Determination of Concentration Using Spectrophotometry

1. Determination of Concentration Using Spectrophotometry Lab 11

2. Outline • Purpose • Qualitative Distinction • Spectrophotometry • Beer’s Law • Calibration Curve • Procedure • Points of Interest • Waste • Safety Concerns

3. Purpose This experiment demonstrates the linear relationship between the absorbance and the concentration of a colored solution. Beer’s Law will be used to determine the concentration of a sample for which the concentration is unknown.

4. Qualitative Distinction • Chemical solutions owe their color to light-absorbing species in the solution, whether these are ions or complex molecules. For example: • The green color of today’s solution could be due to Ni2+ (it is in fact food coloring, not Ni2+). • The color of cranberry juice is due to anthocyanins.

5. Qualitative Distinction • The color we see is the color of light transmitted, or “getting through” the solution. We see the color of light “left over” after some wavelengths have been absorbed. • Today we will see green because the solution absorbs red wavelengths (650 nm) and what’s left over appears green.

6. Qualitative Distinction • The intensity of the color is proportional to the concentration of the absorbing chemical species. • We can do a qualitative distinction by eye, or • We can do a quantitative measurement by spectrophotometry.

7. Spectrophotometry • Spectrophotometers shine a light through the sample. • Some detect light from only one wavelength and some detect light from all visible wavelengths. The MicroLAB™ spectrophotometer emits and detects light from sixteen different wavelengths. • The light that is not absorbed by the sample, but transmitted instead, hits a light detector.

8. Spectrophotometry Transmitted Light Incident Light I0 I Light Source Wavelength Selector Sample Detector b b = 22.45 mm or 2.245 cm

9. Spectrophotometry • The spectrophotometer calculates the percentage of light transmitted. • It then uses an algorithm (formula) to convert percent transmittance to absorbance. • Transmittance: %T = x 100% • Absorbance: Abs = log

10. Standard Solutions • Today you will make up several solutions of known concentration. • There is a known relationship between the concentration of samples and the absorbance of samples at a given wavelength. • This relationship is commonly referred to as Beer’s Law.

11. Beer’s Law • Abs = ε b C where Abs = absorbance (no units) ε = molar absorptivity (M-1cm-1) b = path length (cm) C = concentration (M) These measurements all take place at the wavelength at which our absorbing species absorbs light. By plotting Beer’s Law on a graph, we can establish a calibration curve with our standard solutions.

12. Plotting Beer’s Law If we were to plot an extended range of Absorbance vs. [Colored Solution], M we would notice a linear response in a limited region of the plot only. (Why?) We pick this region for our calibration curve.

13. Calibration Curve Plotting Abs vs. [Colored Solution], M yields: • This is called a “calibration curve.” • For y = m x + b • Abs = m [colored solution] + b • Abs = ε b c • Note: • The “b” in y = mx + b refers to the y- intercept of the graph. • The “b” in Beer’s Law refers to the path length light has to travel through your sample. • DO NOT confuse them!

14. Calibration Curve • After the calibration curve is plotted with your standard solutions, the calibration curve equation can be used to calculate the concentration of an unknown solution, IF: • the unknown solution contains the same color-absorbing species as your known (“standard”) solutions • the absorbance of the unknown solution is known or can be established • the absorbance of the unknown solution falls within the absorbance range of your standard solutions (otherwise any concentration calculations are invalid)

15. Questions • What should you do if your unknown solution has an absorbance value that falls above your calibration range? • What should you do if your unknown solution has an absorbance value that falls below your calibration range?

16. Procedure To obtain the calibration curve: • Prepare a series of colored solutions of known concentration (“standards”). • The absorbance of each solution is measured. • Absorbance versus concentration is plotted. To find the unknown concentration: • Using the calibration curve equation and the absorbance of the unknown solution, the concentration of the unknown solution can be calculated: Unk Abs = m [Unkcs] + b, therefore, [Unkcs] =

17. Points of Interest • MicroLAB™ colorimeter operation • Your instructor will run through the basics. • Cuvet handling • Wipe your cuvets down with a dry KimWipe before insertion into the interface. • Blank Solution Selection • See the next slide

18. Blank Solution • A blank solution is used before the first sample is inserted into the spectrophotometer. • The blank solution is most always the solvent of your solutions. • The solvent may contain species that can absorb light at the same wavelength as the analytical wavelength for your analyte. • The spectrophotometer is zeroed out with the blank solution (Abs = 0 or %Trans = 100%) • The blank solution therefore corrects for the matrix effects of the solvent.

19. Safety Concerns • Reagent: • Food coloring • Health Considerations: • Avoid contact with skin and eyes. • Do not inhale vapor or spray. • Do not ingest.

20. Waste • All waste solutions can be disposed down the sink, with plenty of water.

21. Next Week – Thanksgiving Break • No labs. • Your instructor will let you know when to submit your Lab 11 Report. • Come prepared for the quiz. • Study pages 297 – 317 carefully. • Bring your lab manual and goggles. In 2 Weeks – Skill Evaluations