1. SURVEY OF CHEMISTRY LABORATORY I SPECTROPHOTOMETRIC DETERMINATION
CHEM 1151L
SPECTROPHOTOMETRIC DETERMINATION
OF CHROMIUM

2. CAUTION!!!
BE SURE TO WEAR SAFETY GLASSES AT ALL TIMES IN THE LABORATORY NO EXCEPTIONS TO THIS RULE!

3. SPECTROPHOTOMETRY
- To measure the absorbance of different concentrations of chromium Students will - Prepare chromium-EDTA complex solutions (colored complex) - Determine the wavelength of maximum absorbance of the colored complex (λmax) - Determine the concentration of an unknown chromium solution

4. CHROMIUM SOLUTION
- The chromium ion (Cr3+) react with EDTA (ethylenediaminetetraacetic acid) to form a colored complex - Heat is required for reaction to go to completion - The complex absorbs light at particular wavelength and reflects others - Intensity of color depends on the concentration of chromium - The color intensity can be used to measure the concentration of solutions

5. BEER’S LAW - The absorbance of radiation (light) is related to the
Wavelength of radiation
and
Concentration of the absorbing species
- The equation for the relationship between the optical absorbance and concentration is known as
Beer’s Law

6. BEER’S LAW A = kbc - A is the optical absorbance
- k is a constant known as the molar absorptivity
- b is the pathlength (distance through which light passes sample)
- c is the concentration (molarity) of the solution

7. BEER’S LAW
A = kbc
- Optical absorbance (A) is measured using the spectrophotometer
(read directly from the instrument)
- The spectronic 20 will be used for this experiment
- The small test tube that holds the sample is known as the cuvet
- The diameter of the cuvet = 1 cm
- Implies the pathlength (b) for this experiment = 1 cm
- Beer’s law reduces to A = kc

8. DETERMINATION OF THE WAVELENGTH
PROCEDURE I
DETERMINATION OF THE WAVELENGTH
OF MAXIMUM ABSORBANCE
(λmax)

9. PREPARATION OF Cr3+-EDTA COMPLEX
- Set up a hotplate at your work station - Fill a beaker with water (about half-filled) - Place the beaker of water on the hotplate to heat up

10. PREPARATION OF Cr3+-EDTA COMPLEX
- Use the buret for volume measurements - Obtain a regular large test tube (not the small test tubes near the instrument) - Measure 5.00 mL of chromium nitrate solution and 5.00 mL of EDTA solution into the test tube

11. PREPARATION OF Cr3+-EDTA COMPLEX
- Place the test tube in the boiling water bath for at least 10 minutes

12. PREPARATION OF Cr3+-EDTA COMPLEX
- Remove and cool the solution by placing the test tube in cold water

13. SPECTRONIC 20
- Refer to the “General Procedure for Spectrometer Use” in Appendix II - Turn on the Spectronic 20 instrument to warm up for at least 5 minutes before use

14. λmax DETERMINATION
- Obtain two clean cuvets (small test tubes near the instruments) - Fill one cuvet with EDTA solution - Fill the other cuvet with the prepared colored complex - Fill each to at least three-quarters full

15. Click on picture to play movie
λmax DETERMINATION
- Avoid holding the sides of the cuvets (do not leave finger prints on them) - Carefully clean the sides with kim wipes
Click on picture to play movie

16. Click on picture to play movie
λmax DETERMINATION
- Set the instrument to 350 nm - Insert the EDTA cuvet into the sample holder - Mark on the cuvet should be in line with mark on the sample holder - Make sure instrument is in the absorbance (A) mode - Zero the instrument
Click on picture to play movie

17. Click on picture to play movie
λmax DETERMINATION
- Remove the EDTA cuvet - Insert the COMPLEX cuvet into the sample holder - Mark on the cuvet should be in line with mark on the sample holder - Read and record the abosrbance displayed
Click on picture to play movie

18. λmax DETERMINATION
- Change the wavelength to 360 nm - Insert the EDTA cuvet into the sample holder - Mark on the cuvet should be in line with mark on the sample holder - Zero the instrument

19. λmax DETERMINATION
- Remove the EDTA cuvet - Insert the COMPLEX cuvet into the sample holder - Mark on the cuvet should be in line with mark on the sample holder - Read and record the absorbance displayed

20. λmax DETERMINATION
- Repeat steps at wavelength increments of 10 up to 600 nm - Zero instrument with EDTA cuvet each time wavelength is changed - Clean cuvets with kim wipes each time before inserting into the instrument - Always aline marks on the cuvets and the sample holder

21. ABSORBANCE VS WAVELENGTH GRAPH
- Use the Chart Wizard in Excel - Plot a graph of absorbance verses wavelength - This is not a straight line so do not join the points with a line - Only plot the points on scatter and print it out - Use pencil to join the points with your free hand - Determine the wavelength corresponding to the maximum absorbance This is the λmax of the Cr3+-EDTA complex

22. DETERMINATION OF THE RELATIOSHIP BETWEEN ABSORBANCE AND CONCENTRATION
PROCEDURE II
DETERMINATION OF THE RELATIOSHIP
BETWEEN ABSORBANCE AND CONCENTRATION
OF THE Cr3+ AT THE λmax
DETERMINATION OF THE CONCENTRATION
OF AN UNKNOWN Cr3+ SOLUTION

23. HOMEWORK EXERCISE
Do the following calculations before coming to the next class
- Calculate moles of Cr3+ for test tubes 1 through 5
- Calculate molarities (M) of Cr3+ for test tubes 1 through 5
- Complete the table with moles and molarities of Cr3+

24. HOMEWORK EXERCISE Use the dilution method: M1V1 = M2V2
Molarity (concentration) of stock Cr3+ solution = M1 = mol/liter
Final volume after mixing for each test tube = V2 = mL
For example
Mixing 2.50 mL (V1) of stock Cr3+ solution with 7.50 mL EDTA
2.50 mL Cr mL EDTA = mL total solution = L
Moles of Cr3+ = Stock concentration (Molarity) x Volume of Cr3+ (liters)
= moles/liter x liter
= mole of Cr3+
Molarity (M2) = Moles Cr3+ / Total volume of solution (liters)
= mole/ L
= M (or moles/liter) Cr3+
M means Molarity or moles/liter

25. HOT WATER BATH
- Set up a hotplate at your work station - Fill a beaker with water (about half filled) - Place the beaker of water on the hotplate to heat up

26. PREPARATION OF KNOWN SOLUTIONS
- Use the buret to measure volumes of solutions - Obtain five large test tubes and label them 1 through 5 - Carefully measure out the Cr3+ and EDTA solutions directly into the test tubes using volumes in the table that was designed as the homework assignment

27. PREPARATION OF AN UNKNOWN SOLUTION
- Obtain an unknown sample of 5.00 mL Cr(N03)3 of an unknown concentration - Record the unknown number on your data sheet - Add 5.00 mL of EDTA solution to give you a total of mL

28. HEAT SOLUTIONS
- Place all the test tubes (knowns and unknown) in the boiling water bath for at least 10 minutes - Remove any caps on the tubes before heating

29. COOL SOLUTIONS
- Remove and cool the solutions by placing the test tubes in cold water

30. SPECTRONIC 20
- Refer to the “General Procedure for Spectrometer Use” in Appendix II - Turn on the Spectronic 20 instrument to warm up for at least 5 minutes before use

31. ABSORBANCE DETERMINATION
- Obtain one clean cuvet (small test tubes near the instruments) - Fill the cuvet with EDTA solution - Fill to at least three-quarters full

32. ABSORBANCE DETERMINATION
- Avoid holding the sides of the cuvet (do not leave finger prints on them) - Carefully clean the sides with kim wipes

33. ABSORBANCE DETERMINATION
- Set the instrument to the determined λmax - Insert the cuvet into the sample holder - Mark on the cuvet should be in line with mark on the sample holder - Make sure instrument is in the absorbance (A) mode - Zero the instrument

34. ABSORBANCE DETERMINATION
- Remove the cuvet - Pour out EDTA into a waste beaker - Rinse cuvet twice with bits of solution #1 (pour rinsing solution into the waste beaker) - Fill cuvet with solution #1

35. ABSORBANCE DETERMINATION
- Wipe sides with kim wipes - Insert cuvet into the sample holder - Mark on the cuvet should be in line with mark on the sample holder - Read and record the absorbance displayed

36. ABSORBANCE DETERMINATION
- Repeat steps for the rest of the test tubes including the unknown - Use the same cuvet for all the samples - Always pour out used sample back into its tube and rinse twice with the next solution before refilling (do not rinse with water) - Clean cuvet with kim wipes each time before inserting into the instrument - Always aline marks on the cuvet and the sample holder - Wavelength remains at the λmax - Clean up when done and pour all solutions into the appropriate waste bottle

37. ABSORBANCE VS WAVELENGTH GRAPH
- Use the Chart Wizard in Excel - Plot a graph of absorbance verses wavelength - This is the Beer’s Law line graph - Line is expected to be a straight line so join the points with a trendline (the best straight line) - Include the equation of the trendline

38. ABSORBANCE VS WAVELENGTH GRAPH
- Use the equation of the trendline and the optical absorbance of the unknown to determine the concentration of the unknown
- Also estimate the concentration of the unknown visually from the graph
- Include the two graphs in your laboratory report