SCHA-319
Separations Lab
Release Date 15 March
GAS CHROMATOGRAPHY WITH A MASS SELECTIVE DETECTOR - (GCMSD)
PURPOSE
(1) To illustrate the power and limitations of GC-MSD. In this experiment, a mass-selective
detector (MSD) is used as the detector for a capillary gas chromatography column. This detector
will provide the mass-to-charge ratio for compounds which elute from the column and which
have been ionized prior to detection.
(2) Data from the GC-MSD experiment will be analyzed in two ways: (1) By examining mass
spectral peaks and their fragmentation components, and (2) comparison of data obtained over a
range of m/e values (intensity versus m/e value; called a mass spectrum) to a library of data.
INTRODUCTION
Mass Spectrometry is an analytical technique that can reveal specific, characteristic, structurally
related information about a compound. Most often, the questions that attract attention to mass
spectrometry are qualitative in nature. For example, "Have I successfully synthesized a C19
sterol," or "What is the structure of the by-product from my synthesis reaction?" or "Is the
compound that I isolated from plasma really norepinephrine?" Mass spectrometry can usually
answer these questions.
The compound for mass spectroscopic analysis is injected into a high vacuum where the
molecules can move freely in the evacuated space. Positive ions are commonly produced by
electron ionization (EI), electron impact on the gaseous molecules of the sample. The most
important ion sought in the mass spectrum is the molecular ion (the ionized, intact molecule)
because it is a direct indicator of the molecular weight of the compound. Yet during the process
of ionization considerable excess energy may be transferred to the molecular ion, which,
depending on its stability (related to its structure), may decompose into various fragment ions.
The resulting fragment ions define subsets of atoms, which may be related to functional groups,
or structural components of the original molecule. The array of fragment ions represented by
peaks in a mass spectrum is often called a fragmentation pattern. While an indication of
molecular weight is usually sufficient to answer questions that will confirm the presence of a
given compound, it is much better to compare the major peaks in the experimental spectrum to
those in a reference spectrum of the authentic compound. Careful study of the fragmentation
pattern is especially important when the mass spectrum is to be used to elucidate molecular
structure or to distinguish between two or more structural isomers.
While some fragmentation of the sample molecules is necessary for structure evaluation, many
compounds are so effectively fragmented by EI that no appreciable population of molecular ions
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survives to give an indication of the molecular weight in the mass spectrum. Chemical
ionization (CI), involving ionization of the sample molecule by collisions with ions of a reagent
gas (for example, the ions produced by electron ionization of methane), is a more "gentle"
ionization technique which often produces abundant protonated molecular ions. The resultant
spectrum provides a good indication of molecular weight but may have insufficient
fragmentation for functional group and structural evaluation.
Of course, in order for mass spectrometry to be helpful the ions that are studied must originate
from a pure source of the sample of interest, for to consider ions that are unrelated to the
compound could be worse than having no mass spectral data at all. It is essential to insure that a
sample contains no material that will produce unexpected ions which could interfere with
interpretation. Thus, the combination of gas chromatography and mass spectrometry (GCMS) is
often the method of choice. The gas chromatograph can separate the compound of interest from
other potentially interfering compounds based on the compounds differing volatility and present
it to the mass spectrometer in purified form over a time interval sufficient to obtain the mass
spectrum.
Per unit quantity of sample, mass spectrometry perhaps delivers more information concerning a
molecule than does any other spectroscopic technique. Although mass spectrometry rarely solves
a problem "single-handedly", it often provides the critical complimentary data that finally
"crack" an identification problem. For a complete identification of a sample compound 0.1 to 1
micrograms of the sample may be required. If all that is needed is detection and verification of
the presence of an already-identified compound the sample requirements are less severe
(sometimes requiring only femtogram quantities).
Although mass spectrometry has been used successfully in the determination of many
compounds (usually having a molecular weight of less than 1,000), one consistent obstacle is
whether a compound of interest is amenable to vapor phase analysis. However, techniques such
as field desorption (where a sample molecule is desorbed from the surface of a probe or electrode
as an ion when it enters a high electric field), and chemical derivatization to increase thermal
stability and volatility, extend the applicability of mass spectrometry to increasing numbers and
types of compounds.
Finally it should be noted that there are three basic types of mass sensitive instruments
encountered commonly in analytical chemistry laboratories. The first type is the magnetic sector
mass spectrometer (see Figure 1).
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Figure 1: Schematic of sector magnet mass spectrometer
The ion stream from the source is passed by a wedge shaped magnet. The ions curve in response
to the magnetic field and separate into several ions streams based on their individual trajectories
in response to the magnetic field. At any given magnetic field strength (or, accelerating
potential) only one stream has appropriate trajectory (based on it m/e value) to successfully
survive the curved "track" to the detector. All other ions will collide with the walls of the track
or with the detector slits and will not reach the detector to be recorded. It is common to sweep
the magnetic field strength at constant accelerating potential to bring the different ion streams to
the detector. The magnetic field sweep can be accomplished very quickly and thus an entire
mass spectrum, covering a wide m/e range, can be recorded in a short time frame.
The second type is the quadrupole device which form a class of non-magnetic mass
spectrometers and mass selective detectors (see Figure 2).
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Figure 2: Schematic of quadrupole design
Our HP 5973 Mass Selective Detector is a quadrupole device. It employs a combination of dc
and radiofrequency (rf) potentials as a mass "filter". The quadrupole typically consists of 4
cylindrical, parallel rods (10-25 cm in length) situated symmetrically in a square arrangement.
Rods diagonally opposite each other are connected, in pairs, to dc and rf generators. Positive
ions extracted from the ions source are accelerated into the quadrupole along the longitudinal
axis of the four rods. The ions are influenced by the combined dc and rf fields. In order for an
ion to reach the detector it must traverse the quadrupole without colliding with any one of the
metal rods. For any level of rf/dc voltage only ions of a specific m/e avoid collision and reach
the detector. The entire mass spectrum is obtained as voltages are swept from a pre-established
minimum to a maximum. In our MSD the rf/dc voltages are stepped in a manner that
corresponds to 0.1 amu m/e jumps.
Lastly, there are time-of-flight devices which record the time it take for an accelerated ion to
travel from the ion source to the detector (see Figure 3).
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Figure 3: Schematic of time-of-flight mass spectrometer
Supplies (In flammable cabinet in A143)
1) Alkane mixture
2) Known Mixture (in Pentane)
Alkanes
Octane
Nonane
Decane
Undecane
Dodecane
Tridecane
Alkenes
1-Octene
1-Nonene
1-Decene
1-Undecene
1-Dodecene
1-Tridecene
Alcohols
2-Methyl-1-butanol
1-Hexanol
Cyclohexanol
1-Heptanol
Benzyl alcohol
1-Octanol
Ketones
MIBK
2,4-Dimethyl-3-pentanone
2-Heptanone
Cyclohexanone
Acetophenone
1-Phenyl-1-propanone
3) Unknown Mixture of 3 or 4 compounds from the above list
GC Syringe (10 µL) Sign out of Stockroom
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Procedure
This section includes a number of questions. Answers to these should be included in the report.
These may be placed in either the experimental or discussion sections, as considered appropriate.
I.
Instrument Start-up and Introduction
1) Turn on the computer and monitor. The instrument should already be on.
2) Double click on the “RIT Chemistry” icon on the “Windows NT desktop”. If it isn’t
there, click on “Start”, “Programs”, “RIT Chemistry”, and then “RIT Chemistry”.
3) Click on “Method”, then “Load”. Ask your instructor for the correct Method Name.
Click on it then click on “OK”.
4) Click on “Method”, then “Edit Entire Method”. Click on “OK” until the
“Instrument/Edit/Inlet” screen appears. The Inlet Button is highlighted in yellow.
Record the parameters below in your notebook and describe what each means in
your lab write up. Click on “OK” after you record these values.
a. Split Flow
b. Split Ratio
c. Total Flow
d. Pressure
e. Heater Temperature
f. Gas
g. Calculate the Septum Purge from the above values.
h. Also, record the Carrier Gas (He) cylinder pressure and feed pressure (40-60
psig.)
5) Many dialog boxes will be presented. Most of these should be left unchanged (Use
the "OK" button). The purpose here is to gain familiarity with the instrumentation.
Please consult with the TA if any questions arise concerning any of the information.
Use the software help feature to answer the questions concerning these parameters.
These should be included in the report. Please make a note of the following critical
parameters.
a. Solvent Delay = 0.00 min. - What is the purpose of this parameter?
b. MS Scan Parameters Mass Range: Low = 20 High = 300 Scans/sec = 3.3. What
do these values signify? How are they related?
c. Column Information
d. Temperature Information (Injection and Mass Detector).
e. Oven Program Initial Temperature Setpoint = 45o C, Initial Time = 2.0 min., Final
Temperature = 150o C, Final Time = 1.0 min. Make sure that these parameters
are correct. There should also be a second line in the temperature program that
raises the temperature to 280o C.
f. Library Search Parameters – Use the default Library Name.
g. Save your method with a name other than the one that you started with!
II.
Tuning the Mass Spec.
1. Click on “View”, and then “Manual Tune”.
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2. Click on “Autotune”. What compound is being used? Give the full chemical
name, not just the initials. Also what masses are selected for tuning? What
fragments does each of these three masses represent? Why must the mass spec
be Auto Tuned?
3. When the instrument tune is complete click on “File”, then “Save Tune Values”.
Click on “OK” to save it as “Atune”.
4. Click on “View”, “Instrument Control”, and “Yes” at the prompt.
III.
Determination of Solvent Delay and tm
1. Run your method by clicking on “Method”, then “Run”.
2. Enter a data file. Use "A name you will remember". If you end it with a number then
this value will be updated on subsequent runs.
3. Press the “Run Method” button.
4. The software will give you a dialog box telling you what to do and signaling that the
computer is ready.
5. Press the [Prep Run] key on the GC instrument. Wait until the screen on the GC
signals that it is ready for an injection.
6. Rinse a 10 l syringe with 5 full volumes of pentane. Load the syringe with 10 µL
of air - Inject the air into the instrument. The injection should be a smooth quick
motion. The syringe should be removed quickly and smoothly after injection.
7. Press [START] on the GC immediately after removing the syringe.
8. Allow the run to progress for about 4 minutes beyond your first peak. Use the
pentane peak to set your solvent delay. Modify your method and save it with the new
solvent delay. (Why did you see a peak went you injected only air.) Determine tm
and the required solvent delay.
IV.
Analyze the Samples
1) Modify your method for the new solvent delay.
2) Run your method by clicking on “Method”, then “Run Method”. The software will
give you a dialog box signaling that the instrument is ready.
3) Press the [Prep Run] key on the GC instrument. Wait until the screen on the GC
signals that it is ready for an injection.
4) Rinse the syringe with 5 volumes of pentane. Rinse the syringe with 5 volumes of the
mixture.
5) Load the syringe with 1 µL of air, then 2 µL mixture and then pull the plunger all the
way back to load about 7 µ of air. This gets the sample out of the syringe needle
which will avoid volatilization of sample from the needle. Inject the sample and
press [START] on the GC.
6) The software will then present a dialog box concerning the solvent delay. DO NOT
override the solvent delay. After clearing this dialog box, the chromatogram will
begin to plot, as soon as the solvent delay time is over.
7) When the run is complete the software will process the chromatogram and the
associated mass spectral data. It will then output a library search report.
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8) Perform one injection for the known mixture and the unknown mixture. Be sure to
change the data file name (should be automatic) for each of the injections. Change
the Sample Name for each mixture as well (not automatic).
9) You may notice that some of the known mixtures, and maybe the unknown
mixture as well, show more peaks than expected. Why is this?
10) Run the mixture of alkanes in an isothermal run. I would suggest you attempt to run
it at about 100 C.
V.
Analyze the Data
1) Click on “View”, then “Data Analysis (offline)”.
2) Click on “Chromatogram”, and then “Integrate”. This will integrate and label the
times on the peaks.
3) The mouse pointer should control a vertical line. If this is a cross, then click on
“Tools” and then “Reset View” to obtain a vertical line.
4) Expand the first peak by starting at the top left corner of the peak, with the mouse
pointer, and while holding down the left mouse button, drag a rectangle around the
whole peak. Let go of the left mouse button and that peak will expand.
5) With the mouse, bring the vertical line so it intersects the top of the peak. Double
click with the right mouse button and a Mass Spectrum will appear at the bottom.
6) To print, click on “File” , “Print”, and “TIC & Spectrum”.
7) Place the mouse pointer anywhere in the Mass Spectral window and double click with
the right button of the mouse. A series of library matches will appear in the window
with a match quality. What does this match quality mean? Click on “Print” in this
window. Click on “Done” in this window.
8) While placing the mouse pointer anywhere in the top window, double click with the
left button of the mouse to view all the peaks again.
9) Repeat steps 5-8 to identify the rest of the GC peaks.
10) Use Extract ion chromatograms to find any missing peaks in your known mixture.
VI
Results
1)
Identify the unknown compounds. Include the three independent pieces of
evidence to substantiate the identification. Acceptable evidence includes: library
search results, retention time match to a known compound, and interpretation of
mass spectra (by fragmentation pattern).
2)
Were you able to find all the compounds present in the known and unknown
mixtures?
3)
For the alkane mixture prepare a plot of I vs. log tr’ . Calculate the retention time
for n C15 alkane using this plot.
4)
For the alcohol in your unknown you should assign the six largest fragment peaks
to a potential fragment structure. (Table)
VII.
Questions/Discussion
1)
Would it be possible to identify any of the unknown compounds using the GC
alone? What does the mass spectrometer add to the analysis?
2)
Should you rely on the library matches alone? Why or Why not?
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VIII.
Report
1)
As part of your results section. Prepare a big Table with each of the compounds
in the mixture. Your Table should have the compound name, its’ molecular
weight and boiling point. Also include a column to indicate how you identified
the compound. Spectra match, retention time match or any other method or
combination of methods you might have used.
2)
In your results and discussion make sure to talk about how one might better
separate unresolved compounds.
3)
Make sure to discuss both GC and Ms theory in your report.
4)
Calculate the resolution between 1-nonane and 1-nonene.
5)
Calculate the tr , tr’ and k’ for octane.
6)
Calculate the value of N and H values for octane and tridecane.
7)
Attach the answers in the body of your procedure sequentially in an appendix.
VIII.
References
1)
Junk, G. A.; Richard, J. J., Anal. Chem., 1988, 60, 454.
2)
Skoog, D. A.; Leary, J. J.; Principles of Instrumental Analysis, Fourth edition,
Saunders College Publishing, 1992, Chapter 18.
3)
Watson, J. T., Introduction to Mass Spectrometry: Biomedical, Environmental,
and Forensic Applications, Raven Press, 1976.
Appendix 1
A Partial List of Common Fragment Losses.
A more exhaustive list of fragment losses can be found in the references below.
1H
38 C3H2, C2N, F2
14 CH2
39 C3H3, HC2N
15 CH3
41 CH2=CHCH2
16 O, NH2
42 CH2=CHCH3, CH2=C=O
17 HO
43 C3H7, CH3C=O
18 H2O
44 CH2=CHOH, CO2, N2O, CONH2
19 F
45 CH3CHOH, CH3CH2O , CO2H, CH3CH2NH2
20 HF
46 (H2O and CH2=CH2), CH3CH2OH
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26 CHCH, CHN
27 CH2=CH , HCN
47 CH3S
48 CH3SH
28 CH2=CH2, CO
49 CH2Cl
29 CH3CH2 , CHO
52 C4H4
30 NH2CH2, CH2O, NO, C2H6
53 C4H5
31 OCH3, CH2OH, CH2NH2
54 CH2=CH-CH-CH2
32 CH3OH, S
55 CH2=CHCHCH3
33 HS , ( CH3 and H2O)
56 CH2=CHCH2CH3
34 H2S
57 C4H9 , C2H5CO
35 Cl
60 C3H7OH
36 HCl, 2 H2O
63 CH2CH2Cl
37 H2Cl
71 C5H11
73 CH3CH2OC(=O)
74 C4H9OH
75 C6H3
76 C6H4
77 C6H5
78 C6H6
References
1.
Hamming, M and Foster, N., Interpretation of Mass Spectra of Organic Compounds. New
York, NY. Academic Press.
2.
McLafferty, F. W., Interpretation of Mass Spectra. Mill Valley, CA. University Scientific
Books.
3.
Silverstein, R, Bassler, G., and Morrill, T., Spectrometric Identification of Organic
Compounds. New York, NY. John Wiley and Sons. Inc
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4.
Harris, D, Quantitative Chemical Analysis, New York, NY, W.H. Freeman, 6 th ed.
2002. Chapter 22.
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