GC/MS ANALYSIS
2003 REVISION
OBJECTIVE: The volatile components of a foodstuff such as wine are
isolated and concentrated by headspace analysis. The components
in the concentrate are separated by capillary gas chromatography
and the mass spectrum of each component is measured. The mass
spectra are used to identify the components.
REFERENCES:
Chapter 2 in R. M. Silverstein, G. C. Bassler, and T. C. Morrill,
Spectrometric Identification of Organic Compounds, 5th. ed.,
Wiley, New York.
F. W. McLafferty, Interpretation of Mass Spectra, 3rd. ed.,
University Science Books, Mill Valley, California, 1980.
Mass Spectrometry Data Centre, Eight Peak Index of Mass Spectra,
2nd. ed., Aldermaston, U.K., 1974.
W. G. Jennings, ed., Applications of Glass Capillary Gas
Chromatography, Chromatography Science Series, Volume 15, M.
Dekker, New York, 1981.
H.-D. Belitz and W. Grosch, Food Chemistry, 2nd. ed., Springer
Verlag, Berlin, 1987.
"Solid Phase Microextraction: Theory and Optimization of
Conditions", Technical Note T198923 (Bulletin 923), Supelco,
Bellafonte, Pennsylvania, 1998.
http:sigma-aldrich.com/Brands/Supelco_Home/Technical_Library/
Literature.html
"Solid Phase Microextraction Troubleshooting Guide", Technical
Note T101928 (Bulletin 928), Supelco, Bellafonte, Pennsylvania,
2001.
http:sigma-aldrich.com/Brands/Supelco_Home/Technical_Library/
Literature.html
CHOICE OF METHOD:
Two methods of isolating the volatile components are now available
in the experiment: headspace analysis (properly called purge-andtrap with our adaptation) and solid-phase microextraction (SPME).
In the former, nitrogen is passed through or over the sample, the
volatiles are trapped and dissolved in a small volume of methanol.
Multiple injections are possible in this mode but the sample is
diluted by the solvent. In the latter, an adsorptive fiber is
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immersed in the sample or a slurry of the sample and the analytes
are adsorbed on its surface. The fiber is injected manually in
the gas chromatograph and all of the adsorbed molecules are
quickly evaporated. This approach is very effective in
concentrating the sample and is not limited to the most volatile
components. However, a separate preparation is required for each
injection.
CHOICE OF SAMPLE:\
Discuss the selection of you sample with the instructor before you
begin the experiment. Poor results are guaranteed with a poor
selection of unknown.
The experiment should work with any odiferous foodstuff.
Excellent results have been obtained with vintage wines although
any odiferous beverage should be amenable to attack. However, if
the beverage easily forms a foam, one must use the SPME method.
Solids can also be studied for headspace analysis by chopping up
the material and placing the pieces in the washing bottle. In the
case of SPME, one can either create an aqueous slurry with a
blender and insert the fiber in the slurry or place the chopped-up
pieces of the material in a closed vial and insert the fiber into
the vial.
Fresh mint, pineapple, orange rinds, coffee, and ripe
bananas have yielded excellent results. Attempts to characterize
chocolate failed. Use fresh, ripe material. Fruits lose most of
their volatiles in the canning process. Bay leaves plucked off a
California Mountain Laurel yield far more results than dried
leaves from a store-bought bottle.
METHODS OF ISOLATION OF THE VOLATILES
You will chose the method of isolation in your pre-lab meeting
with the instructor.
I) HEADSPACE ANALYSIS:
The apparatus used to isolate and concentrate the volatile
components of the sample is sketched in Figure 1 at the end of
this document. Nitrogen is passed through a washing bottle filled
with the sample. The exit stream contains the volatile components
which are trapped by a column filled with a solid support, Tenax
in our case. Chromosorb 101 and 102 are also available as solid
supports. After the volatiles have been collected, the column is
heated and backflushed and the released material is collected in a
trap cooled with liquid nitrogen. The components used in each step
of the collection and the conditions are given in Table 1.
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Table 1
Suggested Parameters for Each Step of the Headspace Analysis
__________________________________________________________________
Step Washing
Position
Level
Heater
Length
Trap
Bottle
of the
of the
(Variac)
of the (used
(used
Collection
Flowmeter
Setting
Step
or not)
or not)
Tube(1)
Ball
(min.)
__________________________________________________________________
1
on
A
10.0
off, 0
60
off
2
off
A
7.5
off, 0
30
off
3
off
B
7.5
on, 35
30
on
4
off
B
7.5
on, 35
30
off
__________________________________________________________________
(1) B indicates that the B end of the collection tube is connected
to the nitrogen flow (the orientation shown in Fig. 1). A
indicates the reverse orientation.
The procedure consists of the following steps.
Step 1. Fill the washing bottle with the sample. If it is a
liquid, use ca. 100 ml. If it is a solid, the sample should be
finely divided. The A end of the sample collection tube is
connected to the outlet of the washing bottle and nitrogen is
allowed to flow through the washing bottle for 60 minutes. Water
and other volatile components are swept out of the bottle and
trapped on the column.
Step 2. The washing bottle is removed from the system and the
nitrogen flow is continued for another 30 minutes. Most of the
water is swept out of the collection tube during this period. The
bulk of the sample should remain at the A end of the tube since it
is at room temperature.
Step 3. The collection tube is reversed so that the B end is
connected to the outlet of the flowmeter and the U-shaped trap is
connected to the A end. Purge the trap with the nitrogen flow for
2 minutes. Then cool the trap with liquid nitrogen in a small
Dewar flask and turn on the heater. At the elevated temperature,
the sample will be purged from the collection tube. Collect the
sample for 30 minutes.
After the sample has been collected, light the oxygen-natural gas
torch and then disconnect the trap from the collection tube but
keep it immersed in liquid nitrogen. Quickly seal off at the
upper constriction in the trap with a glass torch, cover the open
end of the collection tube with Parafilm, and remove the trap from
the liquid nitrogen after the hot sealed-off end has cooled. Cut
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the trap at cutting point one. This point should be above the
condensate in the trap. Wash the material collected with a few
drops of methanol. Invert the trap so that the solution collects
at the sealed end. Cut the tube at cutting point two and transfer
the solution to the mini MS sample tube which has small plastic
feet. Place the sample tube in a sample vial and cap the vial
with a cover with a septum. If any material remains in the
collection trap, cover the open end with parafilm.
N.B. You do not want to use too much methanol to dissolve the
sample. Premeasure how many drops of methanol are required to
almost fill the mini sample tube. Use this and no more to
dissolve the condensate.
Step 4. After the trap has been removed, continue the flow
through the heated collection tube for another 30 minutes to
insure that all material has been swept from the tube. Turn off
the heater and allow the tube to cool before the system is
disassembled.
This procedure can be modified depending on the sample. Consider
the case of analyzing mint leaves. In contrast to wine or a juicy
orange, the water content is not high. Hence, one can dispense
with the Tenax trap and directly collect the volatiles in the Utube cooled in liquid nitrogen.
II) SOLID-PHASE MICROEXTRACTION
This handout will briefly describe the use of our SPME unit which
in its first use gave excellent results on coffee. Read the
technical notes on the Supelco Web site for details. The working
part of the SPME unit is a fragile fiber that is coated with an
absorptive coating,presently 75 m of Carboxen/
polydimethylsiloxane (CAR/PDMS. When the unit is not in use or is
being injected through a septum, the fiber is withdrawn into a
protective needle. During the adsorption or desorption step, the
fiber is extended. The following simplified procedure has worked
well.
A) Removal of material pre-adsorbed on the fiber.
The previous user should have cleaned the fiber but one should not
make this assumption.
1) Move the sample changer on the GC-MS unit to the
side.
2) Load the method spme_bg.m
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3) Turn off the flow saver and change the temperature
of the GC injector from 280C to 300C.
4) Make sure that the fiber is withdrawn into the
protective needle. The Adjustable Depth Gauge should be
at the 3.0 position.
5) Manually inject the needle through the septum on the
gas chromatogram.
6) Expose the fiber to the stream of hot helium in the
injection port by pressing down on the plunger of SPME
fiber holder and locking the plunger.
7) Wait 60 minutes for full desorption to occur.
8) Unlock and release the plunger on the SPME fiber
holder. The fiber is now protected.
9) Remove the unit from the injection port of the GC.
10) Lower the temperature of the injector to 280C and
enable the flow saver.
(You will also repeat these steps at the end of the
experiment.)
B) Confirm that the fiber is clean with a background run.
This step will be a practice run on the use of the Hewlett-Packard
GC-MS. Refer to the following section that is written for a
headspace analysis but note the changes to apply to SPME. In
particular, you will be performing a manual rather than an
automatic injection.
1) Move the automatic sample holder off to the side if you
have not already done so.
2) Run the autotune procedure. You only need to do this once
each day.
3 Load the method spme_bg.m.
4) Set parameters and prepare the instrument for manual
injection. The instructor will review the instrumental
parameters at this time. This outline will take you through
a maze of menu options and mouse clicks.
Method
Edit Entire Method
Instrument/Acquisition (Check this option.)
(The other options should not be checked.)
OK
Sample Inlet: GC
Injection Source: Manual
Injection Location: Front
Check the box labeled Use MS.
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OK
Real Time Plot
Signal 1
Attenuation: 0
Offset: 10%
Time: 5.0 min.
(These are the default values.)
OK
MS Tune: ATUNE.U
(You are selecting the file for the
tuning that was defined in the autotune
sequence.)
OK
You will now be prompted to view and
change the GC settings.
OK
You will now be prompted to review and
change the MS SIM/SCAN settings. Set the
solvent delay to 0.1 minutes. There will
be very little solvent to be desorbed
from the fiber so a very short delay is
possible. Consequently, you will not
miss rapidly eluting components.
OK
Finally save the method. Don't change the
path or the extension. The file name should
not exceed 8 characters. Do not use spme_bg
for the file name.
5) To execute a run, complete the following sequence of
steps.
a) Mouse clicks and keyboard entries.
Method
Run
Provide a unique file name (up to 8 characters
but don't change the extension D or the path.)
Run Method (Here, don't click on OK.)
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b) At first a message "Waiting for GC" will appear. The
GC is being is initialized. Do not bypass this step.
Be patient and wait. The message will disappear when
the GC is ready.
c) Wait for a second message window dealing with manual
injection to appear. Press as prompted the Prep Run
button on the righthand keypad of the GC.
d) At first the message on the GC will indicate that it
is not ready. Wait until the message changes to "STATUS
Ready for Injection".
e) Insert the SPME holder as instructed in step A above.
Press down on the plunger and immediately press the
Start button on the GC.
f) You have initiated a run. After 30 seconds of
exposure of the fiber to the flow of hot helium in the
injection port, retract the fiber and remove the SPME
holder from the GC inlet. The length of the exposure
time is a parameter that you might change.
g) After the GC run has been completed, examine the
total ion chromatogram and the mass spectrum at selected
elution times. Determine if further preconditioning of
the fiber is necessary.
C) Collection of adsorbables from the sample.
1) Unless necessary, we shall store the sample
vial or beaker. Using a clamp, mount the SPME
the beaker or vial. Adjust the height so that
does not contact the sample but the fiber will
plunger is depressed.
in an open
holder above
the needle
when the
2) Depress and lock the plunger.
3) Allow 15-30 minutes for adsorption to occur.
4) Retract the plunger to protect the delicate fiber.
5) Analyze the adsorbed material with the GC-MS as described in
step B above. The first run should be conducted with the
conventional 40:1 split. Use the first run to plan for further
runs. Since a quantitative analysis is not our goal, cleaning of
the fiber between runs is probably not necessary.
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D) When you are finished with the analysis, clean the fiber as
described in step A above.
USE OF THE HEWLETT-PACKARD GC-MS FOR HEADSPACE ANALYSIS:
The components of the concentrate are separated on a capillary
glass column whose temperature is varied according to a linear
program. The mass spectrometer is used as a detector for the gas
chromatograph.
1) Place the sample vial prepared above in one of the slots of the
automatic sample changer.
2) Start the MS software and autotune the mass spectrometer.
Select the standard spectra feature that optimizes the
spectrometer for generating spectra useful in library searches.
3) Define the method. That is, set up the instrument for a run.
Normally the protocol is defined so that no single component
exceeds 250 ng. Consider the following example which will be
assumed to apply in our case. Suppose that a 1 wgt-% solution of
an unknown in methanol is prepared. A 1 l sample of the solution
(this is a typical injection volume) would contain 10 g of the
unknown, a value far in excess of the 250 ng. A split ration of
at least 40 is required. That is, only 1/40’th of the outlet gas
stream from the gas chromatograph is passed on to the mass
spectrometer and the remainder is rejected. Use a split ratio of
40 for the first run and decrease it if the signal-to-noise ratio
is poor.
a) Load the c160_vin default method.
Here are some of the features of the method. The instructor
will review all of the parameters during the prelab
orientation.
i) helium flow rate: 1 ml/min
ii) initial column temperature: 70C
iii) time at 70C: 2 min.
iv) ramp rate for the column: 10/min.
v) final column temperature: 200C
vi) total length of the run: 20 min.
vii) mass range: 40-600 amu
viii) split ratio: 40:1
ix) selective ion monitoring at 88 (This is appropriate
for the analysis of wines since the Maclafferty
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rearrangement yields a strong peak here for ethyl esters
of straight-chain aliphatic acids.)
x) solvent delay: 1.5 min. (This number is very
important as the high voltages in the mass spectrometer
are not turned on until after the solvent peak has
eluted from the column. The number is is determined
manually by measuring the time between injection and a
rise in the forepump pressure. The value given above is
for methanol under the specified operating conditions
and must be checked for each solvent and set of
operating conditions.)
b) Modify the method as appropriate.
c) Save the method under a unique name that you select. Do
not change the path or extension.
d) Run the method. Provide a unique file name for the
results and the slot number for the vial.
e) Analyze the results and conduct additional experiments.
There are a number of scenarios.
i) None of the peaks are strong. Conduct another run
with a lower split ratio. If the peaks are very weak,
consider running splitless, i.e. all the GC effluent
enters the MS.
ii) The strong peaks elute early and are followed by
weak peaks. Consider an additional splitless run BUT
increase the solvent delay time to slightly longer than
the last strong peak.
iii) The strong peaks elute in the middle of the run and
are preceded and followed by weak peaks. Two additional
runs are required. For one, apply the strategy just
discussed. Run splitless but increase the solvent delay
to exclude the strong peaks. This run will better
characterize the weak but slowly eluting components. To
improve the sensitivity of the weak components that
elute early, run splitless but design the schedule so
that the mass spectrometer is shut down before the
strong peaks elute. A post run (i.e. the GC component
of the run continues even though the MS is turned off)
is required to elute all the components from the GC
column before the next run.
3) Analyze the results. Average the mass spectrum of each GC peak
over the center of the peak and apply background correction. If
you suspect that the peak has two components, compare the mass
spectrum at the front and the rear of the peak. What would happen
if two substances elute with slightly different elution times only
one peak is resolved? Perform a library search and identify the
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components. Our computer has a subset of the NIST MS database.
Use a visual comparison to assist in making your decision.
Chemical common sense has its place. Would tetraethylsilane or
atropine be likely components of wine?
REPORT:
Your report should contain the following components:
1) a list of all identified components, their relative amounts,
and the basis for the identification. Relate the components to
the known chemistry of the material analyzed. For example, orange
rinds contain terpenes and the isoprene rule applies. The
excellent NIST Web page that is cited by MolData provides links to
the full NIST MS database. The NIST link is found on the Physical
Chemistry page. The SDBS Japanese database cited on the Organic
Chemistry page of MolData is superb. The URL for MolData if you
have forgotten is pages.pomona.edu/~wsteinmetz/moldata.html.
2) an assignment of the principal peaks of the 4 most abundant
components. If you have 4 or less components, present an
assignment of all components. The cited works on mass
spectrometry will be helpful in this step. A sample assignment is
provided with this handout.
3) a comparison of your analysis with what others have observed.
The excellent monograph by Belitz and Grosch is an excellent place
to start. Some use of the primary chemical literature is
expected.
gcms.doc, WES, 9 Jan. 2003