Gas Chromatography Method Development: Determination of an Anisole Mixture

Introduction:
Background:
A musty taste and/or odor is one of the reasons consumers will reject a wine, therefore the wine
industry needs to ensure that there are reliable analytical methods in place to detect the chemical
compounds responsible for this musty odor. Many of the compounds shown to be responsible for so
called cork taint are chlorinated anisoles. These compounds form from chlorophenols which can be
present as a product of water disinfection or from the pesticides present in the wood used in the
wineries. It is also possible that the cork itself contains chlorophenols due to cork manufacturer
disinfection practices. The chloroanisole forms in the cork due to the action of microorganisms which
convert the cholorphenol to the less toxic chloroanisole by o-methylation. The chloroanisole will then
leak from the cork into the wine. The sensory perception ( levels at which off-flavor can be detected) is
quite low, ranging from as low as 1 ng/L to 50 ng/L
1
and so a sensitive analytical method is needed to
detect these compounds in wine. In this experiment, three of the halogenated anisoles that have been
shown to contribute to the musty odor in wine will be determined by gas chromatography mass
spectrometry (GC-MS).
1
Riu, M., Mestres, M., Busto, O. and Guasch, J.: Quantification of chloroanisoles in cork, using
headspace solid-phase microextraction and gas chromatography with electron capture
detection. J. Chromatogr. A,
.
(2006) 1107, 240-247
Instrumental:
Gas chromatography (GC) is a separation method that is particularly useful for the analysis of mixtures
of volatile compounds. Used extensively in the environmental industry, the method is also used in the
pharmaceutical, food and agricultural industries for the evaluation of contaminants and residues. All gas
chromatographs (this is what the instrument is called) require a detection system (more commonly
called a detector) to identify the analytes. There is an extensive array of detection systems for GC;
including flame ionization (FID), electron capture (ECD) and mass spectrometry (MS). In capillary GC,
upon introduction into the injector, a sample is volatized and then pushed along a capillary column
(silica tube that is several meters long and has a very small diameter) by virtue of a carrier gas. The
components of the sample are separated along the column according to their relative boiling points and
detected at different times using one of the detection systems mentioned above. In this instrument, the
compounds are detected by mass spectrometry by virtue of their mass to charge ratio. In this ion trap
mass spectrometer, the gaseous compounds are bombarded with electrons, which results in the
formation of ions which in turn move toward the detector at different rates based on their mass. One of
the significant advantages of GC-MS its two dimensional identification capability: a compound can be
identified by comparing the analyte retention time on the chromatogram (this is what the output graph
is called) to that of a standard, and by its mass to charge ratio which will typically be the same as its
molecular mass.
In this experiment, we will investigate the effect of various GC parameters on the separation and
retention of anisoles and anisole mixtures.
Procedure:
Sample Preparation:
Using the information in the introduction (which is based on previous research) determine an
appropriate concentration for each analyte for the development of a useful method. Prepare a solution
of each analyte at the appropriate concentration and one sample containing a mixture of the three
analytes (equal amounts of each). Analyze each sample by GC-MS using solvent as the blank. If the
peak height or area of your analyte is significant, dilute the sample and inject it into the GC to obtain a
lower/smaller peak.
Sample Analysis:
Set up the following method on the GC.
Carrier gas flow rate: 1.0 mL/minute
Injector temperature 260
o
C
Oven temperature program:
Initial temperature 40
o
C hold for 2 minutes
Ramp to 265
o
C at 12
o
C/minute
Hold at 265
o
C for 1 minute
Sample volume 2.0 L
On the MS portion include a solvent delay of sufficient time to ensure the solvent peak does not appear
in the chromatogram. Leave other MS parameters at settings found.
Method Development
Do each change independently.
1. Change the initial temperature to a value approximately equal to that of the analyte with the
lowest boiling point.
2. Change the ramp rate for the oven to 15
o
C/minute.
3. Change the mode to acquiring in split mode using a split ratio of 25
4. Change the carrier gas flow rate by decreasing and increasing it, do not use a value lower than
0.5 mL/minute or one higher than 3 mL/minute.





Reporting:
Include the answers to the following questions in your results and discussion section. Ensure that the
chemical structure of each analyte is present in your report.
1. Describe and explain how each change to the GC-method affected the chromatogram. The
factors to include in the discussion are retention time, peak shape (peak height and width) and
resolution (if applicable).
2. Using a chromatogram of each analyte of the lowest concentration, determine the detection
limit of the unmodified chromatographic method by hand (1. Use the height of the peak as
the signal and the average height of the blank in the region as the noise to calculate the signal to
noise ratio. 2. Take the detection limit as the concentration of the analyte that would yield a
signal of 3 x S/N).
3. Comment on the effect of changing the parameters on the signal to noise ratio. This may be a
qualitative or a quantitative discussion. Regardless of the method you choose, your discussion
must be THOROUGH.
4. Using the library search feature of the instrument, determine and report the probability of the
identification of each analyte. Considering the results, state at least one limitation of mass
spectrometry as a detection method.