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(𝑡𝑅

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

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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: [email protected] 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.

mailto:[email protected]

http://www.scientific.org/tutorials/articles/gcms.html

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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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