Laboratory
Exercise 10:

Absorption Spectra
of Colored Dyes

Purpose

This is the first in a series of exercises on UV/Vis spectrophotometry. This exercise will introduce you to the operation of our spectrophotometers and also to concepts relating to color and light absorbance. As you explore the tasks in this exercise you will:

• Learn how to operate the Spec 20 and DU64 spectrophotometers
• Learn how to generate an absorption spectrum
• Look at the relationship between light absorption of specific wavelengths and the color of a sample.
• Learn about the relationship between transmittance and absorbance.

Brief Background Review

A. Color And The Wavelength Of Light

Spectrophotometry is discussed in detail in your textbook. A brief summary of some concepts relevant to the laboratory exercise is given below.

Light is energy moving through space. As light moves through space, it can be understood as moving like waves on a pond, with crests and troughs. The distance from one crest to the next is called the wavelength of the light. Our eyes perceive light to be of different colors depending on the wavelength of the light, as summarized in Table 1. Light with slightly shorter wavelengths is called ultraviolet (UV) light. UV light is not visible to our eyes but it can be detected by some spectrophotometers.

Because color is subjective, these wavelengths are approximations.
The particular ranges here are based on various sources.

Consider liquid solutions. A solution will appear to be a particular color, depending on which wavelengths of light are absorbed by the solution and which pass through it, as summarized in the table below.

Note: Colors that appear alongside one another on this table are said to be "complementary" colors.

B. Absorbance And Transmittance

Inside a spectrophotometer, light of a specific wavelength is shined on a sample that is usually liquid. The sample contains a substance of interest, the analyte. As the light passes through the sample, some of its energy is absorbed by the analyte while the rest passes through. The light that passes through without being absorbed is called transmitted light. The spectrophotometer has a detector that is sensitive to transmitted light. A blank is a reference solution that contains no analyte but does contain the solvent and any reagents that are intentionally added to the sample. The spectrophotometer compares the interaction of light with the sample and with a blank in order to establish the effect of the analyte. Transmittance is defined as:

light transmitted through the sample = transmittance = t
light transmitted through the blank

Percent transmittance is the transmittance times 100%:

% T = t X 100%

Transmittance can range from 0 (no light passes through the sample relative to the blank) to 1 (all light passes through). Percent transmittance ranges from 0% (no light passes through the sample relative to the blank) to 100% (all light is transmitted).

All spectrophotometers actually measure transmittance, that is, the ratio between the amount of light transmitted through a sample and the amount of light transmitted through a blank. However, analysts are generally interested in the amount of light absorbed by a sample. Therefore, it is customary to convert transmittance to a measure of light absorption, called absorbance. Absorbance is calculated based on the transmittance as:

A = -log10 (transmittance)

Absorbance is also called optical density, OD. With modern spectrophotometers, you can adjust the instrument to either read out transmittance or to automatically convert the reading to an absorbance value.

C. Absorbance Spectra

An absorbance spectrum is a plot of the absorbance of a sample versus the wavelength of the light shined on the sample. To prepare such a spectrum, the absorbance of a single sample is measured successively at different wavelengths. Then, the absorbance of the sample is plotted with wavelength on the X axis and absorbance is on the Y axis. The resulting graph will have one or more peaks and valleys that are characteristic of that sample, Figure 1.

FIGURE 1

Figure 1. THE ABSORBANCE SPECTRUM OF RED FOOD COLORING.

Qualitative applications of spectrophotometry use the spectral features of a sample to obtain information about the nature of the sample's component(s). Sometimes it is possible to use an absorbance spectrum like a “fingerprint” to identify an unknown substance. The infrared (IR) spectra of organic compounds are complex and form particularly distinctive “fingerprints”. Organic chemists therefore frequently use IR spectra to identify compounds.

UV/Visible spectra are less complex and distinctive than IR spectra and so are used less commonly for identification of unknown substances. There are, however, situations where information about a sample can be obtained from its UV/Vis spectrum. For example, The U.S. Pharmacopeia includes a number of tests of drug identity in which an absorbance spectrum from 200 to 400 nm is used to confirm the identity of a drug. In these identity tests the spectrum of the test sample is compared to the spectrum of a pure reference material prepared under identical conditions. If the sample and reference have identical spectra, it is likely that they are the same compound. UV/Vis identity tests are frequently used because they are simple, rapid, and the equipment to prepare an absorbance spectrum is widely available. A problem with identifying substances using UV/Vis spectrophotometry is that some compounds whose structures are similar have spectra that cannot be distinguished. Therefore, UV spectra are frequently used in conjunction with other identification methods. An example of a test of identity from the U.S. Pharmacopeia is given in Laboratory Exercise 16.

D. Scanning Versus Non-Scanning Spectrophotometers

In this laboratory exercise, you will use spectrophotometers called Spec 20s. The Spec 20s are single beam instruments that are used only in the visible range of light. Although instruments with this basic design have been used successfully for years, basic single beam spectrophotometers have the disadvantage that the operator must reset the instrument to 100% transmittance (or zero absorbance) with the blank each time the wavelength is changed. Manually selecting each wavelength and repeatedly “blanking" the instrument is tedious when an absorbance spectrum is being prepared. Therefore, manufacturers have developed instruments called scanning spectrophotometers that are capable of rapidly scanning through a range of wavelengths and constructing an absorbance (or transmittance) spectrum. In another part of this exercise you will use the DU64s, which are scanning spectrophotometers.

The DU 64, in common with many other modern spectrophotometers, has three major features that distinguish it from a Spec 20:

• The DU64 can automatically scan absorbances at various wavelengths.
• The DU64 has two light sources, one for visible and one for UV light.
• The DU64 is controlled internally by a computer and can be readily attached to an external computer.

E. Proper Use Of Cuvettes

Cuvettes, or cells, hold liquid samples inside the sample chamber in a spectrophotometer. These cells are expensive, fragile laboratory items so it is important that you use them properly and carefully. Table 3 has information regarding the care and selection of cuvettes.

TABLE 3 PROPER SELECTION, USE, AND CARE OF CUVETTES

Cuvettes are expensive, fragile (except for “disposable” plastic ones) laboratory items. It is important that you use them properly and carefully. Use quartz cuvettes for UV work; glass, plastic or quartz are acceptable for work in the visible range. There are inexpensive plastic cuvettes that are suitable for some UV work. Disposable cuvettes are often recommended for colorimetric protein assays, since dyes used for proteins tend to stain cuvettes and are difficult to remove. Matched cuvettes are manufactured to absorb light identically so that one of the pair can be used for the sample and the other for the blank. Do not touch the base of a cuvette or the sides through which light is directed. Do not scratch cuvettes; do not store them in wire racks or clean with brushes or abrasives. Do not allow samples to sit in a cuvette for a long period of time. Make sure the cuvette is properly aligned in the spectrophotometer. Be certain to only use clean cuvettes. Wash cuvettes immediately after use. Figure 2 shows an effective cuvette-washing apparatus.

There are many types of cuvetes for various applications.                         1 mL Plastic Cuvette. Most plastic curvettes cannot be used for UV work. However, some manufacturers make plastic cuvettes that might be acceptable for some UV applications. Standard quartz cuvettes. 4 - 5 mL capacity     1 mL quartz curvette   50 microliter quartz curvettes. Useful for UV analysis of small volumes in molecular biology labs.             Cuvettes for Spec 20 look like glass test tubes - but they are made of a higher quality glass. Side-arm flask that doubles as cuvette         Bacterial cultures are grown in flask Side-arm is placed in cuvette holder of a Spec 20 Cloth is placed over flask to prevent light from entering sample chamber, absorbace of the bacteria culture is read. Absorbace indicates concentration of bacterial cell s present.

FIGURE 2

Using Cuvette Washer

Figure 2. EFFECTIVE CUVETTE WASHING APPARATUS. This simple cuvette washing set-up requires a source of vacuum. Observe the trap that keeps materials from contaminating the vacuum line. A trap should always be used when any vacuum apparatus is set up. The glass piece that holds the cuvette is available from Fisher Scientific, part number 14-385-946A.

Laboratory Activites

Perform these exercises in pairs or individually. Each person should record the data in their notebooks, prepare their own graphs and tape them into their notebook, and answer the questions individually.

Part A. Preparing Absorbance Spectra Using The Spec 20s

A1. Obtain a sample of red food coloring.

A2. Use milliQ water as your blank (the red food coloring was dissolved in milliQ water).

A3. Check the absorbance and the transmittance of the red food coloring from 380 nm to 680 nm in steps of 25 nm. Remember to zero against the blank at every wavelength.
The directions for using the Spectrophotometer 20 are in Box 1.

A4. Repeat for the other two colors of food coloring, but you do not need to read transmittance for these dyes, only absorbance.

A5. Choose any two colors of food coloring and combine them. Observe and record the resulting color. Then, read the absorbances for this new sample, as you did for the other dyes.

A6. On a single piece of graph paper, plot the absorbance spectra for all three color dyes. (I suggest using colored pens or a computer graphing program). Label the graph completely and tape it into your laboratory notebook.

A7. On another graph, plot both absorbance and transmittance spectra for red food coloring.

A8. On another graph, prepare an absorbance spectrum for the sample that was a combination of two colors.

Part B. Preparing Absorbance Spectra Using The Du 64

Prepare absorbance spectra for all the samples using the DU64 by following this procedure:

B1. Turn on the visible lamp by pushing the VIS key.

B2. Turn on the UV lamp by pushing the UV key.

B3. Before you prepare an absorbance spectrum, learn how to take a single absorbance reading at a single wavelength using the DU 64. The instructions are next to each machine. Measure the absorbance of any of the food coloring samples at any wavelength in the visible region.

B4. Now, learn how to automatically scan a sample and generate an absorbance spectrum. Push ABS key.

B5. Push SCAN key.

B6. Push 700 ENTR. This will be the wavelength at which the instrument will begin scanning.

B7. Push 270 ENTR. This will be the ending wavelength. 750 nm/min should be displayed.

B8. Push STEP button. Step through the options until scan speed is requested. Then push 500 ENTR.

B9. When the computer requests upper absorbance limit, push 2.0 ENTR.

B10. Set 0.0 ENTR for the lower absorbance limit. You are now done editing and are ready to calibrate and then blank.

B11. Push CALB. Wait until the display reads BKG.

B12. Using a quartz cuvette, insert your blank and push READ. This reads and stores the blank at all wavelengths. The display will say SCAN when it is done blanking. Remember to be careful with the quartz cuvettes!

B13. Insert the sample (any color dye) into the same cuvette as you used for the blank. Push READ.

B14. The machine will scan the sample and print out the spectrum.

B15. If you push SCAN you will exit from the scan mode. However, let’s not exit; rather, let’s look at the REPLOT mode. In the REPLOT mode, the spectrophotometer automatically prepares another absorbance spectrum for you. You can change the axes of the graph, expand a particular region, etc. The computer in the spectrophotometer "remembers" your data until another spectrum is run.

For example, you might replot a specific narrow wavelength range and a narrower absorbance range.

1. Push 450 ENTR. This will change the starting wavelength on the plot.
2. Push 425 ENTR. This will change the ending wavelength on the plot.
3. Push STEP until display reads RPLT. Push ENTR.
4. Push 1.0 ENTR and 0.1 ENTR to change the upper and lower limits on the plot. Push READ. A new plot should now appear.
5. Select a wavelength range and an absorbance range where you will see a peak, and use replot to expand the peak.
6. REMEMBER TO COME OUT OF THE REPLOT MODE BEFORE GOING ON TO A NEW SAMPLE.

B16. REMEMBER TO TURN OFF THE LAMPS WHEN YOU ARE FINISHED!! These bulbs are expensive to replace!!

B17. Photocopy the graphs for each person. Label them completely, include title, date, your name(s), and a brief caption. Tape them securely into your laboratory notebooks.

Part C. Confirming The Relationship Between Wavelength And Color

Table 1 shows the relationship between the wavelength of light and the color it appears to be. You can reproduce this table using a Spec 20 by viewing the light exiting the monochromatator. (It is not possible to do this with a DU64 because the light turns itself off whenever the sample door is opened.) You may find that the color you see is not exactly the same as Table 1. That is because color is somewhat subjective and different people sometimes name colors differently.

C1. Place a piece of chalk that is about 15-20 mm into a Spec20 cuvette. Place the cuvette into the sample holder. The chalk will reflect the light exiting the monochromator so that you eyes can see it.

C2. Dial in wavelengths at 10 nm increments from 380 to 780 nm. For each wavelength, record the color of light reflected by the chalk.

C3. Prepare a table like Table 1, but with your own observations. Compare your table to Table 1.

Discussion Questions

1. Discuss and explain the spectra for the three dyes. Are they the same or different? Why?
2. Do the spectra you obtained match your predictions before the laboratory? Explain by referring to your results.
3. Do any of the spectra have more than one absorbance maxima? Explain the significance of this. Do any of the spectra have an absorbance peak in the UV region? If so, does this UV absorbance affect the dye’s color?
4. Based on your results and information in lecture and text, why does red dye appear to ours eyes to be red? Why is yellow dye yellow?
5. Compare the spectra generated by the DU64 and the Spec 20. What differences do you observe? How do you explain these differences?
6. What is the function of a blank? (Look up the answer in your textbook.) Why do we need to use a blank every time we change the wavelength on the Spec 20 but do we not need to do this with the DU64?
7. For a given sample, what is the relationship between absorbance and transmittance? Explain by referring to your data and your graphs.
8. What happens to the spectra when two food colors are combined? Is the resulting spectrum a combination of the two original spectra?

Materials To Hand In

• Copies of lab notebook pages (before leaving the laboratory).
• Your graphs (or photocopies of them).
• Answers to discussion questions.

Mmaterials And Preparations

Dilute the food coloring before using it in the Spec20 because undiluted food coloring absorbs far too much light to be readable in the spectrophotometer. This dilution can be done “by eye”; it is not necessary to know the concentration of dye for this activity. A few drops in a one liter flask is a good beginning.

Contact Us:

Lisa Seidman
lseidman@matcmadison.edu
(608) 246-6204

Jeanette Mowery
jmowery@matcmadison.edu
(608) 243-4307