1. Molecular Spectrometry (UV and Visible) Part 1: Absorption

2. Instrument Evolution Beckman DU

3. Replaced prism with grating

4. Turner Spectrometer Spec 20 as well

5. Dual Beam High resolution Cary and Perkin-Elmer

6. PDA Hewlett Packard

7. Fiber Optic Probes Ocean Optics

8. Signal Expressions E out = Φ λ (Ω/4π) T m T s R λ G Φ λ = source spectral power (W/nm) T m = monochromator throughput (nm) T s = Sample transmittance R λ = Detector Responsivity (A/W) G = Gain of electronics (V/A)

9. Signal Expressions T m = (WH/A eff ) Δλ eff T op W= slit width (cm) H= slit height (cm) A eff = Effective area of source image (cm 2 ) Δλ eff = Effective bandwitdh of the monochromator (nm) T op = Transmittance of optics

10. Signal Expressions T s = e -εbc = E sam /E ref E ref = Output voltage for a blank E sam = Output voltage for sample

11. Signal Expressions A = -log (E sam /E ref ) = -log (T s ) = ε b c Notes: 1. Since UV-Vis absorbance bands are much wider than the mono- chromator bandwidth, Beer’s Law assumes that A does not depend on Δλ eff. 2. ε is wavelength dependent, but does not depend on any other instrumental parameter.

12. A = -log (E sam /E ref ) = -log (T s ) = ε b c

13. A = ε b c Design Cell to Maximize b

14. Deviations from Beer’s Law 1. Optical a) slit width b) wavelength selection c) stray radiation 2. Chemical a) high analyte concentration b) pH dependence c) mixtures

15. Deviations from Beer’s Law Slit Width

16. Deviations from Beer’s Law

17. Wavelength Selection

18. Deviations from Beer’s Law Stray Radiation Effect No Stray Light 10% Stray Light

19. Deviations from Beer’s Law High Analyte Concentrations

20. Deviations from Beer’s Law pH Dependence

21. Deviations from Beer’s Law Mixtures: Absorbance is Additive

22. Deviations from Beer’s Law Mixtures: Solve multiple equations!

23. Deviations from Beer’s Law Mixtures: When all else fails, Separate the mixture components

24. UV Absorbance Detector for HPLC

25. Final Note on UV-Vis Signals The Absorbance (Analytical Signal) does not depend on the magnitude of the source signal (except for the deviations discussed above). The Noise in the measurement often DOES depend on the source signal level.