1. INTRODUCTION TO SPECTROPHOTOMETRY
Advancing Science Lab
Gettysburg College
#531, #532, #534

2. BACKGROUND
Spectrophotometry is a method of analyzing that involves how light interacts with the atoms (or molecules) in a sample of matter.
Visible light is only a small portion of the entire electromagnetic spectrum and it includes the colors commonly observed (red, yellow, green, blue and violet). The visible spectrum consists of electromagnetic radiation whose wavelengths range from 400 nm to nearly 800 nm.

3. Why do some substances appear colored?
BACKGROUND
white light is observed, what is actually seen is a
mixture of all the colors of light
Why do some substances appear colored?
When this light passes through a substance, certain energies (or colors) of the light are absorbed while other color(s) are allowed to pass through or are reflected by the substance.
If the substance does not absorb any light, it appears white (all light is reflected) or colorless (all light is transmitted). A solution appears a certain color due to the absorbance and transmittance of visible light. For example, a blue solution appears blue because it is absorbing all of the colors except blue.

4. BACKGROUND
A sample may also appear blue if all colors of light except yellow are transmitted. This is because blue and yellow are complementary colors. (See the color wheel above.)

5. BACKGROUND
The amount of light absorbed by a solution is dependent on the ability of the compound to absorb light (molar absorptivity), the distance through which the light must pass through the sample (path length) and the molar concentration of the compound in the solution.
If the same compound is being used and the path length is kept constant, then the absorbance is directly proportional to the concentration of the sample.

6. Spectrophotometer
A spectrophotometer is used to provide a source of light of certain energy (wavelength) and to measure the quantity of the light that is absorbed by the sample.
Light Bulb Prism Filter Slit Sample Detector

7. Spectrophotometer
The basic operation of the spectrophotometer includes a white light radiation source that passes through a monochromator. The monochromator is either a prism or a diffraction grating that separates the white light into all colors of the visible spectrum. After the light is separated, it passes through a filter (to block out unwanted light, sometimes light of a different color) and a slit (to narrow the beam of light--making it form a rectangle). Next the beam of light passes through the sample that is in the sample holder. The light passes through the sample and the unabsorbed portion strikes a photodetector that produces an electrical signal which is proportional to the intensity of the light. The signal is then converted to a readable output that is used in the analysis of the sample.
Light Bulb Prism Filter Slit Sample Detector

8. Spectrophotometer Absorbance -- a number between 0 and 2
The spectrophotometer displays this quantity in one of two ways:
Absorbance -- a number between 0 and 2
(2) Transmittance -- a number between 0 and 100%.
The sample for a spectral analysis is prepared by pouring it into a cuvette which looks similar to a small test tube. A cuvette is made using a special optical quality glass that will itself absorb a minimal amount of the light. It is also marked with an indexing line so that it can be positioned in the light beam the same way each time to avoid variation due to the differences in the composition of the glass

9. Viewing the Visible Spectrum
Experiment
Viewing the Visible Spectrum
The spectrophotometer is designed to detect absorbances of light at different wavelengths when the light passes through a solution of some given concentration. Some compounds absorb more light at one wavelength than another, so the wavelength must be changed every time a different compound is being analyzed to achieve optimum results from a spectrophotometer. The wave-length of light is selected by adjusting the wavelength dial and read on the wavelength display.
In this lab, the color of light associated with each wavelength will be observed with the eye. The visible range of light is approximately 400 to 700 nm. The very ends of the visible spectrum will also be determined in this experiment. Please note that the accepted symbol for wavelength is the Greek letter lambda ().

10. Viewing the Visible Spectrum
Objective
To observe the color of light emitted by the spectrophotometer
at various associated wavelengths.
Materials Needed:
A piece of white chalk approximately 1-2 cm long
Spectrophotometer
A cuvette/cuvette rack

11. Viewing the Visible Spectrum
Procedure (Best results are obtained by doing this experiment in a dimly lit room)
Cut or rub one end of the piece of chalk to produce a 45o angle.
Place the piece of chalk in a cuvette with the angle end directed up.
Set the wavelength of the spectrophotometer to 425 nm. Be sure the filter switch is set to the left.
Place the cuvette in the spectrophotometer so the angle of the chalk faces to the right of the spectrophotometer.
Open the light slit by turning the transmittance adjustment knob (right knob) clockwise.
Look into the sample compartment and record on the data sheet the color of the light striking the chalk.
Repeat Step 5 increasing the wavelength by 25 nm each time. Continue the process until reaching 675 nm. At 600 nm, move the filter lever (#11 in the diagram) to the right.
While looking at the piece of chalk, slowly increase the wavelength to the point where the color is no longer seen. This is one end of the visible spectrum. Record this wavelength value.
Adjust the wavelength back to 425 nm. While looking at the piece of chalk, slowly decrease the wavelength to the point where the color is no longer visible. This is the other end of the visible spectrum. Record this wavelength value.

12. Viewing the Visible Spectrum
Frequency
(Hertz)
Wavelength (nm)
Observed Color
Notes
End of Spectrum
7.1 x 1014
425
6.7 x 1014
450
6.3 x 1014
475
6.0 x 1014
500
5.7 x 1014
525
5.5 x 1014
550
5.2 x 1014
575
5.0 x 1014
600
4.8 x 1014
625
4.6 x 1014
650
4.4 x 1014
675

13. Spectral Curve of Food Coloring
Background Information:
A visible spectrophotometer can be used to learn why colored solutions appear a particular color. For example, WHY does blue food coloring appear blue? Simply put, the solution is blue because it transmits (and reflects) blue visible light more than it transmits other colors of visible light. In other words, blue food coloring absorbs blue visible light the least and absorbs other colors of light more.
When white light is observed, what is actually being seen is a mixture of all the colors of light. When this light passes through a substance, certain energies (or colors) the light are absorbed while other color(s) are allowed to pass through or are reflected by the substance. This is why some substances appear colored. The color that we see is the combination of energies of visible light which are not absorbed by the sample. If the substance does not absorb any light, it appears white or colorless.

14. 2. Spectral Curve of Food Coloring
A solution appears a certain color due to the absorbance and transmittance of visible light. For example, the blue solution appears blue because it is absorbing all of the colors except blue. A sample may also appear blue if all colors of light except yellow are transmitted (yellow is absorbed). This is because blue and yellow are complementary colors. Any two colors opposite each other on the color wheel (see figure above) are said to be complementary. The wavelength (numbers outside the wheel) associated with the complementary color is known as the wavelength of maximum absorbance. This is because in a colored solution the maximum amount of light is absorbed by the complementary color. Note: cyan = green.

15. Spectral Curve of Food Coloring
How a Spectrophotometer works: Inside the spectrophotometer [See Figure 1] is a light bulb which produces white light. The white light is separated into the different colors of light by either a prism or a diffraction grating (An example of a grating is a CD ROM surface. The reflective surface has tiny grooves etched into it, which separate white light, in a manner similar to light passing through a prism). After the light is separated, it passes through a filter (to block out unwanted light, such as light of a different color) and a slit (to narrow the beam of light--making it in the shape of a rectangle). Next the beam of light passes through the sample that is in the sample holder. The amount of light that passes through the sample is measured and the spectrophotometer displays this quantity in one of two ways:
Absorbance -- a number between 0 and This is a measure of how much light is absorbed by the solution, in other words, now much does not pass through.
Transmittance -- a number between 0 % and 100 %. This is a measure of how much light passes through the solution (this is transmitted light).

16. Spectral Curve of Food Coloring
The spectrophotometer is designed to detect the absorbance of light at different wavelengths (different colors) when the light passes through a solution of some given concentration. Some compounds absorb more light at one wavelength than another, so the wavelength must be changed every time a different compound is being analyzed to achieve optimum results from a spectrophotometer. The wavelength of light is selected by adjusting the wavelength dial and read on the wavelength display.

17. Spectral Curve of Food Coloring
Objectives:
To measure the wavelengths of visible light that various colored solutions absorb.
To plot a graph of wavelength versus absorbance and determine the maximum wavelength (λmax) for each sample.
Using this information we will reason as to why each solution appears a particular color.
Materials:
Visible spectrophotometer Distilled water in squeeze bottle
Food coloring (red, blue, yellow and green) Tissues or Kimwipes
Cuvettes Excel graphing program
Cuvette rack Plastic wrap

18. Spectral Curve of Food Coloring
After you collect the data: handout-you will do the following data analysis
Data Analysis: Now you will plot your data on a graph. (See separate instructions for Excel if you are using a computer to plot your data. Create a scatter graph and then choose the option that connects the dots to draw the curve). Wavelength is plotted on the x-axis and Absorbance is plotted on the y-axis. Remember to put units on your axes and to give your graph a title that tells the purpose of the graph.

19. 3. PHYSICAL AND CHEMICAL CHANGE
BACKGROUND:
Physical change - a change to a substance, such as boiling, freezing, or breaking, that does not result in the formation of a new substance.
Chemical change - a change to a substance, such as burning or rusting, that results in the formation of a new substance.
Color change, bubbles and production of gas, heat and/or light given off, the formation of a precipitate, and odor changes are often observed as chemicals undergo change. Unfortunately, many of these clues may be observed for both chemical and physical changes. The best indication of a chemical change is evidence that a new substance, with different properties from the original substance, has formed, but even this is not always directly observable.
One of the properties of most materials is the wavelength(s) of light that they will absorb. In this lab, you will measure the absorbance by obtaining a spectral curve of the materials before they are mixed and again after the mixture has been formed. The spectrophotometer will help you determine whether the color change shows formation of a completely new substance (a chemical change), or simple physical combination of two substances (a physical change).

20. PHYSICAL AND CHEMICAL CHANGE
OBJECTIVES:
To produce spectral curves of solutions before they are combined and after they are combined.
To determine, by using the spectral curve, whether the color observed is a new product or a mixture of the original solutions that were combined.
To determine whether changes resulted in a chemical change or a physical change.

21. PHYSICAL AND CHEMICAL CHANGE
MATERIALS:
Spectronic Spec 20D spectrophotometer
Computer with printer Pipettes
Graphing software KimWipes
Distilled water
Red and blue food color
1.0 M Hydrochloric Acid
Red cabbage or black bean juice
Graduated cylinder
Cuvettes Cuvette rack
Spectral Curve for Red Food Color
Spectral Curve for Blue Food Color

22. PHYSICAL AND CHEMICAL CHANGE
PROCEDURE:
Mix 5 drops of red and 5 drops of blue food coloring into a cuvette and then dilute with about 5 ml of distilled water. Mix the solution by carefully tapping the bottom of the cuvette as demonstrated by your teacher.
Fill a second cuvette about halfway with distilled water. This will serve as a “blank”.
Obtain a spectral curve for the food coloring mixture from a wavelength of 350 nm to a wavelength of 675 nm and record the absorbance values in Data Table 1.

23. PHYSICAL AND CHEMICAL CHANGE
Data Table 1. Absorbance of Food Coloring Mixture
Wavelength (nm)
Absorbance
350
525
375
550
400
575
425
600
450
625
475
650
500
675

24. PHYSICAL AND CHEMICAL CHANGE
Add ______ (determined by the instructor) drops of red cabbage or black bean juice to a cuvette and dilute with about 5 ml of distilled water.
Prepare another cuvette using ______ drops of red cabbage or black bean juice, dilute with about 5 ml of distilled water, and add a few drops of acid to the juice until you see a color change. Mix the solution by carefully tapping the bottom of the cuvette.
Obtain spectral curves for the juice, and juice/acid mixture and record them in Data Table 2.

25. PHYSICAL AND CHEMICAL CHANGE
Data Table 2. Absorbances of Juice And Juice/Acid Mixture
Wavelength (nm)
Absorbance Juice
Absorbance
Juice/Acid
350
525
375
550
400
575
425
600
450
625
475
650
500
675

26. PHYSICAL AND CHEMICAL CHANGE
Make scatter type graphs of your data, using graphing software or by hand, plotting wavelength on the X axis and absorbance on the Y axis. Compare the spectral curve of the mixture to the spectral curve(s) of unmixed substances to answer the following questions. (If using graphing software, use overlay techniques to compare the graphs. Instructions for Excel graphing are attached to this lab.)
Answer questions on question sheet with your handout.