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STUDENT OBJECTIVE

Students learn to pipette and use balances. Percentage error is introduced. Students also learn to use a spectrophotometer to obtain an absorption spectrum for bromophenol blue. Dilutions of the stock dye solution are used to construct a standard curve. Using this curve, students determine the concentration of an unknown solution. Students explore variations in data and simple statistics.

EQUIPMENT	AMOUNT
	(Class of 24 with 8 groups)
Spectrophotometers (400 nm—600 nm)	8/lab
Balance (minimum sensitivity 0.1g)	2/lab
Analytical balance	Instructor use only
Materials
Pipettes, 5 ml x 0.1 serological	2/student
Beakers, 50 ml or 100 ml (for water)	2/student
Suction device, 10 cc disposable syringe with 1" rubber tubing attached (see lab manual fig. 4.3)	1/student
Cuvettes (color coded for dye and water)	16/lab
Tube racks, nylon or plastic (for cuvettes)	8/lab
Beakers, 100 ml (coded for dye)	8/lab
Beakers, 100 ml (coded for distilled water)	8/lab
Pipettes, 10 ml serological (coded for dye)	8/lab
Pipettes, 10 ml serological (coded for water)	8/lab
Test tube racks	8/lab
Test tubes	72/lab
Squeeze bottles with distilled water	8/lab
Squeeze bottles with dilute bleach	2/lab
Squeeze bottles with detergent	2/lab
Tissue, blotting	8 boxes/lab
Spectrophotometer lamps (spare)	4/lab
OPTIONAL
Variety of laboratory dispensing devices—Mohr, serological, volumetric, automatic, etc.	Display
SOLUTIONS
Distilled water	1 liter/lab
Bromophenol blue (20 mg/liter) (CBS#84-9082) *	1 liter/lab
Unknown: students determine dye concentration	200 ml/lab

*Please refer to the Appendix for name and address of supplier.

PREPARATION

One Week before Lab

1. The stability of spectrophotometers should be checked and bulbs replaced when needed. Older instruments should be turned on for 15 minutes to reach a steady state before students use them. Where there is an erratic reading, the bulb may need replacing.
   
   
2. Stock bromophenol blue preparation:

   Weigh accurately 20 mg bromophenol blue and add to a 1 liter volumetric flask; dilute to mark with distilled water. Mix well. Color will be wine red. Solution should have an absorbance of about 1 at 580 nm. Stability: one month.

   Students will find that the maximum absorbance for bromophenol blue will be 580—600 nm.

   If 20 mg cannot be weighed accurately, make a 10x solution and dilute.

3. Bromophenol blue "UNKNOWN" solutions preparation:

NOTES

1. Bromophenol blue will stain hands and clothing. (Commercial bleach removes stains.) Old newspapers should be spread to prevent permanent staining of work benches.
2. To lessen the chance that students will cross-contaminate reagents in the laboratory, it is a good idea to color code all pipettes and solution containers. Use colored autoclave tape: containers and pipettes for each stock solution should be marked with a color code for that solution.
3. To prevent the students from interchanging the materials in the two parts of the exercise, balances and glassware for pipetting should be located in one area of the classroom while all materials for spectrophotometry should be in a box (plastic crisper) next to each spectrophotometer.
4. Students should be encouraged to clean and rinse all glassware during the exercise. Several wash bottles of detergent and bleach can be provided at the sinks. A tube brush may also be available but make certain it is a size larger than the cuvettes so that students cannot accidentally use it to clean the cuvettes. Use only bleach, strong detergent, and/or distilled water to clean and rinse cuvettes. The use of a brush in a cuvette can scratch it and yield incorrect readings. If bleach is used, thoroughly rinse glassware and cuvettes at least five times before proceeding with the exercise.
5. Students often confuse cuvettes with test tubes if they are of similar size and shape. If possible, use test tubes that look distinctly different than your cuvettes.
6. A tray of various types of dispensing devices can be displayed to show students the variety available. In addition, volumetric flasks can be shown to help discuss precision and accuracy in quantitative chemical procedures.
7. Check spectrophotometers for stability often. Before attempting repair have students show you their blanking procedure. Most "bad specs" are operator error. Most malfunctions of spectrophotometers occur with the lamps or the shutter. Erratic zeroing usually indicates need for lamp replacement. If the needle on the meter does not return to zero after sample removal, the shutter should be checked for cleanliness. Occasionally, deposits from broken cuvettes will build up and cause the hinge to stick in the open position. If students do not always place cuvette into the spectrophotometer with index line in same position, slight variations in readings will occur.

CLASSROOM SUGGESTIONS

1. Students enjoy this exercise, especially the opportunity to try their skill in determining the concentration of an unknown. They can complete the exercise in 2 to 2 1/2 hours.

   Those seeking a challenge could determine the absorption spectrum of a carotenoid extract. They can prepare their own extract by blending half of a carrot (4") in a spark-free blender with 100 ml of 70% ethanol until a slurry is made. Filtering with a Buchner funnel should produce a good extract. The use of ethanol as the blank will help reinforce their understanding of the use of a spectrophotometer.

2. We have found it instructive to place an extra cuvette at each work station. This cuvette contains a strip of white paper. In a dim room, students can insert the cuvette into a spectrophotometer and look into the cuvette while turning the wavelength adjustment knob. This demonstrates the colors of the visible spectrum at different wavelength settings.
3. A minimum homework assignment would be to hand in the absorption spectrum and standard curve graphs, calculation of means and standard deviations from class data for water weight.
4. Check out the links for this lab topic at http://auth.mhhe.com/biosci/genbio/dolphin/ You will find useful materials for developing your lab introduction or summary, and in some cases, you may want to tell students to connect to a partiuclar site for further information.

ANSWERS TO CRITICAL THINKING QUESTIONS

1. The optical properties of each cuvette in a set are the same, or "matched," so that readings can be compared without worry that the glass in the cuvette somehow affected the results. However, fingerprints on the outside of the cuvette can affect results and need to be wiped off before the cuvette is placed into the spectrophotometer. The volume of sample must be at least to the horizontal line on the cuvette, otherwise the beam of light may not pass through the sample, producing erroneous results. As well, the cuvette must be properly aligned within the spectrophotometer so that the same glass surface is always exposed to the light beam. To do this, the vertical line etched onto the glass cuvette should be aligned with a notch inside the sample well.
2. Refer to Appendix C—In a "normal" frequency distribution the "mean" coincides with the "mode" (the most frequently occurring value) and is also the median value. One standard deviation includes 68% of the data (that is +34% and —34% for a total range of 68%).
3. Grading on the curve is based on student performance. An instructor will create a histogram of the numerical scores earned by students in a class. An average will be calculated and made equivalent to a particular grade, say C. A standard deviation will be calculated and those above the high cutoff may be assigned A’s and those below the low cutoff may be assigned F’s. There are no hard and fast rules, but this type of analysis is based on using the concepts of averages and standard deviations.

SUPPLEMENTAL MATERIALS

Use of a Spectrophotometer, 21—minute video cassette. Columbus, OH: EPA Instructional Resources Center. #VC-57