Laboratory Activity: Teacher Notes

Activity 1: Introduction to the Spectrophotometer

Major Chemical Concept

The quantity of light absorbed by a solution is commonly expressed either in terms of percent transmittance (%T) , as in Part I of this activity, or in terms of absorbance (A). Absorbance is defined as:

Transmittance (T) is usually expressed as %T = Tx100%, although it actually is defined as:

I 0 is the incident light and I is the transmitted light. Absorbance may be obtained from %T by the equation:

This last equation is used to convert %T to A. The absorbance scale is a logarithmic scale. As absorbance increases, scale markings get closer together, and the meter becomes more difficult to read. Thus it is frequent practice to read %T and convert to A. For convenience a table showing %T and the corresponding absorbance (A) is included in the Appendix.

For quantitative work the absorbance is more useful than the transmittance because it is directly proportional to concentration. The relationship is known as the Beer-Lambert Law and is:

where a (molar absorptivity or extinction coefficient) is a constant for a given absorbing chemical species, b is the radiation path length through the sample (measured in cm), and c is the stoichiometric molar concentration (mol/L) of the absorbing species. Thus for a given absorbing species at a specific wavelength in a given sample holder of fixed path length (b), a plot of A versus concentration is a straight line if the Beer-Lambert Law is obeyed.

To determine if the Beer-Lambert Law is obeyed over a given concentration range by a given species, measure absorbance as a function of concentration, using the same test-tube for all of the measurements. Plot absorbance vs. concentration; check the linear nature of the curve. If the curve is linear, then the slope of the line may be calculated and used to determine the concentration by dividing the absorbance by the slope. An alternative method of determining concentration from the calibration curve is to mark off the measured absorbance on the ordinate, draw a line perpendicular to the ordinate until it intersects the curve, then drop a perpendicular line to the abscissa from the intersection point. The intersection of the latter line and the abscissa gives the concentration. In practice, only the absorbance and concentration are variables and are in direct proportion, so A µ c, and A = k´C where k´ is the slope of the curve A vs. c.

Level

Part I of this activity can be used for all levels of students. For basic students, it might be best for you to collect the data with the assistance of several students. For general students, groups of two or three should be able to collect the data for Part I, but Part II might be better done as a class activity. Honor students should be able to do the entire activity.

Expected Student Background

This activity will probably be the first exposure students have with instrumentation. The experimental procedure is rather straightforward and should not pose great difficulty to students. By this point in the course, students should have the experience with routine laboratory procedures involving the use of glassware, how to clean glassware, and so forth.

Time

Teacher preparation time to mix the 0.020MCr(NO 3 ) 3 solution and set out the necessary equipment is about one hour. The spectrophotometers should be checked prior to use. The estimated time for students to perform the activity is50 min to complete Part I. Additional time up to 50 min may be required for Part II, particularly if students are required to prepare their own calibration curves.

Safety

Read the Safety Considerations in the Student Version . Safety goggles should be worn during the activity. The chromium(III) nitrate solution is moderately toxic and should not be poured into the drain. Students are instructed to return the solution actually used in the measurements back into the original container. If there is some possibility that the solution supply will be contaminated, then an alternate disposal procedure should be devised. If it has not been contaminated, the solution may be saved for later use. There are several sources referred to in SourceBook that give recommended disposal procedures (see Safety section). Local and state regulations must be followed. In view of the disposal problems, only the actual volume of solution needed with a modest excess should be prepared. Caution students to wash their hands with soap and water at the end of the activity

Materials (For 24 students working in pairs)

Nonconsumables

6 Spectronic 20 " spectrophotometers (per class)

24 Small test-tubes (13-mmx100-mm) or 24 glass cuvettes

12 Plastic wash bottles

Tissue paper (Kimwipes " or equivalent)

Buret, 50-mL

Volumetric flasks, 50-mL and 100-mL (5 of each) or graduated cylinders, 50-mL and 100-mL

Consumables

0.020M Chromium(III) nitrate solution, 250 mL (2.00gCr(NO 3 ) 3 . 9H 2 O per 250 mL solution)

Distilled water

Graph paper

Advance Preparation

You should perform the activity beforehand to be familiar with the procedure. Check the spectrophotometers to be certain they are operating correctly. Allow a 20 min warm-up period prior to use.

To prepare the solution, use 2.00 g Cr(NO 3 ) 3 per 250 mL of solution. Weigh the solute carefully. The unknowns are prepared by successively diluting the 0.020M solution using 100-mL volumetric flasks to bring the volume Cr(NO 3 ) 3 in Column 3 to final volume (Column 6). Use these solutions to prepare a calibration curve and student unknowns. Use a buret to measure all volumes. Have the 0.020M Cr(NO 3 ) 3 solution available in several plastic bottles, at least one per spectrophotometer. Make the dilutions according to the following table.

Pre-Laboratory Discussion

Demonstrate for students the techniques involved with using a spectrophotometer. In particular show them how to clean, fill, and place the test-tubes (or cuvettes) in the instrument. Show them how to set the percent transmittance to 0 and 100%. A transparency master showing a front view of the Spectronic 20 " with the controls identified is in the Appendix. Show students how to use the calibration curve to determine an unknown concentration.

Students should have little difficulty in preparing a data table. For Part I a simple three column table is needed. Part II needs a simple table to record the wavelength used, the percent transmittance recorded and the calculated absorbance. If necessary, help students design a data table.

A useful activity for the pre-laboratory portion of the activity is described in Demonstration 5 in the Atomic Structure module. A beveled piece of white chalk is placed in a sample tube in the cell holder of the Spectronic 20. Looking "down the tube" allows one to see the color of the selected light radiation. It is suggested that students view the color every 25-50nm in the visible range. The monochromator that is part of the Welch ChemAnal " System is another good way to show the colors of light used. Mount the monochromator separately on the optical bench, and use a white piece of paper as a screen to view the colors corresponding with light selected every 25-50nm wavelength. One advantage of the ChemAnal " monochromator is that the lid swings out of the way so that the "works" inside can be viewed directly while the instrument is being used, removing some of the "black box" aspects of the instrument.

Teacher-Student Interaction

Once students begin the laboratory activity, circulate among them and watch that correct techniques are being used. Pertinent questions may be asked at this time. In particular students need to be aware that the test-tube/cuvette needs proper alignment in the sample holder, that 0 and 100% transmittance needs to be checked during usage at each new wavelength, and that care should be exercised in reading the instrument's meter.

Anticipated Student Results

The following table shows typical data obtained in Part I of the activity. The data show one transmittance maximum at 500nm, thus the graph of the data also show one peak at 500nm wavelength corresponding to an absorbance minimum. Two absorption maxima at about 415nm and about 580nm are present. For Part II, the student should measure the absorbance at 580 nm, and then use the calibration curve to measure the unknown concentration to within ±5% of the accepted value [see Percent Transmittance (%T) to Absorbance (A) Conversions in the Appendix for conversions].

Answers to Implications and Applications

1. Two absorption maxima appear at the transmittance minima of about 415nm and 580nm.

2. The absorption maxima correspond to violet and orange light (400-450nm and 600-650nm, respectively).

3. On the basis of the answer to Question 2, a red colored solution would absorb a complementary color. In the case of Cr(NO 3 ) 3 the green color of the solution results because it absorbs its complementary magenta color and transmits green. Thus we expect that a red colored solution would transmit red light.

4. Two uses of an absorption spectrum are to identify an unknown substance and to determine the concentration of a solution of a known substance.

5. A sample substance in solution when placed in the path of light between the monochromator and detector of a spectrophotometer may transmit some, all, or none of light. Light absorption occurs at wavelengths at which the energy of light photons corresponds to the energy needed to excite electrons in atoms, ions, or molecules in the sample.

6. Although the unaided human eye can detect subtle differences in the intensity of colored light, it is difficult to quantitatively measure the differences. Thus, the spectro-photometer is an extension of the sense of sight because it enables the measurement of color intensity to be done routinely and accurately.

Post-Laboratory Discussion

During the post laboratory discussion, help students to graph A vs. l (nm). To measure the concentration of unknowns, absorbance maxima must be known in order to set the correct wavelength for the procedure. It would be useful to students to point out that the concentration of a substance is directly proportional to the area under the peaks in an absorbance spectrum.

For Part II, help students use the A vs. concentration graph. Have several copies and a transparency of the graph available. Use a hypothetical absorbance and the transparency to show how to read the concentration from the graph. Find the absorbance on the vertical axis and draw a line parallel to the concentration axis beginning at this point until it intersects the graph. Then draw a line from the intersection parallel to the absorbance axis until it intersects the concentration axis. The intersection gives the concentration of the unknown.Figure 6. Calibration curve of absorbance vs. concentration for Cr(NO 3 ) 3 at 415 nm with example of unknown determination.

Extensions

1. Students can be instructed in how to prepare their own calibration curve for this activity. This preparation would give them practice in quantitative subdilutions and calculation of dilution concentration. Each group of students could be assigned one subdilution, and data could be pooled.

2. Other solutions lend themselves well to this kind of study. The absorbing species in the chromium(III) nitrate solution is Cr(H 2 O)6 3+ ion. When iron(III) chloride is dissolved in 0.1M HCl to form a 0.10M solution, the ion FeCl 4 -is formed. A 0.30M Cu(NO 3 ) 2 solution that is also 2.0M in NH 4 NO 3 forms the Cu(NH 3 ) 4 2+ ion. These solutions can be used for a study similar to the one in this activity.

1. Laboratory technique may be assessed by noting how the experimentally determined concentration compares with the accepted value of the unknown concentration. Good technique would give a result within ±1%. A result within ±5% is quite acceptable and might be given a grade of "A." A result within ±10% would be a "B," and a result within ± 15% would be a "C."

2. Use a laboratory practical test to assess instruction. One possible test is to give students a solution of unknown concentration along with a calibration curve and the wavelength to use. Grade on the basis of how closely the student's obtained value compares to the known concentration ofthe solution.

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