive gases such as N2 and H2 can be used2. It is of significance to note that

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there is currently a global He shortage making He a less attractive carrier gas.3 When
the gas reaches the column (many types depending on analysis), typically packed with
solids coated with a dense viscous liquid, the gas is in the stationary phase.4
Although GLC is typically used for sample separation with a tandem instrument
used for analysis, GLC is capable of making both quantitative and qualitative
determinations using information such as retention time and voltage output.4 There are
usually two “flavors” of columns employed in a lab: a polar and nonpolar column.
Specific physicochemical properties are tweaked in these columns as they are
manufactured for the unique lab applications. The unique column properties will
determine the retention time of each compound in a sample. For example, more polar
compounds will experience an increased interaction with a polar column resulting in a
longer retention time.4 Retention times of each compound are compared to the
retention time of a prepared standard and similar retention times may indicate a match
and compound identification.4 Factors impacting the retention times can include such
descriptors as molecular weight, percent ionization, and compound polarity which can
all be adjusted to (mis)match the column to alter separation. Resolution can be
increased by increasing the column length, decreasing the column diameter, changing
the column temperature, and adjusting the column flow rate by the carrier gas.
Quantitative measurements using GLC can be determined using an electronic
integrator which measure the areas under the signal intensity peaks.4 Signal intensity
peaks are based on output voltage in the detector. In general, the larger the peak area,
the higher concentration of the compound of interest is present. To move beyond
generalities, the peak areas must be adjusted based on compounds thermal
conductivities and ions produced with a number known as a response factor.4 Weight

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factors and mole factors may also need to be used for more quantitative measurements
using GLC.4 Given the ambiguities in both quantitative and qualitative determinations
using GLC, the technique is typically employed solely as a separation technique feeding
into a separate instrument, like mass spectrometry, for analysis.
High performance liquid chromatography (HPLC) is another separation
technique which can be more or less useful than GLC depending on the application and
instrumental budget. HPLC increases efficiency of typical GLC, allowing for better
throughput.5 HPLC uses high pressures to move samples through the phases of the
instrument faster while still allowing for high resolution. HPLC is built for consistency.
Using a polar/nonpolar solvent mixture, a liquid mobile phase allows for decreased
variation between experiments.5 Using either homogenous (solid-packed stationary
phase) or bonded (liquid stationary phase) column is coupled with a multi-piston pump
driving the sample and solvent for increased sample elution consistency.
Separation efficiency is how well the chromatographic technique is able to
temporally isolate sample compounds’ elution. Separation efficiency depends on a
concept known as a theoretical plate which involves the concept of volatility.5
Theoretical plates are derived from the original meaning in fractional distillation dealing
with the number of plates required for an equilibrium state maintenance in the
separation of a mixture.5 The number of theoretical plates in chromatography are
calculated by eq (1) where N is the number of theoretical plates, tR is the retention time,
and W is the peak width.5
𝑁 = 16(
𝑡 𝑅
𝑊
)2 (1)

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Maximizing the theoretical plates will optimize the separation efficiency.5 For the
greatest separation efficiency, the plate height must be reduced. The plate height is
determined using eq (2) the van Deemter equation where H is the plate height, A is the
constant representing the number of ways a sample can travel through the column,
B
υ
is
the system’s longitudinal diffusion factor, and Cυ is the mass transfer term related to the
adsorption/desorption constant for the analyte to the stationary phase.5
𝐻 = 𝐴 +
𝐵
𝜐
+ 𝐶𝜐 (2)
Since all terms are constants in eq (2), it is necessary to consider the variable all
the constants change with respect to: the flow rate.5 Where a high flow rate will be
effective in decreasing the longitudinal diffusion factor term, the same change will
increase the mass transfer term.5 It is therefore necessary to optimize the flow rate so
that the trade-offs between the mass transfer term and longitudinal diffusion factor
term result in the lowest possible plate height.
Calibration curves are useful so that the systematic variation between GLCs can
be accounted for. Every column is slightly different.5 As columns begin to age with more
samples being run through them, the columns begin to accumulate impurities from run-
to-run. Column impurities change the physicochemical properties that were set at the
time of manufacturing.5 By using a calibration curve with known concentrations of the
compounds of interest, it is possible to account for spectral changes between
experimental runs. GLC was used over HPLC in this experiment since GLC is more
appropriate for volatile compounds, a cheaper instrument, and a more developed

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technique than HPLC.1 Sensitivity of GC-MS and HPLC are comparable.6 However,
greater specificity of GC-MS also makes GC-MS the preferred technique in this
experiment.6
Mass-spectrometry was used in tandem with GLC in this experiment (possible
because GLC will present the sample in a gaseous state for MS analysis). Like GLC, MS
also depends on the sample mass. However, instead of separating the sample based on
the interaction with a column, MS utilizes the ionization in addition to the compound’s
mass for detection. When the gaseous compound enter the ionization chamber of the
MS, a beam of electrons accelerate at each compound resulting in discrete fragments of
each spatially separated group of compounds (from the GLC).2 The charged fragments
then enter a curved vacuum path with a magnetic field allowing for fragments to be
separated based on their mass to charge ratio.7 Now separated ionic fragments then
travel through a mass analyzer, typically by a time-of-flight analyzer or a quadrupole
analyzer. In time-of-flight analyzers, kinetic energy is kept constant and since kinetic
energy is determined by the mass and velocity, the larger ion reaches the transducer
more slowly. Time-of-flight analyzers are more expensive than quadrupole mass
analyzers which rely on alternating voltages to control which ions reach the transducer.7
Too much applied potential will result in bombardment of the ion with the rods of the
quadrupole. When this occurs, the ion annihilates completely due to recombination with
a conductive metal surface.7 MS has high specificity making MS the instrument of choice
for compound identification.
In this lab, GC-MS is used to separate and identify compounds produced in
catalyzed biodiesel synthesis using two unique reaction conditions: 1:6/1 wt% and 1:6/3
wt% (Oil:Methanol, wt%= wt. KOH/wt. oil).

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Methods
Biofuel Synthesis8
12.8ml(+/-0.20) of store-brand Mazola Corn Oil (density = 0.93g/ml) was
combined with 10.0 ml(+/-0.20) of pure methanol and stirred at which a 10ml(+/-0.20)
aliquot was added to conical flask (two conical flasks total, each with a 10ml aliquot). In
conical flask A, 0.5ml(+/-0.7%) of 2.91M(+/-0.0028) KOH in MeOH and 2.5ml(+/-
0.20) of pure MeOH were added to one aliquot yielding a concentration of 1:6/1 wt%
where wt%=wt. KOH/wt. oil. In conical flask B, 1.5ml(+/-1.3%) of 2.91M(+/-0.0028)
KOH in MeOH and 1.5ml pure MeOH was added to the other aliquot. Both conical flasks
were then heated in a water bath at 50.0°C for 30 minutes and stirred via magnets. Oil
phases were aliquoted out and the solution was brought to a neutral pH (tested by
litmus paper) using 6M HCl. Samples were then placed into auto-sampler tubes.
Auto-sampler
An AOC-20i+s autosampler (Manufacturer: Shimadzu) was set using parameters
suggested by the expertise of Susan Gillmor (author). The autosampler pre-set is
provided in Appendix A.
Gas Chromatogram
Gas Chromatography settings were set using parameters suggested by the
expertise of Susan Gillmor (author). The GC pre-set is also provided in Appendix A.

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Mass-Spectrometer
A GCMS-QP2010 with DI mass-spectrometer (Manufacturer: Shimadzu) was
used and set to parameters suggested by the expertise of Susan Gillmor (author). The
mass-spectrometer settings are also provided in Appendix A.
Results
GC-MS data of the two methods used for the synthesis of biofuels revealed
strikingly similar results. A simple comparison of Figures 1 & 2 shows the similarities in
retention times for various compounds isolated by the GC. Tripling the weight percent
of KOH:MeOH for synthesis purposes seemed to effect the concentrations of each
compound generated, but only slightly. Glycerin, for example, was found to have a peak
area (indicative of compound prevalence) of 10297090 and 10687867 for the 1:6/1wt%
and 1:6/3wt% conditions respectively. Retention time also varied between the two
experimental conditions where glycerin interacted with the column for 2.039 minutes
before elution for the 1:6/1wt% condition and 2.308 minutes for the 1:6/3wt%
condition.
Figure 1 GC results for the 1:6/1wt% condition for the synthesis of biodiesel. Note, the
first peak and second peak will be looked at in further detail using their respective MS
graphs.

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Figure 2 GC results for the 1:6/3wt% condition for the synthesis of biodiesel. Note, since
the first peak's MS is nearly identical to that of the 1:6/1wt% condition, only the second
peak will be analyzed in further detail in regards to its MS graph.
GC-MS analysis revealed that regardless of the reaction conditions selected,
glycerin and esters were produced. However, the two reaction conditions produced only
glycerin and hexadecanoic acid, methyl ester in common with relatively high
abundance. Nevertheless, constitutional isomers were produced specific to reaction
conditions. Using only the most abundant compounds analyzed in the database match
at least one pair of constitutional isomers was found: 9-octadecenoic acid, methyl ester
was produced in 1:6/3wt% conditions while cis-13-Octadecenoic acid, methyl ester was
produced in 1:6/1wt% conditions.
Figure 3 Mass-spectrum for the first GC peak (Figure 1) was identified as glycerin by the
MS database match.

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Figure 4 Mass-spectrum for the second peak (Figure 1) was identified as hexadecanoic
acid and methyl ester by the MS database match.
Figure 5 Mass-spectrum for the second peak (Figure 2) was identified as hexadecanoic
acid and methyl ester by the MS database match.
MS analysis (Figures 1-3) revealed relatively few differences between the two
experimental conditions. The MS graphs of hexadecanoic acid, methyl ester (Figures 1 &
2) in both experimental conditions were roughly the same. Both the 1:6/1wt% and
1:6/3wt% conditions revealed similar signal intensities (relative abundances) of
compounds for their respective fragments (m/z Ratio). This is an expected result in MS
because the probability of fragmentation at specific compound sites due to MS does not
vary from compound to compound. The observed intensity differences can be accounted
for due to chance alone. MS of glycerin (Figure 3) also is the least variant of all the
observed compounds because the disproportionally high probability for a single
fragmentation resulting in an m/z ratio consisting of two fragments with m/z 43 and 61.

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Due to low variation and publication restrictions on the number of spectral graphs, only
the MS for one experiment is shown for glycerin (Figure 2).
Discussion
The production of biodiesel has been proposed previously and is given in Scheme
1.8 Given Scheme 1 reveals that fatty-acid methyl esters are produced in conjunction
with glycerin, our results are consistent with theory since regardless of reaction
conditions, glycerin was produced. Glycerin was also the compound with the lowest
retention time in the GC (Figures 1-2) which is consistent with the observation that of
all the product compounds, glycerin has the lowest molecular weight. A low molecular
weight reduces the m/z ratio allowing for faster elution of the compound, thus
explaining why glycerin was the first peak in both MS graphs.
Scheme 1 General reaction for the catalysis of vegetable oil into biodiesel. This
experiment was base catalyzed and used corn oil as the specific vegetable oil.8
The peaks (Figure 3) can be explained based on the fragmentation of glycerin in
the mass-spec. If the fragment charge is one, the m/z ratio can be understood as simply
the mass. Therefore the pear measuring 61 is around 31amu less than the molar mass of
glycerol, thus it is reasonable to assume that the fragmentation resulting in that peak,

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and accounting for isotopes, would be due to the cleavage of a bond between carbon 1
and carbon 2 of glycerol. Explanations of the other peaks would follow similar logic.
Peak broadening is often an issue in GC. Glycerin, for example, was found to have
a peak area (indicative of compound prevalence) of 10297090 and 10687867 for the
1:6/1wt% and 1:6/3wt% conditions respectively. Looking at Figure 1 and comparing to
Figure 2 it is apparent that the height of the peak (signal strength) is vastly different.
The GC of the 1:6/3wt% shows less peak overlap and broadening, indicative of fewer
impurities in the solution, including water.
Retention time also varied between the two GC experimental conditions where
glycerin interacted with the column for 2.039 minutes before elution for the 1:6/1wt%
condition and 2.308 minutes for the 1:6/3wt% condition. Retention time variations
could be caused by a number of reasons including fluctuations in the carrier gas
pressure, changes in voltage due to power demands in the building, or column
degradation resulting in a systematic error. It is unlikely that the column degraded
between the two runs (otherwise we would expect a systematic error) and is thus more
plausible that proximal changes in voltage or carrier gas pressure resulted in the
observed retention time differences.
Other areas of error are apparent in the overlap of signals in the GC. To prevent
the overlap and allow for more careful analysis (better separation), a lower pressure and
longer experimental runtime should be performed, though it should be noted that this
will also decrease throughput.2 The corn oil used in this experiment was also crude
(straight out of the bottle). Distillation and other separation techniques may also reduce
noise associated with the experimental results. The feed lines should also be checked

Machado et al 12
regularly to make sure that the carrier gas is not introducing impurities and causing
increased experimental noise and peak broadening. For increased sensitivity, another
method which could be used is MS/MS, though this analytical method is costly.2
Biodiesel is relevant when the costs of fossil fuels are high.9 The lower power
density provided by biodiesel compared to fossil fuels and the source used are all
important factors to consider when a final life cycle assessment is performed to
determine its net value added to society. Scarcity of land in some regions make the
power density of biodiesel a concern for meeting increasing energy needs. Another
important factor to consider is the initial reactants for the production of biodiesel.
Should a food source, such as corn oil used in this experiment, be use as a fuel, there
may be a decreased supply in food raising costs and having burdensome changes on
lower socioeconomic classes.9
The observation that changing synthesis conditions resulted in different biodiesel
contents is relevant to the field of biotechnology. If the demand for one compound over
another is higher and thus worth more money, one reaction condition may be favored
over another. Although biodiesel from corn oil may not be the answer for the energy
crisis, biodiesel may play even a small role in increasing energy demands. Corn
production continues to rise annually without any indication that this trend should slow
down. The information provided in this report begins to answer questions about the
optimal reaction conditions needed for biodiesel integration through corn oil.

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Author Information
Corresponding Author
** E-mail: jhmachado@gwmail.gwu.edu
Author Contributions
~These authors contributed equally in the lab
^^This author is the course instructor
Notes
The authors declare no conflicts of interest.
References
1. Skoog, D. A., Holler, F. James, & Crouch, Stanley R., Principles of Instrumental
Analysis Sixth Edition. 6 ed.; David Harris: 2007.
2. Douglas, F. GC/MS Analysis.
http://www.scientific.org/tutorials/articles/gcms.html.
3. Magill, B., Why is there a helium shortage? Popular Mechanics 2015.
4. Roberts, R. M., Gilbert, John C., & Martin, Stephen F., Experimental Organic
Chemistry: A Miniscale Approach. The George Washington University ed.; Cengage
Learning: Mason, Ohio, 2002.
5. Barkovich, M., High Performance Liquid Chromatography. In UC Davis
ChemWiki, Online: chemwiki.ucdavis.edu, 2015.
6. Phillips, D. L., Tebbett, Ian R., & Bertholf, Roger L., Comparison of HPLC and
GC-MS for Measurement of Cocaine and Metabolites in Human Urine. Journal of
Analytical Toxicology 1996, 20, 305-308.
7. Nemes, P., Lecture 6 - Atomic Mass Spectrometry. In Instrumental Analytical
Chemistry: CHEM 4122, The George Washington University: 2015.
8. Miller, T. A. L., Nicholas E., Microwave Assisted Synthesis of Biodiesel in an
Undwergraduate Organic Chemistry Laboratory Course. The Chemical Educator 2009,
14, 98-104.
9. Machado, J.-H., Biotechnology: Second Examination. David Morris, P., Ed. The
George Washington University: 2015.

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Appendix A
Setting Value
# of Rinses with Solvent (Pre-run) 2
# of Rinses with Solvent (Post-run) 2
# of Rinses with Sample 2
Plunger Speed(suction) High
Viscosity Comp. Time 0.2 sec
Plunger Speed(injection) High
Syringe Insertion Speed High
Injection Mode 0: Normal
Table 1 Autosampler settings for biofuels analysis.

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Setting Value
Column Oven Temp 125.0 °C
Injection Temp 225.0 °C
Injection Mode Split
Sampling Time 1.00 min
Carrier Gas He Flow Control Mode Pressure
Carrier Gas He Pressure 83.5kPa
Carrier Gas He Total Flow 52.4 mL/min
Carrier Gas He Column Flow 1.01mL/min
Carrier Gas He Linear Velocity 37.8 cm/sec
Carrier Gas He Purge Flow 1.0mL/min
Carrier Gas He Split Ratio 50.0
Program Column Oven Temperature
Total Program Time 23.5min
Column Length 30.0m
Column Thickness 0.25µm
Column Diameter 0.25mm
Table 2 Gas-chromatograph settings for biofuels analysis.

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Setting Value
Ion Source Temp 250°C
Interface Temp 260°C
Solvent Cut Time 1 min
Micro Scan Width 0µ
Detector Voltage Setting Relative to Tuning Result
Threshold Voltage 20kV
Table 3 Mass-spec settings for biofuels analysis

Machado - REPORT GC-MS Biofuels

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