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(ma inlib ) Drona b inol
1. Molecular ion peak (M+) or parent ion peak: the peak that results from the loss of an
electron from the molecule. Since the electron is of negligible mass, the M+ peak gives
you the mass of the original molecule. Note that you may not be able to find a molecular
ion peak in mass spectra of molecules that fragment easily (e.g., aliphatic hydrocarbons).
Furthermore, the M+ peak is considered the mass peak containing atoms of the most
abundant isotope for each element (i.e., 12C, 1H, 16O, 35Cl, 79Br, etc.) and not the less
abundant isotopes (i.e., 13C, 37Cl, 81Br, etc.). This is often one of the highest m/z ratio
ions in an EI mass spectrum. The official name for this peak is the monoisotopic peak.
The second peak in an isotope cluster is called the M+1 peak; the third peak in an
isotopic envelope is called the M+2 peak, and so on. Be aware that co-eluting
compounds, background contamination in the ion source, and portions of the column
stationary phase bleeding into the mass spectrometer can create high m/z ions.
2. Base Peak: the largest peak (highest abundance) in the spectrum. The abundance of each
mass peak in the spectrum is representative of the ions stability in the source, thus the
base peak is almost always the most stable ion. Occasionally, the base peak is the same
as the molecular ion peak, but this not always the case. Since aliphatic compounds tend
to fragment extensively, the base peak is usually a fragment of the original compound.
3. Recall that the atomic weights reported on the periodic table are weighted averages for all
isotopes. The mass spectrometer can distinguish between the isotopes. Thus, use the
mass of the predominant isotope for each atom. For example, the mass of 12C (the most
abundant isotope of carbon with a nominal mass of 12) is 12.0000 (not 12.0107) Da
(Daltons, 1 Da = 1 atomic mass unit), the mass of 1H is 1.0078 and the mass of 16O is
15.9949 (the resolution of the 5973 inert is such that you can round off to the nearest 0.1
Dalton). These differences may seem slight, but they add up quickly in a large molecule
(e.g. for octane, C8H18 has a monoisotopic mass of 114.1409, not 114.0000 (0.1409 =
0.007825 * 18)). The molecular weight calculated using mass values from the periodic
table is called the average mass. This is the molecular weight written on the sides of
reagent bottles and used for stoichiometric calculations.
4. The intensity of a particular ion is proportional to its concentration in the source at any
given time. Thus, a doubling in the intensity of a particular ion implies that the
concentration of the molecular species responsible for that ion has also doubled. This is
analogous to Beers Law used in absorption spectroscopy and enables the analyst to use
the same quantification methodologies used for GC-FID or GC-TCD experiments for
GC-MS. You will calculate response ratios for various fatty acids to allow you to
compare their abundances in commercial cooking oils.
1) Preliminary survey
a. How many carbon atoms are present?
b. Are there any N, Cl, Br, or S atoms?
c. Is the molecule aromatic or aliphatic?
d. What is the molecular weight of the molecule?
2) What functional groups are present?
3) The molecular formula can be determined by piecing the functional groups together.
Hydrogen and oxygen can be filled in as needed.
b) Aliphatics: M = 15 + 14n
c) Benzene: M = 78 (subtract one mass unit for each substitution on the ring)
More examples can be found in textbooks (e.g. Interpretation of Mass Spectra, 4th edition
by F. W. McLafferty and F. Tureek). Sometimes identification is not this straightforward. It is
not uncommon for a hydrogen atom or a methyl group to migrate to form a more stable ion.
Generally, fragmentation will occur where functional groups connect, such as places where
branching occurs or where a side chain is attached to an aromatic ring.
The first thing to notice is that the base peak is M = 94. The M+ peak is M = 136.
Based on the data the molecular weight of the compound is 136. Computing the number of
carbons yields:
There is no significant M+2 peak, so there likely arent any chlorine, bromine or sulfur atoms in
the molecule. Since M+ is even, there is either an even number of nitrogen atoms or none at all.
Notice that there is an intense m/z = 77 peak, which corresponds to a mono-substituted benzene.
This leaves three other carbons to figure out.
There must be an even number of nitrogen atoms present, however since two nitrogen atoms
(m/z = 14) would give a mass of 28, the possibility of nitrogen can be eliminated.
The remaining mass can be accounted for by filling in with oxygen and hydrogen. Obviously the
three remaining carbons are not going to carry 23 hydrogen atoms, however if we use one
oxygen atom:
It is reasonable to imagine seven hydrogen atoms on three carbon atoms. With all of the mass
accounted for the next step is to propose a likely structure. There are several possible
combinations, but only a few actually make sense.
Perhaps the molecule is an alcohol. Recall that alcohols show a 31 + 14n mass pattern. There are
no m/z = 31, m/z = 45, or m/z -= 59 peaks in the spectrum, so it is probably not an alcohol.
How about a ketone? There is an m/z = 43 peak present. However if the molecule were a ketone
then it could not accommodate seven hydrogen atoms.
If the molecule fragmented such that the oxygen was attached to the aromatic ring, but the rest of
the chain broke off, then these fragments would be left:
Generally a hydrogen atom will shift to stabilize an oxyanion, resulting in this fragment:
Referring back to the mass spectrum, m/z = 94 is the most prominent peak, making this structure
the most likely candidate. Thus the final proposed structure is (C6H5)-O-CH2-CH2-CH3.
Clearly, the interpretation of mass spectra takes a lot of finesse. If you were to do this
more frequently, common fragmentation patterns would become much more obvious to you.
Fortunately modern chemists can utilize the on-line spectral libraries of GC-MS systems to
narrow down the likely choices. The Mass Spectrometry Facility has purchased a copy of the
NIST 02 library which contains reference spectra for over 150,000 compounds. Most database
searching programs arrive at their suggestions by comparing the analyte mass spectrum to all of
those in the library. However it is essential to understand how to properly interpret mass spectra
in order to properly assess the validity of a computer-generated database matches.
Although mass spectra databases are very powerful tools for identifying the components
of a complex mixture, they are far from perfect. No database is complete, and there will be times
(especially when synthesizing novel compounds) that the molecule of interest simply will not be
in the database. The NIST 02 library contains over 170,000 entries (150,000 compounds, there
is some redundancy in this database), but there are millions of potential organic compounds.
Another challenge arises when a component of a mixture is present at very low levels. Often the
mass spectra observed will be incomplete. That is, only the most intense fragment ions in the
mass spectrum will show up because the other peaks will be too weak to be considered by the
software. One solution to this would be to inject a more concentrated sample, thus increasing the
concentration of the component in question. This is not always an option however as analysts
are often sample limited in the real world. Furthermore, injection of a more concentrated sample
may foul the column or EI source of the GC-MS significantly shortening the lifetime of those
components. Remember, the concentration of the major components in a mixture will increase
as well.
Figure 3 contains the mass spectra of dodecane, 1-dodecene, and 1-dodecyne and an
unknown, low abundance compound with 14 carbons. Look at the three 12-carbon molecule
spectra and notice the differences in the masses of the low mass (<100) fragment ions.
By comparing the low mass portion of the unknown spectrum to the other three, the
identity should be clear. Check with your AI during the lab period for the correct answer. A
similar analysis can be to identify compounds with shared structure features. In this experiment,
one of the components of olive oil will likely be insufficiently abundant for a proper database
match. You will have to compare its mass spectrum to the mass spectra from olive oils other
components to determine its identity.
Figure 3: Mass spectra of dodecane, 1-dodecene, 1-dodecyne and an
unknown 14-carbon hydrocarbon. Note: the bottom 25% of the 14-carbon
mass spectrum has been deleted to simulate the mass spectrum from a low
abundance compound.
As stated above, the response observed for a given compound is proportional to its
concentration in the gas stream. Unfortunately, not all compounds are ionizable or detectable
with similar efficiencies. This problem is also encountered in optical spectroscopy. Recall that
Beers law contains a proportionality constant (the molar absorptivity, ) that relates how well
any compound absorbs the wavelength in question. If is not readily known, a line relating peak
area and concentration of a compound can be created by analyzing a handful of standards with
known concentrations of the compound in question. This method can become cumbersome as
the number of analytes in a given sample increases.
An alternative to generating a calibration curve for each analyte is computing response
factors. A response factor is a ratio between the observed response (peak area) for a given
concentration of a molecule with the response from the same amount of a closely related
standard (e.g. a stable isotope labeled version or a structural isomer). According to method
8270C from the United States Environmental Protection Agency, the response factor is
computed as follows:
areaanalyte concstd
RF =
areastd concanalyte
The response factor is used to normalize the peak areas of the other analytes using this equation:
area analyte
area corrected =
RFanalyte
In this experiment, you will normalize all response factors to that of palmitic acid methyl ester.
The corrected areas will then be used to compute the compositions of the oils.
The calorie, vitamin, mineral, protein, carbohydrate, and fat content of nearly every food
item sold in the United States must be clearly shown on the package to allow consumers to make
informed nutrition choices. These values are determined using a wide array of analytical
chemistry techniques including atomic absorption spectroscopy, bomb calorimetry, gas
chromatography, gravimetric analysis, liquid chromatography, and ultraviolet-visible
spectroscopy. This particular experiment will use GC-MS to determine the fatty acid profiles of
a handful of commercially available cooking oils.
For most foods, a chemical method is required to isolate the nutrient of interest from the
food prior to analysis (typically homogenization followed by refluxing solvent extraction (aka
Soxhlet extraction), supercritical fluid extraction, solid phase extraction, enhanced microwave
extraction, etc.). The extracted nutrients are often then further modified to make them more
amenable to a particular analysis method (e.g. a chromophore may be added, the nutrient may be
converted to an easily quantified small molecule, the nutrient may be esterified, etc.).
Fats exist as triglycerides, three fatty acids attached to a glycerol molecule with ester
bonds. Fatty acids are long chain carboxylic acids that are isolated from naturally occurring fats.
They can be saturated (fully hydrogenated, i.e. all carbon-carbon bonds are single bonds), mono-
unsaturated (one double bond between carbon bonds in the chain), and poly-unsaturated (more
than one double bond in the carbon chain). All double bonds in natural fatty acids are cis
isomers. The degree of saturation determines many important properties of the fat including
flavor, resistance to oxidation, melting point, and smoke point. Olive oil, which contains more
than 70% unsaturated fatty acids melts at -6 C and has a smoke point of about 210 C. Refined
coconut oil (8% unsaturated fatty acids) melts at 25 C and does not smoke until 232 C. Food
processors often partially hydrogenate vegetable oils to raise their melting points, lengthen their
shelf lives, or change the texture of the finished product. The high temperatures and pressures of
the hydrogenation process can cause isomerization in unsaturated fatty acids, creating trans fatty
acids. Table 1 contains a list of common fatty acids and their sources.
There is an overwhelming body of evidence concerning the health effects of the various
types of fatty acids, and as a result food labels must now list the amount of saturated,
unsaturated, and trans fatty acids in each serving. These effects are summarized in an American
Heart Association publication (http://www.webmd.com/content/article/124/115606). Saturated
fatty acids have been shown to raise low density lipoproteins (LDL or bad cholesterol). Cis
isomers of mono- and poly-unsaturated fatty acids have been shown to lower blood cholesterol
levels. Trans isomers of unsaturated fatty not only raise LDL but lower heart protecting high
density lipoproteins (HDLs or good cholesterol); some researchers consider them worse than
saturated fats. The federal government has mandated that the saturated, unsaturated, and trans-
fat content of all foods be included on the label. It also stipulated that foods with less than 0.5 g
of trans fatty acids per serving could be labeled as having 0 g trans fat.
Table 1: Names, properties, and sources of common fatty acids.
Carbon double
Common Name atoms bonds IUPAC name Sources
Butyric acid 4 0 butanoic acid Butterfat
Caproic Acid 6 0 hexanoic acid Butterfat
Caprylic Acid 8 0 octanoic acid coconut oil
Capric Acid 10 0 decanoic acid coconut oil
Lauric Acid 12 0 dodecanoic acid coconut oil
Myristic Acid 14 0 tetradecanoic acid palm kernel oil
Palmitic Acid 16 0 hexadecanoic acid palm oil
Palmitoleic Acid 16 1 9-hexadecenoic acid animal fats
Stearic Acid 18 0 octadecanoic acid animal fats
Oleic Acid 18 1 cis 9-octadecenoic acid olive oil
Elaidic Acid 18 1 trans 9-octadecenoic acid Hydrogenated oils
12-hydroxy-9-octadecenoic
Ricinoleic acid 18 1 acid castor oil
Vaccenic Acid 18 1 11-octadecenoic acid Butterfat
Linoleic Acid 18 2 9,12-octadecadienoic acid grape seed oil
Alpha-Linolenic 9,12,15-octadecatrienoic Flaxseed
Acid 18 3 acid (linseed) oil
Gamma-Linolenic
Acid 18 3 6,9,12-octadecatrienoic acid borage oil
Arachidic Acid 20 0 eicosanoic acid peanut oil, fish oil
Gadoleic Acid 20 1 9-eicosenoic acid fish oil
5,8,11,14-eicosatetraenoic
Arachidonic Acid 20 4 acid liver fats
5,8,11,14,17-
EPA 20 5 eicosapentaenoic acid fish oil
Behenic acid 22 0 docosanoic acid rapeseed oil
Erucic acid 22 1 13-docosenoic acid rapeseed oil
4,7,10,13,16,19-
DHA 22 6 docosahexaenoic acid fish oil
small amounts in
Lignoceric acid 24 0 tetracosanoic acid most fats
Table adapted from
http://www.scientificpsychic.com/fitness/fattyacids.html
In this experiment, we will saponify the oils to release the fatty acids. Saponification, or
treating the fat with a strong base, hydrolyzes the triglyceride, creating one molecule of glycerol
and three fatty acid molecules. Since the free fatty acids are not very volatile, they will be
converted into methyl esters by reacting them with boron triflouride in methanol. The resulting
fatty acid methyl esters (FAMEs) are then injected into the GC-MS. Mass spectra will be used to
determine the identities of the FAMEs; GC retention times will be used to discriminate between
various FAME isomers.
Gas Chromatography-Mass Spectrometry Experiment
Materials
Procedure
(adapted from J. F. Robinson and J. Neyer-Hilvert in J. of Chem. Educ. 74:1106-1108 (1997))
1) Place a half-filled 250 mL beaker of water onto a hot plate. Bring this to a slow boil.
2) Get small samples of olive oil, 0 g trans fat shortening, and three other fats for analysis.
NOTE: The solids should be scooped out with a spatula and put on weighing paper. You
will need ~75 mg of the solid greases. Be sure to place the glob of grease at the
BOTTOM of the test tube so it can react with the NaOH. Also, you will be unable to
quantitatively transfer the solid greases; this is acceptable as all measurements in this
experiment are relative to the total amount in the test tube. You will need 75 L of each
of the liquid fats.
3) Be sure to CLEARLY LABEL all test tubes, flasks and autosampler vials as all solutions
in this experiment are clear.
4) Pour 3 mL of the NaOH solution into five 8 test tubes.
5) Add 75 L/mg of each fat into a clean test tube and place it in a boiling water bath.
6) Boil them until the solutions are homogenous.
7) Remove the test tubes from the bath and let them cool for 3-5 minutes.
Part 2: Esterifying the free fatty acids
11) Pour 20 mL of saturated NaCl and 25 mL of hexane into five 125 mL flasks.
12) Pour each cooled reaction mixture into a separate 125 mL flask containing the NaCl
solution and hexane.
13) Seal the flask and shake vigorously for 2 minutes. You want to make sure the 2 layers
are mixed very well. Make sure to vent the flask occasionally to release the pressure.
14) Allow the layers to settle for ~1 minute.
15) Decant the hexane layer (the upper layer) into another 125 mL flask containing several
grams of sodium sulfate.
16) Add 25 mL of hexane to the salt/methanol solution and extract any remaining FAMEs
from the salt water and methanol.
17) Combine the 2nd hexane extract with the first extract.
18) Add 20 L of each FAME extract into separate autosampler vials containing 1 mL of
hexane and mix well.
19) Place 1 mL of each standard solution into separate autosampler vials.
20) Take all the autosampler vials and some clean hexane up to the mass spectrometry lab
(A411) for analysis.
21) Setting up the instrument for the experiments: If the software is not already open, log on
to the computer using your network ID and password and start the ChemStation software
by opening Instrument 1 (located on the desktop as well as in the Start menu). Select
Method >> Load and 315_FAME.m (it is in the directory
C:\MSDCHEM\1\315\Methods). This step MUST be completed first.
22) Loading the autosampler: Place the autosampler vials containing the three standard
solutions into positions 1, 2, and 3 of the autosampler tray; put the five vegetable oil
FAME extracts into positions 4-8; put an autosampler vial containing clean hexane into
position 9. Make sure the vial labeled A on the turret is full (nearly to the top) with
clean hexane.
23) Creating the autosampler sequence table: This step creates the table that determines types
of analyses to perform on each sample and specifies the order in which they are to be
performed. The table is generated as follows:
a. Click the Open Sequence: icon (it has a folder and three autosampler vials in it)
and select the sequence BLANK.S from the C:\MSDCHEM\1\315\Sequences
directory.
b. Click on the Edit Sequence icon (it has a pencil and three autosampler vials).
Make sure the data directory is C:\MSDCHEM\1\315\Data\ and the method
directory is set to C\MSDCHEM\1\315\Methods.
c. Select sample type Sample for all entries. The other sample types are only used
with automated data analyses. Enter 1 for the vial. Put a descriptive sample
name in the Sample column (e.g. standard FAME mix or corn oil extract).
Select the method 315_FAME after clicking inside the box under
Method/Keyword. Enter a file name for the run. It is suggested that you use
your initials and a three digit number (e.g. ABC_001). Enter a descriptive
comment (similar to the Sample column) in the Comment/Keyword String.
Leave the other columns (Sample Amt, Multiplier, Level, etc.) blank. When you
are finished, your table row should look something like this:
d. Make a two more entries so the contents of the autosampler vial 1 are analyzed
three times (for computing averages and standard deviations). Make sure to
increment the number in the file name. Create similar triplicate entries for each
vial. You should analyze the blank only once with 315_FAME.
e. Your completed sample table should have 25 entries and should look similar to
the table below.
24) Validate the Autosampler Sequence Table: This step ensures that no errors were made in
typing the names of the methods and that there is enough room on the hard drive to
record your data. Save your completed table by clicking the Save Sequence icon (it has
a red disk and 3 vials on it). Save the sequence in the directory
C:\MSDChem\1\315\Sequences. Give it a file name involving your initials so it will be
easy to find again, if necessary. Click the Simulate Sequence icon (it has a red check
mark and 3 vials on it). Make sure the Full Method button is selected in the Method
Sections To Run box. Then click Run Sequence. Correct any errors that arise and
resave your sequence. Make sure to view and print the verification report so you can
check your notebook entries. Close the sequence verification report after you have
printed it.
25) Start the Instrument: Click on the Run Sequence icon (it has a running figure and 3
autosampler vials on it). Make sure the Full Method button is selected in the Method
Sections To Run box. Then click Run Sequence. The instrument will perform the 26
analyses specified in the sequence table.
At the end of each day
Dispose of any unneeded hexane solutions in the ORGANIC WASTE container. Dispose of the
BF3/methanol/salt water solution in the AQUEOUS WASTE container. If you need to keep a
hexane solution overnight, cap it and seal the top with a small strip of Parafilm. Make sure any
flasks you keep are labeled and stored upright in the refrigerator or freezer rather than a drawer.
Always clean your glassware, the balance area, and your bench space before you leave.
(Note: this section may be performed either in the Mass Spectrometry Facility, A454. See the
Appendix entitled How to use ChemStation in the Library for C315 Students for instructions
on remote access to the GC-MS computer. It also contains a brief introduction to ChemStation.)
26) Load the software and method: Open Enhanced Data Analysis by double clicking on its
icon. Load the 315_FAME.M method.
28) Determine the elution order for cis and trans 9-octadecenoic acid methyl esters: Open the
data file from the oleic acid methyl ester (cis 9-octadecenoic acid) solution. Note the
retention time. Do the same for the elaidic acid methyl ester (trans 9-octadecenoic acid).
29) Determine the response factor for elaidic acid methyl ester: Using a procedure similar to
that in step 26, compute the response factor for elaidic acid methyl ester.
1. Attach at the end of your report one representative chromatogram for each standard sample
analyzed (standard FAME mixture chromatogram, oleic acid chromatogram, and elaidic acid
chromatogram). DO NOT print each chromatogram it is not necessary and wastes paper.
2. Create a table summarizing the chromatographic conditions used for the analyses. Include
the type of column used, the dimensions of the column, the injection volumes, split ratios,
and the temperature program. Column data are posted on the instrument and the temperature
program can be viewed from the Oven icon in the Acquisition window. Discuss how the
method should be modified if the fatty acids had longer hydrocarbon chains.
3. Create a table summarizing the retention times for the FAMEs in the standard mixture.
Include the averages and standard deviations for the retention times for each FAME in the
standard. In separate columns in this table, include the name, concentration (from the
certificate of analysis), peak area (average and standard deviation), and response factor for
each standard FAME.
4. Include in your report the mass spectra for the oleic acid and elaidic acid FAMEs. Describe
how you can determine which of the two isomers is present when only one is found in an
unknown sample.
5. For each of the unknown oils, create a table that lists each of the fatty acid components with
a relative abundance of 0.5% or higher. This table should include name, retention time, peak
area, corrected peak area, and relative concentration (average and standard deviation). Find a
reliable reference and report the amount of each fatty acid known to be present in each oil.
Compare the relative concentration you measured to this known amount. Comment on how
well these numbers agree with each other.
6. Based on the data collected for your unknowns, which oil would you report to be the
healthiest?
7. In the chromatogram for olive oil, you should find a small peak with a retention time of
approximately 7.9 minutes. Include a mass spectrum of this compound in your report and
any evidence that you can use to help identify this compound. Explain the quality of the
mass spectrum and the impact of this quality on the data interpretation.
8. Based on your data table for trans-fat free Crisco, compute the number of grams of trans fat
in one serving (one tablespoon, which is 14 g for liquid oils and 12 g for solid oils/greases).
Is this product truly trans-fat free? Comment on your findings.
9. Other than the balance and the glassware used, what are the major sources of error in
GC-MS? How can these errors be minimized or eliminated? Does the autosampler address
any of these issues? If so, how?
Section Points
Qualifier 25
Introduction 10
Experimental 10
Results and Discussion
FAME Analysis
(a) Chromatographic conditions
Table of chromatographic conditions 5
Conditions for longer chain fatty acid 5
(b) Standard results
Chromatograms 5
Table of results 5
Response factors 5
Oleic & elaidic acid MS 5
(c) Unknown oils
Table of results 10
Comparison to known values 10
Healthiest oil 5
Olive oil peak 5
Trans-fat free Crisco 5
(d) Sources of error 5
Conclusions 5
Literature references 5
Appendix: How to use ChemStation in the Library for C315 Students:
ChemStation is installed on PC # 5 in the library. This PC is located next to the emergency exit
at the far end of the library (near where the journal, Tetrahedron Letters is shelved).
When the PC boots up, double click on the Instrument #1 Data Analysis icon. It should be on
the desktop. If not, it can be found on the start menu in Programs Dept. Sponsored
Chemistry MSD Chem Instrument #1 Data Analysis
You will need to map C315 directory on the GC-MS computer in the mass spectrometry lab as
follows:
Your data will now be on the drive with the letter you selected. For example, if you chose W:,
your data will be found in the directory W:\Data.
You will need to check with library personnel to see where printouts from PC 5 appear.
Quick Guide to Data Analysis on ChemStation:
The left mouse button is used to zoom in on peaks. This feature works in either the
chromatogram or mass spectrum windows. You draw a box around the area you want to zoom in
on. The Y axis does NOT autoscale in the chromatogram window. Whatever is in the box is
what fills the chromatogram window after the zoom. I suggest you start your zoom boxes from
the bottom of the chromatogram. This way you can be sure that you do not cut off the tops of
peaks.
Double left-clicking will undo a previous zoom. If you want to redraw and entire chromatogram,
click on the Draw Chromatogram icon (the icon looks like a little teal chromatogram with no
arrows, letters, or other stuff in it).
The GC method (containing temperature program, split ratio, mass spec conditions, etc.) can be
found by selecting List Header from the File menu. The instrument parameters are found
in the acqmeth.txt window.
The Copy Window item under the Tools menu can be used for copying chromatograms
and/or spectra into other programs. Window 1 is mass spectrum, Window 2 is the
chromatogram.
You can direct the computer to integrate the chromatogram by clicking on the integrate icon. It
looks like a little blue chromatogram with a single, downward pointing arrow on it.
Alternatively you can select Integrate from the Chromatogram menu.
The results of the integration are viewed by selecting the Integration Results icon from the
Chromatogram menu.
The right mouse button is used to select the portion of the chromatogram from which you wish to
extract mass spectra. I suggest you draw the extraction boxes INSIDE chromatographic peaks to
avoid extracting too much noise.
Double right-clicking on a mass spectrum searches it against the selected library. You should
only search SCAN mode data and use the NIST02 library.
The Tabulate option under the Spectrum menu generates a table of masses and intensities
from the displayed mass spectrum. This table can then be copied and pasted into Excel for
further analysis.
ChemStation does have a rather complete set of online help documents which can be accessed
from the Help menu.