There's A Dye In Your Drink

(Bring a computer (floppy) disk to class!)

Light

White light, such as sunlight, is composed of light of several different colors. This is observed by passing white light through a spectroscope, Figure 1. The white light is decomposed into its component colors which are displayed as a continuous spectrum.

Figure 1

A schematic drawing of a spectroscope, showing the spectrum of white light.

According to the wave model of light, light is energy which travels through space as a wave. The wavelength, l, of a wave is the distance between two identical points on the wave, Figure 2. The unit of wavelength in the nanometer, nm.

Figure 2

Light of different colors has different wavelengths, as shown in Figure 3.

Figure 3.

Continuous spectrum of white light

Color

A colored solution appears colored because molecules in the solution absorb only light of certain colors and not others. Red strawberry Kool- AID, for example, appears red because dye molecules in the solution absorb light of all colors except red.

Figure 4.

The amount of light absorbed by the sample is expressed in terms of the absorbance, A, defined as

A = log Io/I

where Io is the incident light intensity and I is the emergent light intensity. The smaller I is relative to Io , the greater is the fraction of light absorbed by the sample and the greater is the absorbance. The absorbance depends upon: the wavelength of the incident light, the concentration of the absorbing molecules, and on the sample thickness.

Absorbance depends on wavelength: The dependence of A on l for red KOOL- AID is shown in Figure 5.

Figure 5.

The absorption spectrum of red KOOL AID superimposed on the continuous spectrum of white light.

The maximum absorbance occurs at 500 nm, and the absorbance is zero at wavelengths above 600 nm. This means that the dye molecules in the KOOL- AID absorb light at wavelengths below 600 nm, which correspond to the colors orange, yellow, green, blue and violet . Red light is not absorbed, and the solution appears red.

Absorbance and sample thickness: The absorbance of a solution increases with the sample thickness. This is evident if we compare the intensity of transmitted light along the long axis of a pitcher of KOOL- AID with the intensity viewed perpendicular to the pitcher axis.

Figure 6.

Absorbance depends on concentration: The absorbance of a solution increases with the concentration of the absorbing species.  Absorption of light is commonly used to quantitate blood glucose levels.  Since this is a blood free lab, we will use Gatorade instead.

The orange color of melon-flavored Gatorade is due to the presence of  food coloring dyes: FD & C Red # 40 and Yellow 6. The maximum absorbance of the Red #40 dye occurs at 502. nm. In this experiment you will prepare a calibration graph of absorbance vs concentration using standard solutions of Red # 40, and then use this calibration graph, together with absorbance measurements on the Gatorade, to determine the concentration of Red # 40 in the drink.

Procedure

Calibration curve: An aqueous stock solution of Red #40 which is approximately 2x10-4 M (the exact concentration will be on the label) will be provided by the Stockroom. Prepare the standard solutions below:

Solution A 5 mL of stock solution diluted to 25 mL.
Solution B 5 mL of stock solution diluted to 50 mL.
Solution C 5 mL of stock solution diluted to 100 mL.
Solution D 5 mL of stock solution diluted to 200 mL.

Prepare Solution A by transferring  5 mL of the stock solution (using a syringe) into a clean 25 mL volumetric flask. Carefully fill the flask to the mark with deionized water. Stopper the flask and mix the solution thoroughly by inverting the flask several times. Repeat the procedure with 50 mL,100 mL and 200 mL flasks, respectively, to prepare solutions B, C and D.

Measure the absorbance of each of the four standard solutions A, B, C and D with the Spectronic 20 spectrophotometer. Follow the instructions below.

1. Be sure that the instrument is turned on and plugged in.  

2. Set the wavelength knob to 502. nm.

3. Using the zero adjust knob on the left side, set the needle to read 0% transmittance (% T) on the top scale of the meter. Nothing should be in the sample compartment.

4. Fill one cuvette with deionized water, wipe it with a tissue, and insert it in the sample compartment with the line on the cuvette aligned with the line on the sample holder. Close the cover.

5. Use the 100% adjust knob on the right hand side to set the needle to read 100% T with the water-containing cuvette in the sample holder. Remove the cuvette and set it aside.

6. Rinse the other cuvette with one of the standard solutions, and fill it with the solution. Wipe it with a tissue, and insert it in the holder. Make sure that the cuvette is properly aligned as before. Read the absorbance to three significant figure on the bottom scale of the meter.

Figure 7. The Spectronic 20 spectrophotometer

 

After you have measured the absorbance of all of your standard solutions.  Make new standard solutions and repeat the measurements.  

Analysis of Gatorade: Dilute 5 mL of melon Gatorade to 25 mL with distilled water.  Measure the absorbance of this dilute solution of gatorade at 502 nm using the procedure outlined in steps 6 to 7, above. Perform this measurement three times.  

Data analysis

1. Calculate the molar concentration of each standard solution.  Remember that C1V1=C2V2.

2. Calculate the average absorbance of each standard solution, and of watermelon-flavored Gatorade. 

3. Put your data for concentration and the average absorbances in a table using Excel.  

Solution Concentration (M) Average Absorbance
A    
B    
C    
D    

4.  Use Excel to make a graph of absorbance vs. concentration for the standard solutions. Use only the data to make a graph, do not highlight any text.  Once you have a graph you can 'right click' on any data point and then select "add trendline", in the the "options" box, select "add trendline to graph".  

5.  This is your calibration graph.  Use this equation to determine the concentration of Red #40 in watermelon flavored gatorade.  Y is the absorbance you measured of the dilute gatorade.  Solve the equation of your trend line for "X"  that will be the concentration of dilute gatorade.

6.  To find the concentration of the original Gatorade.  Use C1V1=C2V2.  C1 is the unknown concentration.  V1 is 5 mL.  C2 is the concentration you calculated in #5 above and V2 is 25 mL.

7. Calculate the average molarity of the Gatorade solution.  

8.  Calculate the average deviation of the Gatorade solution.

Conclusion

Using complete sentences, restate your average value for the concentration of Red # 40 in melon flavored Gatorade.  Why did you need to repeat making your standard solutions?  Was there any differences in the absorption measurements?  What would cause these differences, what are the sources of error for this experiment.  If you added too much water to your diluted Gatorade before you measured its absorbance, would the final molarity be higher or lower than the actual value? 

Last edited by Koni Stone on 10/27/05