Background Information s10

The Absorbance Lab

BACKGROUND INFORMATION:

Light is composed of photons with quantized or specific wavelengths and energies. The longer the wavelength, the lower the energy. Types of light are categorized as: gamma, x-ray, ultraviolet (UV), visible (vis), infrared (IR), microwave, and radio waves, depending on the wavelength of the photon. The light that our eyes can detect, conveniently referred to as the visible region, is a very small section of the light spectrum. Spectrophotometry is the study of the transmission or absorbance of light through a substance. This technology is crucial in research and industry, as it is used to identify unknown compounds and measure their concentration, be it an illicit drug found at a crime scene, a newly synthesized drug created in the laboratory, or a food product ready for market. Some molecules absorb UV light, others visible light, and even others IR light. Transmittance is a measure of the amount of light passing through a substance; absorbance is the amount of light that was captured by a substance. A clear colorless piece of glass has close to 100% transmittance and 0% absorbance of visible light. In colored liquids, for example, the color we see is a result of the different wavelengths absorbed and total amount of light absorbed.

Beer's law states that the spectral absorbance of a sample is directly proportional to the concentration of the sample. If you plot absorbance versus concentration, the resulting graph yields a straight line. The equation for the straight line (termed regression line) can be used to determine the concentration of an unknown solution. Beer’s law is an invaluable technique in accurately measuring concentrations of new pharmaceutical drugs being produced for industry.

First, you will analyze a sample of 1 M stock solution of NiSO4 to determine the absorbance peaks that are present. You will use a Spectrophotometer 200 to measure the absorbance of samples with differing concentrations over the visible-near infrared (NIR) light spectrum (380 – 850 nm). Second, you will test an unknown concentration. Third, you will determine the concentration of an unknown sample using Beer’s law, given known standards.

Purpose: To use absorption of light as a way to determine the concentration of an unknown solution.

Materials: 1 M Nickel (II) Sulfate, Spectrophotometer 200 and cuvettes, Computer, Glassware as needed.

Procedure: KEEP ALL CUVETTES CLEAN WITH WIPES AT ALL TIMES.

1. Learn to use the Spectrophotometer as demonstrated by the instructor.

2. Determine which wavelengths of light is best absorbed by the nickel (II) sulfate. You will need to calibrate at each wavelength. Test the wavelengths in increments of 10 nm.

3. Make a graph of this using Logger Pro (Programs Vernier  Logger Pro)

4. Make a series of 5 solutions which include the original solution, and dilutions which are 80% (0.80M, 60% (0.60M), 40% (0.40M), and 20%(0.20M) of the original concentration. You will need a total volume of no more than 25 ml of each solution. The solutions may be stored in test tubes.

5. Use the computer and record the absorbance of each solution at the wavelength of best absorbance.

6. Create a "line of best fit" using Logger Pro.

7. Determine the absorbance of an unknown solution. Use the line of best fit to determine the concentration of the unknown. Data: As taken in class

Graphs: Include two graphs in your lab report – 1 spectral fingerprint graph, and one Beer’s Law graph. Be sure that labels and titles are correct. Be sure to do a line of best fit for the last graph, ensuring that the information for the line (y-intercept, slope) are shown on the graph so you can determine your unknown.

Calculations: Include any and all relevant calculations, including concentration and dilution calculations.

Conclusion Questions

1. What was the concentration of the unknown?

2. At what wavelengths did your stock solution have maximum absorbance? At what wavelengths did it have minimum absorbance? Discuss how these results are consistent with what the human eye sees visually when looking at the solution. 3. Describe how this lab relates to the structure of the atom. 4. How is this type of spectrum different from the emission spectra in the flame test lab? 5. Describe how you made the 40% dilution. Calibrate your Spectrophotometer: a. Prepare a blank by filling an empty cuvette ¾ full with distilled water. Place the blank cuvette in the spectrometer. b. Select your wavelength using the arrow buttons and wavelength knob. c. Select Go. d. Press the 0.00 button on the Spec, and wait until it is ready.

To create a spectral fingerprint graph: a. Empty the blank cuvette and rinse it twice with small amounts of the stock solution. Fill the cuvette ¾ full with the solution and place it in the spectrometer. Make sure the cuvette sides have been cleanly wiped with a wipe. b. You will start at 350 nm and take measurements every 20 nm. c. Click “Freeze”. d. Record your wavelength and absorbance in Logger Pro. e. Change your wavelength and Re-Calibrate. f. A full spectrum graph of the solution will be displayed, showing wavelengths of high and low absorbances. This is your spectral fingerprint. Note that some areas of the graph contain peak absorbances, and others that contain minima that characterize this substance. a. Select “Save As” from the File menu and save this graph to the K drive. Make sure the graph is labeled and titled properly. b. Determine the wavelength of maximum absorbance.

To create a Beer’s Law graph for concentration analysis: a. Set your wavelength to the maximum absorbance from your spectral fingerprint. b. Re-calibrate. c. Prepare 4 cuvettes containing the 0.80, 0.60, 0.40, and 0.20 M solutions, respectively. Place each into the spectrometer, one at a time. d. For each sample hit the freeze button on the Spec. Enter the concentration and absorbance into Logger Pro. e. When you are done, create a line of best fit. Make sure the statistics are shown for your line of best fit. f. Select “Save As” from the File menu and save it to the K drive. If you have time, save this to a jump drive, and Ms. Michael will print a copy of this absorbance vs. concentration graph. Make sure the graph is labeled and titled properly. This is your Beer’s Law graph. g. Test your unknown, and record the absorbance by hand on paper. You will use this value to calculate its concentration.

To make a graph using Logger Pro a. Click on the “x” data table. Write the name and units. Do the same with “y”. b. Double click on the graph. Under “graph options”, select point protectors and de-select connect points. c. Click on the data table to enter values. d. To change the values on the axes, double click on the last value, and you can enter your own. e. To make a “line of best fit” or a “regression line”, go to “analyze”, and click “linear fit”.