Synopsis

This exercise is designed to introduce the science major to the spectrophotometer and the production and use of the standard curve. The students are introduced to light and given its basic properties and characteristics. The students are then introduced to the spectrophotometer and how it uses light to measure Absorbance and % Transmittance. The use of a blank in spectrophotometry is also presented.

An experiment to determine the Absorbance maximum of Biuret reagent (a protein stain) is performed by the student. The student carries out this experiment, graphs the results using a computer graphing package, and interprets the results.

In a second procedure, the student is then introduced to the concept of a serial dilution and the standard curve. Then a simple serial dilution is carried out by the student, Absorbance is measured and a standard curve for BSA stained with Biuret reagent is generated. The student also measures the Absorbance of a solution of BSA of unknown concentration and then uses their own standard curve to determine the concentration of the unknown.
Specific questions about what they did concludes the experiment.

Technical guide

1. Hazards: none known
2. Reagents
   a. Use a high quality grade of BSA such as Sigma #A-4378. Lower grade BSA tends to not be homogeneous in solution and gives highly variable Absorbance readings.
   
   b. In using the Biuret reagent recipe included in this guide, be aware that Biuret reagent is easily purchased from a number of vendors but the absorbance intensity of the reagent will vary by vendor. We have found that making your own is more reliable. Regardless of your Biuret reagent source, it is best to run a test of the Absorbance level of the various Biuret stained BSA concentrations to make sure that the readings will be on scale. Dilute Biuret reagent if necessary. We have not had any trouble with the Biuret reagent degrading with time.

List of Supplies (for a class of 24, working in pairs)

200 or more cuvettes or very clean test tubes, 13 x 100mm

(Fisher # 14-962-10c borosilicate disposable culture tubes make a good and inexpensive alternative to cuvettes and can be reused until they become hard to clean or scratched)

(Place 45g of sodium potassium tartrate and 15g of CuSO ₄ -H ₂ O in a one liter volumetric flask. Fill to about 3/4 full with 0.2N NaOH and shake to dissolve. Add 5g of potassium iodide, let dissolve and then bring the total volume to one liter with 0.2N NaOH.)

lots of small beakers (so that the students are not constantly getting into the stock solution bottles)
3 unknowns of BSA stained with Biuret reagent (any conc. that is "on scale")

Instructor guide

This exercise is designed to introduce the science major to the spectrophotometer and the production and use of the standard curve. It includes the concepts of the blank in spectrophotometry, the graphical treatment of data and a simple interpretation of the experimental data. Techniques utilized include use of the spectrophotometer, micropipetors, and computers with graphical analysis software. It assumes that the students have been introduced to the use of micropipetors and computers with graphical analysis software in earlier lab exercises. Both the Helms lab manual (1) and the Bloom, Freyer and Micklos lab manual (2) have labs that introduce the use of the micropipets. The Cricketgraph™ graphing software package includes a tutorial. A graphing exercise for use with a graphing software package is included in this package of lab exercises.


Tips to make the exercise run more smoothly

1. Stress student diligence in use of the micropipetors, setting up the experiments and in taking the Spec. readings. It will be reflected in their results. They are prone to making mistakes in setting up their dilutions and need to be monitored fairly closely to avoid using up large volumes of the reagents.

2. General spectrophotometry guidelines

1. Let the spec. warm up for 15 minutes.
2. Tubes need to be at least half full.
3. Make sure that the spec. tubes are clean and free from scratches.
4. Make sure that all of your readings are with the same side of the tube forward (most spec. tubes have a vertical line on the front to help with this)
5. Make sure the solution is free from bubbles.
6. Recheck the "0" Absorbance reading with the blank often to make sure your Spec is free from drift.

3. The Absorption spectrum of Biuret reagent is measured only to 600nm because many specs do not read above this wavelength.The Absorbance maximum is about 580nm or somewhat higher. The students can safely use 580nm for Exercise B and will get good results.

4. A note about setting and interpretation of the spec scale: The generalized idea about 100% Transmittance being no absorption of light and 0% Transmittance being total absorption of light (p. 3 & 4 of the lab exercise) is not completely accurate because the amount of light transmitted at these points depends on how the scale is set. However to explain this fact when learning about the spec for the first time, in my mind, seems unnecessary.


5. We find that the concentration of Biuret reagent listed in this exercise gives us nice, on scale Absorbance readings. However, a prudent precaution would be to run the exercise once before use in the classroom and adjust the concentration of Biuret reagent accordingly.

6. We find that the students have a little trouble applying C ₁ V ₁ = C ₂ V ₂ to the final concentrations in exercise B. Remember that C ₁ is 0.01mg/ml for all tubes, V ₁ changes with each tube and V ₂ is 7ml for all tubes.

Answers to questions

1. Answers to questions on page 8

1. 580nm; 580nm, changes in absorbance will be easy to detect at this strongly absorbed wavelength.
2. Because you want to eliminated the absorbance due to water.
3. Because water and Biuret reagent will have different absorption maxima and the water must be blanked out at that particular wavelength.
4. No, just the relative amount of absorption would change not the maximum, since the chemical composition of the Biuret reagent is not changed.
5. Green; all except green.
6. All available light
7. Since various wavelengths of light are differentially absorbed by a single compound, using more than one wavelength of light would not yield a true reading of the amount of light absorbance that could be compared to any other sample.
8. About 1nm to 380nm.

2. Answers to questions on page 13 & 14

1. Because you want to measure only the Biuret reagent that is bound to protein.
2. In making a standard absorbance scale for the concentration of a certain compound, you have made a scale which can be used over and over again to determine the concentration of that compound in that same assay without having to repeat the entire assay
3. No, different compounds have different absorbance characteristics.
4. No, the background chemicals have not be properly blanked out.
5. There are many scientific examples but applied examples included determination of drugs in urine samples and amount of protein or glucose in urine (although other, more sensitive, tests are routinely used for these applications now).
6. This is the easiest way:
   a. Place one volume of the 0.5M arg in 9 volumes of water.
   b. Place one volume of the 1 in 10 diluted arg in 9 volumes.
   c. Place one volume of the 1 in 100 diluted arg in 9 volumes of water. (This last tube contains 5mM arg.)

Bibliography

1. D.R. Helms. Biology in the Laboratory. 2nd Ed. Worth Publishers. 1994.

2. M.V. Bloom, G.A. Freyer and D.A. Micklos. Laboratory DNA Science. Benjamin/Cummings Publishing. 1996.

If you have any questions or suggestions for improvement, Charlie Hoyt can be reached at (619) 421-6700 x5528 or e-mail at choyt@.swc.cc.ca.us

This lab exercise was developed in part with the support of National Science Foundation (Division of Undergraduate Education) grant # DUE 9552290 and California Community College Chancellor's Office (Curriculum and Instructional Resources Division, Special Projects) grant # FII 95-621-001.

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