1. CHEM 213 Instrumental Analysis Lab Lecture – Fluorescence and Calculations

2. 2 Fluorescence Determination of Quinine in Tonic Water M + h excitation  M*  M + h emission

3. 3 Fluorescence Spectrometer

4. 4 Definitions Emission spectrum – graph of fluorescence intensity vs. wavelength. –Obtained using a fixed excitation wavelength. Excitation spectrum – graph of fluorescence intensity (observed at a constant emission wavelength) vs. the excitation wavelength. –Closely related to absorbance spectrum.

5. 5 (emission) Key features of instrument Two monochromators Emission spectrum usually collected at 90° angle to excitation beam Fluorescence Spectrometer

6. 6 Must calibrate 1.0mL micropipette before making solutions –Similar procedure as for Cu Measure excitation and emission spectra to optimize wavelengths Be very careful to avoid fingerprints on cuvette walls Use Standard Addition Calibration Only have 40 min on instrument so plan ahead and keep on top of things. Tips on Lab Procedure

7. 7 Glucose Experiment Glucose / Sucrose Stock Standard Unknown: Dilute to 50 mL Take 10 mL aliquot, Dilute to 100 mL for analysis

8. 8 Glucose Data 1.139 mg/mL 1.162 mg/mL

9. 9 Glucose by Least Squares Using all Data Points Omitting Outlier AnalyteGraphLSQ (all)LSQ(outlier) Glucose575546568

10. 10 Sucrose Data 3.385 mg/mL 3.654 mg/mL Glucose Data Sucrose Data

11. 11 Sucrose by Least Squares Using all Data Points Omitting Outlier AnalyteGraphLSQ (all)LSQ(outlier) Sucrose176018111734

12. 12 DM·HBr in Cough Syrup Experiment

13. 13 Results Graphical Method 6.896 µg/mL 6.948 µg/mL 6.983 µg/mL × 50/10 – Dilution from original solution For calibration curve, plot AVERAGE CORRECTED ABSORBANCE for each standard.

14. 14 DM·HBr by Least Squares Use Corrected Absorbance Use ALL numbers (not averages) -Makes a big difference in precision of measurement (here error ~doubles) -Makes no difference to actual reported value Order that numbers are entered does not matter -But ensure that the X:Y pairs are correct!!! Here only averages used – NOT a good idea

15. 15 A class exercise… Using the following data calculate [X] in the unknowns by both the graphical method and by least squares. For the least squares, also perform the calculations using only the data from Reading 1 and Reading 2 columns. Try to do it by yourself, especially the graphical method. This is set up as an Assessment for you to practice with on the course website. It is available until Sunday evening.