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Gas Chromatography-Mass Spectrometry (GC-MS)

Objective
- Learn the merits of combining GC with MS
- Become familiar with the operation of a commercial GC-MS
- Using alcohol standards, learn how molecules are fragmented in a MS and how this
fragmentation can be used to identify compounds
- Use GC-MS to attempt to identify two components in a complex sample (gasoline).
References
McLafferty, F. W. Interpretation of Mass Spectra, 4th ed.; University Science: Mill Valley;
1993.
Johnstone, R. A. W.; Rose, M. E. Mass Spectrometry for Chemists and Biochemists, Cambridge
University Press: New York:, 1982.
Skoog, D. A.; Holler, F. J.; Crouch, S. R.; Principles of Instrumental Analysis, 6
th
ed.; Thomson:
California; 2007, Chapter 20.
Kellner, R. A.; Mermet, J.-M.; Otto, M.; Widmer, H.M, Eds.; Analytical Chemistry, Wiley: New
York; 1999, Chapter 9.4.
Introduction
The separation of complex mixtures into individual components is routinely accomplished by the
technique of gas chromatography. Identification of each of these components, however, is not
possible for most detection systems. The unique exception is mass spectrometry, where the
separated species are ionized and analyzed based on their mass-to-charge ratio (m/z). After
compounds are eluted from the capillary column, the effluent enters an ion source region where
it is bombarded with 70-eV electrons inducing ionization and fragmentation of the molecules.
Gradients in the electric field are then utilized to direct the positive ions into the quadrupole mass
filter. Simultaneously a 1.0-MHz AC and a DC voltage are applied to four parallel rods to
control which m/z species are allowed to pass through the mass filter. Finally, ions are detected
by an electron multiplier and recorded as a mass spectrum (intensity versus m/z). Most mass
selective detectors can be operated in one of two modes: scan or selected ion monitoring. In the
scan mode, the mass filter continuously scans a specified range of m/z values in discrete steps
from high to low mass. The resulting chromatogram is often represented as the total ion current
(TIC) or the sum of the ion intensity at all m/z values versus time. Numerous books have been
written discussing the analysis of fragmentation patterns. In this mode, a complete mass
spectrum (over the specified mass range) is acquired every few seconds. In contrast, the selected
ion monitoring mode detects only ions at specified m/z values. This acquisition mode allows for
the maximum sensitivity when analyzing for known compounds, and thus is often used for
quantitative analysis. Because characteristic ion or ions are monitored, co-eluting species do not
interfere with each other and are distinguished by differing ions. The choice of mode is
dependent on the analysis of interest.
2
Chemicals
Standards to prepare: 500ppm 1-hexanol, 500ppm 2-hexanol, 500ppm 3-hexanol,
and 500ppm cyclohexanol
Mixture of equal parts cyclohexanol/1-hexanol standards
Gasoline
Instrument
A Shimadzu GCMS-QP5000 series will be utilized for this experiment. Check and note the
column length, inner diameter, and stationary phase. Instrument and data acquisition will be
controlled with the GC/MS Real Time Analysis software. Two primary windows will be of
interest for this experiment: the GC/MS Real Time Analysis window and the GC/MS Post-Run
Analysis window.
Settings
Injector temperature: 120 C
Oven temperature: 50 C
Detector interface temperature: 280 C
Time: 10 min.
Solvent delay (Solvent Cut Time): 0.0 for air; 0.9 min. for standards with methanol
solvent.
EM Voltage: relative; zero (uses value based on tune)

Mass range: for air: decide based on probable components in air.

for organic: 40-120 (try and avoid the solvent)
Procedure
Before you turn on the instrument, open the oven door and note which information is indicated
on the column (film type, thickness, length, I.D. etc).

Fill each GC rinse vial with methanol. These vials are in the tray located on the auto injector.
Slide the tray out slowly to the right, and fill each vial labeled with an S with methanol.
Carefully slide the tray back in place.

For your samples, pour ~0.5 mL of each standard into a labeled GC vial for your use. Do not put
anything into the stock solutions (pipette etc.) For your air sample, use an empty GC vial (with
cap!).

Instrument: Make sure your gas tank is open (orange helium tank CLOSEST to the GC-MS)
and the instrument is on before starting the GCMS software. The instrument is turned on by
hitting the SYSTEM button and FLOW1-ON-enter). The software login is student and password
Chem480.

3
Tuning: Select Tuning from the menu on the left. Click Start Auto Tuning. Read in the
software manual what this tuning does and check your results with what is expected. Save your
tuning file.

Method: Under the Acquisition Tab select Download Initial Parameters. Select Method Detail
from the main menu. Under each category change conditions as appropriate for the analysis of an
air sample. Check settings under Sampler, GC, and MS. The default method is
chem480method, but each group should save their OWN method file (do NOT overwrite the
default). For a detailed description of these settings refer to the GCMS users manual.

Acquisition: Select Data Acquisition. Using the Sample Login, choose a filename and enter
all other relevant parameters (including your tuning file). Select Standby. The 'Not Ready
Light' will turn off when all temperatures have equilibrated. When the system has
equilibrated, select Start to begin the acquisition. Use the retention time to calculate the
mobile-phase linear velocity (v) and the volumetric flowrate (F = tr
2
v). (The linear velocity
should be approximately 30 cm/s. Do a quick calculation now to confirm that the flow conditions
are correct.) Include your calculated values in your report.

Note: The Program Time under the GC Tab should match the end time for the Mass Spec.

Data Analysis: Select Data Analysis. The most recently acquired data set should automatically
load. Select the Qualitative Icon to see the GC chromatogram and mass spectra. With the cursor
positioned on the peak of interest, double-click to obtain the mass spectrum. Print out these
results and include them in your report. Change the retention times displayed as appropriate and
print this information. Identify two major components in air from the mass spectrum.

Change the solvent delay to 0.9 min. for injection of the standards.

Inject 0.1 L of each of the following standards in methanol: 500 ppm 1-hexanol, 500 ppm 2-
hexanol, 500 ppm 3-hexanol. Determine the mass spectrum for each solute and include
chromatograms/spectra in your report. Is the molecular ion present for each solute? Why or why
not? Identify the major ions in each of the spectra, and clearly explain the fragmentation
pathways. Why are the spectra so different for these chemically similar compounds?

Separately inject and analyze 1-hexanol in methanol (500 ppm) and cyclohexanol in methanol
(500 ppm). Now inject the mixture of 1-hexanol and cyclohexanol in methanol with the same
scan range. Are these solutes fully resolved? From the mass spectrum for each compound,
choose a characteristic ion for cyclohexanol and analyze the mixture again using the selected ion
monitoring technique (see MS parameters). Discuss the difference between the chromatogram
obtained by scanning and that obtained using SIM.

Conduct a Similarity Search on the mass spectra of all of your samples. This is done by right-
clicking on the mass spectrum of interest and choosing Similarity Search. Use the NIST98
library as your database. Confirm the fragmentation pattern and discuss any discrepancies. If for
some reason your sample is not found in the similarity search, explain why in your report (where
are there discrepancies in the mass spectrum?).
4

Reset conditions to temperature program from 40 to 120 at 10C/min. Obtain a small sample of
gasoline. Dilute the sample 100:1 in methanol and inject 0.1 L. Choose two isolated
chromatographic peaks and identify each component. Give clear justification for the
identification based on the fragmentation pattern and report the results of the library search.
Include the structure of each compound in your report.

Note: At several points in this lab you are asked to discuss fragmentation pathways. This
term has specific meaning in mass spectrometry including the origin of the ion, ion structure, any
rearrangements, and the mode of fragmentation. See one of the references regarding
fragmentation and compound identification.

At the end of each day, go to Tools and select Daily Shutdown.
Report
Thoroughly discuss the fragmentation pathways of your samples (air, hexanol samples, mixture,
and gasoline) in your report. Provide molecular structures and fragmentation mechanisms when
necessary.

Discuss your SIM analysis. Why would you use SIM instead of scan mode?

A 28 amu peak is prominent in all your spectra, regardless of sample and is even in your blank or
when you inject nothing at all! You are getting irritated with this interference. What is the
probable identity of this ion and where did it come from? Describe how to test for this problem
and what you would do to eliminate it.

You figure out the 28 amu peak and fix the problem, but then your lab partner points out a pesky
peak at 149 amu. It is also in all of your samples and your blank (but not when nothing is
injected). You are truly irritated now! Again, what is the probable identity of this ion (be
specific!) and where did it most likely come from? Describe an experiment to test your
hypothesis regarding the origin of this interference. Describe what you would do to eliminate this
problem.

Describe how a mass selective detector yields a more definitive identification than flame
ionization detection. Include in your discussion the origin of the signal for MSD and FID
systems.

Give a clear and concise description of how a quadrupole mass selective detector operates as a
mass filter. Be sure to include the necessary vacuum conditions and why they are important.

5
Gas Chromatography-Optimized Separation
Objectives
- Become familiar with the operation of a GC with FID
- Observe how flow rate effects separation efficiency by chromatographic analysis at
different flow rates and construction of Golay plots using decane as the analyte
- Learn how to use Kovats retention index to estimate the carbon number of analytes

Reading
Karger, B. L. An Introduction to Separation Science; Wiley: New York; 1973, Chapter 2.
Skoog, D. A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6
th
ed.; Thomson:
California; 2007, Chapters 26, 27.
Kellner, R.; Mermet, J.-M.; Otto, M.; Widmer, H.M. Analytical Chemistry; Wiley: New York;
1998, Chapter 5.
Introduction
There are a number of factors that must be considered when designing a separation procedure for
GC. Here, the focus will be on optimizing the separation of alkanes by properly selecting the
flow rate and column temperature used. Analysis of peak widths at different flow rates will
allow determination of the minimum plate height. However, time is equally important as
separation quality (In other words, just how long do you want to spend in 480 lab?). Therefore,
you will be asked to find a balance between time and separation quality using flow and
temperature control. Once you have designed a procedure you will be asked to identify (and
quantify) components in an unknown mixture.
From your reading, you should have found that the van Deemter equation is:
H = A + B/u + (C
s
+C
m
)u
This equation is used for packed columns, however, in this experiment you are using an open
tubular column. Therefore, you must use the Golay equation:
H = B/u + (C
s
+C
m
)u

Chemicals
Solution of n-decane in n-hexane
Unknown hydrocarbon mixture containing 3 alkanes (provided by the GSIdont forget
to dilute it before running!)
Instrument
A Hewlett Packard 6890 gas chromatograph with a flame-ionization detector (FID) will be used
for this experiment. Please check and note the stationary phase and thickness, column length and
I.D. by looking inside the oven (length in m, I.D. in mm, and film thickness in m). You may
need to check the company website (Restek) to find the composition of the stationary phase.
6
Computer login: Student password: Chem480

BEFORE YOU HEAT THE GC: Acquire the injection syringe from the dispensing stand and
CHANGE THE SEPTUM on the injection port (front port, septum included with syringe).

Settings:
GC injection port temperature 150 C
GC oven temperature 60-100 C
GC detector temperature 200 C
Column flow rate 1-10 mL/min
Detector flow rate H
2
= 40-50 mL/min, air = 400-450 mL/min
Procedure
On the HP-6890 all flow rates can be set digitally and are actively controlled throughout the
experiment using HPChemStation software. Before starting the software, make sure the GC and
gases are turned on. You will be using hyodrogen, air, and helium (orange tank closest to wall).
Under the Method and Control Window, click the front column icon. Here you will be able to
change setting and ignite the FID. The default method you can use is chem480. Save your
settings by saving your OWN method. Under RunControl--Sample Info, you may change your
sample settings, your file names, and where your files are saved.
Notice that when you set the column flow rate the column head pressure is automatically
calculated and controlled. Using this automated pressure control unit reduces the split ratio (gas
flow onto column / gas flow out split port). This means that more sample is injected onto the
column. To protect the column and detector, we must dilute our samples to 0.1-0.25 vol/vol%.
Before beginning the experiment you must ignite the flame ionization detector (FID). Click the
front column icon and then the detector icon to change FID settings and ignite it. Changing the lit
offset (usually in range from 0 to 2), may help ignition. You may also need to optimize your
detector flow rates. If your FID continually tries to reignite, changing your lit offset may also
help. When the detector is lit you should hear a small pop (if you listen carefully) and the signal
(front detector) should rise from 0.X to approximately 10-30. Once the oven temperatures,
column flow rates and head pressures have equilibrated push <prerun> on the instrument control
panel. Now the GC is ready for a sample injection and the software should give you a green
light/ ready signal to proceed.

n-Decane in n-Hexane. Remember to dilute your sample. Run a series of isothermal
chromatograms over a range of flow rates (at least five). (Remember! You will have to turn
off the FID prior to changing the He flow rate.). Make your runs by injecting approximately 1
L of solution into the inlet. Rinse the syringe about 10 times before each injection with n-
hexane rinse solvent. Adjust the vertical and horizontal scale to show the n-decane peak (the n-
hexane peak will be off scale) and determine the peak width. In the Data Analysis window,
you may access your GC chromatograms and report files, which will contain vital information
such as peak area/width/height. Print chromatograms and report files for your report. From the
retention times and peak widths, calculate N
eff
for the column at each velocity. (Note: HP
Chemstation will give you the peak width in the report file, but you will need to report how peak
widths are determined). See instrument manual if you are unsure.

7
Calculate the dead time of the column for an unretained compound like acetone and use this to
adjust your retention times. Now calculate the effective plate height, H, for each flow rate and
construct a Golay plot. Does your plot suggest a minimum plate height and optimum flow rate?
Include this in your report. Also include a brief discussion of why you constructed a Golay plot
and not a van Deemter plot and describe the difference between the two.

Unknown Mixture. At the optimized flow rate, obtain an isothermal chromatogram of the
unknown mixture. Make sure you have diluted your unknown to the appropriate concentration!
Assign the peaks with familiar retention times. Now estimate the carbon number of the unknown
peak(s) using the Kovats method (look up how to use the Kovats method). Include a Kovats plot
in your report and discuss it. Using these data, make a sample containing a known concentration
of each of your components to check your assignment.

Next, increase the time efficiency of your experiment by designing a temperature-programmed
experiment, discuss your results.

Determine the relative concentrations of the solutes in the unknown. Do NOT simply take the
peak area/total peak area of each unknown component and equate that to % composition. It is up
to you how you will determine your unknown component concentrations. Discuss your technique
in the report. Include error analysis in your report also. Remember the FID detector is sensitive
below 1% v/v, when preparing your standard solutions.

Report
Calculate the theoretical minimum plate height, h
min
, from the following equation:
where r is the column radius and k is the sample capacity (choose k=5). Compare this result to
the one you obtain from the Golay Plot (show the plot) you generate and explain the differences.
Indicate the optimum flow rate on the graph. Discuss the shape of the decane peak, i.e.,
symmetric, tailing, Gaussian. Describe how the peak widths were determined.

Remember to describe all experimental parameters (i.e. temperature program, method, etc.) used
for your unknown determination. Also, discuss why you selected these parameters for the
method you developed. Include a discussion of time efficiency in your results.

Describe your assignment of the peaks in the unknown and the concentrations of each
component. Discuss the results from your Kovats plot. Include an error analysis for your
quantitative determination.

( ) ( ) ( ) | |
2 / 1
2 2
min
1 3 / 11 6 1 25 . 1 k k k r h + + + =
8
Fourier Transform Infrared Spectroscopy
Objective
- Learn the fundamentals of Fourier Transform
- Learn how FT spectroscopy differs from wavelength dispersive spectroscopy
- Learn the advantages of FT methods
- Use FTIR for analysis of polymeric materials
Reading
Skoog, D. A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6th ed.; Thomson:
California; 2007, Chapter 16, 17.
Kellner, R. A.; Mermet, J.-M.; Otto, M.; Widmer, H.M, Eds.; Analytical Chemistry, Wiley: New
York; 1999, Chapter 9.2.
Griffiths, P. R.; de Haseth, J. A. Fourier-Transform Infrared Spectrometry, Wiley: New York,
1986.
Ingle, J.; Crouch, S. Spectrochemical Analysis, Prentice Hall: Englewood Cliffs, 1988.
Shoemaker, D.; Garland, C.; Nibler, J. Experiments in Physical Chemistry, 5
th
ed.; McGraw-
Hill: New York, 1989.
Introduction
The Fourier transform infrared (FTIR) spectrometer is an instrument that has revolutionized the
measurement of spectra in the infrared region. Utilizing an interferometer instead of a
conventional dispersive monochromator, a significant increase in energy throughput and
decrease in scan time are realized. These advantages allow an increased use of signal averaging
to improve the signal-to-noise ratio. A detailed discussion of this instrument may be found in the
reading for this experiment.

In this experiment you will determine the effect of signal averaging and mirror retardation on
spectral noise levels and compare the IR spectra of various polymeric materials in direct
transmission mode and in ATR (attenuated total internal reflectance) mode.
Instrument
A Perkin Elmer Spectrum One FTIR spectrometer with transmission and ATR attachments is
available for these experiments.
Chemicals/Samples
(All samples and microscope slides are in the FTIR drawer)
Styrofoam
Toluene
PTFE film
Polyethylene bag
Procedure
A copy of the procedure for operating the FTIR spectrometer is available in the laboratory.
Review the procedure before beginning the experiment. To familiarize yourself with the operation
9
of the instrument, collect a transmission spectrum of a polystyrene film that is available in the lab
(do not use a sample with a thickness greater than about 38 m). Be sure that you know how to
use the Setup menus before proceeding.

Resolution and Signal Averaging

In this part of the experiment, you will examine the effect of resolution and signal averaging on
spectral noise levels.

From the Instrument menu, choose Scan. Set the resolution to 1 cm
-1
and the number of scans to
1. Collect a Background.

Collect a transmission spectrum using the open beam path (no sample). The spectrum should be
essentially featureless, except for possibly weak bands due to water vapor and carbon dioxide.
This 100% line should be centered at about 100% transmission and display the spectrometer
noise levels. Save the spectrum using a suitable name (i.e. HPL1_1.spa). Following the same
procedure, collect two more transmission spectra at 1 cm
-1
resolution using 4 and 64 scans,
respectively. Save these spectra using suitable names (i.e. HPL1_4.spa and HPL1_64.spa). Take
a new background each time the number of scans is changed.

View the three spectra together on the screen. Set the x-axis limits to display the spectral region
2700 to 2400 cm
-1
(only for the noise analysis). Be sure to view all spectra offset from each
other (split). Note how the noise levels change with the number of scans. Plot these spectra over
the 2700 to 2400 cm
-1
range and measure the approximate peak-to-peak noise levels of each
spectrum. Compare the noise levels of the different scans. Use a few ratios to determine how
the noise level depends upon the number of scans averaged. Compare your results to the
expected ratios based on theory.

Repeat this procedure to determine how spectral resolution (mirror retardation) influences the
spectral noise levels. Collect 100% lines at resolutions of 1, 2 and 4 cm
-1
using 1 scan for each.
Remember to take a new background every time you change the resolution setting. Save the
spectral data using suitable names (i.e. HPL1_1, HPL2_1.spa, HPL4_1.spa) Plot the spectra as
before and measure approximate peak-to-peak noise levels. Determine the effect of resolution on
spectral noise levels and compare to expected values.
Comparison of Transmission vs. ATR Modes
Sample preparation:
Make two PS solutions: dissolve some styrofoam in toluene, and make another solution at twice
the concentration. Record these concentrations for your report. Disperse a small portion of each
solution on the surface of two glass microscope slides (pre-cleaned with toluene). Peel the dry
films up from the glass slides with tweezers and mount on transmission sample holder.

Transmission Sampling. Using the samples provided, acquire transmission spectra of the
polymer samples using the full spectral region.

*You need transmission spectra of:
10
- Both PS films that you made (high and low conc.)
- The polyethylene bag
- The PTFE film

For the polyethylene bag and at least one of the PS samples, you need clear spectra with the apex
of all peaks showing and not bottomed out or cut off at the bottom of the display. You need to
decide how to conduct your experiment to make this happen.

ATR Sampling. Before you begin this portion of the laboratory, be sure that you are familiar
with the basic principles of ATR spectroscopy (Ref. 1, pg. 191-194).

The ATR accessory is in the lab. Consult the instructions for installation and use of the ATR
accessory. These can be found in the Universal ATR Sampling Accessory Users Guide, pp 15 to
21.

Set the resolution to 4 cm
-1
and the number of scans to 128. Collect the background spectrum
with the ATR accessory in place, without a sample present or the ATR shoe in place. After
background collection, mount the Styrofoam sample on the crystal. Apply pressure to ensure
good contact between the sample and the ZnSe crystal, but be careful not to break the crystal.
Use the force gauge to monitor the applied pressure. This can be accessed by clicking on the
Monitor button in the upper left corner of the Scan window. Collect a spectrum. When the
spectrum is displayed, compare it to the transmission spectrum of the polystyrene sample. Look
for vibrational bands appearing in key spectral regions (i.e. C-H and aromatic C=C stretching
regions). If the bands that appear in these regions are weak (S/N ratio is poor), readjust the
sample on the ATR crystal to improve the contact and collect another spectrum.

Use the ATR accessory to obtain spectra of other solid samples.

*You need clear ATR spectra of:
- a piece of styrofoam
- the polyethylene bag

Report
Explain the effects of signal averaging and mirror retardation on spectral noise levels. Define
peak-to-peak noise. How is this different from a S/N ratio?

For all polymer samples, note important energy regions in which bands appear. Draw the
structure of each compound and try to correlate spectral regions with vibrations of specific
functional groups.

Compare transmission and ATR spectra:
ATR of styrofoam vs. transmission of your best PS film
ATR of polyethylene bag vs. transmission of polyethylene bag

Note that the intensity of ATR absorption bands may be skewed in comparison to absorption
11
bands in transmission spectra; absorption bands at lower energy are expected to be more intense
than higher energy bands. Do a little research to find the equation that describes this wavelength
dependence, and include it in your report. Do your results actually agree with this equation? If
not, can you explain why? Explain the different appearance of ATR and transmission spectra. A
few absorbance ratios may help.
12
Normal and Resonance-Enhanced Raman Spectroscopy
Objective
- Learn the principles of Raman spectroscopy and general applications
- Become Familiar with operation of the Raman microscope
- Use Raman to analyze proteins in egg yolk and the carotenoid profile of plant matter
(carrots)
Reading
Carey, P. R. Biological Applications of Raman and Resonance Raman Spectroscopies, Academic
Press: New York, 1982, pp. 71.
Skoog, D. A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6th ed.; Thomson:
California; 2007, Chapter 18.
Harris, D. C.; Bertolucci, M. D. Symmetry and spectroscopy: An introduction to vibrational and
electronic spectroscopy, Dover: New York, 1978. pp 93-97; pp 198-200
Hoskins, L. C. J. Chem. Ed. 1975, 52, 568572.
Introduction
Raman spectroscopy has long had an important role in physical biochemistry. The
relatively low intensity of Raman scattering from water facilitates work in aqueous solution.
Protein and nucleic acid spectra are sensitive to changes in secondary structure. Resonance
enhancement of polyene (carotenoid) and heme chromophores is possible with the most
commonly available lasers. In this experiment, resonance Raman spectra of a carrot and normal
and resonance Raman spectroscopy of an egg are obtained.
Chicken egg albumin consists of water (88%), proteins (11%) and small amounts of
lipids, glucose and inorganic ions. The strongest feature in the Raman spectrum is the protein
amide I band, which is found between 1640 and 1660 cm
-1
. Egg yolk contains 47% water, 33%
lipids, and 17% proteins. The yolk also contains small amounts of yellow-orange carotenoid
pigments, such as retinoic acids. Carotenoids have low energy electronic transitions, so that near-
resonance enhancement is obtainable, even with an HeNe laser. The C=C stretches in the 1500
1600 cm
-1
spectral region and the C-C stretch in the 1100 to 1200 cm
-1
spectral region are
strongly enhanced. The carotenoids in carrots, principally beta-carotene, are different from those
in the egg. The differences are readily seen in the Raman spectra. Individual carotenoids are not
identifiable, because there are many similar compounds present.
Samples
1 Chicken Egg
1 Carrot
2 glass vials (1 dram)
Apparatus
The Kaiser Holoprobe 1000 Raman spectrometer will be used for this experiment. This system is
a totally integrated system for Raman spectroscopy containing a 10 mW HeNe laser (633 nm), a
f/1.8 axial transmission bench equipped with a 1024 x 64 pixel thermoelectrically cooled CCD
13
detector. Both sample excitation and collection of the scattered light is accomplished with a
fiber-optic probe routed through a microscope.

Procedure
Familiarize yourself with the instruction manual located near the instrument prior to beginning
the experiment. Using the features provided with the software will greatly simplify the data
acquisition and processing during this experiment.

Turn on the camera switch located on the backside of the Holoprobe 1000. Turn on the
computer, find the Holograms application and run the program. When the camera has come to
the operating temperature of -20C (temperature should be displayed on the bottom bar of the
Holograms window) turn on the laser key. CAUTION: Do not look directly into the fiber probe
head with the laser power on.

Fill a 1-dram glass vial with isopropanol (use vials from GC/MS, make sure filled to top). Using
the microscope and stage, focus the laser output through the open top of the vial (this can be
achieved by balancing the vial on the microscope lamp and focus through top of vial do not cap
the vial). Then move the probe several millimeters towards the sample so that you are focused
inside the sample container. Practice using the instrument by collecting a spectrum of
isopropanol. NOTE: You will need to close the black curtain and turn out the overhead lights
during spectral acquisition to prevent the detector from saturating.

Sample Preparation
1. Crack open the egg and separate the yolk from the albumin
2. Break the yolk and pour a small portion (approximately 0.250.5 mL) onto a glass
microscope slide. Only enough yolk to cover the slide should be used.
3. Allow the yolk to dry. If available, a drying oven set to low temperature (<40C) can be
used to help dry the yolk faster.
While the yolk is drying:
4. Put several mL of the egg albumin (whites) into a separate vial.
5. The sample of fresh carrot is prepared by cutting with a razor blade or sharp knife to
make a 5 to 10 mm thick disc.

Data Collection
Select the proper conditions to acquire dark spectra and background spectra, and subtract
these from sample spectra. Because both egg components and carrots fluorescence in 633 nm
light, each sample will require multiple acquisitions at short exposure times. Some
experimentation will be required to determine the optimum conditions. An exposure time that is
too long will result in a red Fill indicator on control panel. Note that for the egg spectra, the low-
signal Raman features will be superimposed on a significant fluorescent background. The Raman
may, in fact, not be easily seen until the data has been baseline corrected (see Data Analysis).
On the other hand, the carrot spectra will contain obvious Raman features because of the
resonance enhancement.

1. The albumin spectrum can be obtained through either the side or the top of the filled
vial. Adjust the position of the fiber lens assembly until the focus (narrowest point) of the
14
excitation laser is just inside the wall of the vial.
2. The dried yolk sample will require measurement through the top of the vial with the
laser impinging on the yolk surface at an angle. The focus of the laser should be just at
the surface of the yolk.
3. As with the egg yolk, the focus of the laser should be at the surface of the carrot.
Several measurements of the carrot can be made from various different lateral positions
across the slice yielding a rough map of carotenoid distribution.

Data Analysis
In the egg spectra, the most interesting protein and carotenoid bands are located between
800 and 2000 cm
-1
. This is the only region in which the fluorescence background needs to be
removed. The Raman spectrum from the carrot will be visible even without background
subtraction. Careful subtraction of the background should be done to accurately assess the
difference in the carotenoid bands between the carrot and egg yolk sample. Once you have
selected this region of the spectrum save the file and close all the other data files before
beginning the background correction procedures.
The GRAMS/32 software provides several methods by which a changing baseline can be
removed. Two methods of background (fluorescence) subtraction will be introduced here. First,
select the multi-point algorithm under the Arithmetic : Baseline menu. The program will
prompt you to select points on the spectrum that contain no Raman scattering information, only
baseline. The program will then fit a polynomial function that runs through the points you
selected, and then automatically subtracts that function from the data set.

An alternate method for background removal uses the Arithmetic: Peak Fit utility in
GRAMS/32. The peak fit utility requires that only one spectrum (with only the small area of
interest selected) be loaded. Any additional spectra will be cleared from memory whether or not
they have been saved. When the background is much larger than the Raman signal, fitting the
spectrum to a Gaussian peak can approximate the shape of the background. Because the peak fit
utility operates on all points in the spectrum, whether background or Raman scatter, this
procedure works best when the signal is at least 10 times lower in intensity than the background.

Run the peak fit utility. Use the Options button and set the Use baseline option set to
No and Peak types to same with a Gaussian function. Press OK. Click the right mouse
button approximately 200 to 400 cm
-1
apart across the entire spectral range to enter points to be
used for the line-fitting procedure. Using the Parameters button, set a Low Limit of 200 cm
-1
on
the width of the currently selected peak. Each peak can be selected in succession by using the
Next and Back buttons. When the width limit has been set for all of the peaks, press the OK
button. The limit on the width will prevent fitting to narrow peaks, i.e. to Raman spectral
features.

The number of peaks and lower limit can be adjusted for best results. Select Done,
Iterate, and then Start Fit to begin fitting the data. Select Leave Alone if any warning messages
are given. If many warning messages appear, stop the fit and restart with fewer peaks.

When the final result is obtained, select Quit and Exit (Ignore the message asking you to save
15
results; they were automatically saved). The generated background spectrum is stored in the file
curvefit.spc. It can be subtracted from the original data using Arithmetic : Subtract.

Report
Discuss the two methods of fitting and removing the fluorescence background from the Raman
scattering spectra. Which method gave the smoothest baseline? Which method suffers from the
greatest user bias? Also, based on the data collected and your understanding of the Raman
process, how might the fluorescence background be reduced or eliminated?

Show the background corrected spectra of the three samples. Discuss the appearance of the
Raman spectra of the food samples. Which peaks show evidence of pre-resonant enhancement?
Why is the amide band shifted in the Raman spectrum of the egg yolk sample as compared with
the amide band in the egg albumin sample?

Present your findings on the radial distribution of carotenoid in the carrot cross-section.

Discuss the advantages and disadvantages of this instrument as compared to FT-Raman
spectroscopy using an interferometer optical bench. (Hint: The discussion in Skoog may not be
perfect; be sure to use additional sources)

16
Nuclear Magnetic Resonance Spectroscopy
Objective
- Become familiar with the operation of the FT-NMR instrument
- Acquire 1H spectra of organic compounds for structural determination
- Use NMR to determine equilibrium in an enolization reaction
Reading
Skoog, D. A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6th ed.; Thomson:
California; 2007 Chapter 19.
Kellner, R. A.; Mermet, J.-M.; Otto, M.; Widmer, H. M, Eds.; Analytical Chemistry, Wiley: New
York; Chapter 9.3.
Derome, A. E. Modern NMR techniques for chemistry research, Pergamon
Press: Oxford, 1987.
Harris, R. K. Nuclear Magnetic Resonance Spectroscopy, Longman: Essex, 1986.
Hornak, J. P. Basics of NMR, http://www.cis.rit.edu/htbooks/nmr/, 2003.
Introduction
Fourier-transform, nuclear magnetic resonance (FT-NMR) spectroscopy is the most widely used
instrumental method in modern chemistry. NMR is based on the degeneracy of nuclear quantum
spin states in a strong applied magnetic field. The perturbation of the spin energy by molecular
bonding and neighboring nuclear spins, called the chemical shift, is the source of NMRs utility
to chemistry. The most common use of FT-NMR namely collection of 1H spectra of organic
compounds will be explored in this experiment. The chemical shift and indirect spin coupling
will be explored for cinnamic acid and ethylacetate. The quantitative aspect of NMR spectra will
be demonstrated by determining the position of a chemical equilibrium, specifically the
enolization of acetylacetone.

Chemicals
deutero-chloroform, CDCl
3
(solvent) cinnamic acid, C
6
H
5
CH=CHCOOH
ethylacetate, CH
3
CO
2
CH
2
CH
3
acetylacetone(pentanedione),
CH
3
COCH
2
COCH
3
tetramethylsilane, Si(CH
3
)
4


Instrument
The Bruker AC-200 NMR spectrometer located in Chem 2407 will be used for this experiment.
You should check out three NMR tubes from the dispensing stand. Please refer to the operation
manual for detailed instructions on how to operate this instrument. Be extremely careful since
many students in the department rely upon this instrument for their research. Login: Chem480
password:cheM480.
O O O OH
17

Procedure
For the neat liquid compounds pipet 5 to 6 drops into a clean NMR tube. Dilute the sample with
perdeuterated solvent until the solution fills approximately 3 cm of the NMR tube. For the solid
organic compound place 10 to 15 mg of solid in a clean 1 dram glass vial and dissolve in a
minimum of perdeuterated solvent. Transfer the solution to a clean NMR tube by pipet and fill
the tube to a height of 3 cm with solvent. Place the plastic cap over the end of the NMR to
prevent evaporation.

(Note: It is very important to avoid contamination of the chemicals and solvents used in this
experiment. Never pipet directly from a stock reagent bottle!).

At the NMR console you will find the spinner (a plastic collar) that fits snugly over the NMR
tube. Slide the spinner over the NMR tube to the appropriate place using the depth gauge (also
located near the console). (Note: Be careful not to get oil from your fingertips on the NMR tube
or spinner. Use a Chemwipe to clean the outside of the tube before inserting into the magnet.)
Run the ethyl acetate sample first.

Once the sample has been properly inserted into the magnet the normal procedure is to perform
three steps to achieve optimum magnet homogeneity 1) spinning the NMR tube, 2) set the
deuterium lock parameters, and 3) optimize the shim currents. Before following the instructions
to perform these operations collect one acquisition of ethyl acetate. Set the number of
acquisitions to one, NA=1. After transforming (FT) the free-induction decay signal expand the
screen to the 0 to 4 ppm region to see the line shapes. Print this spectrum for later comparison
after following the spin, lock, shim procedure.

Now that you have followed the proper start-up procedure the line shapes should be more
symmetrical and narrow. The spectrum will need to be phase corrected so that all the peaks are
positive (above the base-line). Locate the TMS peak in your spectrum, this should be the most
up-field (far-right) peak in the spectrum. Center the cursor on this peak (in expanded view
mode) and set the reference mark to 0.0 ppm. Be sure to print this spectrum for comparison.

Collect another free induction decay for ethyl acetate this time set the recycle delay, last delay,
to 10 seconds and the number of scans, NS, to 16 under the acquisition key. Watch the screen as
each acquisition is added to the data buffer. Transform, phase and print this spectrum. You
should be able to comment about the improvement in signal strength relative to the noise (in the
base-line) for the signal-averaged spectrum versus the single acquisition (above). You will use
this spectrum to comment about the peak assignments and spin-spin splitting for your report.

Switch samples now to investigate the cinnamic acid 1H NMR spectrum. Acquire a good quality
(shimmed and locked) spectrum with at least 16 scans. Print the spectrum for your report. You
will be asked to comment on the peak assignments and spin-spin splitting.

Switch the final acetylacetone sample and collect at least three different
1
H spectra, with the
same number of acquisitions. Carefully integrate all the acetylacetone peaks in every spectrum.
You will use the normalized intensities of the methylene and methine peaks to calculate the
18
percent enolization.
Report
Compare the three spectra recorded of ethylacetate, one scan before spin-lock-shim, one scan
after, and the final signal averaged spectrum. Discuss the importance of each step for obtaining
high-resolution NMR spectra.

Discuss the peak assignments and spin-spin coupling for ethylacetate, cinnamic acid, and
acetylacetone. Include labeled molecular structures to aid in your discussion.

Report the average integrated intensity (and standard deviation) for the exchanging peaks in the
acetylacetone spectra. Calculate the percent enolization for this molecule in this solvent and
temperature (clearly explain how you did this calculation). Compare your results to those found
in the literature.

Discuss the applicability of NMR for quantitative measurements. What is the largest source of
error?

Notes on using the Bruker AC-200- TECMAG 200MHz NMR

Precautions
WARNING!!!! Remove your wallet, watches, etc. before approaching the magnet! Never bring
any ferromagnetic items into the magnet room. Be careful with metal objects in the room. Avoid
taking your credit or ATM cards too close to the magnet (approx. 2 feet). No loose staples or
paper clips allowed. People with medical implants should not approach the magnet. People
with pacemakers are not allowed in the room. You must be trained either by the class instructor
or by an NMR staff member.

This instrument is also known as the Bruker AC200. It has a Tecmag DSPect hardware-
computer interface module. The NTNMR software is run under Windows-NT. The magnet
strength is 4.7 Tesla.

Sample Preparation in the NMR Room
At the NMR console you will find the spinner (a plastic collar) that fits snugly over the NMR
tube. Slide the spinner over the NMR tube to the appropriate place using the depth gauge (also
located near the console). (Note: Be careful not to get oil from your fingertips on the NMR tube
or spinner. Use a Chemwipe to clean the outside of the tube before inserting into the magnet.)

Data Collection
On the computer keyboard, press Ctrl-Alt-Del simultaneously, then login.
o Insert the sample tube in the spinner, use the depth gauge to adjust the depth of the NMR
tube in the spinner
o On the SCM (NMR) keyboard, Press the orange button, then Press the Lift button to turn
the lift air on. Put the sample on the column of air on top of the magnet. Press the Lift
off button to lower the sample into the magnet.
19
o Press Spin button to spin the sample. The light on the Spin button should turn on.
o On the NTNMR software window click on the Console button to open the Console
Toolbar window, Click on load, select chem480a.shm file, then Click Open.
o This will load the standard shim set on the SCM keyboard.
o Press Lock Power on the SCM keyboard and set the value between 30 and 35 by turning
the knob on the keyboard. (The range of 30-35 assumes CDCl3 solvent; for Acetone-d6
use 10-15).
o Press Lock Gain and adjust the value between 100 to 104.
o Press the Lock Phase button and adjust the value to 253 (This value may change in the
future; look for a note on the computer with the current value).
o Press Field and adjust the field value till the deuterium signal appears in the middle of
the window. (Feb. 23, 2000 Field = 4241 for CDCl3, Field = 4031 for Acetone-d6).
o Press Lock (the deuterium signal will disappear and the light on the Sweep OFF button
will turn on). The Lock button light will blink.
o If the lock light is blinking, Press Field and slowly adjust the field until the lock light
stops blinking. (Generally, for a solid lock the line should be over one grid line above the
center).
o Now shim the magnet


Shimming Procedure:

Check to make sure that the Fine button has a green light indicating it is engaged. As when
locking, try to maximize the lock signal level by selecting a shim key (see below) and very
slowly adjusting the knob at the bottom of the SCM keyboard.

o If you have difficulty shimming, try reading the latest stored shim file (ask GSI).
o If the lock level disappears off the top of the screen, Press Lock Power and lower the
current value until the lock level is one to two grids below the top.
o Press Z and adjust the value in whichever direction increases the lock level.
o Once the Z value is optimized (i.e. the lock level is maximized in the window) press Z2
and repeat the procedure. Repeat the procedure for the X and Y shims. DO NOTadjust
the Z3 or Z4. Doing so may cause the
1
H signal to be lost.
o After optimizing Z2, X, and Y go back to Z and re-optimize it. Repeat this procedure
(with Z and Z2 or all shims?) until the lock level is completely maximized in the window.
o Press Lock Phase and adjust to maximize lock level
o Press Standby


1
H NMR ACQUISITION

On the NTNMR software click on the File menu. Select Open. Click on small inverted
triangle then Click on NTNMR, then double Click on Data. Select the H1_CDCl3.tnt file
20
(for Acetone-d6 solvent select H1_Acetone.tnt file) and Click the Open button. It will load the
standard proton parameters.

Click on the Dashboard button to check the parameters and make necessary changes
(e.g.number of scans (NS), spectral width (SW), etc.).



Begin with the RG (receiver gain) set at 2. It may be increased at values of 2
n
where n is an
integer.

Click on ZG in the Acquisition Toolbar and wait for the acquisition to finish.






During acquisition, check to make sure the receiver is not saturated. If the FID appears to be
rectangular at the beginning (on the left side) instead of a clean, exponential decay, the receiver
is saturated. Check the receiver gain (rg) under the hardware tab at the lower left side of the
screen. If saturated, wait until the end of the run and change the value to 2 (or 0 if its already at
2) and re-acquire. Do not abort the acquisition If you simply stop the run, the receiver may not
gate properly during the run and you will see no signal. If changing the rg to zero still doesnt
prevent receiver saturation, you need to dilute your sample.


Processing the Data::


Fourier transform: To obtain a frequency-domain spectrum, the data will have to undergo a
Fourier transform. Once the acquisition is complete, hold the SHIFT key (on the keyboard) and
click on the NDFT button. Check the boxes next to 1D Settings, Zero Fill, FT, and set the
apodization to Exponential. Click Set to save the settings. Now, click NDFT without the
SHIFT key to transform your FID. The result should be the
1
H spectrum. Feel free to play
with these settings and observe the effect of each on the resulting spectrum. Press Ctl-F to
perform the Fourier transform in the future.

Phasing: If the spectrum has an uneven baseline and no evident TMS peak (when spinning,
locked, and shimmed), it is possible that the receiver was saturated during acquisition. Adjust
the receiver gain accordingly and repeat the scans.

Press Ctl-H to perform an automatic phase correction. Manual phasing may be necessary. Click
on the Option menu and select Phase Adjustment, it will open the phase adjustment
window to the left side of the spectrum. Adjusting the first sliding bar, Ph0, will adjust the line
shape of all the peaks. The second sliding bar, Ph1, acts as a pivot and has a greater affect on
21
peaks farther away from a selected point. By clicking on the spectrum and then clicking on
set, you can choose such a point. Use these bars to obtain a spectrum where all of the peaks
are upright and have as close to a Lorentzian shape as possible. Click on Apply to save the
settings for printing.

Expanding the spectrum: To expand the spectrum, drag with the left mouse button from one side
of the spectrum to the other side. A black block will appear. Clicking in the black block will
expand the region. To redisplay the full spectrum, Double Click in the small spectrum window
in the upper right corner of the spectrum.

Vertical scale: The vertical scale can be increased or decreased with the UP and DOWN arrow
keys on the keyboard, provided the following yellow button is not depressed.

Base line correction: Type Ctrl-B to perform a base line correction.

Reference: To assign a Chemical Shift expand the region around the peak and put the cursor on
the top of the peak you wish to assign. Use the left and right arrow keys on the keyboard to
move cursor one pixel at a time. Click the Right mouse button in the spectrum window, select
Processing, then select Set Reference; change the scale to PPM and enter the correct
reference value.

Integration: Manual Integration - Highlight a peak as if to expand it; but Click with the right
mouse button, then select Add Integral. Repeat for each peak. OR - Click on the Option
menu and select Integrals. It will open a window on the left side of the spectrum. Expand the
region of interest then Click on the Auto button. Integrals will be drawn in boxes over the peaks
in the expanded region. Drag the left or right edge of the box to adjust the start and end position
of each integral. Integrals can also be adjusted by double Clicking on any Integral box. An
Integral Parameters dialog will open which permits adjustment of all parameters for each
individual Integral.
Slope and Curvature of an Integral can be adjusted by dragging with the double headed arrow
the blue squares at the bottom of the box. The Integral offsett can be adjusted by dragging with
the hand the individual Integral box.

Integral Reference: Double Click on the integral box for the integral to be used as the integral
reference value. Type in the Assigned Value, Click Apply, then close.

Peak Picking: To set the Threshold for Peak Picking Click on the Option menu, select Peak
Pick. This will open a Peak Pick window to edit Level and Noise. The Level Threshold sets a
minimum level for the height of the peaks. The Noise threshold sets the variance between points
needed to define a peak. Click on Calculate, then Click on Apply. OR Click the mouse in
the black region displayed on the spectrum window and adjust the threshold by adjusting the
height of the black window.

Plotting the Data:

To Plot, Click on the File menu and Select Add To Print Preview. The Spectrum,
22
Parameter Window, and the date will appear, along with a four-headed cursor.

Click on the date, and drag it to the top. Click on the Comment button (first button on Main
Toolbar). Edit the comment to create a title. A title can also be created by Right Clicking on
Spectrum region, selecting Label and then clicking Insert. A Dialog box will open. Type the
title and Click OK. To erase this title Right Mouse Click on the title, select Label, then Click
Erase.

Double Click on the blank parameter window. A Printed Parameter dialog box will appear.
Click on Load, then bring up the NTNMR directory, Click on setup and select
Parameters.par file. Click on Open, Click on Apply, then Click OK. Resize the
parameter window with the mouse. Click on Exit at the bottom of print options window.

Click on the Printer button.

To save the spectrum, Click on File and Select Save As. Then click on Tecmag 200
NTNMR data partition. Double click on your folder and type the file name then click on
Save.
Final Details
To eject the sample Press Spin on the SCM keyboard (to turn the spinner off), and Press the
black button located on the left front edge of the console. Press the orange button, then Press
Lift . Pickup the sample, then Press Lift Off to turn the eject air off.
Press Ctrl-Alt Del, then logout of the computer
Common Issues

Spinning

Spin light wont stop blinking It is not going to.. A problem with the sensor causes this to
happen. As long as the actual reading is stable, continue with the run. If the reading is not
stable, pull your sample, re-load it, try again. If it is not stable the second time, do not continue.
Inform Chris Kojiro and your GSI of the problem
Sample wont stop spinning, but the spin light is off The air flow doesnt turn off right away.
On the left-hand side of the console, just underneath the tabletop, theres a small black button.
Press and hold that button. The sample should stop spinning.

Locking

Sample wont lock -- lots of potential reasons
o Check the lock phase, gain, power, and field values and make sure you use the suggested
values as a starting point make small adjustments with the knob when making changes.
You should see the line jump as you near the correct field.
o Check to make sure the Fine button is on
o Check your sample. The tube should be filled with approximately 3-5cm(up top it says
3cm) of sample and there should be no visible solids. If your sample meets those
23
conditions, but it still wont lock, remove some of the sample and add more solvent. Its
possible that there is simply too little deuterium to obtain a lock.
o Turn up the lock gain and power. Make sure that when the Field button alone is
engaged, the signal looks like two sine waves, one increasing in intensity, one
diminishing. They should meet in the middle of the PCI window. If they dont, adjust the
field until they do. The two waves should be reflections of each other. If they signal is
asymmetric, adjust the lock phase slightly to maximize symmetry. Once it is both
centered and symmetric, push Field, then Lock and attempt the locking procedure again.
o Still wont work? Call the GSI


Signal acquisition/Spectral processing

No signal a few possible reasons
Is the lock holding through the run? If not, see the above section. A drifting lock will cause
poor peak shape and potentially no visible signal
Check the receiver gain (rg) value. If it is not a value of 2
n
, you may not see a signal. Set rg
to 2 and try again.
Did you abort a run prior to this acquisition? If so, set the number of scans to 4, the rg to 2,
hit zg, and let the run go to completion. Sometimes after a run has been aborted, the receiver
does not seem to gate correctly. Doing a quick run seems to reset the system.
Load the default shim setting file and re-shim the instrument. Be sure the Fine button is
engaged. A poor set of shims can ruin your signal.
Increase the sample concentration. If you dont have enough nuclei present, you wont get a
signal.
Still wont work? Call the GSI

No TMS peak, uneven baseline Probably a function of a saturated receiver. If the FID appears
to be rectangular at the beginning (on the left side) instead of a clean, exponential decay, the
receiver is saturated. Decrease the receiver gain (remember the 2
n
rule) and try again. See the
directions for more information about the receiver gain. It wouldnt hurt to re-shim either.


Poor peak shape, difficulty phasing Shimming, shimming, shimming. Go through the shim
procedure very slowly and carefully. Make sure you have maximized the signal. Be sure the
Fine button is on while adjusting the shims.

NMR Staff: Dr. Chris Kojiro, 3500A Chemistry Building
734-763-2009; ckojiro@umich.edu
Version 1.1- June 07, 2000; 2001 The Regents of the University of Michigan, Modified by
Kathryn Hughes and Amy Gottfried, 2003.

24
Spectroscopy of Iodine
Objective
Become familiar with operation and capabilities of the UV absorption spectrometer
Use spectrometer to obtain spectra of gaseous iodine
Learn to interpret spectra in terms of electronic and vibronic structure and quantum levels
Reading
Shoemaker, D.; Garland, C.; Nibler, J. Experiments in Physical Chemistry, 5
th
ed.; McGraw-
Hill: New York; 1989, pp 497-507.
Stafford, F. E. J. Chem. Ed. 1962, 39, 626
McNaught, I. J. J. Chem. Ed. 1980, 57, 101.
Barrow Introduction to Molecular Spectroscopy, McGraw-Hill: New York; 1962, Chapter 10.
Herzberg, G. Molecular Spectra and Molecular Structure. I. Spectra of Diatomic Molecules, 2
nd
ed; D. Van Nostrand: New York; 1950, Chapter IV, VII, Sec. 3.
Long, George; Zielinski, Theresa Julia. J. Chem. Educ. 1998 75 1192 (Important!).
Atkins, P. W. Physical Chemistry, 8
th
ed., Freeman: New York, pp. 563-565; 573-581.
Introduction
It is well known that molecular energy transitions can be classified as rotational, vibrational or
electronic depending on how the energy is localized within the molecule. Changes in the
electronic energy almost invariably are accompanied by simultaneous changes in the vibrational
and rotational energies. If the energy emitted or absorbed is in the form of light, the characteristic
wavelengths involved give rise to the vibronic spectrum of the molecule. Since rotational energy
levels are much more closely spaced than the vibrational, and the latter in turn are more closely
spaced than the electronic, the actual appearance of a vibronic spectrum of isolated (gaseous)
molecules as displayed by a spectrograph is a series of groups of lines. Each group constitutes a
band and the whole spectrum is called a band spectrum. Unless the experimental equipment is of
unusually high quality, the rotational lines in an electronic spectrum are not resolved and only
the vibrational structure is visible. In the following discussion, it will be assumed that is the case
and the presence of rotational transitions will not be considered.

The definition of the various energy quantities associated with electronic transitions of a simple
diatomic molecule can be clarified with the help of the schematic diagram in the accompanying
figure (Figure 1). In this figure, energy is plotted along the ordinate and the internuclear
separation of the two atoms along the abscissa. The curves represent the potential energy of the
molecule as a function of the internuclear distance for two different electronic states, the lower
curve applying to the ground state and the upper to an excited electronic state. Energies of the
molecule in the various vibrational states are indicated by the horizontal lines. Allowed energy
values for vibrational states of the electronic ground state can be represented by a series
expression involving a vibrational quantum number, v", as follows:

G(v") = e
e
" (v"+1/2) - e
e
"x
e
" (v"+1/2)
2
+ e
e
"y
e
" (v"+1/2)
3
+ ...[1]

25
The quantities, (e
e
"x
e
"), (e
e
"y
e
"), etc., are referred to as anharmonicity constants. They result
from the fact that the potential energy curves in the energy diagram (Fig.) are most easily
expressed as a power series in the internuclear distance. The terms beyond the first term, which
is the simple harmonic term, are referred to as anharmonic terms. As a consequence of these
anharmonic terms, the vibrational energy levels are not evenly spaced (as they would be for a
simple harmonic oscillator) but converge slowly to a limit, labeled the dissociation limit in the
figure. At this energy, the classical amplitude of vibration of the two atoms is sufficiently large
that the molecule no longer can remain stable but dissociates into two atoms.

These considerations apply to the excited state as well as the ground state. The vibrational
frequency, e
e
', and anharmonic constants, (e
e
'x
e
'), etc., for this state will differ from those of
the ground state since the electron configuration is different. As in the case of the ground
electronic state, the vibrational levels converge to a limit at which the molecule dissociates into
two atoms. If this dissociation limit is higher in energy than that of the ground state, as it is
shown in the figure, one or both of the atoms produced will be in an excited state instead of both
being in their ground state. The quantity, E
a
in the figure represents the atomic excitation energy.
Its magnitude can be obtained from knowledge of the atomic energy levels, providing the
particular atomic excited state involved can be identified. The energy needed to carry a molecule
from its lowest vibrational (ground) state in a given electronic state to the dissociation limit of
that state is termed the dissociation energy and usually is designated by D
o
. This quantity for the
ground state is of considerable chemical interest and thermodynamic importance; in the figure it
is labeled D
o
". It should be clearly distinguished from the quantity labeled D
e
" which is the
energy difference between the minimum of the potential curve and the dissociation limit.

In principle, it would appear that the quantity D
o
" might be determined by observing transitions
from the ground vibrational state to the upper vibrational levels. Such a spectrum should appear
as a series of absorptions in the infrared and near infrared regions converging to a continuum.
Measurement of the frequency of the continuum edge would give directly D
o
". In practice, this
procedure is not feasible since transitions involving a change of more than one unit in the
vibrational quantum number have a very low probability and cannot be observed. Moreover, in
the case of homonuclear diatomic molecules, the absence of a dipole moment causes even the
fundamental transition between the first two vibrational levels to be of such low intensity that it
normally is not observed.

These restrictions on vibrational transitions do not apply when they accompany an electronic
transition and consequently a typical band spectrum consists of a converging series of absorption
maxima representing transitions to successively higher vibrational levels. Most of these
transitions originate from the lowest vibrational state of the electronic ground state and terminate
in the vibrational states of the excited electronic state. The observed convergence limit thus gives
a value for E* shown in the Figure. Since the separation between successive vibrational levels
becomes quite small near the convergence limit, however, it is often difficult to determine
accurately the exact edge of the continuum. Various methods have been used to circumvent this
difficulty and obtain a reliable value for E*. The procedure to be used in this experiment, the
Birge-Sponer method, depends on the fact that the series represented by Eqn. (1) is strongly
convergent allowing third and higher power terms to be neglected. Within this approximation, it
26
can be shown that the difference in successive band maxima, A(Av) in the observed spectrum can
be expressed by Eqn. 2, below. (Since each maximum in the observed spectrum has resulted
from a vibronic transition, Av; the difference between two maxima, therefore, is A(Av)).

A(Av) =e
e
' - 2x
e
'e
e
' (v' + 1) [2]

Here, v' is the upper state vibrational quantum number for the lower frequency maximum. Note
that Eqn. (2) is independent of the ground state vibrational quantum number from which the
transition originates. Since. Eqn. (2) is linear with (v' + 1), an extrapolation can be carried out to
obtain the value of v' for which Av = 0; this, by definition is the dissociation limit. The dissociation
energy of the upper electronic state, D
o
' can be obtained directly from the area under the curve of
Eqn. (2). Alternatively, it can be obtained from the relationship:

2
'
' 4
'
'
e
e
e
o
x
D
e e
=
[3]
Chemicals
A sealed glass cell containing several I
2
crystals.
Instrument
A Shimadzu ultraviolet/visible scanning spectrophotometer will be used for this experiment.
Procedure
The experimental procedures for this experiment are relatively straightforward and involve
recording the absorption spectrum of iodine vapor contained in a sealed glass cell. The vapor is
generated by heating the sealed glass cell in an oven until the iodine is in the gas phase (Heating
is not necessaryit does increase the overall intensity of the bands).

Turn the UV-Vis spectrometer on and allow the lamp to warm up for at least ten minutes. Open
the Shimadzu operating software. You will need to choose appropriate parameters in order to
collect the most resolved spectrum possible try several settings and compare the results.
Report
Determine the wavelengths of the various band maxima in the iodine spectrum and convert to
cm
-1
units by taking reciprocals- include a figure showing these assignments and your spectrum
in the report. (Although this procedure neglects the correction for the refractive index of air, the
error is negligible in this experiment). With the help of the sketch below assign the upper level
vibrational quantum numbers to all observed transitions. Construct the Birge-Sponer plot and
determine the value of v' at the dissociation limit.

From your results, determine E*, D
e
', D
o
', e
e
', x
e
'e
e
', D
o
" and v
o
" (v
o
" is the first vibrational
transition in the ground electronic state) along with their uncertainties. Be sure to show the
calculations used, and describe the physical meanings of these parameters. To determine D
o
",
use the value E
a
= 7603 cm
-1
for the atomic excitation energy of an Iodine atom (Moore, Natl.
27
Bur. Stnds, Circ. #467 (1958)). Using a reasonable uncertainty in the value for E* and a 0.1%
uncertainty in the value of E
a
, calculate the uncertainty in the value of D
o
". Why are transitions
from both the v" = 0 and v" = 1 vibrational states of the ground electronic state observed in the
spectrum? What would be the effect of heating the sample?
Numbering of vibrational transitions in the band spectrum

In the article by Stafford entitled, Band Spectra and Dissociation Energies, errors are present in
the numbering scheme, since he neglected to take into account the fact that the observed bands
arise as a result of transitions from both the v " = 0 and v " = 1 vibrational states of the ground
electronic state. Such an error introduces a discontinuity into the numbering scheme which is
easily obscured, or at least partially so, by the scatter of points, but which nevertheless introduces
some error in the slope. A key to the correct numbering scheme is provided by the sketch below
which shows the correct assignments for a small number of bands to the long wave length side of
the mercury 5461 Angstrom line. The numbers indicate the vibrational quantum number for the
upper state vibrational level and identifies the ground state origin. Data from both series can be
used satisfactorily in a single Birge-Sponer plot if the differences between successive upper state
vibrational levels are correctly plotted against the upper state quantum numbers.



28
Figure 1
29
Notes

You must determine the wavelength location for peaks in the v''=1 series as well as in the v' '= 0
series. You do not need to do separate calculations for each series. Since the Birge-Sponer curve
does not depend on the starting point of the transition, you can pool the data from both series in
making your plot. To just use the v' = 0 series is also sufficient.

The following method for determining E* should be substituted for the method described in the
lab manual.





For a transition to a particular value of v', determine the area under the Birge-Sponer curve from
that value of v' to the x-intercept (as shown above for v'+1=23 or v'=22). This area summed with
the corresponding transition energy from v''=0 to that particular value of v' (= frequency of your
band head at that v' for the v'' = 0 series) will be equal to E*. This can be done for every point in
the v'' = 0 series, yielding a large number of values for E* from which an uncertainty in E* can
be determined (all these calculations can easily be incorporated into a spreadsheet).

30
Fluorescence Spectroscopy
Objective
- Become familiar with operation of fluorescence spectrometer
- Use spectrometer to acquire spectra of quinine under different conditions
- Learn how fluorescence is affected by conditions and interpret in terms of mechanism of
fluorescence
Reading
Lakowicz, J.R. Principles of Fluorescence Spectroscopy, 2
nd
. ed; Plenum Press; New York,
1999.
Skoog, D.A.; Holler, F.J.; Crouch, S.R. Principles of Instrumental Analysis, 6
th
ed.;
Brooks/Cole; Belmont, 2006; Chapter 15.
Willard, H. H.; Merritt, L. L. Jr.; Dean, J. A.; Settle F. A. Jr. Instrumental Methods of Analysis,
7th ed.; Wadsworth: Belmont, 1988.
O'Reilly, J. E. J. Chem. Ed., 1975, 52, 610.
Introduction
Fluorescence is the emission of light when an electron from an excited molecule decays to a
lower electronic state of the same multiplicity. Because most molecules have electrons that are
paired in the ground state, fluorescence involves a singlet-singlet transition. Based on the
following Jablonski diagram, there are several pathways by which an excited molecule may lose
energy.

{
{
{
S
S
T
1
1
2
a a f e
p
e
internal
conversion
intersystem
crossing
{
S
0

Figure 1: Energy-level diagram for molecular ground state (S
0
), first (S
1
) and second
(S
2
) singlet excited states, and triplet excited state (T
1
). a: absorbance; f: fluorescence; e:
external conversion; p: phosphorescence.
31

Fluorescence competes with nonradiative pathways and the longer lived phosphorescence decay.
Quenching occurs when another component acts to favor one of the radiationless pathways, thus
decreasing the likelihood for fluorescence. This can occur by static quenching where
complexation of some component with the fluorophore renders it dark. More commonly,
dynamic quenching occurs where the fluorophore in the excited state interacts with other
components and energy is transferred nonradiatively. In this case, a relationship exists between
the quantum efficiency of the fluorophore in the presence of quencher (|
f
) and in the absence of
quencher (|
f
).

|
f

|
f
= 1+ K
q
[Q]

This expression, known as the Stern-Volmer equation, describes the decrease in the fluorescence
quantum yield as a function of quencher concentration ([Q]). Because the fluorescence intensity
is directly proportional to the fluorescence quantum yield, the Stern-Volmer constant may be
determined from intensity measurements as a function of quencher concentration (3).
Chemicals
Quinine sulfate
Sodium chloride
A sample of flat tonic water (available in the laboratory)
Instrument
An RF-5301PC Shimadzu Spectrofluorophotometer is available in the laboratory.

Procedure

Solution preparation
Prepare a 100 ppm stock solution of quinine in 0.05 M H
2
SO
4
(acid should be available in
laboratory) using quinine sulfate monohydrate as standard material.

Make serial 1:10 dilutions of this standard stock solution in 0.05 M H
2
SO
4
to achieve standards
ranging from 1 ppb to 1 ppm.

Prepare sample solutions as follows: pipet 5.00 mL of tonic water sample into a 250 ml vol.
flask. Dilute to the mark with 0.05 M H
2
SO
4
. Then pipet 5.00 mL of this diluted sample into a
25 mL vol. flask and dilute to the mark with 0.05 M H
2
SO
4
. This will give a final sample
dilution factor of 250.

Prepare 6 solutions containing 1 ppm quinine and 0, 50, 100, 300, 1000, and 2000 ppm NaCl all
in a background of 0.05 M H
2
SO
4
.
32
Instrumental start-up and operation
Turn instrument power on and allow both to warm-up for 5 to 10 minutes.

Familiarize yourself with the operation of the instrument by running the Tutorial beginning on
page 3-1 of the instruction manual. After running the Tutorial successfully, proceed to obtain
fluorescence spectra of your quinine samples.
Excitation and emission spectral acquisition

Fill a clean quartz fluorescence cell with the 1 ppm quinine standard. Place the cell in the cell
holder and position the holder in the proper location (in the light path).

Follow the general procedure of the instruction manual to set instrument parameters to collect
fluorescence excitation and emission spectra. Use the following settings:
(a) Use 5 nm slits (bandwidth) and begin with the SENSITIVITY set on the lowest
setting.
(b) Set the emission wavelength to 0 and collect the fluorescence excitation spectrum
between 700 and 250 nm.

Press the START key to start the measurement. The shutter should automatically open (indicator
light turns off). Check with the TA if this does not occur.

Repeat the fluorescence excitation measurement if the sensitivity needs to be adjusted.

Based on this excitation spectrum, position excitation monochromator to the wavelength that
yielded the maximum signal.

Make certain that the photomultiplier shutter is closed.

Reposition the emission monochromator to a wavelength 10 nm greater than the excitation
monochromator setting and then reopen shutter.

Scan the emission spectrum over a range of 250 nm.

Close the shutter and repeat the entire process using 1.5 nm bandwidth. You may have to
increase sensitivity to see absorption and emission peaks.

Compare spectra obtained with the bandwidth set at 1.5 and 5 nm. Are there any noteworthy
differences? Determine
max
for absorption and
max
of fluorescence for each spectrum.

Include the excitation and emission spectra in your laboratory write-up.
Analysis of quinine in tonic water
You will be running a fixed time scan to determine the fluorescence intensity of samples and
standards.

33
With the shutter closed, change bandwidth to 10 nm and set Excitation wavelength to absorption
maximum observed in previous scanning experiment. Adjust Emission wavelength to maximum
observed in emission spectrum.

Set ACQUIRE MODE to TIME COURSE

Place a cuvette containing 0.05 M H
2
SO
4
into the sample holder and open the photomultiplier
tube shutter.

Zero output using "AUTO-ZERO" key. Close shutter.

Set the scan speed to a longer value (e.g. 2 sec). This is the time the display will take in updating
the value. Keep this rate constant for all runs. Hit the start/stop key and record enough values
from the display so that determination of error is feasible (>15). Ignore the first few displayed
values. Then, place cuvette containing 10 ppm quinine standard in holder and record relative
signal on display. If signal is off-scale, readjust sensitivity.

Whenever you change sensitivity you should always recheck zero value using the blank solution
as the sample (0.05 M H
2
SO
4
). In addition, you should always record the sensitivity factor when
writing down relative fluorescence for a given standard. To obtain data for calibration plot, clean
sample cuvette thoroughly with distilled water, and rinse several times with the next lowest conc.
standard (e.g. 1 ppm). Record relative fluorescence (you may have to adjust sensitivity control).
Repeat this process for all standards (down to 1 ppb). You will probably have to adjust the
sensitivity to the highest value to see a signal for the lowest standard. To construct a calibration
curve, plot log (relative fluorescence) vs. log (conc.). (Note: Relative fluorescence values can be
determined by dividing the display value by an arbitrary sensitivity factor for the detector. This
factor can be determined by dividing the data values obtained for a standard that is n-scale at
both high and low sensitivity.)

Perform similar measurements for diluted samples (in triplicate).

From calibration data, determine the concentration of quinine in samples.

Determine the standard deviation of several blank readings. Using this data, estimate the
detection limits of the method. Clearly discuss the assumptions and reasoning behind your
calculation.
Quenching by chloride ions
Clean the cuvette and fill it with 1 ppm quinine standard containing 0 ppm chloride. Measure the
fluorescence intensity using optimum excitation and fluorescence wavelengths.

Repeat fluorescence measurements for 1 ppm quinine standards containing various chloride ion
levels. Graph this data appropriately and determine the Stern-Volmer quenching constant (K
q
)
for chloride ion. Discuss the assumptions and reasoning behind your determination.
34
Report
What effects do slit-widths have in fluorescence measurements? Which slit-width would give a
lower limit of detection for quinine? What is the relationship between slit-widths and
bandwidths?

Why is quinine such a good fluorophore?

How do your values for quinine in tonic water compare with the suggested levels?

Describe types of quenching. What type of quenching is observed in this experiment?

What are the practical analytical implications of the chloride ion quenching portion of the
experiment?

What effects could CO
2
have on your results if it were present in your sample of tonic water?

35
Surface Tension

Objective
- Become familiar with concept of surface tension and how it varies with solution
chemistry
- Use two different methods to measure surface tension of water and alcohol/water
mixtures

Reading
Shoemaker, D.; Garland, C.; Nibler, J. Experiments in Physical Chemistry, 5
th
ed.; McGraw-
Hill: New York, 1989; pp. 339349.
Daniels, F.; Williams, J. W.; Bender, P.; Alberty, R. A.; Cornwell, C. D. Experimental Physical
Chemistry, 7
th
Ed. McGraw-Hill: Toronto, 1970, pp. 359365; or
Salzberg, H. W.; Morrow, J.; Cohen, S. Laboratory Course in Physical Chemistry, Academic
Press: New York, 1966, pp. 100110.
Central Scientific Co. Bulletin 101 (in binder with other handouts)
Introduction
The interface between phases is of primary importance in a diverse number of systems ranging
from cell membranes to oceanographic systems. It is clear that the properties of the interfacial
region are not accurately described by the properties of either phase individually. The
mechanical properties of a surface are often treated as a hypothetical membrane across the
surface. This membrane is described as being in a state of tension or negative pressure. That is,
surface tension is the force per unit length parallel to the surface that acts to oppose any increase
in surface area. A more complete discussion of surface tension and the Gibbs adsorption analysis
may be found in the references.

In this experiment, the surface tension of water and several aqueous solutions of an aliphatic
alcohol will be measured using the capillary rise method. The data are to be analyzed in terms of
the Gibbs isotherm equation. For comparison, additional measurements will be made with the
more rapid but often less accurate du Nouy torsion balance.
Apparatus
A capillary tube and outer jacket are included in a kit available from the laboratory stockroom.
Water baths and cathetometers are set up on one of the central tables. In place of a previously
used cathetometer, a 3Com Home Connect camera is now connected to a computer so that you
can record images of the level of the liquid and make accurate distance measurements. A du
Nouy torsion balance and platinum ring may also be obtained from the laboratory stockroom
when you come to that part of the experiment.
Procedure
Note: To maker sure that soap residue will not interfere with your results, clean all glass ware
with room temperature nitric acid. Also, use Milli-Q purified water rather than plain distilled
water for best results.

36
Prepare an approximately 0.8 M solution of n-butyl alcohol by pipetting 35 mL of the alcohol
into a 500 mL volumetric flask and filling to the mark. The exact concentration can be calculated
using the density for the alcohol. Prepare by careful dilution 250 mL of each of the following
(approximate) concentrations: 0.4, 0.2, 0.1 and 0.05 M.

The surface tensions of these solutions are to be measured by the capillary rise method using the
general procedure outlined in the text (S, G, S, & N). The capillary tube must be scrupulously
clean since traces of contaminants have disproportionately large effects on the surface tension.
For actual measurements, the capillary and outer tube containing the liquid is placed in a
thermostat regulated to 25 C. After the sample has come to temperature (10 to 15 minutes), an
image of the meniscus in the capillary and the meniscus in the outer tube can be taken with the
camera. Carry out your measurements on the most dilute solution first and proceed to more
concentrated.

Camera: Open the ViVIEWer software. Use the dial on the camera to focus the image. Click the
capture frame icon to take (capture) the picture. Save the image in your own folder as a .bmp
or .jpg file. Open the file with the ImageJ software to manipulate the image. You can compare
distances on the ruler to pixel numbers to more accurately measure meniscus height.

To determine the effective radius of the capillary, the rise of a liquid of known surface tension
should be measured; toluene is preferable to water since traces of impurities have less effect on
the magnitude of the surface tension. Find the surface tension of toluene, record it for your
report, and cite your source. Note: surface tension is temperature-dependent. To eliminate
possible error due to non-uniformity of the bore of the capillary, it is advisable to select some
points of the capillary and adjust the depth of immersion of the capillary so that the meniscus
rises to this same point in each determination. Further errors may be eliminated by using the
same capillary tube throughout this experiment.

Use the ring method to measure the surface tension of water, toluene and your alcohol solutions
at room temperature. Refer to the manual for relevant details. Be sure the platinum ring is flat
and undeformed. For cleaning, it may be dipped in concentrated nitric acid, rinsed in distilled
water and oven dried. Do not touch with the fingers afterwards. To calibrate the torsion wire,
place a small slip of paper across the Pt loop and hang the latter on the horizontal arm of the
balance. Set the scale marker to the zero position and bring the torsion arm to the reference
horizontal position by adjusting the knob at the rear of the balance. Place a 50 mg weight on the
paper and note the increment in reading needed to bring the arm back to its reference horizontal
position. Repeat with several other weights and calculate the torsion constant in terms of dynes
per scale division.
Report
Capillary rise method:
For toluene tabulate the individual heights of rise, the mean height and the calculated radius of
the capillary.

Tabulate the mean heights of rise and the surface tensions of your alcohol solutions. (Assume the
densities of the butanol solutions are the same as pure water)
37

Loop method:
Calculate and tabulate the surface tension values, again based on the average of multiple
measurements.

Best method:
Analyze your data thus far. Choose the data from the method you consider the most reliable and
perform further calculations. Justify your decision.

Plot g versus the logarithm of bulk concentration (or activity), evaluate the slope of the line, and
calculate u, the (excess) surface concentration of solute.

Calculate the total number of molecules per unit area at the surface. (The surface molar
concentration in the absence of adsorption can be taken as the bulk molar concentration raised
the two-thirds power). This may vary for different solutions: why? Is there a limit where this
would no longer be expected to change? Interpret in terms of intermolecular forces and the
differences between surface and bulk environments.

38
ICP Emission Spectrometry

Objective
- Become familiar with the principles of atomic emission spectroscopy
- Become familiar with inductively coupled plasmas as a source for AES
- Learn to operate the ICP instrument
- Observe how nebulizer flow rate and ICP power affect the plasma
- Use the instrument to determine elements in drinking and tap water

Reading
Skoog, D. A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6th ed.; Thomson:
California; 2007, Chapter 11.
Nlte, J. ICP Emission Spectroscopy A Practical Guide, Wiley-VCH: Weinheim, 2003,
Boss, C. B.; Fredeen, K. J. Concepts, Instrumentation, and Techniques in Inductively Coupled
Plasma Optical Emission Spectrometry, Perkin Elmer, 1999.

Contact Person: Carol Carter, 2524.
Introduction
ICP emission spectroscopy (ICP-OES) is one of the most important techniques for elemental
analysis. The sample is atomized and ionized inside an extremely hot plasma. The atoms and
ions are excited within the plasma and emit light of characteristic frequencies that is detected and
used to identify and quantify a particular element. Descriptions of the operational principles and
theory behind the technique can be found in the references (the two latter books are located in
the ICP room). In this lab, you will familiarize yourselves with some of the parameters
important for optimum ICP analysis. You will utilize ICP-OES to analyze drinking fountain and
regular tap water from the Chemistry building.
Apparatus
The Perkin Elmer ICP Optima is located in 2314. Manuals and books are located by the
instrument and there is also an excellent on-line help menu that can be accessed at any time by
pressing F1. BEFORE you begin operating the instrument, check with Carol Carter.

Chemicals

1000 ppm standards of As, Cu, Fe, Na, Pb, Se, and Y are located in 2314. If you would like to
analyze for other elements, check the shelf and speak with Carol for availability of standards.
All samples* should be acidified to 1% v/v nitric acid (trace metal grade) and contain 1ppm Y as
an internal standard (*=except the Na and Y for the bullet test, see below). Also run a blank
(millQ water) and a known sample (provided) in addition to your standards and tap/drinking
water samples.
Procedure
Prior to beginning experiments, it is necessary to replace the sample aspiration tubing. See
procedure in the ICP-OES Users Booklet steps 1-3.
39

Experiment 1: Sodium Bullet Test/Yttrium Bullet Test
When elements are passed through the plasma the image of their emission takes the shape of a
bullet giving rise to the name of this test. The Na/Y bullet test will show the effects on the
plasma and analyte emission resulting from changing the nebulizer flow rate and the ICP power
settings. Sodium emits yellow-orange light (make sure you note the frequency) and Yttrium
emits red/blue light (again note the frequencies and the difference between the different
wavelengths). The plasma can be viewed either axially or radially. Note the best parameters and
apply those for your later experiments.

Detailed Procedure:

1. Login using CHM480 with student as the password.

2. Open Perkin-Elmer Winlab 32 software.

3. Using F10, move the autosampler sipper probe to the rinse station. You may also use
this command to manually sample your Na and Y solutions for the bullet test.

4. Open the Plasma Control window. Turn on the Plas, Aux, and Neb gases, then turn
on the pump. Make sure rinse solution is flowing through the pump tubing, sample
lines, and drain tubing correctly. Click Plasma On.

A. The software will automatically purge the sample introduction system with
argon, start and stop the peristaltic pump and nebulizer flow at the correct
times and ignite the plasma.

5. Aspirate the 1000 mg/L Na solution. Observe the position of the sodium bullet.

6. Click Override Method to allow adjustment of the Nebulizer and Power settings.

A. Adjust the Scott/cross-flow nebulizer flow to 0.7 L/min, then 1.0, then 1.3.

B. Click on Apply after each change of the Nebulizer setting.

C. Observe and note the changes in the bullet position at each nebulizer setting.

D. Set the nebulizer flow to 0.85 L/min.

7. What was observed when the nebulizer flow was changed to the different settings and
why?

8. Adjust the RF power to 1100 watts, 1300 watts, and 1500 watts on the axial/dual
view spectrometer. Click on Apply after each change of the power setting.

9. Note any changes and explain why they occur.
40

10. Repeat procedure for 1000 mg/L Y.

11. Which of the two parameters (RF power or Nebulizer Flow) has the greatest
effect on the bullet?

12. Note the changes in the Red(atom)/blue(ion) emission zones with different
nebulizer flow and power settings during the Y bullet test. Explain these changes.



Experiment 2. Determination of Fe, Cu, As, Se, and Pb in water.

A. Solution preparation. To determine the target elements in water samples, you must
calibrate the instrument for each element studied. To calibrate the instrument you will need to
prepare standard solutions with accurately known concentrations of the analytes, i.e. standards
should be prepared using quantitative glassware. Stock solutions of the analytes are available in
the ICP lab. You should prepare at least three different concentrations of each analyte for
calibration. Since the instrument can determine all elements simultaneously, the standard
solution can contain all of the analytes. (Thus, you can have three standard solutions, each
containing all elements of interest at different dilution.) The maximum concentration levels
allowed under the United States Environmental Protection Agency are: Fe 300 g/L, Cu
1300 g/L, As 50 g/L, Se 50 g/L, Pb 15 g/L [1].
You will need to collect drinking water and tap water samples. These samples should be
acidified (1% v/v) and spiked with internal standard at the same level as standard solutions. You
will also be given a known containing these elements for analysis. Analysis of the known will
let you know if you have calibrated the instrument reasonably well. Finally, a blank of
purified water should be prepared and analyzed.


B. Instrument Method and Sample Analysis. Calibration and analysis will require the
preparation of a method in the software of the ICP-OES instrument. The method instructs the
instrument control computer on the settings, procedures, replications, and standards used in the
analysis. Once the method is entered and started, the instrument will calibrate and analyze
according to these instructions using the solutions you provide it. Consult the ICP-OES Users
Booklet for step-by-step procedures for preparing a method and operating the instrument to
analyze samples. When running your calibration curve, run your standards from HIGHEST to
LOWEST concentration.
Following these procedures, the instrument will prepare calibration curves and report
analyte concentrations automatically. (The instrument will report how much analyte is in the
water sample you provided it. If you diluted your sample in adding the Yttrium and acid, you
must account for these dilutions in the final reported concentration.) Steps 1-3 of the Users
booklet do not need to be repeated if you did them above.

Select more than one wavelength for each element when applicable and compare your
results at different wavelengths is there a difference?
41

C. Detection limit determination. Not all elements will be detectable in the water samples.
Therefore you can only report that they are below the detection limit of the instrument (i.e., you
cannot say that they are zero, but below the minimal amount that you can detect). Therefore,
you will also need to determine the detection limit for your analytes.

Use the following equation to calculate the detection limit of the instrument:
LOD=3*BEC*RSD
0
. BEC is background equivalent concentration (obtained from your
calibration equation) and RSD
0
is the relative standard deviation of the blank. BEC is the
absolute value of the ordinate of the origin of the calibration curve (see figure). How do your
detection limits compare to what you would expect from ICP-OES?

Report
Explain how and why the flow rate and power settings influence the plasma and resulting
measurements. Name at least two additional experimental parameters that influence the results
(use the help menu and manuals to find out).

What is the role of the internal standard? What sorts of possible artifacts/errors can the internal
standard correct for?

Does detection limit depend on wavelength used?

Why did you choose multiple wavelengths, and what factors must be considered in choosing the
wavelength?

Discuss the concentrations of the elements in drinking and tap water. Are these concentrations
accurate and are they within safe levels?

1. Fernandez-Turiel, J. L.; Llorens, J. F.; Lopez-Vera, F.; Gomez-Artola. C.; Morell, I.;
Gimeno, D. Fres. J. Anal. Chem. 2000, 368, 601-606.


42
THE DETERMINATION OF DIFFUSION COEFFICIENTS
BY CAPILLARY ELECTROPHORESIS


INTRODUCTION
In the process of diffusion, molecules and ions move as a result of a concentration
gradient. This may be understood as a manifestation of entropy. A chemical system which
contains regions of different concentrations is more ordered than a homogeneous system. Thus,
the components move until the concentration is equalized throughout.
The parameter which specifies the inherent tendency of a substance to diffuse is called
the diffusion coefficient, D, which is defined by Fick's laws. In Fick's first law, D relates the
concentration gradient to the number of moles, N, of the diffusing substance which cross area A
per unit time, t:

where C/x is the concentration gradient, and the +x direction is perpendicular to A toward the
region of greater concentration. The negative sign indicates that the diffusing species move in the
opposite direction (high concentration to low concentration). The second law of diffusion gives
the change in concentration with time as the diffusion process occurs:

Substitution of units (A in cm
2
, C in mol cm
-3
, x in cm) shows that D is given in cm
2
s
-1
.
Diffusion plays an important role in many physicochemical processes, including
reactions at surfaces (electrochemical reactions, solid-phase catalysis) and very rapid (diffusion-
controlled) reactions in solution. Diffusion is also important in the distribution of nutrients and
pollutants in the environment. In biological systems, movement of species within and between
cells also involves diffusion in many cases; e.g., passage of the neurotransmitter dopamine across
a synapse.
Diffusion is also a major factor in the area of chemical separations, where the aim is to
isolate components in discrete zones; i.e., to create extreme concentration gradients. Thus,
diffusion coefficients appear in many of the equations (e.g., the van Deemter equation
1
)
describing chromatographic systems. Considering the general availability of chromatographic
instrumentation, this presents a possible method for the determination of D values. However, the
equations are usually multi-term expressions because of the complicated involvement of solute
diffusion in both the stationary and mobile phases. To measure diffusion coefficients, it is
necessary to use a simpler system not involving interactions of the solute with several phases. In
this experiment the method of choice is capillary electrophoresis.

CAPILLARY ELECTROPHORESIS
In capillary electrophoresis, the separation site is a very narrow-bore (25 to 100 m) tube
of silica, about one meter or less in length, with the ends in two vessels containing an aqueous
buffer. The buffer reservoirs are equipped with electrodes, and a high DC potential (~10
4
V) is
applied. To observe the separated zones, a UV/Vis source and detector are placed on either side
of the tube about 8.5 cm from the outlet.
43
The overall separation mechanism depends on the different rates of movement of charged
solutes in an electric field. The velocity of an ion in a field of unit strength (one volt/meter) is
called the electrophoretic mobility,
ep
. In CE there is an additional contribution to the overall
movement. The surface inside the capillary has exposed Si-O-H groups, which lose protons at
pH's above about 3. The negatively charged Si-O
-
s sites attract several layers of buffer cations,
which are each surrounded by a sheath of closely held water molecules. When the voltage is
applied, these cations move to the cathode (negative pole), carrying their hydration layers with
them. Because the tube is so narrow, the entire solution system moves in the same direction. This
movement is called the electroosmotic flow. Thus, there are two contributions to the migration of
an ion: its inherent electrophoretic mobility and the overall electroosmotic mobility,
eo
. These
terms are additive for cations, which pass by the detector first, in order of decreasing
electrophoretic mobility. For anions the contributions compete, but except for very small ions
(e.g., chloride), the electroosmotic flow predominates, and anions also move towards the
cathode. They are detected in order of increasing
ep
; those with greater
ep

have a greater
tendency to migrate to the positive pole, and they lag behind in the overall movement to the
cathode. Neutral species move with the electroosmotic flow but they have no electrophoretic
contribution. Thus, they arrive together between the cations and the anions. Unless special
methods are used, uncharged species are not separated from each other.

MEASUREMENT OF DIFFUSION COEFFICIENTS
The diffusion coefficient of a solute is obtained from measurements of the zone
broadening (peak width) caused by the diffusion process. Broadening is expressed in terms of the
uncertainty in the position of the solute; i.e., the solute has a distribution of positions in the tube.
Assuming the peak shape to be Gaussian, the standard deviation, , is one-half the width of the
peak measured between the inflection points, which occur at 0.607 of the maximum height. The
Hewlett-Packard
3D
CE computes w
h
, called the FWHM, the full width at half the maximum
height, which is (2ln2)
1/2
times the inflection-point width
2
. The FWHM is given in time units,
and it must be converted to length from the migration velocity, L
eff
/t
M
, in which L
eff

is the
effective distance of the capillary between the inlet and the detector, and t
M

is the migration time
of the zone center. Thus, the peak variance,
2
, is given by:


The measured variance is the sum of the variances caused by several zone-broadening effects
3
:

In equation 4,
2
diff

corresponds to the zone broadening caused by diffusion, which is given by
the Einstein equation
4
:

The second and third terms,
2
inj
and
2
det
, are the variances due to the finite lengths of
the injection plug (L
inj
) and the detector cell (L
det
). Sternberg
5
showed that these are given by
44

The usual method of sample introduction involves applying a pressure difference, P, to the
injection buffer container for a time t
inj

(usually a few seconds). The value of L
inj

is given by:

in which d is the capillary diameter, L
T
is the total length of capillary between the injection and
outlet ends, and is the buffer viscosity.
6

The next term,
2
diff
, represents electromigration dispersion, which includes band
spreading accompanying migration in the electric field as well as peak distortions due to
differences in the electric fields in the sample zone compared to the background buffer. Mikkers
et al.
6
examined these effects theoretically and showed that migration band spreading is small
compared to
2
diff

if the ratio of concentrations of the solute ion compared to the buffer ion of the
same charge is less than 10
-2
. Peak distortions due to field variations are eliminated by having
equal conductivities in the sample zone and background buffer; i.e., by dissolving the sample in
the background buffer.
Zone broadening can also occur because of thermal gradients, which are caused by Joule
heating from the electrical current. The resulting variance,
2
temp
, is usually small because of the
large ratio of surface area to internal radius of the capillary tube, but Joule heating effects can be
significant with high buffer concentrations and large applied voltages. In this experiment, Joule
heating should not be evident at low applied voltages.
The last term,
2
ia
,

includes other broadening effects, most commonly interactions of the
solute with the capillary wall. As described below, in this experiment wall interactions are
minimized by using a low buffer pH.

Stopped-migration method
The stopped-migration method makes use of two separate experiments. First, the sample
is passed through the capillary using the usual operating conditions, and the values of t
M

and w
h

are determined. Then the sample is analyzed under identical conditions, but with a voltage halt
when the zone has traveled about half the distance to the detector. The solute is allowed to
diffuse under zero-field conditions for about an hour. The voltage is subsequently reapplied and
the resulting w
h

is measured. Assuming identical conditions except for the halt, equations 4 and 5
can be combined to give


in which
1
2
and
2
2
refer to the variances without and with the voltage halt, respectively, and t
halt

is the duration of the halt.

Single-point method
Assuming that
temp
2
,
em
2
, and
ia
2

are all negligible, equations 4, 5, and 6 may be
combined to give:
45


Thus, the value of D may be obtained from
2
and t
M

for a single sample, if L
inj

and L
det

are
known. There is often an error in the value of L
inj

calculated by equation 7, because some sample
enters the capillary when it is placed in the container. Thus, use of equation 9 is subject to
considerable uncertainty.

Graphical method
The uncertainties in the single-point method can be controlled by performing a series of
single-point measurements at different low voltages. A plot of
2

versus t
M

should be linear. The
slope of the line is 2D and the intercept is (L
inj
2

+ L
det
2
)/12.

DOPAMINE
In this experiment the diffusion coefficient of dopamine, 3-hydroxytyramine, will be
determined by the three methods described above.

Dopamine serves physiologically as a neurotransmitter, and its diffusion coefficient is an
important physical property which has been determined by several groups.
8,9
This compound has
a tendency to adsorb onto the capillary wall, especially when the silica surface is negatively
charged. To circumvent this problem, the experiment is conducted at pH 3, where the majority of
the surface oxygens are protonated. As shown in the structure, dopamine is protonated at this pH.
The compound is easily air oxidized, but the problem is less troublesome when dopamine is in
the protonated form. Nevertheless, solutions must be prepared fresh daily with deaerated buffer.

PROCEDURE (These instructions apply specifically to the Hewlett-Packard
3D
CE.)

1. (Note: If the teaching assistant has already prepared the CE system, proceed to step 9). If
necessary, turn on the monitor (far right push-button, labeled with a circled 1) and the printer
(right front bottom rocker switch). Turn on the CE unit (lower left front push-button) and wait
for the yellow indicator light (top right) to go out. (Note: The CE unit must be on BEFORE the
software is activated.)

2. The computer should have Windows-95 active. Click <Start>, <Programs>, <HP
ChemStations>, <Instrument 1 On-line>. After a short wait, the CE screen will appear and the
instrument will proceed through a series of self-tests. DO NOT interrupt the instrument during
this period.

46
3. On the menu bar at the top of the screen, click <Instrument>, followed by <System INIT>
(fourth choice from the bottom) and wait for the red <Not Ready> box to change to the green
<Ready> status. This connects the computer to the CE unit, turns on the D
2

lamp, etc. (Note: The
D
2

lamp and diode-array detector require about 40 minutes for stabilization.)

4. If the reservoir bottle contains filtered Milli-Q water and the waste bottle is nearly empty,
proceed to step 5. Otherwise, click the turquoise <Electrolyte> icon, and select <Change
Bottles>. Install the bottle of filtered Milli-Q water and empty the waste if necessary. Then click
<Done>, and wait for the green <Ready> signal.

5. To clean the tubing between the reservoir and the electrode vials, click the numbered icon
between the <Electrolyte> and <Waste> symbols. Select <Clean Tubes>, and specify vial 45 and
1 minute flush time. Click <OK> and wait for the <Ready> message.

6. On the top menu bar, click <Method>, followed by <Load Method>, and choose <Waterf.m>
(cleans and fills the water vials, and flushes the capillary with water). Click <Run Control> and
<Run Method> (about 12 minutes). (Note: Steps 5 and 6 should have been performed at the end
of the previous lab, but they should always be repeated for proper instrument maintenance.)

7. Click on the turquoise <Electrolyte> icon, followed by <Change Bottles>. Wait for the
pressure to be released. Then replace the water bottle (left one) with the bottle of run buffer (0.05
M phosphate, pH 3, filtered at least once per week). Then click <Done> and wait for the
<Ready> message. Repeat the tube cleaning procedure in step 5, but use vial 46, instead of 45.

8. Load the method <Buffer.m> (cleans and fills the buffer vials and flushes the capillary with
buffer). Then click the <Tray Control> icon (next to the thermometer picture), followed by
<Unload> to remove all vials from the lifts. Click on <A> (vials 1-12). On the CE unit, open the
tray door (tilts out from the bottom), and check to see that vials 1-4 are buffer vials and vials 7-
10 are Milli-Q water. (Note: NEVER turn the vial tray manually; use the computer for tray
positioning.) Close the tray door and click <Done>. Then click <Run Control> and <Run
Method> (about 20 minutes).

9. Use the equilibration time to prepare the dopamine solution. Deaerate the 0.050 M phosphate
buffer with nitrogen bubbling. Pipet 25 mL of the buffer into one beaker and 8 mL of the buffer
into a second beaker.

10. Weigh a 5 mg sample of solid dopamine and dissolve it in the 25 mL of buffer. Pipet 2 mL of
this solution into the 8 mL buffer sample and mix thoroughly. Fill a CE sample vial with the
second solution. The height from the bottom of the vial should be between 1.4 and 1.8 cm.

11. Click on the <Tray Control> icon, enter 25, and click <Get>. Open the tray door and place
the vial in space 25. Close the tray door and click on <Done>.

12. Click <Sequence>, <Load Sequence>, and <Dopamine.s>. Click <Sequence> again and
select <Sequence Table>. On <Line 1>, click in the <Vial> box and enter <25>; in <Sample
Name> enter the vial contents; in <Method> choose <Heather.m> from the pull-down menu; in
47
<Inj/Vial> enter 1; and leave <Sample> in the next box. The remaining columns will not be
used. Click <OK>.

13. Click <Method>, <Load Method>, and <Heather.m>. Again click on <Method> and choose
<Edit Entire Method>. Make sure that all 4 method sections are selected (x in each box). Then
click <OK>.

14. The method screens will sequentially appear. Make the following entries:
Method Information: Change information if necessary; otherwise no changes are needed.
Click <OK>.
Home Values: No changes. Click <OK>.
Conditioning: The following choices should be in effect
<Serial> replenishment and preconditioning
Replenish: <None>
Preconditioning: <None>
Postconditioning: <None>
Make changes if necessary; then click <OK>.
Injecting: No changes. Click <OK>. (25 mbar for 4 s)
Electric: The Polarity is Positive (Inlet [+]; Outlet [-]) and the voltage is 20.0 kV. Click
<OK>.
Time Table: Make sure the Stoptime is 5.0 min and the second line in the white box (line
right above Lastline) says 0.30: voltage: 27.0 kV. If it is necessary to change the
line, click on it and enter the correct data in the pertinent box(es) above the large
box. Then click <OK>.
DAD Signals: (DAD = Diode-Array Detector) No Changes (detection at 280 nm). Click
<OK>.
Signal Details: Click on the line with the black background (only line in the large box).
Make sure End is set to 5.0 min. Then click <OK>.
Edit Integration Events: No changes. Click <OK>.
Specify Report: Make sure that the Destination is Screen, unless printed reports are
required. Then click <OK>.
Area Calculation Mode: No changes. Click <OK>.
Run Time Checklist: No changes. Click <OK>.

15. Click the green <Start> icon (left side of the screen) to initiate the sequence and answer
<Yes> to the warning. After the status arrow enters the blue region, you may display the on-line
electropherogram. Click <View> on the top menu bar. Then click <Online Signals>, and choose
<Signal Window 2>. Use the / arrows on the keyboard to change the full-scale. DO NOT
click Change. (Note: If the on-line plot is closed during a sample, it cannot be retrieved until that
sample is complete. Use the minimize/maximize boxes instead.) At the end of the run, record the
Mig Time and the Width of the Dopamine peak.

16. Repeat steps 14 and 15 with the following changes:
Electric: Change the voltage to 24.0 kV.
Time Table: Change the Voltage to 24.0 kV (both places).

48
17. Continue runs for voltages of 21, 18, 15, 12, 9, and 6 kV. For voltages below 21 kV, it will
be necessary to increase the stoptime to 10 min (18, 15, 12 kV) and 15 min (9 and 6 kV). Change
the Stoptime on the Time Table screen, the End on the Signal Details screen, and the Signal
Detection Time on the Data Analysis screen.

18. For the Stopped-Migration Method, change the Sequence Table to specify method
<Dopalong.m>. (Do not save the changes to Heather.m, unless you are instructed to do so.)
Dopalong.m uses a voltage of 9.0 kV, with a one-hour halt when the dopamine has migrated
about halfway through the capillary.

19. During the Dopalong run, prepare a plot of
2
versus t
M

for the graphical method using data
for voltages between 27 and 12 kV. Also, use data for the 12, 9, and 6 kV runs to calculate three
single-point values. Refer to the Data Analysis section for necessary parameters. Consult with
the instructor to decide if any runs should be repeated.

20. After the experiment is completed, repeat step 11 to remove the vial in position 25. (Note:
Leave all other vials alone.) Click the turquoise <Electrolyte> icon followed by <Change
Bottles>. Replace the buffer bottle with a full bottle of filtered Milli-Q water, and empty the
waste bottle. Then click <Done> and wait for the <Ready> message. Click the numbered vial
icon between the <Electrolyte> and <Waste> symbols, and select <Clean Tubes>. Specify vial
45 and a 1 minute flush. Click <OK> and wait for the <Ready> signal.

21. Load method <Waterf.m>. Click <Run Control> and <Run Method>. Using the vacuum
aspiration set-up, remove the vial contents and rinse it with water.

22. To exit the CE system, click <File> on the top menu bar, and choose <Exit>. Answer <Yes>
to the confirmation question. Turn off the CE module. (Note: Always exit the software BEFORE
turning off the CE module.)

DATA ANALYSIS
1. Use equation 7 to calculate L
inj

and its 95% confidence limits. The total length of the column is
(64.50 0.10) cm, and the internal diameter is (75 2) m. The method specifies an injection
pressure and time of (25 1) mbar and (4.0 0.1) s, respectively. Assume that the buffer
viscosity is the same as that of water at 25 C (assumed errorless). (All uncertainties in this
write-up are given as
95

values.)

2. Use equation 3 to calculate the
2

value for each dopamine sample. The L
eff

is equal to (56.00
0.10) cm. Plot
2

versus t
M

for the 27, 24, 21, 18, 15, and 12 kV runs to obtain D by the
graphical method. Evaluate the 95% confidence limits from the
95

of the slope.

3. Calculate D by the single-point method using data for the 12, 9, and 6 kV runs. Use 1.0 mm
for L
det
. Calculate the average D and the
95
.

4. Calculate D by the stopped-migration method and its
95
. In equation3, use the t
M

for the
without-halt sample to calculate both
1
2

and
2
2
, because supposedly the conditions are identical
49
during forward zone movement. Assume that t
halt

is errorless and that the
95

for
1
2

is the same as
for the unhalted run at 9.0 kV.