Assay Using Spectrophotometry

Blue is the color…

Spectrophotometers are instruments that measure the effect of a sample on a beam of light at a specific wavelength. In biotechnology, we often use spectrophotometers to detect the presence and quantity of particular molecules of interest. For example, we can use spectrophotometer to measure how much DNA is present in an extract, or how much protein is in a sample, or the activity of an enzyme (a protein), or in a purification scheme, how pure is the protein in the sample. To detect proteins and nucleic acids, you need a spectrophotometer that produces light in the ultraviolet (UV) range. UV spectrophotometers are considerably more expensive than the visible range spectrophotometers that we are using in this exercise with colored materials. (But the visible spectrophotometer costs around $2000.00; it is not cheap!)

This activity will use bromophenol blue as the colored solution. You will determine the Amax of this solution first, and then use that experimentally-determined Amax to create a standard curve of bromophenol blue at various concentrations, using a dilutions technique with one of several available volumetric measuring instruments. Next, you will use your standard curve to determine the concentration of a bromophenol blue solution Unknown. Finally, you can pool your class results to compare the accuracy of the different volumetric instruments provided, and you can also determine precision.

SOP Spectrophotometer GENESYS 20

1. Turn power ON (back of machine left side)

2. Warm up 30 minutes.

3. Hit A/T/C until A (Absorbance) shows in display.

4. Use arrows to select desired wavelength (in nanometers – nm).

5. Open sample compartment.

6. Wipe fingerprints and smudges from sample tube using a kimwipe and insert the appropriate blank into cell holder. (water, broth, buffer, etc.) Use cuvette or test tube with 10 mm diameter.

7. Close sample compartment.

8. Press 0 ABS to set blank to zero absorbance.

9. Open sample compartment.

10. Remove blank, insert sample, close compartment, read sample.

Use of Volumetric Measuring Tools

To use the 1-mL transfer pipette, use the bulb on the top of pipette to draw up the liquid sample as well as to deliver the sample.

To use the 1000 ml micropipettor, put a blue tip on the micropipettor, make sure the m/pipettor is set to 1000, depress the plunger on the top of the instrument to first stop to expel the air, insert tip into liquid sample, and slowly release plunger to draw up liquid sample. To deliver liquid sample, insert tip into container, depress plunger to first stop and to second stop, pause, and then remove tip from container before releasing plunger.

To use the serological pipette, remove wrapper and insert pipette into barrel of red pi-pump. Insert pipette into liquid sample. Use the wheel-shaped dial on top of pi-pump to draw up the correct amount of liquid into pipette. To transfer liquid, move the pipette into next container, and either use dial or the release lever on side of barrel on pi-pump to deliver the sample into next container.

Part I. Determination of the A_max of Bromophenol Blue

Amax is defined as the wavelength where a given sample has maximal absorbance.

Materials

Beaker of distilled

Bromophenol blue solution

Spectrophotometer

Cuvettes (or test tubes)

1000 mL Micropipettor and tips

pipette pump with 5mL serological pipette

1mL Transfer pipette

10mL Graduated cylinder

Method

Start by reading the SOP or Standard Operating Procedure for use of the Spec 20 Genesys. This spectrophotometer must warm up for thirty minutes before it is ready to be used.

1. Fill a cuvette with 2.5 – 3mL of distilled water for the “blank” and another

with the Bromophenol blue solution. Use one of the volumetric measuring tools for sample transfer.

2. Open the sample compartment door; insert the “blank,” press the A/T/C soft key

until an A (for Absorption) reads on the LCD screen. Next, using the up or down

key, set the wavelength for 480 nanometers (nm). Finally, press the 0 ABS key

to set the blank.

3. Remove the blank and insert the cuvette containing the Bromophenol blue

solution. A reading will appear on the LCD screen. Record that reading next to

the wavelength of 480 nm in the chart below.

4. Repeat steps 2 and 3, changing the wavelength as indicated by the table. Record

each reading in the chart. Remember to zero the blank at each new wavelength

between each reading.

5. Rinse out cuvettes with distilled water when complete and allow to dry.

Results of Absorbance Spectrum

Wavelength	Absorbance
480 nm
490 nm
500 nm
510 nm
520 nm
530 nm
540 nm
550 nm
560 nm
570 nm
580 nm
590 nm
600 nm
610 nm
620 nm

6. Graph the data on the attached

sheet. The independent variable

is the thing you change

(wavelength). The independent

variable is always plotted on the

X-axis (horizontal). The

dependent variable is the thing

you measure (absorbance) and

should be plotted on the Y-axis

(vertical).

__________________________________(title)

Absorbance
	450 470 490 510 530 550 570 590 610 630 Wavelength in nanometers (nm)

What is the A_max of Bromophenol Blue? _________

Part II. Determine an Unknown Concentration of Bromophenol Blue

In this exercise, you will make what is known as serial dilutions. A dilution is done when you add a solvent (usually water) to a stock solution to make it less concentrated. Using the Amax wavelength you determined for bromophenol blue from previous activity, you will make absorption measurements of known dilutions of the stock 18.6 mM bromophenol blue solution to create a standard curve (actually a line). Then you will graph the data you collect from the dilutions. From this graph, you will be able to determine the concentration of an unknown sample according to its absorbance. This is one purpose for using a spectrophotometer and dilutions in the laboratory.

Method

1. Obtain four numbered cuvettes and one cuvette with distilled water to blank.

2. Using the volumetric instruments assigned to your group (1000 mL micropipette, transfer pipette, 5 mL serological pipette, or 10 mL graduated cylinder) transfer 2000mL of distilled water into each cuvette. Circle which instrument you are using.

3. Add 2000mL of 18.6 mM bromophenol blue to cuvette 1. This cuvette represents a ½ dilution. Mix.

4. Transfer 2000mL from cuvette 1 into cuvette 2. This is a ¼ dilution (1/2 of ½ = ¼). Mix

5. Transfer 2000mL from cuvette 2 into cuvette 3. Mix.

6. Transfer 2000mL from cuvette 3 into cuvette 4. Mix.

7. Remove 2000mL from cuvette 4 and discard down the sink.

8. Set the Spec 20 to the wavelength of highest absorbance that you determined in the previous lab.

Remember to zero the spectrophometer using your blank of distilled water. There is no need to blank between samples.

9. Record the absorbance for cuvettes 1 through 4 and complete the table below.

10. Obtain the sample of Bromophenol Blue of an unknown concentration. Transfer 2000μL of the unknown into a clean cuvette. Find the absorbance using the spectrophotometer. Use your “best fit” line from the graph of tubes 1 through 4 to determine the concentration of Bromophenol Blue in the unknown.

Cuvette number	Dilution	Concentration of Bromophenol Blue (μM)	Absorbance
1	½	9.3 mM
2	¼
3
4
Unknown	----

11. Graph your results below and plot a line of “best fit.” Be sure to label the axes of the graph.

Absorbance vs. Concentration of Bromophenol Blue

Challenge Questions:

1. From the graph above, if the concentration of Bromophenol Blue was 3.5μM, what would its absorbance be? ________________

If your sample had an absorbance of 0.32, what is the concentration of your

sample? _______________

2. How would you describe the relationship between absorbance and concentration?

_________________________________________________________________

_________________________________________________________________

3. List ways spectrophotometry might be useful to scientists.

4. Look over this graph below carefully, and determine WHAT IS WRONG WITH THIS GRAPH:

a. the independent variable should be on the X-axis

b. concentration should be on the Y-axis

c. the intervals of concentration on the X-axis are incorrect

Explain your answer:

Part III. Accuracy and Precision of Volumetric Measuring Instruments

Fill in the chart below, pooling the results of the different measuring instruments used by the class members:

Cuvette number	Dilution	Concentration of Bromophenol Blue (μM)	Abs 10 mL grad cyl	Abs 1 mL tfr pipt	Abs 5 mL ser pip	Abs m/pip
1	½	9.3 mM
2	¼
3
4
Unknown	----

Accuracy – is an expression of how close a measurement tool comes to the true or accepted value – in order to find out how accurate your determination of the Unknown bromophenol blue solution was to the actual value, contact OCCC and ask for the value:________________________________________

Results:

• how did the value YOU determined for the Unknown compare with the actual value:____________________________________
• Was one measuring tool used in class more accurate than the others?

___________________________________________________________________

Precision – (repeatability) is an expression of how close in value are repeated measurements on the same instrument. One simple way of indicating precision is looking at the range of values: range is the different between the highest and lowest values of a set of measurements.

Suggest a procedure that you could use with bromophenol blue, your measuring tool, and the spectrophotometer, to determine precision of your measuring tool. If there is time, perform the experiment and write up the results.

Optional: A more common way to evaluate the variability of a set of measurements would be to calculate the mean and then the standard deviation. The lower the standard deviation, the better the precision. Look up the formulas for calculating mean and standard deviation, perform on your set of repeated measurements, and re-evaluate the precision of your tool.