EXPERIMENT 6

INTRODUCTION TO SPECTROSCOPY
INTRODUCTION
Much of what we know about the structures of atoms and molecules has been learned through experiments in
which photons (electromagnetic radiation—visible light, microwaves, ultraviolet or infrared radiation, radio
waves, etc.) are emitted or absorbed by the atoms or molecules. The energy of a photon is related to its frequency,
, and wavelength, , according to
c
E photon = hv = h (6-1)

where h is Planck’s constant, and c is the speed of light. The energy of an emitted or absorbed photon corresponds
to the change in energy the atom or molecule experiences.
E photon = E = E final − Einitial (6-2)

Whether photons are absorbed or emitted is correlated with the type of energy change the atom or molecule
is undergoing. Thus, for example, a molecule can be raised to an excited electronic state by absorbing a visible or
ultraviolet photon. A molecule already in an excited electronic state can return to the unexcited, or ground state
by emitting a visible or ultraviolet photon. The energies of photons in this portion of the electromagnetic spectrum
correspond to the differences between the ground and excited electronic states. For changes in vibrational or
rotational energy, infrared and microwave photons respectively, have energies corresponding to the differences
between states. Careful analysis of the details of the radiation absorbed or emitted as a function of wavelength
(the absorption or emission spectrum), coupled with the formulation of physical models to interpret and explain
them, has provided a wealth of detailed information about atoms and molecules.
In addition to the structural information that can be gained, studies involving the absorption and emission of
electromagnetic radiation have proven to be extremely useful in other practical ways. For example, even without
knowing why particular wavelengths are absorbed or emitted, we can often use the observed spectra to identify
the substances responsible. This is particularly true in the infrared region for organic molecules, where many
vibrational spectra have been recorded and cataloged and can often serve as “fingerprints” to identify what is
present. In a similar way, the specific wavelengths of visible and ultraviolet radiation emitted by atoms and ions
in a flame or in an electrical discharge can provide an unambiguous means of identification. In fact, a number of
elements were first discovered in this way, when previously unknown emissions were observed. Spectroscopic
measurements are now routinely employed in the analysis of chemical samples.
While measurement of the wavelengths emitted or absorbed can provide a convenient means for qualitative
analysis of samples (i.e., what is present), measurement of how much light is emitted or absorbed can be used for
quantitative analysis (i.e., how much of a substance is present). Carrying out such measurements is sometimes
referred to as spectrometry, from “spectrum measure” rather than spectroscopy. Although quantitative
experiments can be performed using various regions of the electromagnetic spectrum, one of the most useful is
the visible portion, sometimes in combination with the ultraviolet region. In this experiment we will be working
with visible electromagnetic radiation, ordinary light.
 In order to study the emission and absorption of visible light, we will make use of an instrument known as a
spectrometer. A simplified drawing of the main components of our spectrometer is shown in FIGURE 6-1. A
spectrometer is an instrument that accomplishes two main tasks. First, it disperses, or spreads out, the light entering
it into all of the wavelengths or colors present. This can be done with either a prism or a diffraction grating. Our
spectrometer uses a grating. Second, it provides a signal proportional to the intensity of the light of each
wavelength. It does this by directing the dispersed light onto a detector, which provides the electrical signal. In
our spectrometer the detector consists of an array of 2048 tiny diodes arranged in a straight line and positioned so
that the dispersed light is spread from one end of the array to the other. Therefore we actually have 2048 tiny,
individual detectors, and each one has light of a slightly different wavelength, or color, falling on it.
Whatever type of experiment we are
carrying out, the signal from the
spectrometer is always just a set of
blue
values, one from each of the tiny diodes,
mirrors
indicating the intensity of the light
reaching them. (In actual practice we
reduce the amount of data to be handled
by averaging the signals from the diodes
red in adjacent pairs, thereby obtaining 1024
diode array values from the original 2048.)
detector The diodes are more sensitive to red
grating light than blue light, so signals in the
blue end of the spectrum will be
somewhat reduced compared with the
light in red end. However, the signal for any
wavelength is proportional to the
Figure 6-1. Simplified Spectrometer Diagram intensity of the light of that wavelength.
Thus, for example, no matter what the
color, the signal will double if the intensity of the light of that color is doubled.
When we use the spectrometer to measure an emission spectrum, we simply direct the light emitted by the
sample (gas in a discharge tube, flame, etc.) into the spectrometer. The set of values we get from the spectrometer
can then be examined to see what wavelengths of light were emitted. We will only do qualitative emission
experiments, where only the wavelengths, and not the intensities, of the emitted light are important, so the
variation of detector sensitivity with wavelength will not affect its usefulness.
When we use the spectrometer to measure an absorption spectrum, the situation is quite different. We use a
lamp to supply light of all wavelengths throughout the visible region and use the spectrometer to determine the
extent to which light of each wavelength is absorbed. The physical arrangement is shown in FIGURE 6-2. The
sample is contained in a cuvet, a small container with clear windows. I0, is the intensity of the light incident on
the sample as a function of wavelength, . The wavelength variation of I0, is determined by the characteristics of
the lamp. We use a tungsten-halogen lamp, for which the intensity goes through a maximum in the visible region.
I is the intensity of the light remaining after passing through the sample. At wavelengths where the sample does
not absorb, I is equal to I0,. Where the sample does absorb, I is less than I0,. We measure I as shown in FIGURE
 I0, s I
a
lamp m spectrometer
p
l
e

Figure 6-2. Measurement of light absorption as a function of wavelength.

6-2. To measure I0,, we replace the sample cuvet with a reference cuvet, which is identical to the sample in every
way except that it does not contain the absorbing molecules. It is important to measure I0, this way, since some
light could be lost by reflections or by absorption in the walls of the cuvet or by the solvent. Using a reference
cuvet provides the best measurement of the decrease in light intensity due only to absorption by the absorbing
molecules in the sample.
The most important routine use of absorption measurements is for the determination of the concentration of
absorbing molecules. This requires knowledge of the quantitative relationships between the light intensities and
the concentration. This has been worked out experimentally, as summarized briefly in the following. The percent
transmittance, %T, is defined to be
I
%T =  100 % (6-3)
I 0, 

The value of %T has been found to depend on three things. First, the nature of the absorbing molecules
determines what particular wavelengths of light will be absorbed. Second, the longer the path the light travels
through the sample, the greater the fraction of light absorbed (as found by J. H. Lambert). Third, the greater the
concentration of absorbing molecules, the greater the fraction of light absorbed (as found by A. Beer). These three
factors combine quantitatively in the following manner (Lambert-Beer Law, or, sometimes just Beer’s Law),
A =    b  C (6-4)

where  is the molar absorptivity, characteristic of the absorbing molecules, b is the path length of the light
through the sample, measured in cm, C is the molar concentration of the absorbing species, and A is called the
absorbance. A plot of A vs.  is called the absorption spectrum.
Since, as EQUATION 6-4 shows, the absorbance is directly proportional to the concentration of absorbing
molecules, it is the quantity we use to determine the concentration. Unfortunately, absorbance itself cannot be
measured directly. It can be calculated, however, from the observed light intensities, as shown in EQUATION 6-5.

I   100% 
A = log10  0  = log10   (6-5)
 I   %T 
As EQUATION 6-5 shows, the absorbance could be calculated from the percent transmittance, but it is simpler
just to calculate it from the light intensities directly. We use the two sets of values, I0, and I, to calculate A point
by point, for all of the elements in the diode array. Since only ratios of intensities are used in the calculations, the
variations of lamp intensity and detector sensitivity with wavelength do not cause a distortion of the absorption
spectrum.
Data from a representative absorption measurement are displayed in FIGURES 6-3 and 6-4 on the next page.
The raw data, that is, reference (I0,) and sample (I) signals, are displayed in FIGURE 6-3. The absorbance values
(A) calculated from these data are displayed in FIGURE 6-4. Note in these graphs how the maximum in the
absorbance between 600 and 650 nm corresponds to the largest fractional decrease in the sample signal compared
to the reference. Note also that the absorbance is essentially zero from about 700 nm to 1000 nm, although the
reference and sample signals are not zero. They are essentially the same over this range, so the ratio is always
unity, giving an absorbance of zero. The opposite is true at the other end of the spectrum. The sample signal is
always a little less than the reference, so the absorbance is greater than zero.
At the highest and lowest wavelengths recorded, both I and I,0 become very small and thus difficult to
measure. When I and I,0 are poorly known, their ratio becomes very uncertain. The absorbance values calculated
from these ratios become less and less reliable, and this leads to increasing “noise” as the extreme ends of the
spectrum are approached. This effect is clearly evident in FIGURES 6-3 and 6-4. If it were necessary in some
experiment to obtain reliable absorbance measurements in these wavelength ranges, we would have to somehow
alter the spectrometer. We might, for instance, choose a different lamp with more intense emission in the desired
range.

OBJECTIVES
• to measure the absorption of visible light by a solution of an absorbing substance and examine
how the intensity plots result in the calculated absorption spectrum
• to prepare plots of absorbance (at the wavelength of its maximum absorption) vs. concentration
for a series of solutions (Beer’s Law plot) of two different food dye stock solutions, and
determine the molar absorptivity, , of the absorbing species at that wavelength for each food
dye.
• to measure the absorption spectrum for two solutions of unknown concentration and use the
Beer’s Law plots determine their concentrations.
Figure 6-3. Absorbance Experiment Raw Data: Reference and Sample Signals

Figure 6-4. Absorbance calculated from data in Figure 6-3.

EQUIPMENT NEEDED
Spectrometer volumetric flask, 10 mL
Mohr (graduated) pipet, 5 mL cuvets
pipet pump disposable plastic pipets

CHEMICALS NEEDED
aqueous stock solutions of food dyes FD&C blue #1 and FD&C red #40 (concentrations on containers)
Sports Drinks

PROCEDURE
COMMON MEASUREMENT MISTAKES TO AVOID
In order to obtain high-quality, reliable absorption spectra, you must be careful to avoid a number of common
mistakes. The most typical mistake, which will affect your results significantly, is to position the cuvet incorrectly
in the cell holder. The cuvets have two clear sides and two frosted sides; if the cell is positioned so that the light
passes through the frosted sides, the absorption will be too high or too low, depending on whether the reference
cell or sample cell was the one positioned incorrectly. If any of your spectra are significantly above or below zero
at the highest wavelengths, you should redo the spectrum, making sure to align the cuvets properly.
Still other mistakes to avoid include having a smudge or fingerprint in the light path on one of the cuvets.
Another possibility is the presence of an air bubble in the light path, or one of the cells may not be positioned all
the way in the bottom of the cell holder or may have been tilted from the vertical. In short, to achieve good results,
you must be careful in each measurement to have clean cuvets, properly positioned and oriented, with no air
bubbles to interfere with the light path.
Sample Preparation
1. Obtain about 25 mL (no more!) of one the colored stock solutions in a 50 mL beaker. Record its
concentration. Fill one of the cuvets about three-fourths full with distilled water (reference) and the other
with the stock solution (sample).

Setting Up the Workstation

Note: If the workstation is displaying a plot of data from a previous user’s experiment, select New from
the File menu and touch Discard.
2. If the spectrometer is not plugged in yet, do so using the USB port on the side of the workstation. Unplug
any other probes from the workstation.

Zeroing and Calibrating the Spectrometer

3. Touch the green Collect arrow—this will start the lamp warmup process and will zero the spectrometer.
When the warmup is complete, insert the reference cuvet into the spectrometer with one of the clear sides
pointing toward the white arrow to the left of the opening, and touch the Finish Calibration box. Once
the calibration is completed (it will take a few seconds), touch OK.

Collecting Absorbance Spectrum—Sample

4. Place your sample cuvet in the cell holder, clean, properly oriented, and fully inserted. In a few seconds
the absorbance curve will appear on the display.
5. Evaluate the displayed spectrum on the workstation screen—the absorbance at high wavelengths (beyond
the red background of the display) should be close to zero—this is called the baseline. If it is not, make
one of the following corrections:
If the baseline is significantly above zero, remove the sample cuvet to see whether you had inserted it
with the frosted sides pointed toward the white arrow; if this is the case, reinsert the cuvet with the correct
orientation. If the baseline is still high, check to see if the cuvet is smudged or cloudy at the bottom of one
of the clear sides; if this is the case, discard the cuvet, refill a new cuvet with solution, and reinsert the
cuvet into the spectrometer.
If the baseline is significantly above zero, check the reference cuvet for smudges; discard and replace
it if necessary. You will need to recalibrate: touch the red Stop square, select New from the File menu
and touch Discard. Touch the Collect arrow and redo the calibration as in step 3 (you may skip the
warmup when given the choice), making sure the cuvet is aligned correctly.
If the baseline still deviates from zero, consult with your TA to solve the problem.
6. Once you are satisfied with your spectrum, touch the red Stop square.

Absorbance Measurements—Beer’s Law Plot

Preparing the Diluted Solutions
Before preparing the diluted solutions, practice pipetting with the Mohr pipet using distilled water until you
can measure out volumes to the nearest 0.1 mL by controlling the flow with the pipet pump. Measure the position
of the bottom of the meniscus with respect to the graduation marks on the pipet. A good way to measure out 2.0
mL, for example, is to fill the pipet to the 2.0 mL mark and drain it to the 4.0 mL mark. Before using the pipet for
the stock solution, rinse it with a small portion of the stock solution.

Obtaining Absorbance Spectra of Diluted Solutions

7. Pipet 2.00 mL of stock solution into the 10-mL volumetric flask. Using a disposable pipet, add distilled
water to the flask to bring the bottom of the meniscus to the mark on the neck of the flask. Place a small
piece of Parafilm over the top of the flask and invert the flask several times to ensure thorough mixing.
Prepare this solution as carefully as you can, since the quality of your Beer’s Law plot will be determined
to a large extent by how accurately the concentrations are known.
8. Use a small amount of the diluted solution to rinse out the sample cuvet, and then fill the cuvet about
three-fourths full with the solution.
9. Touch the File Cabinet icon in the lower right corner of the workstation screen to start Run 2. Touch the
Collect arrow and obtain the absorbance spectrum for the solution as in steps 4-6 above. Do not discard
any solutions until the end of the lab period when you are satisfied that all of the spectra have been
obtained satisfactorily.
10. Repeat step 7, this time using 4.00 mL of the stock solution. Obtain and save an absorption spectrum for
this solution as in steps 8-9 (you will be on Run 3 now). Be sure to touch the Stop square once you have
obtained the spectrum.
11. Repeat steps 7-9 for two more solutions, using 6.00 mL (Run 4) and 8.00 mL (Run 5) of the stock solution.

Obtaining Absorbance Spectra of Sports Drink

12. Obtain ~ 5 mL of a sport drink (it must be the same color as the solutions you prepared). Touch the File
Cabinet icon to start Run 6. Rinse the cuvet with a small amount of the sports drink, then fill it ¾ full.
 Place the cuvet in the spectrometer and observe the maximum absorbance value. If it is above 1.0, use the
volumetric pipet to deliver 3.00 mL of the sports drink into the volumetric flask. Dilute to the mark with
distilled water, and obtain the absorption spectrum of the diluted solution. (If the maximum absorbance
value is below 1.0, you don’t need to do the dilution step.)

Determining Absorbance Values at Selected wavelength

13. You will now determine the absorbance values at a selected wavelength for each of the five assigned
solutions and your unknown solution. Make sure the data collection has stopped, then touch the box that
should now be reading Run 6. Select All Runs—this will display the absorbance spectra for the stock
solution (Run 1), the 4 diluted solutions (Runs 2-5) and the sports drink (Run 6). Touch the spectrum near
the maximum absorbance—you should see a vertical line appear, and a table of absorbance values at the
wavelength selected to the right of the spectra. If necessary, use the cursor controls to adjust the line to
around the highest absorbance value.
14. Go to your TA’s computer and consult with him or her to make sure the spectra look OK. If they do, you
can email yourself (and your lab partner) a screen shot of the spectra from the computer.
15. Repeat steps 1-14 to obtain absorbance spectra for other color food dye and sports drink, and email the
spectra to your account.

Waste Disposal
Any unused stock solution can be washed down the sink.

RESULTS
A. Absorption Spectra
The spectrometer does not measure absorbance directly; rather it measure the light intensity after the light passes
through the reference cell (I0,) and the light intensity after it passes through the sample containing the
absorbing species (I) and then calculates A at all wavelengths. In this part you will demonstrate your
understanding of how the absorbance values are obtained.
1. Follow the instructions on slide 5 of the postlab PowerPoint.

B. Absorbance Measurements—Beer’s Law Plot

1. Use the instructions in the postlab PowerPoint to create your Beer’s Law plot using Excel for each food dye
solution.
2. From your data and EQUATION 6-4, determine the molar absorptivity, , for each solution at the wavelength
used for this plot. Note: the path length b through the cell is 1.00 cm. Determine the average molar
absorptivity for the substance you investigated.
3. A Beer’s Law plot of absorbance vs. concentration gives a straight line passing through the origin. This has
the form y = mx, where m is the slope of the line. Thus, using EQUATION 6-4, A =    b  C , a plot of
absorbance (y-axis) vs. concentration (x-axis) will give a straight line where the slope = εb. Note this very
important consequence when collecting experimental data: if the data fits a straight line, we can often
obtain information regarding the phenomenon being observed from the slope of the line. Use the slope
of linear regression line from your Beer’s Law plot to determine the molar absorptivity for the substance you
investigated. Note: when a printout lists a value in scientific notation, but is not capable of displaying
superscripts, the symbol ‘E’ is often used to represent a power of 10. For example, a slope value of 1.67 x 102
 would be displayed as 1.67E+02. You must click on the Graph Data link on the report sheet before
entering your slope value, or you will receive an error message.
4. Using the slope value of your Beer’s Law plot, determine the concentration of your unknown sample. Since
A =    b  C and slope = εb (from above), then Aλ = (slope)C. Thus, the concentration of your unknown
can be found by
A
Concentrat ion = (6-6)
slope
provided the absorbance value from the spectrum of the unknown solution is obtained at the same wavelength
as those absorbance values used to generate the slope of the line from your Beer’s Law plot.
 EXPERIMENT 6
REPORT SHEET
Name: _______________________________________ Date:__________

Partner:______________________________________

PART A. ABSORPTION SPECTRA

• Note: you will obtain the data for this section from the plots on the Chem21 report sheet.

Wavelength, nm I0, I A (measured) A (calculated)

PART B. BEER’S LAW PLOT

Food Dye color: Blue concentration ___________________ Wavelength used: ______ nm

Volume of Concentration, Molar

Sample number stock solution, mL mol/L Absorbance Absorptivity
(1) stock xxxxxxxxxxxxxxx
solution xxxxxxxxxxxxxxx
(2) first diluted
solution
(3) second diluted
solution
(4) third diluted
solution
(5) fourth diluted
solution

Average

Determination of Food Dye Concentration in Sports Drink

Color of sports drink ___________________

Absorbance ____________________

Concentration of diluted sports drink (if diluted) _______

Concentration of undiluted sports drink ________

Food Dye color: Red concentration ___________________ Wavelength used: ______ nm

Volume of Concentration, Molar

Sample number stock solution, mL mol/L Absorbance Absorptivity
(1) stock xxxxxxxxxxxxxxx
solution xxxxxxxxxxxxxxx
(2) first diluted
solution
(3) second diluted
solution
(4) third diluted
solution
(5) fourth diluted
solution

Average

Determination of Food Dye Concentration in Sports Drink

Color of sports drink ___________________

Absorbance ____________________

Concentration of diluted sports drink (if diluted) _______

Concentration of undiluted sports drink ________