Laboratory
Exercise 12:

Standard Curves with Food Coloring

Purpose

In this exercise relating to spectrophotometry, you will prepare a series of standards of known concentration for red food coloring. To do this, you will begin with a stock solution of red food coloring and you will progressively dilute it to lower and lower concentrations. You will then measure the absorbance of the standards and plot them versus their concentration to get a linear standard curve. The purpose of this standard curve is to find out the concentration of food coloring in unknowns, provided by the teacher. If you pipette your dilutions accurately, then the absorbances for the standards will all fall on a straight line and the values you get for your unknowns will be correct. If your pipetting is not very good, then the points will not form a straight line and your answers will be “off.” Thus, in this exercise you will:

• Learn how to use the DU64 spectrophotometers to obtain a standard curve.
• Learn how to use a standard curve to determine the concentration of analyte in a sample.
• Practice dilution calculations.
• Improve your ability to pipette accurately.

Brief Background Review

A. Standard Curves and Quantization

In the previous laboratory exercise, you explored the relationship between absorbance and concentration. Now, you will see how this relationship can be exploited for quantitative analysis. Quantitative applications of spectrophotometry have the purpose of determining the concentration (or amount) of an analyte in a sample. Determining the concentration of analyte in a sample is an extremely powerful application of spectrophotometry that is widely used in biology laboratories.

Quantitative analysis with a spectrophotometer typically involves a “standard curve,” which, for spectrophotometric analysis is a graph of analyte concentration (X axis) versus absorbance (Y axis). To construct a standard curve, standards are prepared with known concentrations of analyte. The absorbances of the standards are determined at a specified wavelength and the results are graphed, Figure 1. Given a standard curve, it is possible to determine the concentration of an analyte in a sample based on the sample’s absorbance.

In this laboratory exercise you will first prepare a series of standards of red food coloring. In the previous laboratory exercise these standards were made for you. This time you will prepare them yourself. Then, you will use the standards to construct a standard curve “automatically,” using the DU64. The DU 64 uses software called Quant II to prepare and print out a standard curve. You will then use the Quant II program to determine the concentration of two unknowns.

FIGURE 1

Figure 1. A STANDARD CURVE.

B. Standard Curves: Linear Range

At lower standard concentrations, a plot of concentration versus absorbance forms a straight line. But as the concentration of the standards increases, the plot begins to curve, Figure 2. This is because at a certain point, the samples absorb so much light that the spectrophotometer is “maxed out.” Usually, one works with standards and samples (unknowns) whose absorbances are in the linear range. Standards or samples with absorbances above the linear range are diluted. If your samples have absorbances that fall outside the linear range of the spectrophotometer, then be sure to dilute them. You will then need to calculate the concentration in the original undiluted sample.

FIGURE 2

Figure 2. The effect of stray light If there were no stray light, the relationship between concentration and absorbance would be perfectly linear. Stray light causes the relationship to deviate at higher absorbance levels. The lower the absorbance level where deviation occurs.

Laboratory Activities

Part A. Dilutions

Work with a partner but have each student prepare their own dilutions. Compare your results.

A1. You will be given a sample of red dye at a concentration of 25,000 ppm.

A2. What wavelength should you choose to construct a standard curve for red dye? _____________

A3. Set your spectrophotometer for the proper wavelength.

A4. Check the absorbance of the red dye. ______________________

A5. Oh no! What is the problem here? __________________________

A6. I will tell you that for red food coloring, concentrations in the range of about 1 - 70 ppm will yield absorbance readings that are in the linear range of the spectrophotometer. This means that you must dilute the red dye quite a lot before you can construct a standard curve. There are many dilution strategies. The best one depends on the volumes you need, the glassware you have available, and the pipettes you are using. I will show you one strategy to figure out how to dilute the red dye appropriately. You are welcome to devise your own strategy if you would prefer.

A7. Diluting The Red Dye: (Remember to Mix Well at Each Step!)

• Prepare 10 ml of 1000 ppm stock from the original sample.
  You can approach this as a C₁V₁ = C₂V₂ problem.
  mL of original red dye required _______
  mL water required _______
• Using your 1000 ppm stock, prepare 20 mL of 100 ppm stock.
  mL of 1000 ppm stock red dye required _______
  mL water required _______
• Using your 100 ppm stock, prepare 40 mL of 10 ppm stock.
  mL of 100 ppm stock red dye required _______
  mL water required _______
• Using your 10 ppm, 100 ppm and 1000 ppm stocks, prepare 10 mL of standards of 4 ppm, 6 ppm, 8 ppm, 10 ppm, 50 ppm, 75 ppm, 100 ppm, and 200 ppm. (We expect some of these to be above the linear range for red food coloring.) It is useful to construct a table, such as shown below before you prepare the final standards.

PREPARING STANDARDS
STANDARD	PPM IN STANDARD	STOCK TO USE	ML OF STOCK REQUIRED	ML WATER REQUIRED
1	4
2	6
3	8
4	10
5	50
6	75
7	100
8	200

• Prepare the standards according to the table you prepared.
• Check your work. All the standard tube should have the same volume and you should be able to see the color in the standard tubes get progressively darker as the concentration increases. If not, something is wrong

A8. Prepare a standard curve. Measure the absorbance for each standard. Graph the data with absorbance on the Y axis and concentration on the X axis and have the graph checked by your instructor. The better your pipetting technique, the closer all your points will be to forming a straight line.

A9. Use your standard curve to determine the concentration of food coloring in two or three unknowns. If the concentration of food coloring in the unknowns is too high to be read by the spectrophotometer, then dilute them. Calculate the concentration in the original, undiluted sample by multiplying by the dilution factor.

Part B. Preparing a Standard Curve with the DU 64

Preparing and graphing a standard curve by hand (as you did in the previous laboratory exercise) is time-consuming. So, it is often preferable to do this task on a spectrophotometer that can automatically plot your data. The DU 64 can be used to conveniently prepare a standard curve and to use this standard curve to determine the concentration of analyte in an unknown sample. It takes some patience and practice to learn to use the computer program that controls the DU 64 because it is a relatively old program and it is sometimes awkward. You cannot go backwards in the DU 64 program. Nonetheless, the DU 64s are excellent spectrophotometers and once you learn to use them they are fast and convenient. To prepare a standard curve with the DU64, follow these directions:

B1. Insert the Quant II linear module into the right port of the DU 64.

B2. Press PROG key to enter the directory.

B3. Press the STEP key till the display reads PROG 6:QNT2LIN.

B4. Press R/S key. The DU 64 will begin to run the program to generate your standard curve.

B5. 0 TO EDIT 1 TO RUN: The instrument is asking whether you want to edit or run the program. Press 0, ENTR so that you can edit the program for your standards.

B6. ANAL WL: The program is asking you what wavelength to use. I suggest 517 for the red dye. Type 517, ENTR.

B7. NO OF BKG WL: It is possible to subtract out background automatically with this program. We will not do this. Hit 0, ENTR.

B8. NO. OF REPLICATES: Enter the number of replicates of each sample. Use only one standard at each concentration. Therefore, type 1, ENTR.

B9. NO. OF CELLS: 1 ENTR.

B10. WT:VOL: This is an option we will not use. Using this option, one can compare unknown samples of the same relative composition without obtaining identical weights. Press 0, ENTR.

B11. NO OF STDS: You will begin with 8 standards, therefore, hit 8, ENTR. The instrument will next prompt you to tell it the concentration of each standard. The first is 2 (units are ppm, but the instrument is not "interested"), the second is 4, the third is 6, the fourth is 8, the fifth is 10, the sixth is 50, the seventh is 75, and the last one is 100.

B12. STD 1 CONC: Hit 2, ENTR. Repeat by typing in the value for each standard when prompted.

B13. 0 TO EDIT 1 TO RUN. The program is now back to the beginning, asking you if you wish to edit the program or to run it. If you made a mistake in editing, you can correct it. To edit some more, push 0, ENTR and you will be back in the editing mode. Otherwise, push 1, ENTR.

B14. 0 TO CALB 1 TO RUN: The instrument is now ready to calibrate (blank). Make sure the proper light source is on. Press 0, ENTR to instruct the instrument to calibrate.

B15. INS BLNK R/S TO CALB: The instrument is prompting you to place a cuvette of blank in the holder. Press R/S. The instrument will automatically calibrate. The blank in this case is water.

B16. RUN(0=STDS 1=SPLS): The instrument is asking whether you are going to insert standards or sample. You have not yet run the standards, so hit 0, ENTR.

B17. INS STD 1 PRESS R/S: Place standard 1 in the sample compartment and press R/S key. The instrument will read the standard and print out its absorbance in a table.

B18. INS STD 2 PRESS R/S: The instrument will continue to prompt you for each of the standards. It will stop prompting when all the standards have been read.

B19. 0=STD 1=PLOT 2=SPL: Press 1, ENTR and you should get your standard curve plot. In this case, all the standards are in the linear range of the instrument and should fit a line fairly closely.

B20. 0=STD 1=PLOT 2=SPL: Now run your unknown samples. Press 2, ENTR.

B21. INS SPLS R/S TO CONT: Place the sample in the sample compartment and press R/S key. The sample's absorbance will be read, its concentration will be calculated automatically, and the data will be printed with statistical information.

B22. Repeat with the second sample.

B23. If the concentration of food coloring in the unknowns is too high to be read by the spectrophotometer, then dilute them. Calculate the concentration in the original, undiluted sample by multiplying by the dilution factor.

B24. Repeat steps B1-23, but his time, use only the first (least concentrated) five samples instead of all eight. The purpose of this is to see what happens when some of the standards are too concentrated. In principle, when you remove the more concentrated standards, the values for your unknowns might change slightly. Why?

Discussion Questions

1. Compare your standard curve and your values for the unknowns with your partner. How close were they? What does this tell you about your pipetting technique?
2. Compare the results you obtained using just 5 standards to those obtained with all 8. Which gives more accurate values for the unknowns? Explain what it means to be in the linear range of the standards.
3. Check the answer key to find out the concentration of red food coloring in the unknown samples. How close were your values for the unknowns to what you expected based on the answer key? Calculate per cent errors for each value. Discuss these results. Remember, the whole point of preparing a standard curve is to find the concentrations of analyte in the unknowns.
4. Compare an absorbance spectrum to a standard curve. What goes on the X and Y axis in each case? What is the purpose of an absorbance spectrum? What is the purpose of a standard curve?
5. Calculate the absorptivity constant for red food coloring. Show your calculations and explain.

Materials to Hand In

• Turn in the copies of lab notebook pages from your laboratory notebook before leaving the laboratory.
• All graphs.
• Turn in the answers to discussion questions, either in your lab notebook, or on a separate sheet of paper, as directed by your instructor.

Materials and Preparations

Unknowns for each group. There should be three unknowns whose values range from 2 ppm to 90 ppm.

Contact Us:

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

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