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CHM 5570
Preformed 02.19.2016, 02.26.2016
Turned in 03.08.2016

David Thomas Sloss (With Yahiya Saif, Vishwas Tiwari, Gregory

Rosenhauer, Nivedita Singh, and Brendan Blazejewski )

The use of artificial spices that mimic cinnamon is prolific and ubiquitous in the US market, the
most common being cassia. The purpose of this experiment is to determine whether an unknown
solution of cinnamon extract contained actual cinnamon or an analogue molecule that mimics
cinnamons unique smell and taste. This lab will consist of multiple analytical techniques to
determine the presence or absence of certain molecules.

Using a variety of techniques that includes gas chromatography (GC) in conjunction with a mass
spectrometry (MS), we will create an index that will comprise all ingredients found in both pure
cinnamon, cassia, and aggregate forms. An index will be created and used to analyze locally
purchased produced labeled as containing cinnamon, using GC and MS confirmation. By
alternating between samples containing cinnamon compounds and a sample containing
hydrocarbons (octane to eicosane) the order at which various compounds in the sample are eluted
can be determined. The order of elution will remain constant for the duration of the experiment
and will help identify compounds in unknown samples. Cinnamon and cassia differ in several
areas; cinnamon is characterized by the presence of eugenol and benzyl benzoate as minor
components that are absent in cassia GC. For this experiment a local cinnamon extract will be
tested for any indications that the extract isnt pure cinnamon. Analyte indicative of cassia
contain Coumarin and -cadinene that are either absent or only trace amounts detected with
cinnamon. In addition, 2-methoxycinnamaldehyde, Cinnamyl acetate, Cinnamyl alcohol,
cinnamic acid, benzaldehyde, 3-phenylpropanal, and 2-phenylethanol are common to both and
will also be used to identify the unknown solution.

Gas chromatography is a notable analytical method that separates compounds from complex
samples with a liquid stationary phase and a gas
mobile phase. The rates and orders at which
compounds are eluted from the column depends
on the molecules various chemical and physical
properties, and how said properties interact with
the stationary phase. For part A, the GC is used
in conjunction with a flame ionization detector,
a detector only applicable for hydrocarbon or
organic containing compounds as it works by
carbons ability to form cations and electrons
upon pyrolysis. The electrons generate a current Figure 1: A GC-MS schematic [2]
between electrodes that are adjacent to the flame, the increase in current appears as a peak on the

For Part B, to ensure accuracy a Gas chromatography Mass spectrometry instrument was used
which combines various features of GC and MS to determine the identities of various substances
within a test sample. The mass spectrometer used electron ionization (EI) as a form of ionization.
In EI, the molecules enter the MS and are subsequently bombarded by electrons that causes the
molecule to fragment in a characteristic and reproducible manner. [1] This form of ionization is
considered a hard ionization technique and results in a myriad of fragments with low mass to
charge ratios (m/z). By comparing the pattern of molecular fragmentation to online libraries the
identities of the molecules can be confirmed.

Methods and Results

Part A

A retention index was established by testing 4 standard solutions over the course of 20 to 30
minutes, a time determined to be sufficient for suitable separation of compounds. In lab it was
found that 20-minute time span was sufficient for full separation of all compounds. The HP5
column contained 5% phenyl methanol. The standards were prepared in a solution of acetonitrile,
alkane solutions in hexanes, ranging from octane to icosane (C8-C20). The initial temperature
(Ti) was set at 40C, with a temperature change (T) of 20C/min, and a final temperature (Tf) of
250C with a four minute hold. The inlet temperature was set to 230C and the column was ran
with nitrogen and air. The FID (Flame Ionization Detection) gas used was
hydrogen. The standards were injected with a 10C syringe rinsed with acetonitrile with a total
volume of 1.0C solution per injection. A spectra of abundance as a function of time is
established. Calculation of the retention index required sequential injections of a standard
mixture of n-alkanes and then standards and extracts, so that approximate times of various
carbon chains can be be used to correctly identify standard elution times. Therefore, GC analysis
was performed by alternating the hexanes solution and the standards in the following order: SM-
2, SM-3, SM-2, SM-4, SM-2. The results of GC alone arent sufficient for confident analysis;
thus the experiment was performed in conjunction with FID mass spectroscopy. The results of
the retention index are found below on table 2 in appendix A and the retention index is found

Table 1 Retention Index Library

1 5213.4 5.713 106.121 0.826133807
2 5957.8 7.006 154.13 0.944094064
3 6132 8.609 132.16 0.971698412
Cinnamyl Alcohol
4 5184.4 8.887 134.17 0.821538364
5 4841.3 9.258 164.2 0.767169524
cinnamyl acetate
6 5214 9.893 176.21 0.826228885
7 3904.1 10.053 146.1427 0.618657497
8 Methoxycinnamaldehy
6178 10.6 162.1852 0.978987735
benzyl benzoate
9 6310.6 12.194 212.25 1
Graph 1 shows the linear
relationship between reference Index value vs RT
index values and retention time, 2000
where the x axis is the retention y = 122.93x + 299.3
1800 R = 0.9659
times and the y value is the index
value, calculated from equation 1. 1600
An outliner occurs around 10.50
minutes where the retention index
value calculated for Cinnamyl 1200
Acetate and Coumarin appear to
be reversed. However, it is 6 7 8 9 10 11 12 13
possible that ring formation or
ring breaking may have occurred. This is examined more in the discussion section. Graph 1

Part B

Gas Chromatography-Mass Spectrometry

is used to confirm the identities of
compounds identified from the retention
index. The mass spectrometer was
calibrated by leaking
perfluorotributylamine through the
reference reservoir into the ion source. The
PFTBA is pumped away and the
compound tristrifluoromethyltriazine
(METRI) is injected into the GCT
reference reservoir. This calibration will
correct for any drift that would be
observed due to temperature changes. Our
samples were diluted 100 fold with
acetonitrile, and injected 1 L into Agilent
2 mL auto sampler vials. The vials were
labeled and their position in the auto
sampler were recorded. The scan time was Figure 2
set at 12 minutes. The results of the gas chromatograms for the standard mixture and unknown
are found below, and the mass spectrometry data is located in appendix B. The analysis of the
unknown extract is outlined in detail below.
Figure 2 shows the GC-MS spectra
obtained for the standard mixture
solution. The peaks are marked based on
the retention index, and MS data. The
peaks were analyzed using software in
accordance with the procedure, allowing
the individual peaks to be analyzed from
the MSEI data. The trends in ion
fragmentation were confirmed by
visually examining peak patterns from
the national library.

Figure 3 is the GC-MS spectra for the

unknown solution, determined to be
pure cinnamon. The major peaks just
past the 9-minute mark were determined Figure 3
to be Eugenol and Cinnamyl acetate, the minor peak analyzed was benzyl benzoate which
was eluted at approximately 11.57 minutes. To confirm that the analysis and assignment of the
large peak to eugenol is correct it was crosschecked with EI library data for eugenol, this is
explained below.


The fusion of gas chromatography and Mass spectrometry is a very powerful analytical tool that
allowed us to unquestionably determine that the cinnamon extract contained pure cinnamon.
Because most molecules tested only differed by only a carbon or two certain fragmentation
patterns became quickly evident by working out the structures of the ionized molecule as it is
broken down by electrons. By crosschecking online reference patterns, MS in conjunction with
GC allows structure determination with high confidence.

Based on the retention index established, relative peak area, and MS data, the botanical source of
the cinnamon extract is from Cinnamomum Zeylanicum. This can be confirmed by the presence
of eugenol (App.B, Fig10), trace amounts of Coumarin (App.B, Fig12), and an absence of
cadinene. The elution time was consistent with the index established earlier and the MS data was
confirmed via EI indexes online, the EI information for Eugenol can be found in appendix B,
figure 13.

Based on the relationship between the index values and the retention time, a linear relationship
was established with the form [y=122.93x+299.3] where x is the retention time and y is the
retention index value, from this a suitable retention index window of 20.44 minutes to 35.04
minutes would suffice to identify the various individual compounds using the gas
chromatography method outlined in Method A.
From the 2 prominent peaks in Figure 2, the first is what is suspected to be eugenol, could also
possibly be acetonitrile, the substance used to dilute the standard solutions. The source of
contamination may have been from carelessness. The EI spectral data is attached in appendix B,
figure 13. The second prominent peak could also fit the criteria of dimethyl phthalate, and the
data in table 1, appendix B suggests the patterns are very similar. Dimethyl Phthalate is used in a
variety of applications and can be found in many plastic products. It is possible some of the
cinnamon extract became contaminated.

A strange anomaly is present in the data in table 2, App. A, where the I value calculated for
Coumarin and Cinnamyl acetate are out of numerical order, and its possible the compounds
were misidentified in lab when the retention index was made. This obviously had an impact on
the plot of peak vs I value (App. A, Graph 1.), and by reversing the order of Cinnamyl acetate
and Coumarin the r squared value for the linear trend was increased from 0.963 to 0.966.

There are many advantages of using a GC-MS system, but there are a few disadvantages. First,
the length of time required to accrue sufficient data for the retention index is time consuming. In
addition, there are limits on which types of molecules GC-MS is able to analyze, as nonvolatile,
polar and thermally labile compounds are undetectable. Despite this. GC-MS is a powerful tool
capable of separating and identifying all ingredients in a unknown sample.

Appendix A

Table 1.
PEAK Time Area MW
1 5.713 5213.4 Benzaldehyde 106.121
2 7.006 5957.8 Linalool 154.13
3 8.609 6132 trans-cinnamaldehyde 132.16
4 8.887 5184.4 Cinnamyl Alcohol 134.17
5 9.258 4841.3 Eugenol 164.2
6 9.893 5214 cinnamyl acetate 176.21
7 10.053 3904.1 coumarin 146.1427
8 10.6 6178 2-Methoxycinnamaldehyde 162.1852
9 12.194 6310.6 benzyl benzoate 212.25
8 2.948 164.6 108
9 3.916 206.3 122
10 4.941 235.7 136
11 5.957 255.5 150
12 6.912 272.5 164
13 7.799 281.3 178
14 8.626 301.8 192
15 9.401 324.2 206
16 10.131 344.7 220
17 10.52 352.3 234
18 11.472 385.4 254
19 12.128 434.7 270
20 12.801 415.2 288
1 6.963 575.2 linalool 154.13
2 8.842 3196.4 Cinnamyl Alcohol 134.17
3 9.988 2281.4 2-Methoxycinnamaldehyde 162.1852
4 10.614 8749.4 Coumarin 146.1427
1 5.693 1845.5 benzaldehyde 106.121
2 8.576 1942.77 cinnamaldehyde 132.16
3 9.251 4412.9 eugenol 164.2
4 9.995 8799.26 cinnamyl acetate 176.21
5 12.238 12710.8 benzyl benzoate 212.25
1 5.678 10 benzaldehyde 106.121
2 6.693 147.7 linalool 154.13
3 8.554 115.2 cinnamaldehyde 132.16
4 8.685 105.4 cinn-oh 134.17
5 9.174 7445.5 Eugenol 164.2
6 9.795 164.3 Cinnamyl Acetate 176.21
7 9.838 93 coumarin 146.1427
8 10.046 36.2 2-Methoxycinnamaldehyde 162.1852
9 10.561 43.37 benzyl benzoate 212.25
10 12.137 344.5

Table 2.
Peak Compound Area Time MW Relative areas #carbons I
1 Benzaldehyde 5213.4 5.713 106.121 0.826133807 7 NA
2 Linalool 5957.8 7.006 154.13 0.944094064 10 1203
3 trans-cinnamaldehyde 6132 8.609 132.16 0.971698412 9 1357
4 Cinnamyl Alcohol 5184.4 8.887 134.17 0.821538364 9 1385
5 Eugenol 4841.3 9.258 164.2 0.767169524 10 1425
6 cinnamyl acetate 5214 9.893 176.21 0.826228885 11 1512.14
7 coumarin 3904.1 10.053 146.1427 0.618657497 9 1498.73
8 2-Methoxycinnamaldehyde 6178 10.6 162.1852 0.978987735 10 1557
9 benzyl benzoate 6310.6 12.194 212.25 1 14 1860.4

Equation 1 - I = 100z + 100[TE(X) TE(Z)] / [TE(Z+1) TE(Z)]

[1] - http://www.massbank.jp/jsp/Dispatcher.jsp?type=disp&id=JP003484&site=10
[2] "Critical Mass: A History of Mass Spectrometry". Chemical Heritage
Foundation. Retrieved 23 January 2015.
[3] - Wiley's Scientific, Technical, and Medical Databases: Home. wiley.com