1. Colorimetry
&
Spectrophotometry

2. Useful Terminology Colorimetry is the use of the human eye
to determine the concentration of colored species.
Spectrophotometry is the use of instruments
to make the same measurements. It extends
the range of possible measurements beyond
those that can be determined by the eye alone.
Note: This experiment will demonstrate
both techniques on the same set of dyes.

3. Colorimetry
Visual Observations – Because colorimetry is based on
inspection of materials with the human eye, it is
necessary to review aspects of visible light.
Visible light is the narrow range of electromagnetic
waves with the wavelength of nm.
                                                   
ROY G. BIV
= the mnemonic used to remember the colors of the visible spectrum.

4. Visible light is only a very small portion
of the electromagnetic spectrum.
Note: Frequency (υ) and Energy (E) are directly proportional
whereas Frequency (υ) and Wavelength (λ) are inversely proportional.

5. Electromagnetic Spectrum
Type of Radiation
Frequency Range (Hz)
Wavelength Range
Type of Transition
gamma-rays
<1 pm
nuclear
X-rays
1 nm-1 pm
inner electron
ultraviolet
400 nm-1 nm
outer electron
visible
4-7.5x1014
750 nm-400 nm
near-infrared
1x1014-4x1014
2.5 µm-750 nm
outer electron molecular vibrations
infrared
25 µm-2.5 µm
molecular vibrations
microwaves 3x
1 mm-25 µm
molecular rotations,
electron spin flips*
radio waves
<3x1011
>1 mm
nuclear spin flips*

6. Where h = Planck’s constant & c = speed of light in a vacuum.
Electromagnetic radiation is characterized by its
wavelength, , Frequency,  and energy, E:
E = h= hc /  c =  
Where h = Planck’s constant & c = speed of light in a vacuum.
longer wavelength, lower energy;
shorter wavelength, higher energy.

7. Color Wheel
(ROYGBIV)
Complementary colors lie across the diameter on the color wheel and combine to form “white light”, so the color of a compound seen by the eye is the complement of the
color of light absorbed by a colored compound; thus it
completes the color.

8. Observed Color of Compound Color of Light Absorbed
Observed Color of Compound
Color of Light Absorbed
Approximate Wavelength of Light Absorbed
Green
700 nm
Blue-green
600 nm
Violet
550 nm
Red-violet
530 nm
Red
500 nm
Orange
450 nm
Yellow
400 nm

9. Observed Color of Compound Color of Light Absorbed
Observed Color of Compound
Color of Light Absorbed
Approximate Wavelength of Light Absorbed
Green
Red
700 nm
Blue-green
Orange-red
600 nm
Violet
Yellow
550 nm
Red-violet
Green-yellow
530 nm
500 nm
Orange
Blue
450 nm
400 nm

10. Visual Colorimetry
Intensity: For light shining through a colored solution,the observed intensity of the color is found to be dependent on both the thickness of the absorbing layer (pathlength) and the concentration of the colored species.
←Side view
←Top view
(a.k.a. Bird’s eye view)
For One Color: A series of solutions of a single color demonstrates the effect of either concentration or pathlength, depending on how it is viewed.

11. Visual Colorimetry
←Ratio used
←Purple produced
For more than one color: the ratio of an unknown mixture can also be determined by matching the shade of the color to those produced from known ratios.
In this example, the ratio of a mixture of red and blue can be determined visibly by comparing the mixture to purples produced from known ratios of red and blue.

12. Dilution Factor (constant pathlength)
Recall: C1V1= C2V2
Then for the dilution,
Cdiluted x Vdiluted= Cstd x Vstd
Cdiluted = Cstd x (Vstd / Vdiluted)
Since Vdiluted = Vtotal
Cdiluted = Cstd x (Vstd / Vtotal)
Substituting the volumes:
Cdiluted = Cstd x (3 drops / 8 drops)
If the original concentration is 5.88 ppm,
then:
C diluted = 5.88 ppm x (3 / 8)
C diluted = 2.21 ppm
3 drops of dye std
+ 5 drops water
8 drops total volume

13. Intensity: When the product of the concentration and the pathlength of any two solutions of a colored compound are the same, then the same intensity or darkness of color is observed.
Duboscq visual
colorimeter
Adjustable
Path Lengths

14. Spectrophotometry
Spectrophotometer - an instrument that measures the amount
of light absorbed, or the intensity of color at a given
wavelength.
The intensity of color can be given a numerical value by
comparing the amount of light prior to passing it through
the sample and after passing through the sample.
These quantitative measurements of light absorbed are the
Transmittance and the Absorbance.

15. Main use of Beer’s Law is to determine the concentration
Absorbance
Beer-Lambert Law (a.k.a. Beer's law) - the linear relationship between absorbance and concentration of an absorbing species.
A = abc
A is the absorbance
“a” is molar absorptivity in L/[(mole)(cm)]
Also called “extinction coefficient” or “”;
it is dependent on the material being studied.
“b” is the path length in cm
The diameter of the cuvette or sample holder which is the distance
the light travels through the absorbing sample. “b” is a constant
when the same size cuvette is used for all samples.
“c” is the concentration of the sample in (mol/L)
Main use of Beer’s Law is to determine the concentration
of various solutions.
(We used Beer’s Law to calculate concentration in the equilibrium experiment.)

16. Transmittance is Related to Absorbance
Transmittance is given by the equation:
T = I/Io
where I is the intensity of the light after it has gone
through the sample & I0 is the initial (time = 0) light intensity.
Absorbance is related to the %T:
A = -logT = -log(I/ Io)

17. T= (I/I0) = 10-A %T = (I/I0) x 100 A = -logT = log(1/T)
Equation Summary
T= (I/I0) = 10-A %T = (I/I0) x A = -logT = log(1/T)
Sample Calculation
If %T = 95%, then A = log(100/95) = log(1/.95) = -log(.95)
A =
Note the scale for Absorbance: 9/10th of the scale is from 0-1 and 1/10th is from 1-2.
For this reason, the spectrometers have been calibrated in % Transmittance and
all readings will be taken in %Transmittance.

18. Spectronic 20 (a.k.a. Spec-20)
Spec-20 - A single-beam visible light spectrophotometer.
Tungsten filament lamp emits visible wavelengths of light.
Blank is inserted to adjust 100%Transmittance at each wavelength.

19. Simple Spectrophotometer Schematic
The lamp emits all colors of light (i.e., white light).
The monochromator selects one wavelength and that wavelength is sent through the sample.
The detector detects the wavelength of light that has passed through the sample.
The amplifier increases the signal so that it is easier to
read against the background noise.

20. Spectronic 20 Instructions
(Directions below will be available next to each instrument)
Digital Display
Mode Knob
(set to Trans)
Sample Chamber
1. With sample chamber empty, set desired wavelength then adjust to 0%T with right knob on front panel.
2. Insert blank solution, close lid and adjust 100%T
with right knob on front panel.
3. Insert dye solutions, read and record %T values.
4. Change wavelength*, repeat steps 2-4.
Filter Lever
Wavelength Knob
0-100%T Knob
*NOTE: The filter must be changed periodically to coordinate with the wavelength range studied: blue ( ), green ( ) and orange ( ).

21. Post Lab: 4 Plots of Absorption Data
Plots similar to the 3 below will need to be generated using a computer program such as Excel.
You will also need to make a plot of your unknown blue or red which will look similar to #1 or #2. #1 #2 #3
This plot is shown here simply to demonstrate
the underlying colors of the purple graph.

22. Development of the Spectroscope
Joseph von Fraunhofer’s initial desire was to create a glass lens that did not produce an image that was fringed with a rainbow of colors. He realized the problem was that the glass lens bent some colors more than others. He began searching for a source of light of a single color.
In 1814, he developed a spectroscope to study the spectrum of the light given off by the sun. He was amazed to discover that in the midst of the rainbow of colors was a series of black lines.
These dark lines were later determined to be the result of the absorption of selected frequencies of the electromagnetic radiation by an atom or a molecule.
Joseph von Fraunhofer
(March 6, 1787 – June 7, 1826)

23. Fraunhofer lines observable in the Solar Spectrum
390 nm
700 nm

24. Development of Diffraction Gratings
Fraunhofer also completed an important theoretical work on diffraction and established the laws of diffraction. One important innovation that Fraunhofer made was to place a diffraction slit in front of the objective of a measuring telescope in order to study the solar spectrum. He later made and used diffraction gratings with up to 10,000 parallel lines per inch. By means of these gratings he was able to measure the minute wavelengths of the different colors of light. (Diffraction gratings will be discussed more later.)

25. Gustav Robert Kirchhoff Robert Wilhelm Eberhard Bunsen
Gustav Kirchhoff and Robert Bunsen
Gustav Robert Kirchhoff
(March 12, 1824 – October 17, 1887)
German Physicist
Robert Wilhelm Eberhard Bunsen
(March 31, 1811 – August 16,1899)
German Chemist
Bunsen and Kirchhoff further developed the spectroscope by incorporating the Bunsen burner as a source to heat the elements. In 1861, experiments by Kirchhoff and Bunsen demonstrated that each element, when heated to incandescence, gave off a characteristic color of light. When the light was separated into its constituent wavelengths by a prism, each element displayed a unique pattern or emission spectrum.

26. Emission Spectra Complement Absorption Spectra
The emission spectrum seemed to be the complement to the mysterious dark lines (Fraunhofer lines) in the sun's spectrum. This meant that it was now possible to identify the chemical composition of distant objects like the sun and other stars. They concluded that the Fraunhofer lines in the solar spectrum were due to the absorption of light by the atoms of various elements in the sun's atmosphere.

27. Hydrogen Spectrum – The Balmer Series
In 1885, Johann Jakob Balmer
analyzed the hydrogen spectrum and found that hydrogen emitted four bands of light within the visible spectrum. His empirical formula for the visible spectral lines of the hydrogen atom was later found to be a special case of the Rydberg formula, devised by Johannes Rydberg.
Wavelength (nm)
Color
656.2
red
486.1
blue
434.0
blue-violet
410.1
violet
Johann Jakob Balmer
(May 1, 1825 – March 12, 1898)
Swiss Mathematician &
Honorary Physicist

28. Part A: Calculating the Balmer & Lyman Series
The four bands of light calculated by Balmer can be simply calculated using the Rydberg equation:
 = c / 
Where v = frequency
n = the quantum number
R = (Rydberg constant)
R = 3.29 1015 Hz
1 Hz = 1 s-1
C = (speed of light)
C = 108 m/s
The permitted energy levels of a hydrogen atom.
*These equations will be used on page 159.

29. In 1913, Bohr developed a quantum model for the hydrogen atom.
Proposed the Solar System model of the atom
where the electron in a hydrogen atom
moves around the nucleus only in certain
allowed circular orbits.
Niels Henrik David Bohr
Oct. 7, 1885 – Nov. 18, 1962
Danish Physicist
The Nobel Prize in Physics 1922
for the investigation of the structure
of atoms and of the radiation
emanating from them.
These orbits then correspond to the energy levels seen in the Balmer series. (p 167)

30. PART B: Emission spectrum of other compounds using
The STAR Spectrophotometer.
View the line spectrum through the STAR Spectrophotometer
- point slit towards the light and view to the right.
2. Verify that the scale is lined-up accurately by looking at the fluorescent light. In addition to other lines, you should see a green doublet for mercury at ~570 nm (the scale on the bottom).
3. Measure the line spectrum of the gas tubes set up in Room 201.
Note: The fastest/easiest way to do this is have one partner view the lines
and the other write down the observations.
4. Compare your results with NIST literature values.
For the fluorescent light compare it to the element mercury.

31. Atomic Spectra of Hydrogen & the Noble Gases
Helium
Neon
The Atomic Spectra will be determined for Hydrogen and the Noble Gases by looking at the gas discharge tubes.

32. Atomic Spectra Checkout Gas discharge tubes set up in Room 201.
Colorimetry & Spectrophotometry Checkout
Visual Portion Spec-20s
well plate 5 - cuvettes in a well strips* test tube rack 5 - Beral pipets**
*2 of which need to be at least 9 wells long.
**Don’t have to be returned.
Dyes - Located in Lab: Record Concentrations
Blue std. = _____ ppm
Red std. = _____ ppm
Waste (We are using FDA food dyes and distilled water.)
Atomic Spectra Checkout
STAR Spectroscope Set of Crayons ROYGBIV
Gas discharge tubes set up in Room 201.
(for viewing by STAR spectroscope)

33. For April 21-23 Turn In: 1.) Colorimetry & Spectrophotometry pp 51-58
+ 4 Graphs
2.) Atomic Spectra pp &
Read Over:
“Radiochemistry” pp in Lab Packet
& remember to bring your student id.