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Chemists use many types of modern instrumentation to identify compounds. This week you will use two
types of ultraviolet-visible spectrophotometers, and the GC-MS, gas chromatograph-mass spectrometer.
INTRODUCTION
Laboratory instruments allow us to extend our powers of observation and quantify minute differences
between molecules. For example, while our eyes might tell us that two compounds are slightly different
shades of red, a laboratory spectrophotometer would be able to identify the specific wavelengths at which
those compounds absorb visible light. Such specific information supplied by various laboratory instruments
can be used to determine what is in a sample, how much of that substance is present, separate the
components of a mixture, or even cause a chemical reaction to occur.
Many common instruments are spectrophotometers, that is, instruments that measure the amount of light
that a sample absorbs or emits at a particular wavelength. Before we can understand these instruments,
however, we must first understand the relationship between light and energy. The visible light that we can
see is only a small part of the electromagnetic spectrum.
Different portions of the spectrum vary by wavelength and energy content. The energy of one photon of
any type of light may be calculated from the relationships E = h ν and E = h c / λ where E is energy per
photon, h is Planck's constant, (nu) is the frequency, c is the speed of light, and (lambda) is the
wavelength. Notice that energy is inversely proportional to wavelength. In other words, as wavelength
increases, energy decreases. Red light has the longest wavelength of all visible light, but has the lowest
energy content.
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the ways that the molecule may release this energy is by emitting light with an energy equal to the energy
difference between the two orbitals. (You may recall that Bohr used this concept to develop his model of
the atom.) In order for an electron to move from a lower to a higher energy orbital it must gain energy, and
the molecule may get this energy by absorbing light with that specific energy content. Since most
molecules have energy gaps between orbitals that fall in the visible or ultraviolet (UV) regions of the
spectrum, studying UV-visible spectra of a molecule provides information on the characteristic energy gaps
between molecular orbitals. This information in turn can be used to help identify the molecule.
In addition to the qualitative information, quantitative values can also be derived from a UV-visible
spectrum. The amount of light that any solution will absorb is proportional to the concentration of that
solution. This is expressed mathematically as Beer's Law:
A = abc
where A is the absorbance of the solution, a is the absorptivity constant, b is the thickness of the sample
(width of the cuvette), and c is the concentration of the solution. The concentration of any solution can be
calculated from its absorbance if we know the values of “a” and “b.” The value of “b” is normally 1.00 cm.
When we don’t know the value of “a,” we can find the Calibration Curve for Spec-20
concentration of an unknown solution by comparing its
absorbance to that of solutions with known concentrations. 0.7
Typically we will prepare three or more solutions of known 0.6
concentrations, measure the absorbance of each, and then 0.5
Absorbance
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Mass Spectrometry, MS
Although mass spectrometry can be a stand-
alone technique, our instrument couples gas
chromatography with mass spectrometry in an
instrument called a GC-MS. As you saw in an
earlier experiment, gas chromatography
separates a mixture, allowing each component
to come off the end of the chromatography
column at a different time. This results in a
chromatogram such as the one in Figure 4 in
which a mixture has been separated into five
major components. One way to identify these
components is to send the material coming off
the chromatography column directly into
1 Time →
a mass spectrometer. Figure 4: Gas Chromatogram
In the mass spectrometer, molecules are turned into +1 ions by removing one electron. Then the mass
spectrometer measures the exact mass of each ion that reaches its detector. The mass spectrum of CH3Cl is
shown in Figure 5. The x-axis shows the masses of the particles and the y-axis shows how many particles
of a given mass were detected. How can one compound give multiple mass peaks? Let’s figure out where
these peaks are coming from. Imagine that we have 100 CH3Cl molecules. The molar mass of CH3Cl that
you add up off the periodic table is 50.5, but that is an average. Approximately 75 of those 100 molecules
contain 35Cl and have a mass of 50. The remaining 25 molecules contain 37Cl and have a mass of 52. Some
of these CH3Cl molecules make it to the detector .
intact giving peaks with masses at 52 and 50.
H
Figure 5: Mass Spectrum of Chloromethane Figure 6: Fragmentation of Chloromethane
(M)
100 Cl + C H
80 H
Abundance
60 mass = 35 or 37 H
40 Cl C H
mass 15
20
0
H
H
10 20 30 40 50 60
m/e
mass = 50 or 52 Cl C + H
H
mass = 49 or 51
Many of our 100 CH3Cl molecules, however, don’t make it to the detector intact. Conditions inside the
mass spectrometer cause some bonds to break, creating fragments that then travel to the detector. Some of
the CH3Cl molecules will break a C-Cl bond, others will break a C-H bond. As seen in the fragmentation
scheme in Figure 6, this creates fragments with masses of 15, 35, 37, 49, and 51. The peak at 50 is
considered the “molecular ion” and often labeled (M) because it represents the intact molecule with its most
abundant mass. When we try to interpret the mass spectrum of an unknown, the molecular ion peak is
extremely helpful since it is approximately equal to the molar mass of the unknown compound.
A significant M+2 peak is almost always due to the presence of Cl or Br in the molecule. One Cl atom
gives an M+2 peak about 1/3 as tall as M. One Br atom gives an M+2 peak nearly equal to M in height.
A peak at M+1 is primarily due to 13C (In nature, 1.1% of all carbon atoms are 13C). The number of
carbon atoms in the molecule can be calculated from the height of M+1 relative to the M peak.
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Number of carbon atoms = (M+1 height as a percentage of M) / 1.1
Thus if the M+1 peak is 10% as tall as M, then the number of carbon atoms in the molecule equals
10/1.1 = 9. The masses of the fragments from a spectrum allow us (or a computer) to figure out what the
original, intact molecule must have look like. The way that a molecule fragments in a mass spectrometer
is predictable to some extent. Molecules don’t normally fall totally apart into individual atoms, but rather
break at a couple of natural weak points. By looking at a molecule’s structure or using a model, we can
often see where the molecule is most likely to break apart.
Consider the structure of acetophenone shown in Figure 7 and imagine how it might fragment. The ring
of six carbon atoms is very common in organic chemistry and quite stable. This is not likely to fragment,
but the larger groups hanging off of the ring will be more easily broken off. In particular, it is very
common for a molecule to fragment directly next to a C=O group. We can imagine this molecule
fragmenting in at least two ways: the bond between the ring and the C=O group might break, or the bond
between the C=O and the CH3 might break. Such a fragmentation pattern will give us peaks at 120, 105,
77, 43, and 15. Of course, not every possible fragmentation pattern will be observed, and some bonds
will be more likely to break than others will, so these peaks are not all expected to be of equal intensity.
H .
Figure 7: Fragmentation of Acetophenone O
H C
C
C C
+ CH3
C C
H O mass = 43
H C H
H C C
H mass = 77
C C CH3
C C H O
H C H
H C C
H C C
H mass = 105
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HAZARDS
Although the unknowns are not particularly hazardous, you should still take reasonable care to avoid
prolonged contact of these compounds with the skin or prolonged inhalation of the vapors. The unused
portion of the unknowns should kept in their original containers and returned to the instructor
PROCEDURE
You will be working with an assigned partner. You should work as a team on all aspects of this
experiment and turn in a single lab report for your team. You each should do the normal pre-lab entry in
your own notebook, but once in lab all data will be recorded directly in the data tables at the end of this
experiment. Finish your report before leaving lab – staple your report, spectra, graph, and GC-MS
printouts together, and turn this in before leaving lab. Your instructor will tell you in what order to do the
various parts of this experiment.
On graph-ruled paper (a page from your lab notebook), draw a calibration curve similar to the one in Figure
3 using the absorbance and concentration data for the solutions with known concentrations. Also include the
point (0,0) because you set the absorbance to zero with distilled water. Consult the Appendix for correct
graphing techniques (download the file on graphing!). Your instructor will grade the graphs strictly by the
criteria listed there. Read the concentration of your “Spec-20 Unknown” from the calibration curve and
record this value on the data sheet.
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Part 4: Questions on UV-Visible Spectroscopy
Answer all the questions on the data sheets.
Note: The instructor may choose to have you do the data analysis on the four computers in the entrance
annex to our regular lab.
LABORATORY REPORT
Turn in one joint lab report for your team before leaving lab. Staple together the data sheets that follow with
your UV-Visible spectrum, calibration curve, GC-MS printout pages, and preliminary notebook entry pages.
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LABORATORY REPORT FOR
IDENTIFYING COMPOUNDS WITH UV-VISIBLE AND GC-MS
____________________________
Spec-20 Data:
Solution Identity Concentration, M Absorbance
Blank 0 0
Standard #1
Standard #2
Standard #3
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Part 3: The Colors of Visible Light by Spec-20
(list values for both partners since you may get differing results)
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Part 4: Questions on UV-Visible Spectroscopy
Answer the following using complete sentences. Note that to answer some of these questions, you must
have already completed the parts above.
1. Consult the schematic diagram of a Spec-20 that is given in the Appendix as you answer these questions.
a. Based upon the schematic diagram, describe the path that light travels through the
spectrophotometer.
b. A relatively small portion of the light produced by the source eventually reaches the detector.
Consult the schematic diagram and then describe two reasons for this.
c. Describe two ways in which the scanning spectrophotometer you used in Rm 472 is different from
the Spec-20.
2. What wavelength(s) was/were max for your “Scanning Visible Unknown?” Consult your results from
part 3. What color(s) corresponded to this/these wavelength(s)? Are these the same color as your
unknown? Explain!
3. What is the wavelength of the highest energy light visible to your eyes in part 3? Be careful!
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4. Compare your results for part 3 with those of two other teams. Did you get exactly the same answers?
Did you find the same longest and shortest visible wavelengths? What conclusions do you draw from this?
5. In part 3, you could not see any light striking the chalk at the highest and lowest wavelengths. Does this
mean that no light was reaching the chalk? Explain.
H H H H
m/e
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M(34)
2. The mass spectrum at right is for a compound 100
containing only carbon, hydrogen, and fluorine. 80
Abundance
The M+1 peak is about 1% of M. (This gives 60
specific information! Check the introduction.) 40
Use the molecular ion (M) mass to deduce the 20
formula for the compound. Explain your 0
reasoning. Draw structures of the fragments 12 14 16 18 20 22 24 26 28 30 32 34 36
responsible for the peaks at masses 15, 19, and
m/e
33.
M(112)
100
3. The organic compound whose mass
80
spectrum is shown contains only three
Abundance
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4. The first thing that happens inside the mass spectrometer is that an electron is removed from each
molecule, making a +1 ion. (It is easier to measure the mass of an ion than a neutral molecule, and
the detector is sensitive to ions, not neutral molecules). Does the lost electron significantly affect
the measured mass? Why or why not?
1. According to the chromatogram, how many major components are in the sample? Explain.
2. Suppose that the sample was known to contain two more major components than you just counted. Give
one possible explanation for this discrepancy.
3. With the instructor’s help, use the computer’s software to identify each of the major components by
matching their mass spectra to the library of known mass spectra. Complete the table below, recording the
best identification given by the computer, but also comment on the “quality” of each identification. The
computer supplies a numerical rating for the quality of the match between a peak’s mass spectrum and the
known mass spectrum. A quality of 100 is a perfect match. The computer also gives several alternative
compounds to choose from.
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Your instructor will give you a packet of printouts that includes the chromatogram and a best match
identification for each compound in our mixture. Each of these best match pages shows two mass spectra.
The one at the top is the mass spectrum of a component of our sample, labeled with its retention time. The
mass spectrum at the bottom of the page is the spectrum of a pure sample of the compound identified as the
best match identification. Use these printouts to answer the following questions.
1. a. Find the printout with the best match for the peak at 3.9 minutes. Give the name and structure for this
compound.
b. Think about how this structure might fragment, then draw the fragments that correspond to the mass
spectrum peaks at 114, 85, and 71.
c. Note the four evenly spaced peaks at 85, 71, 57, and 43. What constant mass difference separates
adjacent pairs of these peaks? What small bit of the compound’s structure does that represent?
2. a. Find the printout with the best match for the peak at 5.9 minutes. Give the name and structure for this
compound.
b. The molecular ion peak (M) is at 156. What does the peak at 158 tell you?
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c. Think about how the structure of this compound might fragment, then draw the structure of the
fragment at 77.
3. a. Find the printout with the best match for the peak at 7.81 minutes. Give the name and structure for
this compound.
b. Think about how the structure of this compound might fragment, then draw structures for the
fragments that correspond to mass spectrum peaks at 136, 105, and 77.
c. Compare the mass spectra and structures for the peaks at 7.56, 7.81, and 10.2 minutes. What mass
spectrum peaks are common to all three? What common structural feature explains this?
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