Fall 2014

CHL 311 Instrumental Analysis Manual

M. J. Prushan
Table of Content

CHM 311 Laboratory Schedule ................................................................................................................. 2

Instrumental Analysis Laboratory Policies ................................................................................................ 3
Instrumental Analysis Instrumentation Contract ..................................................................................... 5
MODULE I: SPECTRAL SOURCES AND SIGNAL TO NOISE

Experiment 1 BLACKBODY RADIATION or What’s the difference between a light bulb and the sun? ..... 10
Experiment 2 SPECTROSCOPY TRADING RULES: Signal-to-Noise, Resolution, Ensemble Averaging, Digital
Smoothing ................................................................................................................................................... 12

MODULE II: SPECTROPHOTOMETERS: Instrumentation and Applications

Experiment 3 DETERMINATION OF CHLOROPHYLL IN OLIVE OIL by UV-Visible and Fluorescence

Spectroscopy .............................................................................................................................................. 17
Experiment 4 PERFORMANCE CHARACTERISTICS OF A SPECTROPHOTOMETER or Introduction to
Ultraviolet—Visible Spectrometry (UV-Vis) ................................................................................................ 22

Experiment 5 SPECTROPHOTOMETRIC ANALYSIS OF A TWO COMPONENT SYSTEM: Multi-wavelength

linear regression analysis (MLRA) .............................................................................................................. 32

Experiment 6 FLUORESCENCE Spectroscopy ............................................................................................. 36

MODULE III: ATOMIC ABSORPTION SPECTROMETRY

Experiment 7 ATOMIC ABSORPTION ANALYSIS OF CALCIUM IN MILK .................................................... 45

MODULE IV:CHROMATOGRAPHY

Experiment 8 “IMPOSTER” vs. “REAL” PERFUMES via GC-MS ................................................................. 54

Experiment 9 DRUGS ON MONEY: GC-MS determination of cocaine on paper money ........................... 62

Experiment 10 HPLC: Caffeine Concentration in coffee, tea, energy drinks and soda ...........................

Appendix I INSTRUMENT INSTRUCTIONS .......................................................................................... 65

Appendix II USE OF A MICROSYRINGE ................................................................................................. 70

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 Instrumental Analysis Laboratory Policies

INTRODUCTION
In the laboratory portion of Instrumental Analysis you will be exposed to several different types of
instrumental techniques. In most experiments, you will be investigating the performance characteristics
of the instrument in addition to using it to make a measurement. It is still important, however, that you
continue to use your best analytical technique when preparing standards and samples. Poor technique
will always produce poor results!

LABORATORY MODULES
The laboratory course is divided into four modules, each of which emphasizes a different aspect of
instrumental analysis. Each module consists of 1-3 experiments that relate to the theme of the module.
You will work as part of a 2-person group, but because of limited instrument availability there is a
possibility that you will have to schedule analysis time outside of class.

LAB PREPARATION
The key to efficiency in the lab is preparation. You are expected to read the lab and contact the
instructor
before the lab period for questions and/or clarifications. It is highly recommend that you consult with
your group members before the lab period to divide responsibilities so that your laboratory time is used
most efficiently. It is also important that you are considerate of your lab partners and arrive at lab on
time.

LAB NOTEBOOK
You must keep a lab notebook starting with the first day of experiments. All data must be recorded in
the laboratory notebook, not on loose sheets of paper or on the lab manual. To prevent abuse (i.e. filling
in the notebook at the end of the semester), you must get the signature of the lab instructor each week.

The primary purpose of the laboratory notebook is to record data and experimental details that are not
included in the lab manual. You do not need to rewrite procedures that are already in the laboratory
manual, and you do not need to include calculations.

The notebook will be graded based on the following guidelines:

1. The first few pages of the notebook should be reserved for a table of contents. This table of contents
should be kept up to date.
2. Each page should have a page number and be dated.
3. The notebook must be hard bound.
4. No pages should be removed for any reason.
5. All data entries must be in indelible (i.e. non-erasable) ink.
6. Mistakes and errors should be crossed out with a single line. White-out is not allowed.
7. Try to keep the notebook reasonably neat – you should be able to understand each entry. It is often
helpful to construct tables prior to lab and fill them out as you collect data.
8. Keep your notebook current. It is unethical (not to mention illegal in many circumstances) to go back
and fill in your notebook with data you wrote on loose pieces of paper. Backdating (i.e. going back and
writing the date you think the data was recorded) is also forbidden.

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REPORTS
You must write a report for each experiment. These reports are due one week after completing the lab.

IMPORTANT

• Reports are due at the beginning of the lab period. YOU MAY NOT WORK ON YOUR LAB REPORTS
DURING THE LAB PERIOD.

• You are encouraged to work with your partners on analyzing your data. However all written work must
be individual efforts. Your report must be written in your own words and contain your own calculations
and interpretations. Copied reports will result in a failure in the course.

• Lab reports will not be accepted from anyone that has missed an experiment. You may not simply copy
your partners’ data and turn a report; you must arrange to do the experiment (preferably during
another lab period).

GENERAL REPORT FORMATTING

• Reports must be printed with a high quality printer.

• One member of your group must attach your copies of the raw data.
• Make a cover sheet for every report. See the example on page viii.
• Answer all questions completely and in paragraph form. Do not merely answer “yes” or “no”, but
always give a rationale for your answer.

Here is an example of an answer that would receive full credit:

What is the optimal flame observation height for Zr?
The data in Figure 3, a plot of Zr absorbance as a function of observation height in the flame, shows that
the maximum absorbance occurs at a height of 2.25 cm. Therefore, the best sensitivity for atomic
absorption of Zr occurs at an observation height of 2.25 cm.

• Be sure to include sample calculations. Handwritten calculations are acceptable.

• Sample calculations should be easy to follow.

• When fitting data to a line (e.g. a calibration curve), always use linear regression. Report the equation
for the line and the correlation coefficient (r or r2).
• For all replicate determinations, the average, standard deviation, and % relative standard deviation
must be reported.
• The following are examples of acceptable presentation of data. Note that tables include descriptive
captions and that columns include units.

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The following is an example of a poorly-constructed table. This table does not have a descriptive title,
lacks units, has poor labeling of the samples, has totally ignored significant figures, is poorly aligned, and
has no column for %RSD.

GRAPHING
Good graphical presentation of data is critical for analysis of experimental results.

Listed below are general guidelines for properly plotting and annotating a graph.

1. Choose a scale that best shows the full dimensions of the data and also results in scale divisions that
allow easy interpretation of the data. In other words, do not bunch the data in one corner of the graph.
Most spreadsheets scale graphs automatically, but you can usually re-scale the graph manually. The
origin need not be included in the graph unless you are showing data near the origin.

2. Plot the dependent variable (the one that is a function of the other, such as absorbance as a function
of concentration) on the vertical axis. The independent variable should therefore be plotted on the
horizontal axis. Always label both axes and include units in parentheses.

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3. Each point should be located with a
small distinct data point. Use different
symbols (e.g. circles, squares, etc.) to
distinguish one data set from another.

4. Lines through data sets should not

“connect the dots”. The best method (if
the data is supposed to be linear) is to use
linear regression to determine the best-fit
line for your data.

5. Never let Excel draw a smooth line

through your data. In nearly every case,
these curves are meaningless!

6. When you plot data over several orders of magnitude, it is advisable to construct a log-log plot so that
data at both low and high concentrations are visible. Although you can do this by setting the properties
of the axis in Excel to “logarithmic”, it is usually best to calculate the logarithm of the data before
graphing (especially when you want to make a linear fit to the log-log data).

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 [INSTRUCTOR COPY]
CHL 311 Laboratory
Instrumentation Contract

The instrumental techniques that you will be learning are critical tools in so many areas of science, not
only in chemistry but also in medicine, geology, food science, forensic science, environmental and
agricultural science, material science, and pharmaceutical science as well. You will have a lot of
independence and opportunity to control your own work schedule. However, because of the structure
of the course, much of the responsibility for your education rests with you.

You will also have responsibility at times for the use and care of nearly $500,000 worth of
equipment. Often others will depend on your effort and cooperation. In order for everyone to get
the most out of this course and to protect you and the instrumentation we must agree to abide by
some rules.
Please read the following statements about expectations and use of facilities. Failure to follow
these rules will result in a failing grade in the course.

1. I will follow safe working procedures in lab. If the proper practice is unclear, or questionable in my
opinion, I will ask the instructor about it.

2. I agree not to eat or drink in the lab, nor to bring open beverages or containers of food into the lab.

3. I agree to clean up around the computers, lab benches and instruments whenever I work in the lab.
If I bring reagents, equipment or materials into the lab for my work, I will remove them when I leave for
the day.

4. I will see that the instrument or computer that I use is left in the appropriate idle (or off) condition
when I am done (unless the next user is present to take over).

5. I will sign the operator’s log book after using an instrument and report any problems or special needs
(such as a low gas level) in the logbook or directly to the instructor.

6. Since equipment and supplies are intended for members of this class and other chemistry courses, I
will not remove any materials or equipment from the lab that I did not bring there without express
permission from a faculty member. (Obviously, such things as my data, print-outs and used reagents are
exceptions.)

7. I will not misuse any instruments or equipment in the lab.

8. I will not operate any equipment for which I have not been given instruction in operating.

9. I will be responsible for anyone that I let into the lab and will see that they abide by our class
guidelines for use of any equipment or facilities.

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10. I will work through all of the reading assignments and tutorials.

11. I will reserve use of instruments on the weekly reservation sheet and honor the reservations made
by others.

12. I will cooperate with my lab partner. If the group becomes dysfunctional, it is part of my
responsibility to work things out. If that cannot be done satisfactorily in a short period of time, I will talk
to the instructor about the matter.

Signature _____________________________________

Date: ______________________

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 [STUDENT COPY]
CHL 311 Laboratory
Instrumentation Contract

The instrumental techniques that you will be learning are critical tools in so many areas of science, not
only in chemistry but also in medicine, geology, food science, forensic science, environmental and
agricultural science, material science, and pharmaceutical science as well. You will have a lot of
independence and opportunity to control your own work schedule. However, because of the structure
of the course, much of the responsibility for your education rests with you.

You will also have responsibility at times for the use and care of nearly $500,000 worth of
equipment. Often others will depend on your effort and cooperation. In order for everyone to get
the most out of this course and to protect you and the instrumentation we must agree to abide by
some rules.
Please read the following statements about expectations and use of facilities. Failure to follow
these rules will result in a failing grade in the course.

1. I will follow safe working procedures in lab. If the proper practice is unclear, or questionable in my
opinion, I will ask the instructor about it.

2. I agree not to eat or drink in the lab, nor to bring open beverages or containers of food into the lab.

3. I agree to clean up around the computers, lab benches and instruments whenever I work in the lab.
If I bring reagents, equipment or materials into the lab for my work, I will remove them when I leave for
the day.

4. I will see that the instrument or computer that I use is left in the appropriate idle (or off) condition
when I am done (unless the next user is present to take over).

5. I will sign the operator’s log book after using an instrument and report any problems or special needs
(such as a low gas level) in the logbook or directly to the instructor.

6. Since equipment and supplies are intended for members of this class and other chemistry courses, I
will not remove any materials or equipment from the lab that I did not bring there without express
permission from a faculty member. (Obviously, such things as my data, print-outs and used reagents are
exceptions.)

7. I will not misuse any instruments or equipment in the lab.

8. I will not operate any equipment for which I have not been given instruction in operating.

9. I will be responsible for anyone that I let into the lab and will see that they abide by our class
guidelines for use of any equipment or facilities.

8
10. I will work through all of the reading assignments and tutorials.

11. I will reserve use of instruments on the weekly reservation sheet and honor the reservations made
by others.

12. I will cooperate with my lab partner. If the group becomes dysfunctional, it is part of my
responsibility to work things out. If that cannot be done satisfactorily in a short period of time, I will talk
to the instructor about the matter.

Signature _____________________________________

Date: ______________________

9
 Experiment 1: BLACKBODY RADIATION or What’s the difference
between a light bulb and the sun?

The color of light produced by a hot object depends on its temperature. This has to do with the fact that
more energetic light produced a different wavelength of light. The wavelength, frequency, and energy of
light are related by

and

The amount of radiation given off by a hot object depends on two things, how hot it is, and what
wavelength you are observing. The following equation describes this behavior.
2hc2
I hc , I is the Intensity or spectral density
 (e
5 kT
 1)
where h is Planck’s constant, c is the speed of light, k is Boltzmann’s constant (1.38 x 10-23 J K-1) T is the
Kelvin temperature and  is in nm.

Procedure.

1. Using an Ocean Optics Spectrometer, record the visible spectrum of the sun and a incandescent
bulb (tungsten light bulb).
2. Save the data points of the spectrum and open the data files using Excel.
3. Make a plot (I vs. ) the Blackbody Radiation function. Plot the spectral density for 4000, 5000,
6000 K on the same graph. What changes can be seen as the temperature increases?
4. Open the data files (of the sun and the tungsten lamp) using Excel. Now plot the Blackbody
Radiation function on the same graph. [NOTE, you might have to place a multiplier in the
equation, such that both the function and the dataset are on the same scale.]
5. Using the curve fitting functions of Excel, fit your data of the sun and the tungsten lamb with the
Blackbody Radiation function. [THE FOLLOWING IS A GUIDE, the data does not necessarily have
to be in these cells, but you do need to be consistent]
(a) To use the curve-fitting function, label cells along the top of the sheet with A then place a
“guess” value for the TEMPERATURE in the cell.
(b) Place the data to be fit in the first two columns. In the third column, enter the Blackbody
equation, referencing to the A, B and C values at the top of the sheet
(c) Label the fourth row (sum differ squares), enter the following function: ($B4-$C4)^2

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 (d) Label the fifth column (R^2) and enter the following equation : (($B4-$C4)/$B4)^2
(e) Enter the label : sum R^2 in G4 and enter and equation which sums your R^2 values (SUM())
in H4.
(f) Enter the label : sum sums in G5 and enter and equation which sums your sum
diff squares values (SUM()) in H5.
(g) open the solver window (Tools, solver), make sure the window looks something like what is
shown below:

Reference the cells that you want to

change to fit the data

(h) click the solve button… and watch the magic happen!

6 . What temperature gives the best fit in each case? State not only the temperature but the
error in the values. Look up values for the temperature of the sun and the tungsten lamp, how
do your experimental values compare?
7 Which of the two “give” off more visible light. What is the range of light that our eyes can see?
How does this overlap with the tungsten lamp and the sun spectra? Does this provide evidence
for why our eyes evolved to see particular wavelengths?

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 Experiment 2.
SPECTROSCOPY TRADING RULES:
Signal-to-Noise, Resolution, Ensemble Averaging, Digital Smoothing

Introduction

Using instrumentation of all kinds involves compromises. This lab is designed to

illustrate some of the compromises involved in performing measurements with
spectroscopic instrumentation: to demonstrate some of the “trading rules”.
Before performing tasks illustrating these concepts, let's briefly discuss these terms,
which you can read more about in the text.

RESOLUTION: The most straight forward definition of resolution is in terms of the

difference in frequency (wavenumber, cm-1) or wavelength (nm) between two absorbance
peaks that can be just separated by the instrument. All other factors being equal, the
greater the resolution the better to detect absorbances (peaks) as close together as
possible.

SIGNAL-TO-NOISE: In the text, S/N is defined as 1/RSD of a recorded signal.

This can be thought of as the “root mean square” signal-to noise ratio. You will determine the
rms S/N of the Fourier transform infrared spectrometer under a variety of instrumental
conditions. All other factors being equal, the greater the S/N the better to detect the weakest
absorbances, or absorbances at lower concentration levels.

SIGNAL AVERAGING: Also called ensemble averaging, it's a way to enhance the S/N
ratio by acquiring multiple spectra and obtaining the average of the result. The signal increases
to the first power of the number of spectra averaged but the noise, being random, increases to
the square root of the number of spectra averaged. Thus the signal increases faster than the
noise as multiple spectra are averaged resulting in an increased signal-to-noise ratio.

SMOOTHING: Also know as digital filtering (which is one way to smooth data that will be used in
this laboratory). This is another way to enhance the S/N ratio of the spectrum.

As you will see, there are trade-offs which must be considered when obtaining any
spectrum. You always want a high S/N, but you need sufficient resolution to obtain a
representative spectrum for your needs in a reasonable period of time. You must pick the
appropriate conditions which best fulfill your needs, and the only way to accomplish this
is to understand how S/N, spectral resolution, smoothing and signal averaging interact to
change the data acquisition time and spectral result.

A Short Introduction to the Spectroscopy

The absorption of electromagnetic radiation by ions and molecules serves as the basis
for numerous analytical methods of analysis, both qualitative and quantitative. Studies of

12
absorption spectra provide knowledge concerning the formula, structure, and stability of
many chemical species, as well as establish the most favorable conditions for analysis.
Because the energy of a molecule is the sum of many individual types of energy,

Emolecule = Eelectronic + Evibrational + Erotational + Etranslational + others

absorption of a photon can increase molecular energy in a variety of ways. In UV-Visible

spectroscopy, the energy of the photon corresponds with electronic energy transitions of
molecules and ions. Thus UV-Visible spectroscopy is a type of electronic absorption
spectroscopy. In infrared spectroscopy, the energy of the photon corresponds with vibrational
energy levels of molecules. Thus infrared spectroscopy is a type of vibrational spectroscopy. It is
usually safe to say that more detailed structural information can be derived from vibrational
spectroscopy than from electronic spectroscopy.
In UV-Vis electronic absorption spectroscopy in liquids, the absorption peaks are
very broad. The broad peaks result from a phenomenon called “collisional broadening”. In the
liquid state, molecules are contantly colliding and interacting with one another. This causes a
near continuum of vibrational and rotational energy levels superimposed on top of the
electronic energy levels; resulting in broad absorption bands over a range of wavelengths.
These collisions are much less frequent in the gas phase, so one can see individual vibrational
peaks superimposed on top of the electronic peaks in gas phase UV-Vis spectroscopy (assuming
the instrumental conditions of obtaining the gas phase spectrum affords sufficient resolution).
Similar phenomena occur in the infrared region, which is the part of the electromagnetic
spectrum in which you will be working in this laboratory. Relatively broad peaks are observed in
the infrared spectra of liquids, because a near continuum of rotational energies are
superimposed on vibrational energy levels. Collisional broadening in the liquid phase makes it
impossible to detect the individual rotational transitions. In the gas phase, you will see both
vibrational and rotational transitions occurring as a series of sharp peaks. You will see this for
gas phase CO2 in the atmosphere, and see changes in these gas phase spectra as you change the
spectral resolution. You should conceptually understand what the implications are of spectral
resolution in obtaining the gas phase spectra, and how the other variables of ensemble
averaging, digital smoothing and resolution interact with one another to form some of the
spectroscopic trading rules. Although we are using the FTIR spectrometer to illustrate the
spectroscopy trading rules, it should be emphasized that any spectroscopic instrumentation will
show the same types of trends. Since the FTIR is being used however, a few points which may
otherwise cause confusion should be discussed. The FTIR is a single-beam instrument. The
instrument components lab is designed to clearly show the difference between single-beam
and double-beam instruments. A measurement of the % transmittance (from which absorbance
can be calculated) actually requires 2 measurements.

%T = (I/Io) x 100

where I = intensity of light at a given wavelength that the detector senses when the
sample is in the source beam, and Io = intensity of light at the same wavelength that the
detector senses when a “blank” is in the source beam. In a single beam instrument an

13
absorbance or % transmittance spectrum is obtained by first making measurements on a
blank to obtain Io as a function of wavelength or frequency. This is a single beam
spectrum. Then a sample is put into the source beam and measurements obtained to obtain I
as a function of wavelength or frequency. This is also a single beam spectrum.
By taking the ratio of I/Io as a function of wavelength or frequency one obtains a transmittance
spectrum.

With the FTIR, when you collect a background spectrum, you obtain a plot of Io as a function of
frequency. If you look at this spectrum you will see that the y-axis is not %T
or absorbance, but emittance. This is simply a spectrum of instrument signal intensity as
a function of frequency. When you obtain a sample spectrum, you obtain a plot of I as a
function of frequency. You obtain a transmittance or absorbance spectrum by performing
the appropriate mathematics to both I and Io. In this lab you collect both single beam
spectra I and Io under identical conditions, that is no sample in the source beam in either
case. In the resulting transmittance spectrum the deviation from 100%T is a direct
measurement of the random error or noise of the measurement.

In steps 6 and 7 of the procedure you are plotting single beam spectra of CO2, that is a
plot of Io as a function of frequency. You are able to do this because of the CO 2 in the
air. Since what you are seeing here are single beam spectra, this is not a noise
measurement (although noise always exists in the measurement). The differences you are
seeing in steps 6 and 7 are primarily a result of differences that the instrument detects for
the CO2 signal. Do not interpret the results in steps 6 and 7 as coming from differences in
the amount of noise in the spectra.

PROCEDURE
1. Your instructor will start up the Nicolet FTIR spectrometer, and briefly go over the operation
of the instrument and the use of the software with you. You shouldn’t have to do anything with
the instrument except type at the keyboard and plot spectra. Any questions or problems see
the instructor.

2. With 1 scan, collect a background spectrum at 0.5 cm-1 resolution. With 1 scan, collect a
sample spectrum at 0.5 cm-1 resolution. Find the S/N ratio of this spectrum
between 2200 – 2000 cm-1. The Nicolet software will find the RMS noise within the
displayed range for you. The S/N ratio is 100% T/RMS noise. Save this spectrum (100% T line) to
disk. (Note: if your spectrometer’s software does not perform this calculation, students can
manually obtain the peak-to-peak S/N ratio by taking 100 divided by the difference between the
high and low points in the spectrum.

3. Now take this spectrum which you just calculated the S/N for, and perform a digital
smoothing routine. First perform a 5 point smooth and calculate the S/N of this smoothed
spectrum from 2200 – 2000 cm-1. Recall the unsmoothed spectrum and repeat the smoothing
process using 9, 13, 17, 21 and 25 point smoothing routines. After each smoothing routine
calculate the new S/N ratio and recall the unsmoothed spectrum. Tabulate your data and make

14
a plot of S/N ratio versus number of smoothed points. In your write-up, comment on the affect
of smoothing on the S/N of this spectrum.

4. Averaging 8 background and sample scans perform experiments at the following

resolutions: 0.5 cm-1, 1 cm-1, 2 cm-1, 4 cm-1, 8 cm-1, 16 cm-1 and 32 cm-1. You will need
to run a background spectrum at each resolution for this to work. No need to smooth any
of these however, just calculate the S/N of these spectra. The S/N should be calculated in
three separate regions: between 3000-2800 cm-1; 2200-2000 cm-1; and 600-400 cm-1.
Make a plot of spectral resolution (x-axis) vs. S/N ratio (y-axis) for the three spectral regions.
Compare the S/N ratio not only as a function of resolution, but also as a function of spectral
region. Why do you think the S/N varies from region to region?
(Hint: compare single beam signal intensities in these three regions and see if you can devise a
reasonable explanation.) Discuss the results and also discuss how spectral acquisition time
varies with resolution.

5. You already have the S/N data for 8 scans at 4 cm-1 resolution from part 4. Scan both
background and sample spectra at this same resolution using the following number of scans in
both sample and background files:

Number of Scans S/N (2200-2000 cm-1) Spectral Acquisition Time

1
4
16
64
256
512

Plot the # of scans signal averaged (x-axis) vs. S/N ratio (y-axis). Fit this data to a
power function. This is done in Excel by adding a trendline and choosing a power fit.
Annotate your plot with the equation. Discuss the functional form of this relationship,
and how your result compares to the result which you would expect from theory. Discuss
how spectral acquisition time varies with signal averaging, and the ultimate effects on
S/N.

Now for the affect of resolution and smoothing on a single beam gas phase CO 2
spectrum. This is separate from the S/N portion of the lab earlier, but is related in
terms of the spectroscopy trading rules.

6. Obtain a backround spectrum by co-adding 16 scans at 0.5 cm-1 resolution. Display

this spectrum between 2400 - 2200 cm-1. What you are seeing are a series
vibrational/rotational transitions for gas phase CO2 in the spectrometer which is in the
atmosphere. If you look around in other regions of the spectrum, you will also see many sharp
peaks for gas phase water around 3500 and 1600 cm-1. Save this gas phase spectrum (this is not

15
an absorbance or transmittance spectrum because it is a single beam spectrum) between 2400
and 2200 cm-1 to disk. Now repeat the smoothing routines which were done for the 100% T line
in Step 3. Don’t calculate S/N of the smoothed spectra as before, but observe what the
smoothing does to your CO2 spectra. In your lab write-up discuss the affect of digital smoothing
on the spectrum of gas phase CO2. In light of your results on smoothing earlier (Step 3) in terms
of S/N, discuss the trade-offs and compromises involved in using digital smoothing. Plot
appropriate spectra which illustrate what you observe.

7. Obtain two more spectra of gas phase CO2 at 8 and 32 cm-1 resolution, respectively.
Plot spectra to illustrate how resolution affects the gas phase CO2 spectrum. In your lab
write-up, discuss the affect of resolution on the gas phase spectrum of CO2. In light of
your results on resolution with respect to S/N and spectral acquisition time, discuss the
various considerations one must take into account when choosing a suitable resolution to
acquire a spectrum.

For your report

Suggestions of what should be included in the report have been given throughout the
procedure. Remember to always cross-reference figures and plots in your write-up.

Questions
Here are some issues which should probably be addressed directly in your write-up.
Many of these have already been mentioned in the procedure section in italics. This list
is not all inclusive.

1. What is the relationship between S/N and the number of scans co-added to obtain
spectrum? Why does this occur? Are there any disadvantages to co-addition?

2. What is the affect of smoothing and the number of smoothed points on S/N ratio?

3. What is the affect of smoothing on the gas phase spectrum of CO2?

4. What is the affect of resolution on the gas phase spectrum of CO2?

Most importantly, answering this question thoughtfully would make a fine conclusion to
the lab write-up.

5. EXPLAIN THE VARIOUS TRADE-OFFS AND CONSIDERATIONS ONE MUST MAKE IN BALANCING
S/N, RESOLUTION, ENSEMBLE AVERAGING, AND DIGITAL SMOOTHING WHEN CONSIDERING
INSTRUMENTAL CONDITIONS IN OBTAINING A SPECTRUM.

16
Experiment 3.

Determination of Chlorophyll in Olive Oil by UV-Visible and

Fluorescence Spectroscopies

Olive oil is made by pressing or extracting the rich oil from the olive fruit. It seems like a simple
matter to press the olives and collect the oil, but many oil extraction processes exist for the many
different types of olives grown around the world. To complicate things further, there are also various
grades of olive oil, and carefully selected groups of officials meet to define and redefine the grading of
olive oil. To help make our experiment a more scientific and less political exercise, we will winnow our
investigation of olive oil down to a manageable few variables. After processing, olive oil comes in three
common grades: extra virgin, regular, and light. Extra virgin olive oil is considered the highest quality. It
is the first pressing from freshly prepared olives. It has a greenish-yellow tint and a distinctively fruity
aroma because of the high levels of volatile materials extracted from the fruit. Regular olive oil is
collected with the help of a warm water slurry to increase yield, squeezing every last drop of oil out of
the olives. It is pale yellow in color, with a slight aroma, because it contains fewer volatile compounds.
Light olive oil is very light in color and has virtually no aroma because it has been processed under
pressure. This removes most of the chlorophyll and volatile compounds. Light olive oil is commonly used
for frying because it does not affect the taste of fried foods, and it is relatively inexpensive. The visible
light absorbance spectrum of chlorophyll gives interesting results. The chemistry of chlorophyll (some
references site four types: a, b, c, and d) creates absorbance peaks in the 400–500 nm range and in the
600–700 nm range. The combination of visible light that is not absorbed appears green to the human
eye, but different sources of chlorophylls will have different ratios of these peaks, which create various
shades of green. The ability of chlorophyll to soak up light energy across a wide swath of the visible
range helps power photosynthesis at optimum efficiency in plants. In this experiment, you will have two
primary goals. First, you will analyze the various grades of olive oil to determine the absorbance peaks
that are present and the relative amount of chlorophyll found in each grade. You will use a
Spectrometer to measure the absorbance of the olive oil samples over the visible light spectrum. You
will then test an unknown sample of olive oil and grade it as extra virgin, regular, or light.

OBJECTIVES
In this experiment, you will:
Measure and analyze the visible light absorbance spectra of three standard olive oils: extra
virgin, regular, and light.

 Measure the absorbance spectrum of an “unknown” olive oil sample.

 Identify the unknown olive oil as one of the three standard types.

 Compare the results with those obtained via fluorescence spectroscopy

17
PROCEDURE

1. Connect the Spectrometer to the USB port of your computer.

2. Start Logger Pro. If it is already running, choose New from the File menu.

3. Obtain small volumes of the three standard and one unknown olive oils. Transfer enough of one olive
oil sample to fill a cuvette 3/4 full. Place a lid on the cuvette and mark the lid. Prepare all of your
samples in this way so that you have four cuvettes of olive oil with labeled lids.

4. Calibrate the Spectrometer.

a. Prepare a blank by filling an empty cuvette 3/4 full with distilled water.
b. Choose Calibrate ► Spectrometer from the Experiment menu.
c. When the warmup period is complete, place the blank in the Spectrometer. Make sure to
align the cuvette so that the clear sides are facing the light source of the Spectrometer.
d. Click Finish Calibration, and then click OK.

Part I Comparing Three Grades Of Olive Oil and Identifying an Unknown

For Part I of this experiment, you will calibrate the Spectrometer with distilled water. Your goals
are: (1) to compare the absorbance spectra of the different grades of olive oil; and (2) to identify
the grade of an unknown sample of olive oil.

5. Conduct a full spectrum analysis of an olive oil sample.

a. Place one of the olive oil samples in the Spectrometer.
b. Click COLLECT. A full spectrum graph of the olive oil will be displayed. Review the graph to
identify the peak absorbance values. Click STOP to complete the analysis.

6. To save your data, choose Store Latest Run from the Experiment menu.

7. Repeat Steps 5–6 with the remaining olive oil standard samples.

8. Repeat Step 5 with the unknown. Note: Do not store the last run.

9. Examine the plots of the olive oil samples. Save your experiment file.

10. Rinse and clean the cuvettes and other oil-bearing containers with isopropyl alcohol.

Part II Comparing the Chlorophyll Concentration of Regular and Extra Virgin Olive Oil

In Part II, you will use the light grade of olive oil to calibrate the Spectrometer and presume that
light olive oil contains no chlorophyll. Next, you will compare the chlorophyll content of the regular
grade with the extra virgin grade.

11. Set up a new file and calibrate the Spectrometer using light olive oil.
a. Choose New from the File menu.
b. Prepare a blank by filling an empty cuvette 3/4 full with light olive oil.
c. Choose Calibrate ►Spectrometer from the Experiment menu.

18
 d. When the warmup period is complete, place the light olive oil blank in the Spectrometer.
Make sure to align the cuvette so that the clear sides are facing the light source of the
Spectrometer
e. Click “Finish Calibration”, and then click OK.

12. Measure the absorbance spectrum of regular and extra virgin olive oil.
a. Remove the cuvette of light olive oil from the Spectrometer and replace it with the cuvette
of regular olive oil.
b. Click COLLECT. A full spectrum graph of the regular olive oil will be displayed. Note the
slight difference in the plot as a result of using the light olive oil as the calibration blank.
Click STOP.
c. To save your data, choose Store Latest Run from the Experiment menu.
d. Measure the absorbance spectrum of the extra virgin grade in the same way.

13. Save your experiment files.

Part III Fluorescence Spectroscopy of Chlorophyll in Olive Oil

Chlorophyll is a fluorescent molecule. Fluorescent molecules can absorb light of one

wavelength and then reemit light at a new and longer wavelength of light. As you have seen
in this experiment already, chlorophyll absorbs light in the violet and blue regions of the spectra. If you
were to shine a violet or blue light through a sample of extra virgin olive oil, you would see the oil turn
red in color. The intensity of the red color is an indication of how much chlorophyll is in the olive oil.

14. Shine the light from a UV Flashlight through a cuvette containing extra virgin olive oil. Does the
sample that is hit by the light turn red in color? Repeat this test for regular olive oil, light olive oil, and
your unknown. Could you use this method to determine if a sample of olive oil is really extra virgin olive
oil? Could you use this method to determine the grade of any sample of olive oil?

Fluorescence spectroscopy is another method that can be used to determine the quality of
olive oil. In fluorescence spectroscopy, a sample can be “excited” with a chosen wavelength
of light and the resulting fluorescence from the sample can be measured and quantified.
The SpectroVis Plus from Vernier Software & Technology can be used for this purpose.

15. Follow the directions below to measure the fluorescence of all of your olive oil samples using the
SpectroVis Plus.
a. Use a USB cable to connect the Spectrometer to your computer. Choose New from the
File menu.
b. Place the cuvette containing the extra virgin olive oil into the cuvette slot of the
Spectrometer.
c. Choose Change Units ►Spectrometer from the Experiment menu and select Fluorescence
405 nm.
d. Choose Set up Sensors ►Spectrometer from the Experiment menu and change the sample
time to 150 ms.
e. Click COLLECT. A full spectrum graph of the fluorescence of the oil will be displayed. Note
that one area of the graph contains a peak at approximately 675 nm. This peak is from
chlorophyll. Click STOP.

19
16. Adjust the sample time to increase or decrease the size of the fluorescent peak. If the peak
intensity is above 1, decrease the sample time by 10 ms and collect a new fluorescent spectrum.
Continue to decrease the sample time until the peak is fully visible. If the fluorescent peak is below 0.3,
increase the sample time by 10 ms and collect a new fluorescent spectrum. Continue to increase the
sample time until the peak fluorescent amplitude for the chlorophyll is above 0.8.

17. Once you have a nice peak, store your data by choosing Store Latest Run from the Experiment menu.

18. Collect full spectra from the remaining olive oil samples. Do not adjust the sample time.

19. Save your experiment files.

DATA ANALYSIS

Part I Comparing Three Grades of Olive Oil and Identifying an Unknown

1. Describe the graph of each of the standard olive oil solutions. Emphasize the differences between
each grade of olive oil, identifying the absorbance peaks and other distinguishing features.

2. Compare the absorbance spectra of the three grades of olive oil with the sample graph in Figure 1.
What evidence is there that regular and extra virgin olive oil contain chlorophyll while the light grade of
olive oil does not?

3. Identify your unknown olive oil as extra virgin, regular, or light. Explain your choice.

Figure 1.

Part II Comparing the Chlorophyll Concentration of Regular and Extra Virgin Olive Oil

4. Which grade of olive oil, regular or extra virgin, contains the greater amount of chlorophyll?
Use your absorbance spectrum graphs to speculate how much more chlorophyll one
grade contains compared to the other.

20
 Part III Fluorescence Spectroscopy of Chlorophyll in Olive Oil

5. Compare the fluorescent spectra of the three grades of olive oil. The peak that is visible at
approximately 675 nm is from chlorophyll. Which sample has the largest peak in this region?

6. Using the fluorescence from the known olive oil samples as your standards, determine the
quality of your unknown olive oil sample.

7. Compare your results using fluorescent spectroscopy to your results using traditional
spectroscopy. Is one method better than the other?

21
EXPERIMENT 4

PERFORMANCE CHARACTERISTICS OF A SPECTROPHOTOMETER or

IInnttrroodduuccttiioonn ttoo U
Ullttrraavviioolleett—
—VViissiibbllee SSppeeccttrroom
meettrryy ((U
UVV--VViiss))

Objective: Investigate several characteristics of a commercial spectrophotometer and compare different

cuvette materials.

Introduction: spectroscopy In this experiment you will become familiar with different features of
commercial ultraviolet-visible (UV/vis) spectrophotometers. The prefixes ultra and infra mean above
and below, respectively. Hence, the energy of ultraviolet radiation lies just above the visible violet light
(i.e., ultraviolet means above visible violet light in energy). Likewise, infrared means below visible red
light in energy. Because the energy range for a typical UV analysis is right next to the energy range of
visible light, many instruments, including ours, operate in both the UV and visible regions. In this lab
period, you will learn how to prepare samples for analysis and how to obtain data from the UV-Vis
instrument. The samples are compounds that might be found in explosives. They are nitroaromatics or
compounds that contain nitro groups (-NO2) bonded to an aromatic ring. In this case, the aromatic ring
is toluene. You will examine the output of the instrument in the wavelength region between 200 and
900 nm (nanometers). One nanometer is 1 x 10-9 meter. Background Theory for UV Absorption13 In
terms of energy, the IR region lies below the visible region, which lies below the UV region. Thus, IR is
strong enough to cause atoms within a molecule to vibrate, but not strong enough to cause electrons to
change orbital locations. UV radiation between 200 and 400 nm is strong enough to cause loosely held
electrons to change locations. These electrons can be either the non-bonding electrons (n-electrons) of
aldehydes or ketones, or they can be the -electrons of conjugated  systems. Figure 1 shows typical
structures of compounds that contain n-electrons.

Figure 2 shows compounds that contain conjugated systems.

22
1

Ultraviolet spectroscopy is one of several forms of spectroscopy that we will study this semester.
Accordingly, it is important that you understand the capabilities and limitations of each of these forms
of spectroscopy. The words spectroscopy and spectrometry have different meanings. A spectroscope is
an instrument, and spectroscopy is the use of a spectroscope. Spectrometry means the measurement of
a spectrum. One generally measures wavelengths or frequencies, or a spectrum of them. We will use
the electromagnetic spectrum to gain information about organic molecules.

The modifier ultraviolet means that the information will come from a specific region of the
electromagnetic spectrum called the ultraviolet region. The electromagnetic spectrum includes all
radiation that travels at the speed of light c (3 x 1010 cm/sec). The electromagnetic spectrum includes
radio waves, which have long wavelengths, x-rays, which have short wavelengths, and visible light,
which has wavelengths between those of radio waves and x-rays. All of these waves travel at the speed
of light. We normally describe these waves in terms of their energy. Of the three kinds mentioned, x-
rays are most energetic, visible light next, and radio waves least energetic. Thus, the shorter the
wavelength is, the greater the energy of an electromagnetic wave.

Electromagnetic radiation (EMR) has a dual nature; it has the characteristics of both waves and particles.
Both forms of EMR are important. From the wave nature of the waves we get the wavelength () or
distance between two crests. The wavelength is related to the frequency (), how many wavelengths
pass a given point in a given time, by the velocity of the wave c. From the particulate nature of EMR, we
get the energy E of a given wave, which is proportional to its frequency. Plank’s constant h turns the

proportionality into an equation. The mathematical relationships among these variables are shown
below.

Frequency and Wavelength: ν = c/λ or λ = c/ν or c = νλ

1
McMurry, Organic Chemistry, 5th Edition, pp 543-548.

23
 Energy and Frequency: E α ν or E = hν

Energy and Wavelength: E = hc/λ

Visible light includes the rainbow colors red, orange, yellow, green, blue, indigo, and violet. A handy
acronym for these colors is ROY G BIV, which most people remember from grade school. Note red is at
the low-energy end of the visible spectrum and violet is at the high-energy end. These facts allow us to
quickly understand the terms infrared and ultraviolet. The prefix infra means below, and the prefix ultra
means above. Thus, infrared radiation is outside the visible range and lies just below red on the energy
scale. That is, infrared radiation is less energetic than visible light. Ultraviolet radiation is outside the
visible range and is just above violet on the energy scale. Thus, infrared literally means “below” red (in
terms of energy), and ultraviolet means “above” violet (in terms of energy). The UV energy of the sun is
what gives ride to sunburns.

Figure 3

We learned in general chemistry that visible yellow light is observed when sodium ions are heated in a
Bunsen burner. The heat excites some ground-state electrons to higher energy levels, then when the
electrons “fall” back to the ground state, they “emit” energy that corresponds to the energy difference
between the energy states (orbitals) where the electrons are found. When this energy difference falls
within the energy range of visible light, we can see it as a color. In the case of sodium, we see yellow
light. Thus, the light results from the emission of energy by the electrons as they fall from higher to
lower energy states (orbitals). Note that it takes the same amount of energy to make the electrons jump
from the lower to higher states as the amount of energy the electrons emit when they fall from higher
to lower states. We generally add more energy than is absolutely necessary for the transition to ensure
that the transition occurs. When we add energy to a system, we give it a positive sign (endoenergetic).
When a system gives off energy, we give it a negative sign (exoenergetic).Just as heat causes some of
sodium’s electrons to move to higher energy states, ultraviolet radiation causes electrons in certain
organic compounds to move from their ground state locations to orbitals of higher energy. The energy

24
of the ultraviolet light acts just like the energy of the heat. In this case, the molecules are said to
“absorb” ultraviolet radiation. A measurement of this phenomenon is called an absorption spectrum as
opposed to an emission spectrum. When electrons move from lower to higher energy levels, we call the
movement an electronic transition. Thus, the basic interaction between UV light and organic
compounds is that UV light causes electronic transitions in certain organic structures. That is, for a given
molecule, an electron changes orbital locations because the energy of the UV light forces it to change
locations.

The organic compound is dissolved in a solvent that does not absorb UV light. Such a solvent is said to
be transparent to UV light. The sample (compound in its solvent) is placed in a cuvette. A cuvette is a
sample holder that has very precise dimensions. The cuvette is placed in an ultraviolet
spectrophotometer. The instrument produces ultraviolet light over a range of wavelengths between 200
and 400 nanometers (nm), and the UV light is split into two equal beams. One beam is directed through
the solution of the organic compound (the sample) and the other beam is directed through the solvent
(the reference). The two beams are called the sample beam and the reference beam. A nanometer
equals a millimicron (mμ), which is sometimes still used by chemists to report wavelengths. As the UV
light passes through the sample, the instrument records a plot of absorbance (A) versus wavength (λ). In
other words, the instrument measures how much UV light is absorbed (the absorbance A) and where
the light is absorbed (the wavelength λ) for the specific sample. In this lab, we will obtain a UV spectrum
(plot of A vs. λ) for a sample of the organic compound toluene (methylbenzene) dissolved in hexane.
From the plot we will find the wavelength where the maximum absorbance occurs and record the
wavelength as λmax and the absorbance as a raw number. We call λmax the wavelength of maximum
absorbance. Thus, after you obtain your plot or printout of A vs. λ, all you will record on your data sheet
from the printout is λmax and A at λmax. We will then make use of certain relationships that govern how
much UV light can be absorbed by a sample. Namely, that the amount of light absorbed (A) is
proportional to how many molecules or the concentration (c) of molecules that are absorbing light, and
how far the UV light must pass through this concentration or the path length l. These relationships are
shown below.

Aαc

Aαl

Aαcxl

This last proportionality says that the absorbance A is directly proportional to the concentration of the
sample and to the path length (width of the cuvette). This is why the dimensions of the cuvette must be
precise. The proportionality is useful for one given concentration or sample. A far more useful form of
the relationships above is the Beer-Lambert equation, which makes the proportionality into an equation
by addition of the proportionality constant ε (pronounced epsilon)

A = εcl

Beer-Lambert Equation

25
Like λ and ν, ε is a Greek letter. However, it is simply a constant that makes the above proportionality an
equation. The constant ε does not vary. So various concentrations of toluene measured in different size
cuvettes would give the same value of ε. Therefore, our laboratory exercise will include calculating ε.
The constant ε is called the molar extinction coefficient.

Therefore, the concentration of the sample must be in moles per liter (mol/L). The standard path length
is 1 cm. Thus, most cuvettes, including those in our lab, are exactly 1 cm wide where the light passes
through. These conventions ensure that everyone calculates ε the same way.

Figure 4. Cuvettes and Price List

We will learn that compounds such as benzene that contain conjugated π systems absorb UV light very
strongly (i.e., ε is typically 5,000 to 30,000). Whereas aldehyde and ketones, which contain isolated
carbonyl groups, absorb UV light weakly (i.e., ε is typically less than 100). The spectrum may be run on a
very small sample, but small amounts of impurities that absorb UV must be avoided.

Example problem: One milligram of a compound of molecular weight 140 is dissolved in 20 mL of

ethanol. The UV of the sample is measured in a 1-cm cuvette. The maximum absorption is 0.50 recorded
at 248 nm. Calculate the value of ε.

26
Solution: The concentration in mol/L = [(0.001)/140]mol/.012 L = 0.0060 mol/L ;

ε = A/cl = 0.50/(0.0060)(1.00) = 83.

Acids and acid derivatives, which contain a heteroatom next to the carbonyl might absorb UV radiation,
but not measurably in the 200-400 nm range. Therefore, except for aldehydes and ketones, compounds
that contain only carbonyl groups generally do not absorb UV radiation. Thus, UV spectroscopy enables
us to identify a conjugated π system or the carbonyl group of an aldehyde or ketone by the value we
find for ε. A strong absorption corresponds to ε > 1,000, and a weak absorption to ε < 100. If log ε = 5, is
the UV absorption strong or weak? Does it represent an aldehyde or a conjugated system? Figure 5
shows the simulated UV spectrum of a conjugated ketone. Note that there are two maxima in the curve.
There is a strong absoption (ε > 1,000) and a

weak absorption (ε < 100). The strong absorption is due to a conjugated π system (i.e., C=C-C=O,
alternating double bond, single bond, double bond). The weak absorption is due to the presence of a
ketone carbonyl group in the compound. The carbonyl groups contain non-bonding electrons. Hence,
the strong absorption is due to a π to π* electronic transition and the weak absorption is due to an n to
π* transition.

Figure 5. UV spectrum of an α,β-unsaturated ketone.

π Electrons and Conjugated π System

UV radiation has more energy than IR radiation; therefore, UV radiation interacts with compounds
differently than does IR radiation. IR radiation makes molecules vibrate (i.e., twist, bend, scissors, etc.),
whereas UV radiation causes a loosely held electron within a molecule to change locations. Because
electrons can only exist in orbitals, the change in location is from one orbital to another orbital. The
process of changing orbital locations is called a transition. We could say that when we walk from the
first floor to the lab on the third floor that we transition to the lab, but the word transition is usually
reserved for the movement of a particle such as an electron from one orbital location to another. When

27
an electron changes orbital locations, we call the process an electronic transition. Electrons do not
change location spontaneously; they need help. The help appears in the form of UV radiation. UV
radiation is just energetic enough to cause certain loosely held electrons to move from one orbital to
another orbital but not energetic enough to cause an electron to be ejected from the molecule.
Radiation of higher energy than UV radiation such as X-ray or Gamma radiation is sufficiently energetic
to eject electrons. When a negatively-charged electron is ejected, a cation or positively-charged particle
is left. Therefore, high-energy radiation is called ionizing radiation. Of course, if a human is subjected to
ionizing radiation, the result can be a radiation injury. Thus, when you get a dental x-ray, a lead-
containing protective apron is draped over you to protect your body from the ionizing radiation.

The Instrument: See Quantitative Chemical Analysis, 6th Ed. Pg 462-463

You will perform checks on the following performance characteristics

• Wavelength calibration – How accurate are the reported wavelengths?

• Linearity – Is the photometric response linear over a reasonable absorption range?
• Resolution – How small of a wavelength difference can be detected?
• Effect of Slit Width – How does the exit slit of the monochromator affect the absorption spectrum?
Each of these is discussed in further detail below.

Wavelength Accuracy
The wavelength accuracy of a spectrophotometer is the correctness with which the wavelength of light
reaching the sample matches the wavelength reported by the instrument. When a wavelength is
selected, the monochromator’s grating (Grating 2 in Figure IB-1) is rotated so that the specified
wavelength is centered on the exit slit of the monochromator. Errors can come from two sources. First,
imperfections in the positioning system might cause errors in the grating rotation, which in turn causes
errors in the wavelength reaching the sample. Second, the light reaching the sample is not a single
wavelength, but rather a band of wavelengths. The width of this band is the spectral bandwidth (SBW)
of the instrument, and is defined as the width in nanometers of the band of light leaving the
monochromator measured halfway between the baseline and the peak intensity. Wavelength errors
tend to increase with SBW. Wavelength accuracy is verified using calibration standards. In general the
two standards that are used are theD2 source lamp lines and a holmium oxide (Ho2O3) filter. Both are
relatively stable and have several well-defined peak for calibration purposes. In this experiment we will
use the holmium oxide filter.

Linearity
Photometric linearity is the ability of the optical detector to generate a change in electrical output that
is proportional to a change in incident light intensity. The detector must exhibit photometric linearity
over the entire operating wavelength range of the spectrophotometer and over a range of light
intensities.
Photometric linearity will be assessed using a series of standards of either Ni2+, Co2+, or Cu2+. (Of course

it is important to realize that the solutions must be made correctly in order to truly assess the
spectrophotometer’s linearity. Errors in solution preparation will appear as instrumental errors.)

28
 Resolution and the Effect of Spectral Bandwidth

Resolution refers to the extent to which two peaks or bands (e.g., spectral, chromatographic, etc.) can
be separated spatially such that peak overlap is minimized. The limiting resolution of the
spectrophotometer depends upon the narrowest SBW that can be achieved. For high resolution work a
very narrow SBW is required. A solution of toluene in hexane will be used to assess the resolution of the
instrument. The SBW of an instrument is a function of the exit slit width (w) and the reciprocal linear
dispersion (D-1) of the monochromator. For a given value of D-1, the SBW is given by

SBW = wD−1 (4)

The reciprocal linear dispersion of a spectrophotometer is usually fixed, so SBW is controlled by

changing the slit width. Using the spectrometer manual, determine D-1 in nm/mm and SBW range in nm.
Smaller values of SBW (i.e. a smaller slit width) produce higher resolution spectra. But because the light
intensity reaching the sample also decreases with slit width, spectra recorded with a small SBW also
exhibit decreased signal-to-noise ratios. Conversely, larger values of SBW produce spectra with less
noise (more light reaches the sample) but also with less resolution. The effect of SBW on the absorption
spectrum of benzene vapor will be investigated as part of this experiment.

Safety Considerations.
 Benzene is a carcinogen. Do not open the cuvettes containing the benzene vapor.
Hexane and toluene are flammable and toxic.
 Dispose of hexane, toluene in the appropriate waste container.

Procedure.
This experimental procedure has several different parts, but they do not need to be done in
sequentially.

NOTE: This procedure does NOT need to be performed every time you use the spectrophotometer.
However, it is important to realize that just because the readout from the instrument indicated that 500.00-
nm light is being measured, this does not absolutely guarantee that this is the case. If wavelength accuracy is
of primary concern, then you should verify the spectrophotometer’s calibration by measurement of the
absorption lines of holmium glass or emission lines of the deuterium (D2) lamp.

 Before using the instrument, prepare the following solutions. (Note that some of these will be
provided for you; check with your instructor)
1. 0.020 %v/v Toluene in Hexane (UV grade)
2. 20-500 mM Ni2+, Co2+, or Cu2+ solutions (see introduction to Module I)

 Have appropriate blanks ready for each solution.

 Use quartz cuvettes for all components, unless otherwise noted.

29
 1. Wavelength Calibration, and Resolution

Holmium Glass Method. This method allows you to verify the wavelength calibration at three
different wavelengths, 460.0 nm, 360.9 nm and 279.4 nm. The performance is considered
satisfactory if the wavelength errors of the holmium glass absorption lines are within ±1 0.3 nm

a. With the Holmium Glass Filter in the sample compartment, record the spectrum
between 250 and 500 nm. at a 0.5 nm bandwidth, 10 nm/min scan speed, and a data
interval of 0.1 nm.
b. b) Determine the wavelengths of maximum absorbance using the Peak Pick Table. How
does the spectrum compare with the \wavelength values reported above?

Resolution. Record the spectrum of the toluene/hexane solution over the full range of the instrument
for a range of slit widths low to high. At what point can the “fine structure” no longer be seen? How is
this related to the resolution of the instrument?

2. Effect of slit width

Place one drop of benzene in the bottom of a clean dry quartz cell and seal the cell. Do not get benzene
on the walls of the cell. Put cell in the sample compartment; no reference is necessary.
Set up the instrument to scan between 300 and 200 nm using a data interval of 0.200 nm (the scan rate
should automatically adjust to 120 nm/min). Record the absorption spectrum of benzene vapor in the
provided cuvette at spectral band widths (SBW) of 0.2, 0.5, 1.0, and 2.0 nm. Also record the spectrum
using the Ocean Optics diode array spectrophotometer.

Q1. What is the effect of spectral bandwidth on the absorption spectrum of benzene? Comment in terms
of resolution and in terms of signal-to-noise ratio.

Q2. Estimate the SBW for the diode array spectrophotometer by comparing the benzene spectrum to the
spectra recorded at different SBW on the Unicam.

Q3. When might a small SBW be necessary? When might a small SBW be a disadvantage?

3.Absorption properties of cuvette materials

On a single graph, overlay spectra (recorded with the Unicam instrument over the entire uv-vis range) of
cuvettes made of glass, plastic, and quartz. Leave the reference cuvet empty when recording this
spectrum and use a reasonably fast scan rate (~700 nm/min). Also, be sure to record a blank spectrum
(i.e. no cuvette in either holder). Include the overlaid spectra in the report.

4. Linearity
Find the λ max of Ni2+, Co2+, or Cu2+ (whichever ion you selected for preparing the set of 5 solutions).
Record the absorbance of each solution at the λmax you determined. Make at least 3 measurements of
absorbance for each solution. Each time you make a replicate measurement, be sure to remove and
replace the cuvette. (This will help to account for positioning errors.)

30
Q4. Does the spectrophotometer respond linearly to concentration? Explain how you determined this and
how you can quantify the linearity of the response.

1. Nitroaromatics.

a. Place the reference and sample cuvettes in the instrument in the correct locations.

b. Obtain the spectrum from 200-900 nm and save the data points of the spectrum. Note that our
instrument scans both visible and ultraviolet regions of the electromagnetic spectrum.

c. Repeat the procedure for using the other solutions of the nitroaromatic (in three solvents total)

For each of your nitroaromatic solutions , write out the appropriate UV data in the format you would
expect to see in a scientific journal (e.g., nitroaromatic: λmax 250 nm(ethanol), ε 12,000).

Determine whether the change in solvent resulted in a bathochromic, hypsochromic or no shift for the
nitroaromatic compound. (Show all spectra on the same graph, and clearly label). What conclusion(s)
can you draw from your data about the nature of the transitions in the compound?

Also to be include in the Report:

1. The report should contain a schematic diagram of the internal workings of the instrument.

2. For what types of molecules is UV-VIS spectroscopy most useful? For which is it not? As a
consequence, what are the best solvents for UV-VIS spectroscopy?
3. What is meant by bandwidth? As you narrow the bandwidth of the UV-VIS spectrometer, what might
you expect to happen to the spectrum?
4. Labeled copies of all the spectra and peak data.
5. Discuss in what situations would an accurate calibration of the UV-VIS be most important?
6. Discuss how changing the bandwidth changes the spectrum of the benzene vapor.
7. Compare the different optical material used to make cuvettes. What wavelength ranges is each
material useful for?
8. Explain what is meant by lambda max (λmax).

9. What information do you get from a molar extinction coefficient?

10. Draw the structure of cyclohexanone and show its non-bonding electrons. What value of ε do you
expect for cyclohexanone?

11. Draw a bond-line structure of toluene. What value of ε do you expect for toluene?

12. Why do the two examples in Figure 4 both have two maxima in their UV spectra?

31
13. A student dissolves 1.00 mg of a solid (140 g/mol) in 10.00 mL of ethanol. What is the concentration
of the solid in mol/L in the ethanolic solution?

14. One milligram of a compound of molecular weight 160 is dissolved in 10 mL of ethanol. The UV of
the sample is measured in a 1-cm cuvette. The maximum absorption is 0.60 recorded at 240 nm.
Calculate the value of ε.

15. Given a sample of acetophenone, predict what kind of UV spectrum you expect.

32
EXPERIMENT 5.

Spectrophotometric Analysis of a Two-Component System with

Overlapping Spectra

A number of methods have been developed to determine the composition of a binary mixture
spectrophotometrically. Most of these are directed at mixtures where one component can be isolated
from the other or they require a Beer’s law experiment to measure the molar absorptivity of each of the
substances in the mixture. However, Blanco1, et. al. described a method of resolving mixtures with
overlapping spectra, called Multi-Wavelength Linear Regression Analysis or MLRA, without determining
molar absorptivities or complicated mathematics. Using Blanco’s method, the composition of a binary
mixture with overlapping spectra can be resolved with only three measurements, the absorbance of a
standard solution for each component, and the unknown mixture itself.

Here’s how MLRA works: Assuming additivity, the absorbance of a mixture is the sum of the
absorbances of its components. If we have a mixture consisting of two components, 1 and 2, with an
unknown concentration of C1 and C2, then: Absorbance of the unknown mixture, Amixture = A1 + A2 but
applying Beer’s law: A1 = 1 b C1 and A2 = 2 b C2. Substituting: Amixture = 1 b C1 + 2 b C2. However, the
absorbances of standard solutions of the same substances will follow the same Beer’s law relationship
and have the same molar absorbance, , and one centimeter path length, b, as the unknown solutions
under the same conditions. Therefore, we can write:
Astandard 1 = 1 b Cstandard 1 and Astandard 2 = 2 b Cstandard 2

Rearranging these relationships: and

Substituting, or

Dividing by Astandard 1 and simplifying we obtain:

33
 Therefore, a plot of vs. will give a slope of and a y-intercept of

. That is, the concentration of the unknown component in the mixture 2, equals the slope

times the concentration of the standard solution for component 2. Likewise, the concentration of the
unknown component in the mixture 1, equals the product of the intercept times the concentration of
the standard solution for component 1, or simply C 2 = m Cstandard 2 and C 1 = b Cstandard 1
Let’s apply this method to the analysis of the metals in a United States five-cent coin, A.K.A., a nickel. To
this end, the absorption spectra of the standard solutions with known concentrations and that of the
coin are obtained as three overlapping curves on a single graph.

Procedure

1. Prepare solutions of the standards by dissolving 0.5 grams of nickel wire in dissolved in 15 mL of 1:1
nitric acid and diluted to 50 mL with deionized water. The copper standard solution is prepared similarly,
by dissolving of 0.2 grams of copper in 5 mL of the 1:1 nitric acid solution and again, diluted to 50 mL
with deionized water. The U.S. five cent coin solution was prepared by dissolving 0.13 grams of a nickel
5 cent coin in 5 mL of the same nitric acid solution and diluted to 50 mL.

2. The spectrophotometer, calibrated using a distilled water blank, is used to measure the absorbance
vs. wavelength for each of the three solutions prepared above.

3. Now that the absorbance of each solution has been measured, remove from consideration
absorbance values which are the most unreliable (those at the extreme ends of the spectra or those
outside the optimum 0.15 - 0.70 absorbance range3).

4. Use the procedure described above and in reference 1, calculate the concentration of the
components of the mixture using the slope and intercept of the line of best fit found in step 10. Use this
information to determine the percentage composition of the metals in a U.S. five-cent coin based on the
MLRA. Be sure to include quantitative error analyses of your results. Compare your results with the
composition stated by the US mint.

34
References
1
Blanco, J. Chem. Ed, 66, 189 (February, 1989)
2
Willard, Hobart H., Lynne L Merritt, and John A. Dean, Instrumental Methods of Analysis, 4th Ed., D.
Van Nostrand Company, 1965, p. 90.
3
ibid., p. 91

35
 FLUORESCENCE SPECTROSCOPY
INTRODUCTION
Fluorescence is a spectrochemical method of analysis where the molecules of the analyte
are excited by irradiation at a certain wavelength and emit radiation of a different wavelength.
The emission spectrum provides information for both qualitative and quantitative analysis. As
shown in Figure 1, when light of an appropriate wavelength is absorbed by a molecule (i.e.,
excitation), the electronic state of the molecule changes from the ground state to one of many
vibrational levels in one of the excited electronic states. The excited electronic state is usually
the first excited singlet state, S1 (Figure 1). Once the molecule is in this excited state, relaxation
can occur via several processes. Fluorescence is one of these processes and results in the
emission of light (Refer to Figure 1 during the following discussion).

Figure 1: Electronic transition energy level diagram.

Following absorption, a number of vibrational levels of the excited state are populated.
Molecules in these higher vibrational levels then relax to the lowest vibrational level of the
excited state (vibrational relaxation). From the lowest vibrational level, several processes can
cause the molecule to relax to its ground state. The most important pathways are:

1. Collisional deactivation (external conversion) leading to nonradiative relaxation.

36
 -9
2. Intersystem Crossing (10 s): In this process, if the energy states of the singlet state
overlaps those of the triplet state, as illustrated in Figure 1, vibrational coupling can occur
between the two states. Molecules in the single excited state can cross over to the triplet
excited state.

3. Phosphorescence: This is the relaxation of the molecule from the triplet excited state to the
singlet ground state with emission of light. Because this is a classically forbidden
transition, the triplet state has a long lifetime and the rate of phosphorescence is slow
-2
(10 to 100 sec).

4. Fluorescence: Corresponds to the relaxation of the molecule from the singlet excited state
-8
to the singlet ground state with emission of light. Fluorescence has short lifetime (~10
sec) so that in many molecules it can compete favorably with collisional deactivation,
intersystem crossing and phosphorescence. The wavelength (and thus the energy) of the
light emitted is dependent on the energy gap between the ground state and the singlet
excited state. An overall energy balance for the fluorescence process could be written as:

where E is the energy of the emitted light, E is the energy of the light absorbed by the
fluor abs
molecule during excitation, and E is the energy lost by the molecule from vibrational
vib
relaxation. The E term arises from the need for the solvent cage of the molecule to
solv.relax
reorient itself in the excited state and then again when the molecule relaxes to the ground
state. As can be seen from Equation (1), fluorescence energy is always less than the absorption
energy for a given molecule. Thus the emitted light is observed at longer wavelengths than the
excitation.

5. Internal Conversion: Direct vibrational coupling between the ground and excited electronic
states (vibronic level overlap) and quantum mechanical tunneling (no direct vibronic
overlap but small energy gap) are internal conversion processes. This is a rapid process
-12 -8
(10 sec) relative to the average lifetime of the lowest excited singlet state (10 sec) and
therefore competes effectively with fluorescence in most molecules.

Other processes, which may compete with fluorescence, are excited state isomerization,
photoionization, photodissociation and acid-base equilibria. Fluorescence intensity may also be
reduced or eliminated if the luminescing molecule forms ground or excited state complexes
(quenching). The quantum yield or quantum efficiency for fluorescence is therefore the ration
of the number of molecules that luminesce to the total number of excited molecules. According
to the previous discussion, the quantum yield (φ) for a compound is determined by the

37
relative rate constants (kx) for the processes which deactivate the lowest excited singlet states,
namely, fluorescence (kf), intersystem crossing (ki), external conversion (kec), internal
conversion (kic), predissociation (kpd), and dissociation (kd).

B. EXPERIMENT SUMMARY
In this experiment:

1. the excitation and emission spectra for the fluorescent dye fluorescein will be measured.

2. the effect of concentration and instrumental bandwidth on the fluorescent signal will be
studied.

3. quinine in tonic water will be determined fluorimetrically using a calibration curve and
standard addition.

C. EQUIPMENT

The instrument used in this experiment is Hitachi Spectrofluorometer.

Optical System

38
A 150 W xenon lamp is used as the light source. The bright spot of the xenon lamp after being
collimated into a beam, is focused via a concave mirror onto the excitation slit assembly
through the entrance slit . Part of the beam, which is then dispersed to a spectrum via the
diffraction grating assembly, is directed out of the exit slit , passes through a collecting lens
assembly, and impinges on the sample cell. For light source compensation, a portion of the
excitation light is reflected by a beam splitter quartz plate to a Teflon reflecting plate. The
scattered light from Teflon plate is directed to a monitor photomultiplier. The emitted light
from the cell is passed through a lens, and directed into the emission monochromator,
consisting of the slit assembly and a diffraction grating assembly. The spectral light is reflected
from a convex mirror and directed to the measurement photomultiplier.

D. EXPERIMENTAL
D.1. Start-Up
See the instructor to learn how to start up the instrument. Prepare solutions while the
instrument is warming up.

D.2. Solutions
i. Tonic water solutions
1. Solution TW10:
Dilute the tonic water by a factor of 10 in 0.1 M H SO . Pipette 10.0 mL of tonic water into a
2 4
clean 100 mL volumetric flask and fill to the mark with 0.1 M H SO .
2 4
2. Solution TW 200:
Pipette 5.0 mL TW10 into a clean 100 mL volumetric flask and filling to the mark with 0.1 M
H SO . Calculate how many times the original tonic water has been diluted.
2 4
ii. Solutions for Standard Addition Method
Pipette 5 mL of TW10 solution into each of five 100 mL volumetric Flasks. Dilute the first
volumetric flask to volume with 0.1 M H SO . Then pipette 1 mL of the stock 10 ppm quinine
2 4
solution to the second volumetric flask and dilute with 0.1 M H SO . Repeat by pipetting 2, 3,
2 4
and 5 mL to the third, fourth and fifth volumetric flask respectively and again dilute each to
volume with 0.1 M H SO . Calculate how many times the original tonic water has been diluted.
2 4
iii. Fluorescein solution (1000 ppm in 95 % ethanol)

D.3. Excitation and Emission Spectra of Fluorescein

Fluorescence spectroscopy can yield low detection limits, high sensitivity and high
specificity. The high specificity is largely due to the fact that fluorophores exhibit specific
excitation (absorption) and emission (fluorescence) wavelengths. These wavelengths can be
determined via the collection of two spectra, an excitation spectrum and an emission spectrum.
Although the approximate excitation and emission wavelengths for many molecules are known,
these wavelengths should generally be optimized for the specific conditions employed.

39
In this section, the excitation and emission wavelengths for fluorescein will be determined by
collecting excitation and emission spectra.

Fluorescein (CAS No.: 2321-07-5, MW: 332.31)

Absorption max: 498 nm

Fluorescence max: 518 nm

D.3.1 Cell Handling

Absorption cells (cuvettes) should receive the same care given a lens or other optical
component. The optical surfaces of cells that are placed in the light beam must be absolutely
clean, or serious errors in spectrophotometric measurements will result.

In the handling of cells, the following well-known rules should be followed without exception.

1. Never touch the optical surfaces of the cell. Contact with the skin will invariably leave a film that,
though invisible to the eye, will change the light transmission and reflection characteristics of
the cell windows, especially in the ultraviolet region.

2. Handle cells only at the top portions of the side plates that do not face the optical axis.

3. When filling cells with sample solutions, a dropper, or preferably a pipette, should be used
rather than direct pouring from a beaker or test tube.

4. Rinse the cell with several portions of the solution before filling. Avoid overfilling the cell.

5. Do not spill liquid on the outside of a cell. Before inserting a cell into the holder, carefully wipe
the cell windows with a clean lens tissue or suitable absorbent lint-free disposable wipe.

40
6. Always orient cells in the same direction in the cell holder. When using a matched pair of cells,
always use the same cell for the reference.

7. For the disposable plastic cells, solvents like methanol and ethanol can be contained for a
maximum time of 5 min. Never use the plastic cells for toluene.
D.3.2 Emission Spectrum

Note: For the fluorescein analysis, the plastic disposable cuvettes are used.

Find fluorescein’s approximate absorption maximum (498 nm).

With the excitation wavelength fixed at 498 nm, obtain the emission spectrum between 350 and
670 nm.

Scanning Speed: medium

Slit Width: Ex = 3.5 and Em = 3.5

You may need to adjust these.

Fill a plastic cuvette with the solution of 10-ppm fluorescein in 95% ethanol. Fill the cuvette with a
disposable pipette. Place the cuvette into the sample compartment, and close the cover of the
spectrofluorometer.

Ask your instructor how to obtain an emission spectrum.

Locate the exact wavelength of the maximum.

D.3.3 Excitation Spectrum

Repeat the same procedure as for the emission spectrum, ensuring that the following parameters
have been adjusted.

Em Wavelength: the value you have just obtained.

Ex Wavelength: 350 - 670 nm

Once the parameters are correctly set, run the spectrum. Locate the excitation maximum.

D.4. Inner Filter Effect

In this experiment, you will monitor the fluorescence intensity of Fluorescein as a function of
concentration. Prepare the following solutions: 0.1, 1.0, 10 and 100 from the stock 1000 ppm
Fluorescein in 95% ethanol. You will measure the relative fluorescence intensity over a set
period of time.

41
Ex Wavelength: maximum measured above
Em Wavelength: maximum measured above
Slit Width:
Ex = 3.0
Em = 3.0
Fill a disposable cuvette with the blank solution ( i.e., EtOH) and insert it into the holder.
Run the blank, which will be used to correct the remaining data for background radiation.

List the average fluorescence intensity for each concentration below. (Remember to record the
sensitivity setting used for each solution)

Concentration (ppm) Average Fluorescence Intensity

0.1
1.0
10
100
1000

D.5. Bandwidth Effect on the Quality of the Spectrum

Note: For the bandwidth analysis, the quartz cuvette is used.

The bandwidth of a monochromator is defined as the span of monochromator settings (in units
of wavelength) needed to move the image of the entrance slit across the exit slit [4].

The bandwidth of the spectrofluorometer can be changed by adjusting the width of the
excitation and emission slits. For this study, the fluorescence intensity of 5000 ppm anthracene
in toluene is measured at different slit widths to observe this effect. Fill the quartz cuvette with
5000 ppm anthracene in toluene, and insert it into the cell holder.

Spectrum Type: Emission

Ex Wavelength: 380 nm
Em Wavelength: 390 - 600 nm
Scanning Speed: high

Obtain the emission spectrum using the following sets of slit widths.
Slit Width:
Ex = 3 ; Em = 3
Slit Width:
Ex = 10 ; Em = 3
Slit Width:
Ex = 10 ; Em = 10

42
D.6 Analysis of Quinine in Tonic Water- Calibration Curve Method
Note: For the quinine analysis, the plastic disposable cuvettes are used.
In this section, a calibration curve of quinine will be constructed using a series of standard
solutions you will prepare.
The concentrations of these solutions are 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 ppm.
The excitation and emission maximums for quinine are 350 and 445 nm respectively. The exact
maximums may differ from those given above. You may want to determine what they are
exactly under our experimental conditions.
Change to the following settings:
Ex Wavelength: 350
Em Wavelength: 445
Slit Width:
Ex = 3 ; Em = 3

Run a blank solution sample before beginning with the standards.

Place the first standard in the cell holder and obtain an intensity measurement. Repeat with the
remaining standards to complete the calibration curve. Then, insert the TW200 example you
prepared into the cell holder and obtain an intensity measurement. List the intensity values in a
table.

D.7. Analysis of Quinine in Tonic Water – Standard Addition Method

Another method of quantification, standard addition, involves adding varying quantities of a
standard to a constant concentration of unknown. These are the solutions that you
prepared at the beginning of the lab. Adjust the parameters as follows:
Ex Wavelength: 350
Em Wavelength: 445
Slit Width:
Ex 3 ; Em 3
Insert a blank solution in the sample holder and run the blank to correct for the background.
Measure the intensities of all five solutions that you have made using the same procedure
described in section D.4.
List the intensity values in a table.

E. DATA PROCESSING AND QUESTIONS

1. Considering its molecular structure, why would you expect fluorescein to be highly
fluorescent?

2. What causes the inner filter effect for fluorescein? How can errors due to this effect be
reduced or eliminated?

3. What effects on the spectrum do the excitation and emission bandwidth have? Why?

43
4. From the values obtained in the calibration curve analysis, determine the concentration (in
ppm) of quinine in the tonic water.

5. Starting with F=Kc (where, F is fluorescence intensity; K is a constant which depends on the
quantum efficiency of the fluorescent process; and c is concentration: derive an expression
relating fluorescence intensity to concentrations and volumes in the standard addition
method.

6. Using the expression you derived, plot a linear curve with your standard addition data. Using
the slope and intercept of the plot (and any other known values that you might need)
calculate the concentration of quinine in the tonic water. Do not determine the
concentration graphically!

7. Do the results from the calibration curve agree with those by standard addition? If so what
does this prove? If not, what does this prove, and which do you think is correct?

8. When would standard addition be more suitable than a calibration curve for quantitative
analysis?

9. In this experiment, you optimized literature excitation and emission wavelength maxima
prior to actual analysis. How would you go about determining and optimizing excitation and
emission wavelength maxima for an analyte without any literature data?

F. REFERENCES

1. J. E. O'Reilly, J. Chem. Ed. 1975, 52, 610.

2. Skoog, Holler and Crouch. Chapter 15.

3. R. D. Baun, Introduction to Instrumental Analysis, McGraw-Hill, NY, 1987, Chapter 11.

4. Skoog, Holler and Crouch. Chapter 7.

44
Experiment 6.

Atomic Absorption Analysis of Calcium in Milk

Purpose
This experiment will use atomic absorption spectroscopy to analyze milk for the element
calcium content using the Beer’s Law Method. The measured calcium content will be compared with
the USDA values.

Introduction
See the following link for general background on Atomic Absorption Spectroscopy (AAS) :
http://ewr.cee.vt.edu/environmental/teach/smprimer/aa/aa.html

Milk is a complex mixture of emulsified lipids and proteins and other dissolved components such
as lactose, vitamins, and minerals (namely calcium). In milk, calcium exists both as an ion and as Ca
9(PO4)6 bound to the milk protein, casein. Casein exists as spherical micelles in milk, primarily due to the
hydrophobic nature of the protein. The micelles clump together due to the interactions between
Ca9(PO4)6 and casein (Figure 1). Approximately 75% to 90%1 of the calcium present in cow’s milk is
bound to casein as described above.

Figure 1. The Calcium Phosphate –Casein Complex

Milk undergoes several processing stages before being sent to market. The first stage is
clarification, standardization, and separation. The milk is centrifuged to remove fat and solid impurities.
Cream is reintroduced to the purified milk if necessary (to make non-skim milk). The second stage,
pasteurization, eliminates most bacteria from milk by heating it over an extended period of time. The
third process, homogenization, reduces the size of fat globules in milk. Finally, the milk is fortified with

45
nutrients such as vitamins A & D. It is important to note that the calcium concentration of raw milk is
essentially unchanged after processing. Therefore, the calcium concentration in milk depends on the
calcium present in the cows’ food sources.

A few considerations are necessary before one can determine the total calcium content in milk. First, it
is necessary to dissociate the calcium ions from the calcium-casein complex. This is typically
accomplished by acid precipitation of casein. Acid liberates calcium from casein, causing the insoluble
phosphoprotein to precipitate. Precipitation of the protein also means that it can be easily removed to
prevent clogging of the spectrophotometer. A final consideration is the potential of interference caused
by the formation of calcium-phosphorus and calcium-sulfur compounds. These compounds cannot be
easily dissociated in the flame. Lanthanum or strontium are added to compete with calcium for
phosphorus, to prevent formation of Ca-P molecules2. Sodium, magnesium and potassium can also
cause interference, but only at concentrations of 500 ppm or more, higher than concentrations typically
found in milk 3. Methods and theory related to the use of the atomic absorption spectrophotometer can
be found in Perkin Elmer Users Guide.

Materials

Trichloroacetic acid, Sigma-Aldrich (100% w/v, 6.1N)

Lanthanum chloride solution (5% w/v solution)

1000 ppm calcium standard soln.

Milk (Wawa brand – skim, 1% milk fat, 2% milk fat, whole)

Perkin-Elmer Model AA 4000 atomic absorption spectrophotometer

centrifuge and centerfuge tubes

micropipetters- P-20, P-200, P-1000

Procedure
Preparation of Standards and Blank for Beer’s Law Plot:

(All solutions and subsequent dilutions are to be made with deionized water)

Prepare a series of calcium standards (1-6 ppm) from the provided 1000 ppm calcium standard
solution. (for X ppm, use X * 10 L of Ca stock). For example, the 1 ppm calcium standard is
prepared by mixing 10L of the 1000 ppm Ca stock + 120L TCA + 1mL lanthanum and diluted up

46
 to 10mL. Prepare a blank by taking 120L of the TCA solution + 1mL of the lanthanum chloride
solution and diluting to 10mL.
Preparation of the Milk Samples: Prepare the Skim, 1% low fat, 2% low fat, whole milk samples using
the procedure below

1. 500 L milk + 1.2 mL TCA dilute to 10mL

a. Shake for 15 minutes.
b. Centrifuge, 2500 RPM, 5 min.
c. Transfer 300 L of the supernatant liquid to a clean labeled tube, add 500L
of the lanthanum chloride solution and dilute to 5 mL with water.

Prepare a duplicate sample using one type of milk, without the added lanthanum chloride solution. Use
this solution to test the effect of the La on the atomic absorption of calcium.

Instrumental Procedure:

See your instructor for an overview of the Perkin Elmer Atomic Absorption Spectrometer. Set the
following instrument parameters : slit size 0.7, monochromator wavelength 422.7 nm.

A) Preparation of the Instrument. Verify that an calcium lamp has been installed in the instrument and
that water is present in the drain system float assembly container. Open the vent for the exhaust above
the instrument and close the hood door. The acetylene tank pressure should be 75 psig or greater. We
use welding grade acetylene in which the acetylene is dissolved in acetone. When the total tank
pressure drops below 75 psig, the partial pressure of acetone in the tank becomes unacceptably high
and the tank must be replaced. Set the monochromator and the slit width to to the appropriate
settings.

B) Turning on the Instrument and Preparation for Measurements.

l) Turn on the instrument. The power switch is located on the right-hand side of the instrument.
The message SELF TESTING MODE will appear on the display. When this message disappears, the
instrument has passed a series of diagnostic tests and you may proceed.

2) Define the operating parameters by pressing the Param Entry function key. You will be prompted
to define a series of parameters in the given order. After each entry of a parameter you will need to
press the Entry function key to proceed to the next parameter.

a) Lamp Current. Enter 45, the operating current in mA for the calcium lamp. It is important
that you have verified that an calcium lamp is present before completing this step. A 45 mA

47
 current might damage other types of lamps.

b) Integration Time. Enter 1. Each measurement will be made for 1 s. You may wish to change
this number later. The allowed range for the integration time is 0.1-60 s.

c) Replicates. Enter 5. Each measurement will be repeated 5 times. You may wish to change
this number later. The allowed range for the number of replicates is 1-99.

d) Calibration Type. Enter 1 for non-linear. The 3100 allows three types of calibration: non-
linear (1), linear (2), and the method of additions (3).

d) AA technique. Enter 1 for flame. The other options apply to accessories which we do not
have.

e) Standard 1. Enter the concentration of the first (most dilute) standard in µg/ml. Use the
format nn.mm. For example, if the concentration is 2.12 µg/ml, enter 02.12 .

f) Standard 2. Enter the concentration of the next most concentrated standard in µg/ml.

g) Standard 3-n. Continue the process for your n standard solutions.

h) Standard n+l. Do not enter a number in this case. Simply press the Enter key.

3) Optimize the power reaching the detector. Press the Energy function key. The display will show
the energy screen. The Counts (CTS) and Energy (EN) parameters provide a measure of the amount
of radiation reaching the detector. Maximize Counts by making small adjustments in the
wavelength setting. The adjustment of the wavelength should be small. The final wavelength should
not be 247 nm. There is an iron line at 247 nm but it has lower intensity than the 248 nm line. If the
bar graph which gives a visual measure of Counts goes off scale, the gain will be automatically
adjusted. If the gain is not automatically adjusted, press the Gain function key to bring the bar
graph back on scale. Once the wavelength has been adjusted, adjust the two black alignment
screws on top of the lamp for maximum Counts. When the optimization procedure has been
completed, record the values of Counts and Energy. Then convert the instrument to Automatic
Baseline Correction mode by pressing the AA-BG function key. You will hear the movement of a
mirror in the optics and the AGC routine will be applied.

4) Ignite the flame. Use common sense and pay attention to the details outlined below.
Acetylene is a hazardous combustible gas that can be used safely if one follows the lab protocol.
Before you ignite the flame, put on your safety goggles and continue to wear them until you
turn off the flame.

48
 a) Open the values of the compressed air and acetylene cylinders. Adjust the air (oxidant) outlet
pressure to 60 psig and the acetylene (fuel) outlet pressure to 14 psig.

b) Turn the Oxidant Switch on the pneumatics control panel to Air. Adjust the air flow to 4 units
on the right (Oxidant) flowmeter using the Oxidant Control Knob. Adjust the acetylene flow to 2
units on the left (Fuel) flowmeter using the Fuel Control Knob. Since there is a strong coupling
between acetylene flow and acetylene pressure, also adjust the acetylene pressure with the
acetylene regulator to 13 psig. The acetylene pressure should not exceed 15 psig.

NOTE: If the gases are left on for 15 sec without ignition or if the acetylene pressure drops
below 12 psig, the red interlock light comes on and the system turns off the gas flows. If this
happens, turn the gas control switch to Off and then back to Air.

c) When the gas flows and pressures have been adjusted, press the Ignite button until the flame
appears. When gases are first turned on, the flame may not light the first time due to air in the
supply lines. If the flame does not ignite, turn the gas control switch to Off, then back to Air,
and press the Ignite button.

C) Measurement of Absorbance. Fill the 5" test tubes provided with blank solution (distilled water),
wash solution (distilled water), the standards in order of increasing concentration, and the samples.
This should be done prior to adjusting the gas flows and igniting the flame so that you can go directly
from ignition to measurement. For a given run attempt to place the capillary aspiration tubing in a
reproducible manner in each test tube and try to have the height of the solution the same in each test
tube. Check the acetylene pressure and maintain it close to 13 psig.

Data Collection

A) Preparation of the Instrument. Verify that an calcium lamp has been installed in the instrument and
that water is present in the drain system float assembly container. Open the vent for the exhaust above
the instrument and close the hood door. The acetylene tank pressure should be 75 psig or greater. We
use welding grade acetylene in which the acetylene is dissolved in acetone. When the total tank
pressure drops below 75 psig, the partial pressure of acetone in the tank becomes unacceptably high
and the tank must be replaced. Set the monochromator and the slit width to to the appropriate
settings.

B) Turning on the Instrument and Preparation for Measurements.

l) Turn on the instrument. The power switch is located on the right-hand side of the instrument.

49
The message SELF TESTING MODE will appear on the display. When this message disappears, the
instrument has passed a series of diagnostic tests and you may proceed.

2) Define the operating parameters by pressing the Param Entry function key. You will be prompted
to define a series of parameters in the given order. After each entry of a parameter you will need to
press the Entry function key to proceed to the next parameter.

a) Lamp Current. Enter 45, the operating current in mA for the calcium lamp. It is important
that you have verified that an calcium lamp is present before completing this step. A 45 mA
current might damage other types of lamps.

b) Integration Time. Enter 1. Each measurement will be made for 1 s. You may wish to change
this number later. The allowed range for the integration time is 0.1-60 s.

c) Replicates. Enter 5. Each measurement will be repeated 5 times. You may wish to change
this number later. The allowed range for the number of replicates is 1-99.

d) Calibration Type. Enter 1 for non-linear. The 3100 allows three types of calibration: non-
linear (1), linear (2), and the method of additions (3).

d) AA technique. Enter 1 for flame. The other options apply to accessories which we do not
have.

e) Standard 1. Enter the concentration of the first (most dilute) standard in µg/ml. Use the
format nn.mm. For example, if the concentration is 2.12 µg/ml, enter 02.12 .

f) Standard 2. Enter the concentration of the next most concentrated standard in µg/ml.

g) Standard 3-n. Continue the process for your n standard solutions.

h) Standard n+l. Do not enter a number in this case. Simply press the Enter key.

3) Optimize the power reaching the detector. Press the Energy function key. The display will show
the energy screen. The Counts (CTS) and Energy (EN) parameters provide a measure of the amount
of radiation reaching the detector. Maximize Counts by making small adjustments in the
wavelength setting. The adjustment of the wavelength should be small.. If the Counts goes off
scale, adjuct the gain. Once the wavelength has been adjusted, adjust the two black alignment
screws on top of the lamp for maximum Counts. When the optimization procedure has been
completed, record the values of Counts and Energy. Then convert the instrument to Automatic
Baseline Correction mode by pressing the AA-BG function key. You will hear the movement of a

50
 mirror in the optics and the AGC routine will be applied.

4) Ignite the flame. Use common sense and pay attention to the details outlined below. Acetylene is
a hazardous combustible gas which can be used safely if one follows the lab protocol. Before you
ignite the flame, put on your safety goggles and continue to wear them until you turn off the
flame.

a) Open the values of the compressed air and acetylene cylinders. Adjust the air (oxidant) outlet
pressure to 60 psig and the acetylene (fuel) outlet pressure to 14 psig.

b) Turn the Oxidant Switch on the pneumatics control panel to Air. Adjust the air flow to 4 units
on the right (Oxidant) flowmeter using the Oxidant Control Knob. Adjust the acetylene flow to 2
units on the left (Fuel) flowmeter using the Fuel Control Knob. Since there is a strong coupling
between acetylene flow and acetylene pressure, also adjust the acetylene pressure with the
acetylene regulator to 13 psig. The acetylene pressure should not exceed 15 psig.

NOTE: If the gases are left on for 15 sec without ignition or if the acetylene pressure drops
below 12 psig, the red interlock light comes on and the system turns off the gas flows. If this
happens, turn the gas control switch to Off and then back to Air.

c) When the gas flows and pressures have been adjusted, press the Ignite button until the flame
appears. When gases are first turned on, the flame may not light the first time due to air in the
supply lines. If the flame does not ignite, turn the gas control switch to Off, then back to Air,
and press the Ignite button.

C) Measurement of Absorbance. Fill the 5" test tubes provided with blank solution (distilled water),
wash solution (distilled water), the standards in order of increasing concentration, and the samples.
This should be done prior to adjusting the gas flows and igniting the flame so that you can go directly
from ignition to measurement. For a given run attempt to place the capillary aspiration tubing in a
reproducible manner in each test tube and try to have the height of the solution the same in each test
tube. Check the acetylene pressure and maintain it close to 13 psig.

Data Collection
a) Enter the data collection mode by pressing the Data function key. The message on the screen
should read ABS (MEAN):------ (mean absorbance), SD:------ (standard deviation), RSD:------
(relative standard deviation).

b) Aspirate the blank and let the flame stabilize for 30-60 seconds. Then press the A/Z
(absorbance zero) function key. Five measurements of the absorbance of the blank solution will

51
 be made. Each measurement will require 1 s. The instruments’ microprocessor will use the
average of the 5 measurements to adjust the absorbances of the other solutions.

c) Read the absorbances of the standard and unknown solutions by dipping the aspiration tubing
in the solution, waiting a few seconds, and pressing the Read function key. Five measurements
of the absorbance will be made. Each measurement will require 1 s. The final display will give
the average absorbance and the standard deviation of the measurement. After aspirating a
solution, aspirate some of the wash solution for a few seconds before moving on to the next
solution. Collect the data in a random order.

d) If any of the samples gives an absorbance value that is well beyond the absorbance value for
the most concentrated standard solution, make an appropriate dilution of the solution and
measure the absorbance for the diluted solution.

Shut-Down. When you have completed all you measurements, aspirate wash solution for
several minutes. This will purge the burner of acid solution. Then turn the gas control switch on
the Pneumatics Control Panel to Off. Close the valve on the acetylene cylinder and give the
acetylene regulator several clockwise turns so that the pressure exceeds 15 psig. Note the
flame is out at this point. Turn the gas control switch to Air to allow the acetylene to bleed. The
red interlock light on the Ignite button should turn on. When it does, turn the gas control switch
to Off and then to Air. Repeat this procedure until the acetylene pressure has been minimized.
The high pressure gauge should read close to zero and the low pressure gauge should read ca.
10 psig. Now give the acetylene regulator several turns in a counter-clockwise direction to
release pressure on the diaphragm. Close the valve on the air cylinder. Note that the air valve
is the last one closed and that the fuel line pressure is reduced as much as possible. This is
always the procedure in working with combustible gases. Finally, turn off the power switch on
the instrument

5. Perform the following steps: lamp alignment, burner alignment (use tap water, which is calcium-rich),
Nebulizer optimization and Fuel/air ratio optimization (again using tap water).

1. Again using the instruction manual as a guide, obtain the measurements of the blank, the
Beer’s Law standards and the milk samples.

Include in the Report:

1. Sketch the main components of an atomic absorption spectrophotometer.

2. What is the U.S. RDA of calcium (in ppm)?

4. Outline the method used to prepare milk samples including all observations.

52
5. Prepare a Beer’s Law plot of your standards and determine the best fit line or curve for the data and
plot it (trendline with R2 value). Determine the amount of Ca in each of your milk samples from your
calibration curve. Determine the error in your readings by performing a regression analysis with Excel
(add-in, regression analysis). Plot the residuals and discuss.

6. Compare the absorbance readings of the milk samples with and without added LaCl3.

7. Report the amount of Ca (ppm) in your samples. Gather the entire classes data for each type of milk.
Report the average ± 95 % confidence interval. Compare the class average to the value reported by the
USDA. Report the % difference (don’t forget to propagate the error).

8. Is the calcium concentrations between each of the four types of milk (skim, 1% , 2% and whole)
statistically significant* ? Do you notice any correlation? If yes, explain.

* To determine whether or not a significant difference existed between the calcium content of different

types of milk use Excel’s ANOVA test (a single-factor analysis of variance) This statistical test is used

typically to analyze populations of data where more than two different treatments of one factor are used,

e.g. four different milk types 3

References

1. Hurley, Walter L. Department of Animal Sciences, University of Illinois.

http://classes.aces.uiuc.edu/AnSci308/index.html. Jan. 26, 1997.

2. “Dairy Science and Technology”. University of Guelph.

http://www.foodsci.uoguelph.ca/dairyedu/home.html.

3. Devore, Jay L. Probability and Statistics for Engineering and the Sciences, 4th ed.

Duxbury Press: New York, 1995.

53
Experiment 8.
"Imposter" vs. "Real" Perfumes via GC-MS

from J. Chem.Ed, vol. 81, pg. 87-89, 2004.

INTRODUCTION
The goal of this experiment is to identify "active" ingredients in different fragrances. We
would also like to compare name brand fragrances to their imitation counterparts and see if
both fragrances really contain the same active ingredients. There are essentially 3 concepts
involved in this laboratory exercise besides the underlying fundamentals of gas
chromatography (GC).
They are: headspace analysis, temperature programming, and mass spectral detection for the
purpose of analyte identification. In GC, a sample is typically injected into the inlet with a
syringe. Usually the sample injected is a liquid, which is quickly vaporized in the hot inlet.
Gaseous samples may also be injected. In the food, flavor, and cosmetics industries smells are
very important. This is one example of the utility of headspace analysis. In headspace analysis,
the vapor above a sample is drawn into a syringe and injected into the GC. The rationale for this
type of sampling is that the molecules responsible for scent are those present in the vapor
phase above the sample. If you smell a cup of coffee, the molecules that reach your nose are
the volatile ones. We will use this technique to investigate the components responsible for the
scent of real and imposter perfumes.
In gas chromatography (GC), components of a mixture are separated on the basis of their
interaction with a stationary phase within the gas chromatography column. In this experiment,
the sample is first injected into a sample inlet, volatilized, and then swept by a carrier gas
through the column into a detector. The column is actually a narrow glass capillary in which the
walls are coated with a nonpolar liquid film. Components of a mixture are therefore separated
based on polarity as well as volatility, with more polar, volatile compounds eluting from the
column more quickly than less polar, less volatile compounds.
As you might imagine, temperature dramatically influences the degree to which components of
the mixture interact with the stationary phase. By changing the temperature of the oven in
which the column resides, we have some control over the separation process and can
manipulate it to achieve more rapid or more effective separations. Temperature programming
is accomplished by ramping the temperature of the column during a separation. The advent of
computer-controlled instrumentation has made temperature programming routine.
When a mass spectrometer (MS) is used as the detector, eluate from the GC column is
immediately swept into an ion source in which the sample is ionized and the abundance of each
ion is measured as a function of mass to charge ratio (m/z). Since the charge of most ions
formed is +1, the m/z is often referred to simply as the atomic mass of the ion generated.
Electron impact ionization is a very “hard” ionization technique, meaning that it involves
sufficient energy to not only ionize a molecule, but also fragment the molecule into many
smaller pieces. The pattern by which a molecule fragments is characteristic of the structure and
functional groups of the molecule. For example, molecules containing an ethyl group often lose

54
the CH2CH3 when ionized. Fragmentation patterns are therefore very useful for qualitatively
determining the structure of the original parent molecule!
Once the positively charged ions are formed, they are accelerated into a mass analyzer,
which separates ions of different masses. There are two methods of obtaining data using GC-
MS.
The first method is to scan a specified range of m/z ratios. The data can then be plotted as the
total ion abundance verses time. This plot, called a Total Ion Chromatogram (TIC) looks like
a “regular” chromatogram with peaks at various retention times. Each peak is the sum of the
abundance of all ions (regardless of mass) as a function of time. One can also analyze the same
data to determine the masses of the fragments that are eluting from the GC column at a
specific point in time. In this case, abundance is still plotted on the y-axis, but m/z is plotted on
the x-axis to generate a mass spectrum. The fragmentation pattern contained in the mass
spectrum is indicative of the compound, and ideally enables one to qualitatively identify each
compound as it elutes from the GC.
A second method of acquiring data on the GC-MS is called Selective Ion Monitoring, or SIM. In
SIM mode, one would select a few m/z ratios that are unique to the compound of interest. The
mass filter is then set to detect those select fragments with specific m/z ratios, ignoring all
others. This mode minimizes the chance that co elution of an interferent will compromise the
detection of a primary species of interest. It also provides maximum sensitivity because the
detector spends more time looking for those particular m/z fragments. SIM mode is both more
sensitive and selective, but the ability to identify analytes based on mass spectral libraries is
sacrificed because an entire mass spectrum is not collected.

EXPERIMENTAL METHODS
Hazards note: Some perfumes may be irritating and even toxic to people with severe allergies.
It is recommended that liquid perfumes be dispensed into septum vials in a fume hood using
gloves. Keep perfumes away from extreme heat and open flames.

If your instructor has not already done so, tune the mass spectral detector.
Obtain 2 small septum vials. In one vial place approximately 0.5 mL of a perfume sample. In the
second vial, place approximately 0.5 mL of the corresponding imposter perfume. As you
dispense the real and imposter perfumes, smell them, describe the scents and identify any
olfactory differences.
Select the appropriate stored method file to control the GC-MS instrument. Check each
parameter to become familiar with the operating software and be sure your fellow comrades
didn’t change it!

Injector: we will be making manual injections.

Inlet: temperature 250 °C; split mode; split ratio 50:1
Mobile Phase Flow Rate: 1.0 mL/min
Oven (controls column temp): 140 °C hold for 20 min
Auxiliary (transfer line from GC to MS): temperature 280 °C
MS detector: 3.00 minute solvent delay
Scan mode from 20 to 500 m/z

55
Step by Step Instructions for Data Collection and Evaluation
1. Once you’ve become familiar with the software and confirmed your operating conditions,
prepare to inject 1 μL of a liquid perfume sample. Let the instructor show you how to inject the
first time. Collect a Total Ion Chromatogram (TIC) of the sample. Make note
of the directory in which you save this and all subsequent chromatograms.

2. After you have collected the first TIC, modify the operating parameters to include a
temperature ramp during the run according to the following:
a. Initial Temp. 60 oC
b. Hold it at 60 oC for 2 minutes
c. Ramp at 20 oC/min up to 250 oC
d. Hold at 250 oC for 3 minutes.

3. Inject the identical sample as in step 1 - 1 μL of liquid perfume sample.

4. Clean the syringe with methanol or ethanol.

5. Inject 1 μL of the imposter perfume under identical conditions.

6. While chromatograms are being acquired, you may begin analyzing chromatograms already
collected. Be sure to make the following part of your analysis:
a. Compare the isocratic (fixed temp) and temperature programmed chromatograms.
b. Zoom in on the chromatograms to investigate small time windows with multiple peaks.
c. Display mass spectra for several peaks.
d. Remember each molecule results in a unique fragmentation pattern. Therefore, the pattern
from the mass spectrum can be used to identify the molecule. You can compare your mass
spectra with those in a mass spectral library. The probability that your mass spectrum matches
one in the library should be noted. A probability greater than 90 indicates that there is a high
probability that the two compounds are the same, while probabilities less than 50 indicate that
substantial differences exist between the unknown and reference mass spectra.
e. While looking at the mass spectrum for one of the peaks in a congested area of the total ion
chromatogram, make note of the m/z of two or three of the most abundant fragments. You will
use these to perform selected ion monitoring (SIM) later in this experiment.
f. Using the mass spectral library search feature identify 5 of the components of the perfume
sample. Only trust matches that have a probability of 90 or better. Report what scent the
volatile "active ingredient” of your fragrance provides. Does it provide a floral, woody or spicy
"note?" This will require a little literature searching. Several great articles about this have
appeared in Journal of Chemical Education, Analytical Chemistry and Chemical and Engineering
News recently. The Sigma Aldrich Catalog of Fragrances and Flavors is an excellent resource
for this type of information; as is the internet, particularly a site by the Good Scents Company!
g. View the perfume and imposter on the same set of axes in order to better determine
compositional differences between the two samples.

7. Set the MS detector to operate in SIM mode at the m/z chosen in step 6e. All other

56
conditions should remain identical.
a. As before, inject 1 uL of the genuine perfume.
b. When this SIM chromatogram is complete, return the MS to operate according to
the original SCAN settings.
c. Compare the SIM and TIC chromatograms.

8. Before doing headspace analysis of the perfume, obtain a 50 or 100-μL gas-tight syringe.
Change the inlet mode to:
a. Pulsed Splitless
b. Inj. Pulse Pressure: 30 psi
c. Pulse time: 1.0 min.
d. Purge Flow: 50 mL/min.
e. Purge Time: 0.9 min.
i. Note: If your instrument does not have automated pulsed splitless capability, you may want
to warm both the samples and the gas-tight syringe in a 40 oC oven to increase headspace
concentrations of perfume components.

9. Perform headspace sampling by inserting the syringe into the septum, but not under the
surface of the liquid. Draw 50 μL of the VAPOR above the liquid surface into the syringe. Inject
the gas sample into the GC. Collect a TIC.
a. View the headspace chromatogram and the liquid chromatogram on the same plot.
Based on a comparison of these chromatograms, what can be said about the volatility of most
of the components of this mixture?

10. Collect a headspace TIC of the imposter to the perfume you have selected.
a. Overlay the real and imposter headspace chromatograms. Identify (using the mass spec
library) as many components as you can that are present in the real perfume but missing from
the imposter, or vice versa. Why do you think these are missing? OR Why do you think these
are present?

11. Clean both syringes with methanol or ethanol.

Guide for the Lab Report

Submit a Title and Abstract.
Focus on the benefits of using a mass spectral detector in the theory section.
You should include separate model numbers for the GC and the MSD (mass spectral detector).
The procedure should focus primarily on how you obtained the headspace sample.
Data to present:
TIC of perfume – isocratic
TIC of perfume – T programmed
TIC of imposter – T programmed (these could be overlaid)
SIM chromatogram (identify the major peak)
Table of Retention Times, Peak Identities, Characteristic Odors (if found)
TICs of real and imposter headspace samples (can be overlaid).

57
Identity of extra or missing components in imposter perfume.

Results and Discussion

How might extra components contribute to the scent? How would the missing components
affect the scent? Does your analytical evaluation support your olfactory observations?
Compare the SIM and TIC chromatograms. Consider the y-axes on these chromatograms.
Which provides a larger signal for the analyte on which you focused for the SIM? Why does
SIM mode offer greater sensitivity?

58
Directions for using Agilent GC (6890) with MSD (5973)
If the computer isn’t on, log-in.
Check to make sure the gas line is open and set to at least 20 psi. You
may need to adjust the pressure later to satisfy the pressure needed by your method.
From the desktop, double-click “1 GCMS” icon. Three windows will open – leave all
three open. We will assume that the MSD is already pumped down and ready. If it is
not, please see your instructor for help.

Tuning the instrument

If it has been more than 24 hours since the instrument has been tuned, you need to
perform a quick tune to confirm the settings.
1. In the “Instrument Control” window, choose “View – Manual Tune.”
2. In the “Manual Tune” window, choose “Tune – Quick Tune.”
3. The MSD will click on and perform a scan of three ions from a standard stored in
the instrument. It will adjust to make the peaks as narrow as possible and as
accurate as possible.
4. If the scan completes, you are ready to move on. If it does not, please see your
instructor.
5. Go to “File – Save Tune Values” to save the tune. Choose “atune.u” for the file
name.

Setting up a method
In order to run the instrument, you will set up a method in Chemstation.

1. If you are not in the “Instrument Control” window, open it by choosing “View –
Instrument Control.”

2. If you plan to use a stored method, choose “Method – Load” and choose your
method. If you plan to start a new method, choose “Method – Edit Entire
Method.”

3. In the “Edit Method” window, make sure that only “Method Information” and
“Instrument/Acquisition” are selected. Select “OK.”

4. In the “Method Information” window, enter any comments about the method and
make sure that only “Data Acquisition” is selected. Select “OK.”

5. In the “Inlet and Injection Parameters” window, make sure that “GC” is chosen as
the inlet, “Manual” is chosen as the injection source (unless an auto-injector is to
be used), “Front” is chosen as the injection location and an “x” is in the box next
to “Use MS.” Select “OK.”

6. Now you will edit each step of the analysis. These parameters can also be edited

59
individually later by choosing the icons on the “Instrument Control” screen. The
parameters you will most likely want to edit are:
a. Inlets: Two options are available, split or splitless. For a “Split” run,
choose “Split” and set the ratio below. For a “Splitless” run, choose
“Splitless.” On this screen you should set the heater temperature, type of
carrier gas, pressure and total flow for the inlet.
b. Column: For Mode, choose “Constant Flow.” For Inlet, choose “Front.”
For Detector, choose “MSD.”
c. Oven: On this screen, you can set the starting temperature and any
temperature gradient using the table of times and temperatures. Any
changes you make will be expressed in the graph of temperatures at the
top of the screen. Make sure that your temperatures will not go over the
“Oven Max” chosen on this screen.
d. Aux: This refers to the MSD. On this screen you should set the
temperature for the transfer line to the MSD.

7. After editing all of the instrument parameters, select “OK.”

8. In the “GC Real time plot” window, make sure that no signals are selected. These
would only apply if you were using a different GC detector. Select “OK.”

9. In the next window, make sure to select “atune.u” or the name of the tune file you
have saved previously.

10. In the “MS SIM/Scan Parameters” window, you can set up the parameters for the
mass spectrometer. The instrument can be run in “Scan” mode for a total ion chromatogram
(TIC) or in “SIM” mode for selected ion monitoring.
a. Set the solvent delay. Three minutes is usually sufficient. This delay keeps the MSD from
turning on until after the solvent in a liquid sample has passed. If you are injecting a gaseous
sample, it is unnecessary.
b. Scan: If you would like to adjust the range of m/z ratios that the instrument will monitor or
the amount of time it will spend on each m/z, do so in the table at the bottom of the screen.
Most likely, you will not need to adjust this table.
c. SIM: If you know the compounds you will be detecting and have their mass spectra, you may
use this screen to choose specific ions to be detected at different times during the run.
11. After selecting “OK,” you will be given a place to save your method. Type in a method name.
All names used in this program must be 8 characters or less.

Running a Method
Make sure your method is loaded.
1. Choose “Method – Run” in the “Instrument Control” window.
2. Choose a name for your file. To find a list of files and folders, type “?” in the “File name” box.
3. Fill in the rest of the boxes with information about your run.
4. Choose “Run Method” (not “OK”) to start the method.

60
5. When “GC Ready” window appears, prepare your sample to be injected. See your instructor
for information about appropriate volumes used. Make sure to get rid of any bubbles in the
syringe.

6. On the GC itself, press the “Prep run” button. When the “pre-run” light turns on, quickly
inject your sample by piercing the septum in the front inlet and pressing the plunger of the
syringe. Immediately press the “Start” button on the GC.

7. Do not choose “override solvent delay” unless you are injecting a gas into the GC. Overriding
the solvent delay when injecting a liquid sample will greatly reduce the life expectancy of the
detector. Ignore the window.

8. Your chromatogram will appear in the “Total Ion” window. Double click on it to expand the
window.

Data Analysis
During a run, from the “MS Top/Enhanced” window, choose “Open New Data Analysis.”
Otherwise, open a data analysis window by choosing “View – Data Analysis (offline).”

1. If you would like to analyze your chromatogram as it is being collected, choose “Take
Snapshot” under the “File” menu. Otherwise, choose “Load Data File” under the “File” menu.

2. To display the mass spectrum at any point in a chromatogram, double right click on a point in
the chromatogram. The spectrum will be displayed below. You may also average several points
together by drawing a rectangle with the right mouse button clicked.

3. To search the library for a given spectrum:

a. Choose “Spectrum – Select Library.”
b. Type “?” in one of the boxes. Scroll down to choose “Wiley275.L” as the
library to search.
c. Double right click on a spectrum and a list of potential library matches will be displayed along
with the probability of a match (“Qual” column –nearer to 100 is better). To find out more
information about a certain choice, highlight the compound and then click the “Text” button.

4. To determine the area of the peaks in the chromatogram:

a. Select “Chromatogram – Integrate.” The retention time of the peaks will be listed next to
each peak.
b. If too many or too few peaks are chosen, go to “Chromatogram – Select Integrator.” Choose
“RTE Integrator.” Choose “Chromatogram – MS
Signal Integrator Parameters” and change the minimum peak area detected (increase if too
many peaks are chosen, decrease if too few were chosen). Integrate again by selecting
“Chromatogram – Integrate.”
c. To display the areas of each peak, choose “Chromatogram – Integration Results.”

61
Experiment 9

Gas Chromatography : Drugs and Money

For background, obtain the paper: Sleeman, R. et. al. Analytical Chemistry, 2000, 72, 397A-403A.

The average life of a $1 bill is 18 months and up to 10 years for larger denominations such at $100’s.
Researchers have found that over 80% of money in circulation in the US is contaminated with
microgram amounts of cocaine, primarily from cross contamination in counting and ATM machines, but
ultimately comes from direct contact with cocaine during drug use and trafficking. We will analyze
money for the presence of cocaine using GC-MS. The procedure involves extracting the cocaine from the
bills followed by injection of the extract on the GC-MS and subsequent analysis of peak area and
abundance.

Procedure:

1.) Obtain 3 $1 bills and 2 of other denominations (these may be foreign if you have them) before lab.
You may beg and borrow (stealing is not recommended) from friends, professors etc. Be sure to return
the money to its rightful when you are finished. Your chemistry professors will probably be interested in
the results of the analysis, but please don’t stiff them out of their cash. ;-)

2.) Label 5 centrifuge tubes with dollar amount. (i.e $1 a,b,c,d etc)

3.) Place 4 mL of methanol into each of 5 15 mL centrifuge tubes.

4.) Wearing gloves, fold the bills accordion style so that they will fit into the test tube and have good
contact with the solvent.

5.) Add the bill to methanol using tweezers. Cap and shake the tube vigorously for 4 minutes. You can
also vortex the bill.

6.) Using tweezers, pull the bill out of the solution, but leave it at the top of the centrifuge tube. Using a
1 mL pipette, rinse the bill by dripping 1 mL (exactly) of methanol on it to remove any cocaine that might
remain on the bill surface.

7.) Remove the bill, shaking off the excess into the centrifuge tube.

8.) Rinse all bills with water and allow them to dry before giving them back to their

owner.

GC-MS procedure/parameters:

1.) Set the GC to the parameters as follows:

Sample inlet temperature: 270 oC

62
Temperature program: 130 oC for 1 min, ramp at 12 oC/min to 280 oC

He carrier flow: 2 mL/min

Injection type : Splitless

Ion Source Temperature: 230 oC

Injection Volume : 2L

Solvent delay : 8 minutes

2.) Obtain a few milliliters of methanol in a beaker to rinse your syringes.

3 .) Run each of your samples, 2 L injections.

4.) For each spectrum:

a. On the total ion chromatogram (TIC): the peak position of cocaine marked, area of the peak auto
integrated, integrated area of the background of the peak. Consult the instructor for a demonstration on
how to get the background number.

b. On the extracted ion chromatogram (EIC): total abundance of the peak for cocaine in each sample,
total abundance at the same position for a blank.

Data Analysis:

6.) Explain in your report how and why methanol is a good choice for an extraction solvent.

7.) Explain the fragmentation pattern seen in the cocaine EIC.

8.) Was there a correlation between the relative amount of cocaine found and the size of the
denomination?

Note: Extracted ion chromatography is a post-run data manipulation that allows the experimenter to
view the chromatogram as if only one (or a limited set) of ions had been monitored. True single-ion
monitoring is conducted by setting up the mass spectrometer before the analysis so that it collects only
that ion, offering significant improvements in the counts obtained.

63
Experiment 10

64
 APPENDIX I

INSTRUMENT OPERATING INSTRUCTIONS

Nicolet Avatar FT-IR

Operating Instructions for the Ocean Optics UV-vis Spectrometer

HP GCD Gas Chromatography Mass Spectrometer

65
 Directions on using the Nicolet Avatar FT-IR

1. The IR and computer should already be turned on. If they are not, turn them on and let
the laser warm up for about 30 min

2. Open the Omnic software (double-click icon on desktop).

3. Set the number of scans by clicking Collect->Experiment Setup. Click on the Collect tab
and change the number of scans as needed (default is 32).
4. Put your background sample (either a clean salt plate or pressed KBR) on the sample

holder. Click on the Collect Background icon and click OK to start

the scan.
5. After collecting the background you don’t need to add it to the data window (ie.
click no).

6. Put your sample in the holder. Click on the Collect Sample icon . Type the
name of your sample and click OK to start the scan.
7. When the sample is done being scanned, click YES to add the spectrum to the
data window.
8. Save your spectrum by clicking on File->Save As and enter the filename and
where you want to save it (hard drive, ZIP disk, etc).

Data Analysis:
1. To toggle between absorbance and transmittance mode, click on the appropriate

icon: .

2. To do a baseline correction, click on the . You MUST be in absorbance

mode to do baseline correction, but you may go back to transmittance after this
correction completes.
3. To label your peaks click on the icon (lower left). Click on each peak of
interest and press enter to mark it with the wavenumber value. If you pick a
wrong peak, click on the wavenumber value then press delete and then enter.
Your spectra can be printed by going to File->Print.
5. More detailed information about acquisition can be printed out with the spectra.
The templates for these can be found under the Reports menu.

66
 Operating Instructions for the Ocean Optics UV-vis Spectrometer

Initializing Instrument
1) Make sure USB spectrometer is attached to PC and is plugged into power wall socket. You may need
to restart computer if it is not installed.
2) Double click on OOI icon to start spectrometer program. Make sure that white spectrometer box has
power switch in off (center) position. Make sure you are in scope mode (S is clicked on toolbar).
3) Hit dark current store button (the black light bulb at left side of second toolbar).
4) Turn on lamp (switch white box to on position) and set the spectrometer values [average = 6, boxcar =
10, flash delay = 100 ms]. Adjust integration time so the maximum signal is <4000.
5) Press the yellow light bulb icon to store an initial reference background and then switch the screen to
absorbance mode (A icon on the right side of bottom toolbar).

RECORDING A SPECTRUM
1) Prepare a sample solution that has a visible, but pale color. If your sample is too concentrated the
peaks will be off scale. You do not need to know the absolute concentration of your solution for this
measurement.
2) If computer is on, the spectrometer program is running, and you are in absorbance mode (A is clicked
on the toolbar) and the screen is constantly updating itself. If it is unchanging, click on the camera icon
(static snapshot mode) to bring it back to real time acquisition.
3) Insert a blank (cuvette and solvent) and hit the store reference button (yellow light bulb icon).
4) Replace the blank cuvette with your sample cuvette and the spectrum should immediately appear
and constantly keep updating.
5) Click on the camera icon to get a snapshot and stop the spectrum from updating.
6) Print out your spectrum in landscape mode. Make sure to label it with your sample name!
7) Use the cursor keys to move the green cursor line to your peaks of interest and record the
wavelength on your printout. Use the scale key (button with 4 arrows like compass points) to expand
the spectrum to see if there are any low intensity features.
8) Save your processed spectrum to a file with a descriptive label. The program automatically adds an
extension (.master.absorbance). This file is in ASCII/text format and you can read the data into graphing
programs like Excel or Kalideograph and plot your spectra.
9) Unclick the camera button, remove your sample, and leave the program running for the next user.

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HP GCD Gas Chromatography Mass Spectrometer Instructions

Click on GCD Top to start program

To tune, go to Maintenance, System Verification. Then you want to do a Tune,
But NOT Acquisition and Review Data.

To set a method, go to Method, then Edit.

Do not set any temperatures above 300C!!!
Set parameters.
Injection temperature: Temperature of the injection port.
Initial temperature: Temperature of the oven upon injection.
Initial time: Amount of time that the oven stays at the initial temperature.
(For an isocratic run, you don’t need to set any other temps. Make sure all rates are
zero.)

Rate box: For use with gradient runs. Set a temperature to increase to, a rate of
temperature increase (0-70C/min), and a time to hold that temperature constant in

the oven. All of these go across on one row of the table. You can set more than

one step of the gradient.

Flow rate: The rate of the gas flow through the column. Generally keep it around
0.7 mL/min.

Mass range: The range of mass to charge ratios the mass spectrometer detects.

Save the method.

Go to Inject, then One Sample.

Type in name to save your file as, operator name, name of your sample, etc.
Hit run, then get your sample ready. Rinse syringe w/ sample several times. Pull up
about 1 μL of sample into the syringe followed by ~3 μL of air. When red light on GC

goes off, you can inject. Hold syringe in injection port for ~5 s, then push down

plunger to inject and hit start on the GC. Hold syringe in place for ~15 s, then remove.

Rinse out the syringe several times with solvent.

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Review Data to see the chromatogram.
To zoom in on a peak, click and drag a box with the left mouse button.
To view the mass spectrum of the peak, double click with the right mouse button at the
center of the peak.

To identify the peak, go to Identify, then Search Spectrum.

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 APPENDIX II USE OF A MICROSYRINGE

Liquid samples can conveniently be injected into a gas chromatograph (GC) with a microsyringe.
These syringes are calibrated so that a fixed volume of liquid remains in the needle before and
after the injection. However, in the GC the sample is usually injected into a heated zone where
some of the liquid vaporizes from the needle, thus delivering a larger sample than intended.
Following are the manufacturer's suggestions on the use of a microsyringe.

A. Filling
Grasping only the syringe flange and plunger, pump out the air, and overfill the syringe. Then, grasping
the flange and plunger, hold the syringe perpendicular to one's master eye and against a white
background. Bring the syringe plunger just to the top of the scribe line of the desired volume. Lightly
blot the needle, being careful not to pull liquid out of the syringe needle. Now back off the plunger,
pulling the liquid from the needle into the syringe. The total liquid is now read. Insert the needle
through the rubber septum and quickly discharge the volume. Rapidly withdraw the needle. Now
withdraw the plunger and read the residual liquid. The difference in the initial and final volume is the
liquid injected.

B. General Comments
A few pumps with the liquid being transferred will give adequate lubrication. (The use of grease as a
lubricant generally results in a split glass barrel.) If the plunger is withdrawn completely from the syringe
wipe carefully before replacing. Perspiration and soil from one's fingers will very often cause a sticky
plunger or occasionally will even freeze it solid in the glass. In using the syringe as outlined above, we
have found it necessary to keep fingers from the syringe barrel. The syringe is filled and handled by the
flange and the plunger button. Thus, the heat of the hand does not throw off the measurements, which
can cause variations as high as 0.3 μL. By observing the preceding techniques, one can routinely
calibrate to the accuracy of the laboratory balance, which is 0.1 milligrams or 0.1 μL when using water.

C. Cleaning
Detergent and water, glacial acetic acid, nitric acid, or common laboratory organic solvents will clean
quite well. If a plunger is withdrawn and handled or exposed to dust, carefully wipe it with tissue before
replacing it in the syringe. In this course it will be sufficient to flush the syringe with a volatile organic
solvent and then dry it by pumping the plunger several times.
NOTE: Since there is a positive pressure of helium gas flowing through the gas chromatograph at all
times, it is imperative that you keep hold of the syringe plunger, otherwise the plunger is easily forced
out of the syringe, negating the results of that particular sample.

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