EXPERIMENT 9

IDENTIFYING COMPOUNDS BY UV-VIS AND GC-MS rev 5/11

GOAL
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.

A chemical substance will absorb only certain characteristic

amounts of energy and thus certain wavelengths of light. The
substance absorbs these particular wavelengths/energies of light
because they correspond to the energy differences between
various states of the molecule. For example, in Figure 1 we see
a molecule that has two states, one with higher energy than the
other. The molecule will absorb light with an energy that exactly
matches the energy gap between the two states shown. The
molecule is not able to absorb light that is slightly higher or
slightly lower in energy, only the light with the correct Figure 1: Energy transition between two states
energy to match the gap between the states. Because the
state of a molecule can be changed in many ways, and
because we have many ways of observing these changes, a great number of different types of spectroscopy
have been developed.

Ultraviolet-Visible Spectroscopy, UV-Vis

The energy needed to move an electron from one orbital to another is equal to energies of light in the visible
and ultraviolet portions of the electromagnetic spectrum. When an electron moves from a higher energy
orbital to a lower energy orbital, the difference in energy between the two orbitals must be released. One of

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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.

The human eye is able to distinguish among wavelengths in the

visible region. When all the visible colors - red, orange, yellow,
green, blue, indigo, and violet - are present, the eye sees white. If any
color is missing, the eye sees that color's complement, the mixture of
colors that are left. Figure 2 shows a small complementary color
wheel. Colors directly opposite each other are complements. For
example, red and green are complements. If red is missing from a
mixture of visible light, the eye will see the light as green. This
means that green-colored compounds appear that way because red is
missing from the light they reflect. In other words, they must absorb
light in the red region. A compound that absorbs light around 470 nm
is absorbing blue light and will appear orange to orange-yellow to the Figure 2: Complementary Color Wheel
human eye.

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

plot a calibration curve of absorbance vs. concentration,

0.4
such as the one shown here. After measuring the
0.3
absorbance of the unknown solution, its concentration can
0.2
be read from the graph. For example, if the solution of
0.1
unknown concentration had an absorbance of 0.60, we
0
would consult the calibration curve, and find a
0 0.05 0.1 0.15 0.2 0.25
concentration of 0.18 M.
Concentration, M

Figure 3: Calibration Curve Graph

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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

mass = 120 + CH3

C C
H C H mass = 15

H mass = 105

Mass Spectrum Summary

1. Molecular ion (M) gives mass of the intact molecule

2. M+2 peak indicates presence of
Cl if M+2 height is 1/3 M height
Br if M+2 height is approximately equal to M height
3. M+1 peak indicates number of carbon atoms
# C = (M+1 height as a percentage of M) / 1.1
4. Molecules fragment at key point, breaking off groups
e.g. next to rings, C=O groups, etc.

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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.

Part 1: Scanning Visible Spectroscopy

Take your unknown labeled “Scanning Visible Unknown” to room 472 as directed by the instructor, who
will demonstrate the correct use of the scanning UV-visible spectrophotometer. Run the visible spectrum
of your compound and measure the absorbance at the highest point of each peak (the absorbance at each
max). Print your spectrum. Identify your unknown by comparing its max (the wavelength where its
absorbance is greatest) to those given in the list of possible unknowns posted in lab.

Part 2: UV-Visible at a Fixed Wavelength–the Spec-20

Find the concentration of your unknown labeled “Spec-20 Unknown” using a Spec-20 in the regular lab
room. Consult the Appendix for detailed instructions on the use of a Spec-20 (download the file from the
usual web site). Take three clean, dry beakers to the reagent bench and get 5 mL of each of the Spec-20
standards. Be sure that you record the concentration of each solution that is given on the dispenser and also
the wavelength at which measurements are to be made. Following the instructions from the Appendix,
record the absorbance of each of the standards, and also that of your “Spec-20 Unknown.” Be sure that you
do not accidentally dilute any of your solutions by using wet glassware. Do not attempt to dry out the Spec-
20 cuvette, but rather rinse the cuvette with a small volume of your solution and then discard this rinse
solution down the drain. (These solutions are not hazardous.)

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.

Part 3: The Colors of Visible Light by Spec-20

Use the Spec-20 in the regular lab room to find the colors that correspond to wavelengths in the visible
portion of the spectrum. Obtain a special Spec-20 cuvette that contains an angled piece of chalk. Place this
in the Spec-20 and leave the sample cover open. Set the wavelength control to 700 nm. Record the color of
the light that you see on the chalk. Now change the wavelength by 10 nm. Record the color you see.
Continue recording the colors of light that you see for wavelengths between 750 and 400 nm. Also record
the wavelength of both the longest and shortest wavelengths that were visible to your eye.

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Part 4: Questions on UV-Visible Spectroscopy
Answer all the questions on the data sheets.

Part 5: Learning to Interpret Mass Spectra

Carefully read pages 3 and 4. Note the summary box on page 4. Use this information to complete the
questions on the data sheet. Models are available in the lab to help you visualize the molecules.

Part 6: Using the GC-MS

Go to the GC-MS in Room 472 as directed by your instructor, who will guide your work for this part.
Because the GC is time-consuming to run and you have already run this same instrument in the Crime
Solving Chromatography experiment, you will spend your time doing a computerized analysis of a sample
that has already been run and saved as a data file. Be sure to staple the GC-MS printouts to your report.
With the help of the instructor, do the following.
1. view the parts of the instrument
2. at the computer, position the cursor on each of the major peaks
3. retrieve the mass spectrum for each major peak
4. do a computerized library search to identify each major peak
5. record this information on your report sheet (Questions 1 and 3)
6. get a packet of printouts including the chromatogram and the identifications of the major peaks
7. answer the remaining questions

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

Team Members: ____________________________ Date __________

____________________________

Part 1: Scanning UV-Visible Spectroscopy

“Scanning visible unknown” number _________

Color of “Scanning visible unknown” solution _________

max, nm _______________ _______________ _______________

(Your unknown may have only one peak, or several peaks.)

Absorbance at max ______________ _______________ _____________

(If your spectrum has more than one max, record the absorbance at each)

Identity of unknown ______________

ATTACH your visible spectrum to your lab report

Part 2: UV-Visible at a Fixed Wavelength–the Spec-20

“Spec-20 Unknown” number _______ Wavelength for measurements _________

Spec-20 Data:
Solution Identity Concentration, M Absorbance

Blank 0 0

Standard #1

Standard #2

Standard #3

Your Spec-20 Unknown XXX

Concentration of your “Spec-20 Unknown” ______________

(read from the graph you made, and include units)

ATTACH your calibration curve to your lab report

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Part 3: The Colors of Visible Light by Spec-20

Wavelength Color Wavelength Color Wavelength Color

750 nm 630 nm 510 nm

740 nm 620 nm 500 nm

730 nm 610 nm 490 nm

720 nm 600 nm 480 nm

710 nm 590 nm 470 nm

700 nm 580 nm 460 nm

690 nm 570 nm 450 nm

680 nm 560 nm 440 nm

670 nm 550 nm 430 nm

660 nm 540 nm 420 nm

650 nm 530 nm 410 nm

640 nm 520 nm 400 nm

Longest wavelength visible to your eye :

_________ nm (first student) and _________ nm (second student)

Shortest wavelength visible to your eye :

_________ nm (first student) and _________ nm (second student)

(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.

Part 5: Learning to Interpret Mass Spectra

(Consult pages 3-4, and especially the summary box on Page 4, for help with these questions.)
H H H H
1. The mass spectrum at right is for n-butane,
H C C C C H
the structure shown. Imagine how this
molecule might break apart. Draw structures 100
Abundance

H H H H

of the fragments responsible for the peaks at 80

masses 15, 29, 43, and 58. 60
40
20
0
10 15 20 25 30 35 40 45 50 55 60

m/e

10
 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

elements. Determine the molecular 60

formula of this compound. Start by 40 M+2

considering what the height of the M+2 20
peak tells you. The M+1 peak is just 0
under 7% of the height of the M peak. 40 60 80 100 120
You do not need to determine the
m/e
structure of this compound, just its
molecular formula.

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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?

Part 6: Using the GC-MS

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.

Peak retention time Best computer ID of the Compound Comments on Quality of ID

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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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