Chem 483 Manual Experiments

8/3/2019 Chem 483 Manual Experiments

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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 thisfragmentation 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

thed.; 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 whereit 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 massfilter. 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 massselective 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 beenwritten 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 selectedion monitoring mode detects only ions at specified m/z values. This acquisition mode allows forthe 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 isdependent on the analysis of interest.


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Chemicals

Standards to prepare: 500ppm 1-hexanol, 500ppm 2-hexanol, 500ppm 3-hexanol,

and 500ppm cyclohexanol

Mixture of equal parts cyclohexanol/1-hexanol standardsGasoline

Instrument

A Shimadzu GCMS-QP5000 series will be utilized for this experiment. Check and note thecolumn 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-RunAnalysis window.

Settings

Injector temperature: 120 COven temperature: 50 C

Detector interface temperature: 280 CTime: 10 min.

Solvent delay (Solvent Cut Time): 0.0 for air; 0.9 min. for standards with methanolsolvent.

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)

ProcedureBefore 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.


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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 yourtuning 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 anair 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 () and the volumetric flowrate (F = r2). (The linear velocityshould 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 automaticallyload. 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 andprint 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 whynot? 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 chromatogramobtained 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 forsome reason your sample is not found in the similarity search, explain why in your report (where

are there discrepancies in the mass spectrum?).


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Reset conditions to temperature program from 40 to 120 at 10C/min. Obtain a small sample ofgasoline. 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, anyrearrangements, 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 whennecessary.

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 problemand 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 yourhypothesis 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 amass filter. Be sure to include the necessary vacuum conditions and why they are important.


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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 atdifferent 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

thed.; 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 forGC. 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 asseparation 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 + (Cs+Cm)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 + (Cs+Cm)u

Chemicals

Solution ofn-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.


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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 CColumn flow rate 1-10 mL/min

Detector flow rate H2 = 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 tochange 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 yoursample 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 (gasflow 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 litoffset (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 alsohelp. 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 on the instrument control

panel. Now the GC is ready for a sample injection and the software should give you a greenlight/ 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 informationsuch as peak area/width/height. Print chromatograms and report files for your report. From the

retention times and peak widths, calculate Neff 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 peakwidths are determined). See instrument manual if you are unsure.


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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 andconstruct 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 unknownpeak(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 thepeak 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 techniquein 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, hmin, from the following equation:

where r is the column radius and k is the sample capacity (choose k=5). Compare this result tothe 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 themethod 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 yourquantitative determination.

2/122min 13/116125.1 kkkrh


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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: NewYork; 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

thed.; McGraw-

Hill: New York, 1989.

Introduction

The Fourier transform infrared (FTIR) spectrometer is an instrument that has revolutionized themeasurement 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 thereading for this experiment.

In this experiment you will determine the effect of signal averaging and mirror retardation onspectral 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)

StyrofoamToluenePTFE 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


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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 touse 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 to1. 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 eachother (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 eachspectrum. 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 onspectral 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 eachsolution 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:


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- 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 todecide how to conduct your experiment to make this happen.

ATR Sampling. Before you begin this portion of the laboratory, be sure that you are familiarwith 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 ATRaccessory. 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 theMonitor 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 stretchingregions). 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 specificfunctional 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


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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 wavelengthdependence, 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.


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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 andelectronic 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 normaland 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 proteinamide 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 arestrongly 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 Egg1 Carrot

2 glass vials (1 dram)

Apparatus

The KaiserHoloprobe 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), af/1.8 axial transmission bench equipped with a 1024 x 64 pixel thermoelectrically cooled CCD


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detector. Both sample excitation and collection of the scattered light is accomplished with a

fiber-optic probe routed through a microscope.

ProcedureFamiliarize 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 dataacquisition and processing during this experiment.

Turn on the camera switch located on the backside of the Holoprobe 1000. Turn on thecomputer, 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 theHolograms 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 capthe vial). Then move the probe several millimeters towards the sample so that you are focusedinside 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 Preparation1. Crack open the egg and separate the yolk from the albumin2. 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 (


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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 thelaser 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 positionsacross 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 beremoved. 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 haveselected 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 ofinterest 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 thespectrum 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 mousebutton approximately 200 to 400 cm

-1apart across the entire spectral range to enter points to be

used for the line-fitting procedure. Using theParameters button, set aLow Limitof 200 cm-1

on

the width of the currently selected peak. Each peak can be selected in succession by using theNext 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 spectralfeatures.

The number of peaks and lower limit can be adjusted for best results. Select Done,

Iterate, and then Start Fitto begin fitting the data. SelectLeave 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 Quitand Exit(Ignore the message asking you to save


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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.

ReportDiscuss 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 thegreatest user bias? Also, based on the data collected and your understanding of the Ramanprocess, 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-Ramanspectroscopy using an interferometer optical bench. (Hint: The discussion in Skoog may not be

perfect; be sure to use additional sources)


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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


California; 2007 Chapter 19.

Kellner, R. A.; Mermet, J.-M.; Otto, M.; Widmer, H. M, Eds.;Analytical Chemistry, Wiley: NewYork; 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 usedinstrumental 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 organiccompounds 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, CDCl3 (solvent) cinnamic acid, C6H5CH=CHCOOH

ethylacetate, CH3CO2CH2CH3 acetylacetone(pentanedione),

CH3COCH2COCH3

tetramethylsilane, Si(CH3)4

InstrumentThe 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 sincemany students in the department rely upon this instrument for their research. Login: Chem480

password:cheM480.

O O O OH


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ProcedureFor 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 fillthe 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 tubeor 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 performthree 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 ofacquisitions 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 moresymmetrical 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 viewmode) 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 aseach 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 usethis 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 different1H 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


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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, andacetylacetone. 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 andtemperature (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 magnetstrength is 4.7 Tesla.

Sample Preparation in the NMR RoomAt 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 CollectionOn 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 NMRtube in the spinner

o On the SCM (NMR) keyboard, Press the orange button, then Press the Liftbutton to turn

the lift air on. Put the sample on the column of air on top of the magnet. Press the Liftoffbutton to lower the sample into the magnet.


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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 ClickOpen.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-d6use 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 thefuture; 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 ofthe 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 Lockbutton light will blink.

o If the lock light is blinking, Press Field and slowly adjust the field until the lock lightstops blinking. (Generally, for a solid lock the line should be over one grid line above thecenter).

o Now shim the magnet

Shimming Procedure:

Check to make sure that the Fine button has a green light indicating it is engaged. As whenlocking, 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 thecurrent value until the lock level is one to two grids below the top.

o Press Zand 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 andY shims. DO NOTadjustthe Z3 orZ4. Doing so may cause the

1H signal to be lost.

o After optimizing Z2, X, andY go back to Zand 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 levelo Press Standby

1H NMR ACQUISITION

On the NTNMR software click on the File menu. Select Open. Click on small invertedtriangle then Click on NTNMR, then double Click on Data. Select the H1_CDCl3.tnt file


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(for Acetone-d6 solvent select H1_Acetone.tnt file) and Click the Openbutton. It will load thestandard 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 2n

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 receiveris 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 notgate 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) andclick on the NDFT button. Check the boxes next to 1D Settings, Zero Fill, FT, and set theapodization to Exponential. Click Set to save the settings. Now, clickNDFT without theSHIFT key to transform your FID. The result should be the 1H spectrum. Feel free to playwith these settings and observe the effect of each on the resulting spectrum. Press Ctl-F toperform 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. Adjustthe receiver gain accordingly and repeat the scans.

Press Ctl-H to perform an automatic phase correction. Manual phasing may be necessary. Clickon the Option menu and select Phase Adjustment, it will open the phase adjustmentwindow to the left side of the spectrum. Adjusting the first sliding bar, Ph0, will adjust the lineshape of all the peaks. The second sliding bar, Ph1, acts as a pivot and has a greater affect on


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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 thesettings for printing.

Expanding the spectrum: To expand the spectrum, drag with the left mouse button from one sideof 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 arrowkeys 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 assigna 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 tomove 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 PPMand enter the correctreference value.

Integration: Manual Integration- Highlight a peak as if to expand it; but Click with the right

mouse button, then selectAdd Integral. Repeat for each peak. OR - Click on the Optionmenu and select Integrals. It will open a window on the left side of the spectrum. Expand theregion of interest then Click on theAutobutton. Integrals will be drawn in boxes over the peaksin 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 eachindividual Integral.

Slope and Curvatureof an Integralcan 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 withthe 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, ClickApply, then close.

Peak Picking: To set the Threshold for Peak Picking Click on the Optionmenu, select PeakPick. 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 onApply. OR Click the mouse inthe 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,


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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 MainToolbar). 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 ClickOK. To erase this title Right Mouse Click on the title, select Label, then ClickErase.

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 selectParameters.par file. Click on Open, Click on Apply, then Click OK. Resize theparameter window with the mouse. Click on Exitat the bottom of print options window.

Click on the Printerbutton.

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 onSave.

Final Details

To eject the sample Press Spin on the SCM keyboard (to turn the spinner off), and Press theblack button located on the left front edge of the console. Press the orange button, then Press

Lift. Pickup the sample, then Press Lift Offto 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 problemSample 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 suggestedvalues 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 theFine button is ono 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


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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 isasymmetric, adjust the lock phase slightly to maximize symmetry. Once it is both

centered and symmetric, push Field, thenLockand 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 causepoor 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 rgto 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 isengaged. 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 2n

rule) and try again. See thedirections 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; [email protected] 1.1- June 07, 2000; 2001 The Regents of the University of Michigan, Modified byKathryn Hughes and Amy Gottfried, 2003.


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Spectroscopy of Iodine

Objective

Become familiar with operation and capabilities of the UV absorption spectrometer

Use spectrometer to obtain spectra of gaseous iodineLearn to interpret spectra in terms of electronic and vibronic structure and quantum levels

Reading

Shoemaker, D.; Garland, C.; Nibler, J. Experiments in Physical Chemistry, 5th

ed.; McGraw-

Hill: New York; 1989, pp 497-507.

Stafford, F. E.J. Chem. Ed. 1962, 39, 626McNaught, I. J.J. Chem. Ed. 1980, 57, 101.

BarrowIntroduction 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.199875 1192 (Important!).

Atkins, P. W.Physical Chemistry, 8th

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 theelectronic 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 energylevels 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 aband 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 caseand 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 accompanyingfigure (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 themolecule 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 seriesexpression involving a vibrational quantum number, v", as follows:

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


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The quantities, (e"xe"), (e"ye"), 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, whichis 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 thefigure. 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', and anharmonic constants, (e'xe'), etc., for this state will differ from those of

the ground state since the electron configuration is different. As in the case of the groundelectronic 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, Ea in the figure represents the atomic excitation energy.

Its magnitude can be obtained from knowledge of the atomic energy levels, providing theparticular 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 Do. This quantity for the

ground state is of considerable chemical interest and thermodynamic importance; in the figure it

is labeled Do". It should be clearly distinguished from the quantity labeled De" which is the

energy difference between the minimum of the potential curve and the dissociation limit.

In principle, it would appear that the quantity Do" 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 Do". 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, inthe 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 terminatein 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, theBirge-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


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can be shown that the difference in successive band maxima, () in the observed spectrum can be expressed by Eqn. 2, below. (Since each maximum in the observed spectrum has resulted

from a vibronic transition, ; the difference between two maxima, therefore, is ()).

() =e' - 2xe'e' (v' + 1) [2]

Here, v' is the upper state vibrational quantum number for the lower frequency maximum. Notethat 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 v = 0; this, by definition is the dissociation limit. The dissociation

energy of the upper electronic state, Do' 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

eo

xD

[3]

Chemicals

A sealed glass cell containing several I2 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 isgenerated 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 spectrumin 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 anddetermine the value of v' at the dissociation limit.

From your results, determine E*, De', Do', e', xe'e', Do" and o" (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 Do",

use the value Ea = 7603 cm-1 for the atomic excitation energy of an Iodine atom (Moore, Natl.


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Bur. Stnds, Circ. #467 (1958)). Using a reasonable uncertainty in the value for E* and a 0.1%

uncertainty in the value of Ea, calculate the uncertainty in the value of Do". 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 groundelectronic 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 introducessome 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 statevibrational levels are correctly plotted against the upper state quantum numbers.


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Figure 1


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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 thelab manual.

For a transition to a particular value of v', determine the area under the Birge-Sponer curve fromthat 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 inthe 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).


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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 offluorescence

Reading

Lakowicz, J.R.Principles of Fluorescence Spectroscopy, 2nd

. ed; Plenum Press; New York,

1999.Skoog, D.A.; Holler, F.J.; Crouch, S.R.Principles of Instrumental Analysis, 6

thed.;

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 alower 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 loseenergy.

{

{

{

S

S

T

1

1

2

a a f e

pe

internalconversion

intersystemcrossing

{S0

Figure 1: Energy-level diagram for molecular ground state (S0), first (S1) and second

(S2) singlet excited states, and triplet excited state (T1). a: absorbance; f: fluorescence; e:

external conversion; p: phosphorescence.


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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 othercomponents 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 1Kq [Q]

This expression, known as the Stern-Volmer equation, describes the decrease in the fluorescencequantum 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 bedetermined from intensity measurements as a function of quencher concentration (3).

Chemicals

Quinine sulfateSodium 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 H2SO4 (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 H2SO4 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 H2SO4. Then pipet 5.00 mL of this diluted sample into a

25 mL vol. flask and dilute to the mark with 0.05 M H2SO4. 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 H2SO4.


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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 spectrumbetween 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.


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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 maximumobserved in emission spectrum.

Set ACQUIRE MODE to TIME COURSE

Place a cuvette containing 0.05 M H2SO4 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 displayedvalues. 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 H2SO4). 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 bedetermined 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 yourcalculation.

Quenching by chloride ions

Clean the cuvette and fill it with 1 ppm quinine standard containing 0 ppm chloride. Measure thefluorescence 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 (Kq)

for chloride ion. Discuss the assumptions and reasoning behind your determination.


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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 andbandwidths?

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 CO2 have on your results if it were present in your sample of tonic water?


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Surface Tension

Objective

Become familiar with concept of surface tension and how it varies with solutionchemistry

Use two different methods to measure surface tension of water and alcohol/watermixtures

Reading

Shoemaker, D.; Garland, C.; Nibler, J. Experiments in Physical Chemistry, 5th

ed.; McGraw-Hill: New York, 1989; pp. 339349.

Daniels, F.; Williams, J. W.; Bender, P.; Alberty, R. A.; Cornwell, C. D. Experimental PhysicalChemistry, 7

thEd. McGraw-Hill: Toronto, 1970, pp. 359365; or

Salzberg, H. W.; Morrow, J.; Cohen, S. Laboratory Course in Physical Chemistry, AcademicPress: 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 rangingfrom 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 thesurface. 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 ofthe 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 previouslyused 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 stockroomwhen 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.


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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 thiswould no longer be expected to change? Interpret in terms of intermolecular forces and the

differences between surface and bulk environments.


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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


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 CoupledPlasma 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 andions 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 inthe 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 bypressing 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 asan 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.


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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 Yttriumemits 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 andapply 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 usethis 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 turnon the pump. Make sure rinse solution is flowing through the pump tub

Documents

Chem 483 Manual Experiments