Lab: Spectrophotometry
Cranberry Juice Quality Control

Background

For detailed discussion of how light is used in the analysis of solutions to find concentration, please see the accompanying handout. The idea behind this mini-lab is to introduce students to the techniques of spectrophotometry. Cranberry juice will be diluted to various concentrations on a percent-by-volume basis. A calibration curve based on the absorbance of light at a specific wavelength will be used to do ‘quality-control’ on two samples of an apple-cranberry juice blend.

Materials

• LabQuest software on a computer
• Vernier SpectroVis Probe
• cuvets with covers
• 100% cranberry juice
• distilled water

• prepared standard samples of cran. juice
• prepared samples of cran. juice ‘unknowns’
• disposable beral-type pipets

Safety

The chemicals used in this activity are all of negligible toxicity and are used in dilute solutions. Even so, it is best to wear standard laboratory safety equipment such as splash goggles and long pants. Tie back long hair and do not wear dangling jewelry in the lab.

Part I:

Procedure

In this part of the lab you will collect data showing the absorbance of light at a range of wavelengths by the cranberry juice.

1. Pick up a cuvet by touching the grooved sides. Handling the smooth sides will put fingerprints on it which could have a small effect on the data collection. Obtain enough cuvets to take a sample of each of the labeled standards. Make sure to keep track of which sample is which by using clear labels of your own. Wipe each cuvet dry once it is filled.
2. Plug the SpectroVis Plus unit into the USB port of the computer and start the Logger Lite or Logger Pro software.
3. Click on the “Experiment” menu, select Calibrate and click the word Spectrometer.
4. A dialog box will pop up to inform you that the lamp is warming up. Do not skip this step, it only requires 90 seconds.
5. Fill a cuvet with tap water and place a cap on it. Wipe it dry. Once warm-up is complete place the cuvet into the SpectroVis device so that the smooth sides face the white circle and white arrow. Click “Finish Calibration”.
6. Once Calibration is complete, click OK.
7. Click on the “Experiment” menu again, select “Set Up Sensors” and click on the word Spectrometer.
8. In the dialog box that pops up change the “Wavelength Range” so that it begins at 350 nm and ends at 750 nm. Click the X to close the dialog box.
9. Click the “Wave” button at the top of the screen. Ensure that “Absorbance vs. Wavelength” is selected and then click OK.
10. Place the cuvet containing the 10% cranberry juice solution into the SpectroVis with the clear sides facing the white icons. Click the “Collect” button at the top of the screen and wait for data collection to begin. It will complete in very little time. Click the “Stop” button at the top of the screen. Then click the “Store” button to save the data.
11. Once you have stored all the data set, save the Logger file in a place where you can access it again in the future. Do not save it to the hard drive of the device you are using! Instead, save it to your own network folder or online cloud storage service. You can only use the file with the Logger software (a free version may be downloaded).
12. Next, start MS Excel. In the Logger software select all of the numerical data using the mouse and paste these data into a spreadsheet starting in Cell A2.
13. Add labels at the top of each column: Wavelength (nm) and Absorbance.
14. Create a scatter plot graph of these data with a smooth line and no data markers. Add appropriate titles to the axes and the graph overall. This will be your graph for the absorbance spectrum of cranberry juice.

Part II:

Procedure

In this part of the lab you will collect data to allow you to build a calibration curve according to Beer’s Law (A = εbc). This allows you to establish a direct proportion between the absorbance of light at a particular wavelength to the concentration of juice in percent. The absorptivity constant (ε) is sometimes given as a but in most publications is given as shown here.

1. Click the “Wave” button at the top of your screen. Select Absorbance vs. Concentration. If desired, change “Column Name” to Percent and adjust “Short Name” and “Units” as well.
2. To the right will be a display of the absorbance spectrum of the cranberry juice. The software will automatically select the wavelength of maximum absorbance. Check that it is the wavelength at which cranberry juice is strongly absorbing but at which apple juice does not absorb light.
3. Click OK. Click the “Collect” button at the top of the screen. Remove the cuvet from the spectrophotometer instrument and place a cuvet with the standard solution with the highest concentration into the device. In the lower-left-hand corner a display shows the absorbance of the sample in the spectrophotometer. Wait until the value is steady and unchanging and then click “Keep”. Enter the concentration of the sample when prompted then click OK.
4. Remove the cuvet and replace it with the one with the next smaller concentration. Repeat the data collection procedure for each of the other samples: wait, click “Keep”, and enter the concentration. As you generate data the software will automatically create a graph. If all is well all of the points will fall along a straight line.
5. Once you have finished collecting data click “Stop”. Then click “Store”. Save your Logger file.
6. Copy the newly generated data and paste it into Excel. Label the columns ‘Concentration’ and ‘Absorbance’. Concentration should be to the left of Absorbance so that it will be on the x-axis.
7. Highlight the data and create a scatter-plot with no lines connecting the data points. Add all appropriate titles and adust the axes as necessary to make a well-formatted graph.
8. Use the Excel software to generate a linear trendline. Display the equation and the R² value on the graph. Move the equation to the upper-right-hand corner.
9. Obtain samples of the unknowns (mixtures of pure cranberry juice with apple juice and water and sugar). Measure their absorbances and record the data for later analysis.

Post-lab Questions

Answer the following questions in a typed document on a separate paper. As part of your report you must include your formatted and labeled graphs and the answers to the questions below. This lab does not require a formal lab report.

1. Describe and interpret the graph of the absorbance of light as a function of wavelength (the absorbance spectrum) by the cranberry juice.
2. When you look at the absorbance spectrum you will see that the graph has high values for wavelengths of light that are strongly absorbed by the substance. What part of the spectrum is absorbed by cranberry juice and how does this account for the color we see when we look at it?
4. What is the absorptivity constant that you measured for cranberry juice in this lab?
5. Does apple juice absorb light significantly at the wavelength we used to analyze the concentration of cranberry juice? Explain what effect this has, if any, on the analysis of cranberry juice concentration of a cran-apple juice mixture.
6. If apple juice really did absorb a significant amount of light at the analysis wavelength how would this have affected your measurements of cranberry juice concentration in the juice blend? Would you have over- or under-estimated the concentration of cranberry juice?
7. What was the concentration of cranberry juice in the two unknowns? Show your work for the calculation to find the percentage using the equation generated as a trendline in Excel.