Name _____________________________________________________ Date _____________________
NORTH ALLEGHENY SENIOR HIGH SCHOOL
Department of Chemistry
AP Chemistry
An Introduction to Spectrophotometry
INTRODUCTION/THEORY
One of the most common ways to measure absorbance of visible light in the lab is through the idea of
spectrophotometry, or quantitatively monitoring the absorbance/transmittance of incident light in a sample of
matter. This lab will provide an introduction to the most common instrument used in the science of
spectrophotometry, the Spectronic 20 spectrophotometer.
Spectrophotometric methods have their basis in the production or interaction of electromagnetic radiation
with atoms and molecules. They are commonly characterized by the type of electromagnetic radiation involved.
Some examples are x-ray, ultraviolet, visible, infrared, microwave, and radio frequency methods. For this
experiment we will restrict our discussion to visible light or radiation with a wavelength between 380 nm and 750
nm.
Light can be described in terms of waves and particles. In discussing spectrophotometry it is most
convenient to view light as a stream of discrete particles of energy called photons. The energies are proportional to
the frequency of the radiation according to the equation E = h, where h is Planck's constant and  is the frequency.
Three possible situations occur when white light strikes an object. The light may be reflected by, absorbed by, or
transmitted through the object.
Absorption of visible light is the origin of color. Absorption is the process by which chemical species
selectively remove certain frequencies of electromagnetic radiation. The energy of the photon then converts the
atom or molecule to a more energetic or excited state. After a brief period (10–6 to 10–9 seconds) this excitation
energy is lost, most often as heat. Absorption is therefore a nondegradative process. Species which absorb visible
light will appear to the eye as the complementary color of the region of the spectrum which has been absorbed (See
Figure 1). If more than one color is absorbed, the response of the eye determines which colors are perceived. If no
visible light is absorbed the sample appears colorless.
The wavelengths of light which are preferentially absorbed are characteristic of a particular atom or
molecule, and this knowledge can be very useful in identifying an unknown species (qualitative analysis). Although
this experiment just provides us with an introduction, in the following experiment we are interested in quantitative
applications of light absorption (determining how much material is present in an unknown sample). Long ago it
was observed that the concentration of a colored solution is related to the intensity of its color, meaning that a more
concentrated solution absorbs more (transmits less) light and appears to be more intensely colored. If a series of
colored solutions of known concentration were available, we could determine the approximate concentration of an
unknown by visual comparison. This method of analysis is known as visible colorimetry. The usefulness of this
method is limited by the relatively poor accuracy of the eye as a detector. This limitation was overcome by the
development of the phototube as a detector. The name photometry was given to methods using this type of detector.
The basic components of a spectrophotometer are shown in figure 2. The source of the visible light in the
Spec 20 comes from a tungsten lamp which emits radiation over the high UV, visible, and near IR region of the
electromagnetic spectrum. The radiation then passes diffraction grating or prism to obtain a source of
monochromatic light (light of a single wavelength or color) and a photoelectric detector to measure the intensity or
power of light transmitted by a sample. Spectrophotometry measures the intensity of light as a function of
wavelength (See Figure 2).
Spectrophotometers do not directly measure absorbance. They measure the intensity of the transmitted
beam. First a zero adjustment is made in the absence of the sample to correct for the small amount of current which
flows through the detector in the absence of light (dark current). Then a blank or reference is placed in the sample
container (test tube) and the meter adjusted to read 100%T. This is to compensate for small reflective losses from
the test tube and any absorption by the container or the solvent in which the sample is dissolved. The test tube is
then filled with the sample and the intensity of the transmitted beam is measured. Transmittance is defined as the
ratio of the intensity of light transmitted through the sample (I) to that transmitted through the blank (Io) or T = I /
Io, and percent transmittance, %T, is equal to (100)(T). Absorbance is logarithmically related to the percent
transmittance according to the relationship A = log (100 / %T) (See Figure 3).
Choosing the wavelength at which an experiment is run is based on a number of considerations. First of all,
a slight shift in wavelength should not result in an appreciable shift in absorbance (See Figure 4). Secondly the
species whose concentration you wish to determine should be the only absorbing species at the wavelength you
choose. Finally the greatest accuracy is obtained if a wavelength is chosen which gives absorbance readings ranging
from 0.2 – 0.7 in the concentration range of interest. For the above reasons the wavelength of maximum absorbance
as determined from a curve of A vs.  is usually chosen as the working wavelength. In the second part of this
experiment a working wavelength will be determined for a chromium(III)nitrate system to be studied. From there,
development of a calibration curve will allow us to determine the concentration of an unknown solution.
Quantitative applications of spectrophotometry have their basis in a series of observations first noted around
the middle of the eighteenth century as to the factors which affect the magnitude of A or absorbance. Lambert in
1760 found that when a beam of monochromatic light enters a sample, the decrease in the intensity of the light as it
passes through the sample is directly proportional to the thickness b of the sample. In 1852 Beer established a
similar relationship between the decrease in intensity and the concentration c of the absorbing species. The
combined Beer-Lambert law (more simply known as Beer's Law) is most frequently written as A = bc where
A = absorbance
 = proportionality constant known as the molar absorptivity – It is a constant for a given
species at a given wavelength. (Related to the probability of a photon being absorbed
and causing an electronic transition.)
b = path length or thickness of the sample in cm
c = concentration of the absorbing species in moles/liter
In an experiment such as the one you are performing,  and b are held constant so that a plot of A vs. c will yield a
straight line going through the origin with a slope equal to b. Beer's Law is not applicable at all concentrations or
in certain systems. Deviations occur in situations where the solution is too concentrated causing the solute species
to interact with one another or where there is a chemical interaction between the absorbing species and the solvent.
Spectrophotometry using ultraviolet or visible radiation is one of the most useful tools available today
for quantitative analysis. This is due to its wide applicability, high sensitivity in the concentration ranges of 10–
7 to 10–4 M, moderate to high selectivity, good accuracy with relative errors of typically 1-3% in concentration
measurements, ease, and convenience.
FIGURE 1
1
Lamp
Lens A
Entrance Slit
2
Lens B
4
Occluder
5
Grating
Sample
To Meter
Filter
3
Light Control
Phototube
Cam Follower
Arm
Exit Slit
Ca
m
Components of a Spectrophotometer
1. Stable source of radiant energy. A tungsten filament source is useful in the visible region.
2. Monochromator – Limits radiation to a particular narrow band of wavelengths.
3. Nonabsorbing containers of constant thickness for the sample.
4. Phototube to convert radiant energy to an electrical signal.
5. Meter or other signal detector.
FIGURE 2:
Relationship between
T, %T, and A
FIGURE 4: A model of the Spectronic 20
FIGURE 3: Selecting a working wavelength
PROCEDURE
(For much of parts one and two in the procedure refer to figure 4 above, a spectrophotometer.)
I. Care of the Test Tubes:
1. The pair of test tubes used for a colorimetric determination is matched in size so that the light paths
through the blank and solution are almost identical. Great care must be taken to avoid scratching the
tubes since scratches will cause diffusion of light.
2. Never use a brush to clean the test tubes. If the tubes seem to be unclean , they should be washed with a
detergent solution and rinsed well with tap water and then distilled water.
3. Do not touch the lower part of the test tubes (the portion through which light will pass) with your
fingers.
4. Choose one test tube and use only it for the "blank" (distilled water). Fill this tube about half-full of
water.
5. The second tube is used for the aqueous solutions. The tube should be rinsed with two small portions of
the solution before the 3 ml sample of the solution is placed in the tube. Use only this second
tube
for the sample solutions.
6. Each tube should be carefully wiped with a soft tissue after water or a solution has been placed in it.
7. The tube is inserted into the sample holder of the Spectronic-20 in this manner:
o Grasp the test tube by the upper rim.
o With the index line on the tube facing the operator, gently push the tube in to the holder.
o Line up the index line on the tube exactly with the index line on the holder.
When the determination has been completed, carefully rinse the second test tube with distilled water.
Store both test tubes in the test tube rack.
II. Operation of the Spectronic-20:
1. Turn the power switch and zero control knob (left front) clockwise so the red pilot lamp lights.
Allow the instrument to warm-up for five minutes.
2. Turn the zero control knob (left front) until the needle on the meter lines up with 0% T on the
scale. There should be no test tube in the holder and the lid on the sample holder should be
tightly closed. This setting should not vary during the experiment, for it is independent of
wavelength and sample.
3. Fill the “blank” tube half full of distilled water. Carefully wipe the lower portion of the test tube with a
tissue and place the tube in the sample holder, aligning the marks on the tube with your reference point.
4. Turn the wavelength scale knob to the value you want to begin with.
5. Rotate the light control knob (right front) until the meter reads 100% T. This step eliminates
any absorption due to the water at the given wavelength.
6. Remove the "blank" and insert the carefully wiped tube half filled with solution. Close the lid of the
holder after aligning the tube in the proper manner.
7. Record the observed absorbance in your log.
III: An absorbance spectrum
In the first part of the experiment you will measure an absorption spectrum of a chromium(III) nitrate solution
within the visible range of 350 nm to 700 nm. You will use this to determine a working wavelength for the
procedure in part IV. Fill your test tubes about 2/3 full of solution for the analysis below.
1. Obtain 20 ml of a 0.0200 M chromium(III) nitrate solution to analyze. This will be provided for
you.
2. Set the wavelength on the spectrophotometer to 350 nm.
3. Follow steps 1-7 in part II above for your data collection, using your solution.
4. Repeat step 3 above for each of the following wavelengths: 350, 375, 400, 425, 450, 475, 500, 525,
550, 575, 600, 625, 650. A data table will be helpful here. Note: you must set 100% T using
the blank each time you collect a data point
5. Record absorbance for each wavelength tested.
6. Looking at your data, determine the working wavelength you will use for an experiment involving
chromium(III) nitrate. Note this in your log.
7. Plot a graph of Absorbance vs. wavelength for the chromium(III) nitrate solution.
IV: Variation of Absorbance with Concentration. The Calibration Curve
1. Obtain about 20 mL of each of the following chromium(III) nitrate solutions: 0.0500 M, 0.0400 M,
0.0300 M, 0.0200 M, and 0.0100 M.
2. Set up the Spectronic-20 at the working wavelength as determined in part III. Complete steps 2-5 in
part II one last time to determine the Spec 20 is zeroed and ready to go. The wavelength scale should
not be touched again during this determination.
3. Perform steps 5-7 (part II) with each of the five solutions (0.0100 M to 0.0500 M). Record the
absorbance for each solution. Be sure to rinse the sample tube as described in Part I with each new
solution.
4. Plot A vs concentration on a graphing program that you may be familiar with (Remember 0,0 should
be a part of this graph). From this plot determine the slope of the line and the molar absorptivity
constant  This plot will provide you with a calibration curve for an analysis of an unknown Cr(III)
solution.
V: Analysis of an Unknown Chromium (III) Solution
-
Obtain approximately 20 mL of an unknown Cr(III) solution and determine the absorbance of this
solution at your working wavelength. Using your calibration curve (and more importantly, the
equation of the curve), determine the concentration of the unknown solution. Be sure to record the
number of your unknown solution for identification in the calculations and conclusion.
CALCULATIONS
The following are items that should be 'calculated' in this lab.
-
an absorbance vs. wavelength curve should be plotted, with the working wavelength for the
experiment noted on the graph.
-
a calibration curve should be constructed for the chromium(III) nitrate solutions tested. The
equation of the line should appear on the graph, with the slope and molar absorptivity constant
calculated.
-
the concentration of the unknown solution should be determined using the equation of the
calibration curve
-
ask Mr. Davis for the actual concentration of the unknown. Determine the percent error of the
concentration.
CONCLUSION
Summarize the results of the experiment in a brief paragraph. Begin by telling us the purpose of the
experiment and the basic experimental procedure used to collect the data. Give us the results you found,
commenting on the results as you feel necessary. Finally, with your percent error as your foundation, provide
any sources of error that would explain the difference in your calculated concentration versus the accepted
concentration.