1. HPLC Detectors UV-Vis Fluorescence Derek Jackson CHM410/1410 October 22, 2009 djackson@chem.utoronto.ca

2. HPLC Detectors Once a mixture of compounds has been separated by HPLC, how do we detect them? Requirements for an HPLC detector Good sensitivity (high signal, low noise) No interference from mobile phase Must be able to work in a liquid phase environment

3. HPLC Detectors List of more common HPLC detectors Refractive Index UV-Visible Fluorescence Conductivity (for ion chromatography) Mass Spectrometry

4. Refractive Index Detector “Legacy” bulk property detector Almost universal, rugged Low sensitivity (high ppm), no gradient programs possible

5. UV-Visible Detector Most common detection method along with mass spectrometry Detects solute analytes by their absorbance of light at various wavelengths More sensitive than refractive index, depends on specific analyte and wavelength Less sensitive than mass spec, compound must absorb in the UV-Vis, mobile phase cutoff

6. Molecules and Light Why is our universe coloured? Absorbance - compounds absorb light of specific wavelengths and reflect or transmit all others Emission - compounds emit light after converted to a higher energy state (ex: fluorescence, phosphorescence)

7. Molecular Orbital Theory Molecular orbitals exist at different energy levels; bonding orbitals (sigma/pi), non- bonding orbitals and anti-bonding orbitals Molecular absorption occurs when photonic energy causes promotion of an electron to a higher energy orbital, different types of transitions possible

8. Molecular Orbital Theory σ (sigma) – orbital has symmetry about the bonding axes, lowest energy π (pi) – only one orbital plane passes through both nuclei involved n (non-bonding) – orbital involved is not involved in bonding, usually a lone pair, higher in energy σ *, π * (anti-bonding) – nodal planes exist between nuclei, high in energy, usually unpopulated in stable molecules

9. FORMALDEHYDE σ π n Molecular Orbital Theory

10. BENZENE π π* Molecular Orbital Theory

11. Absorption occurs when light of a specific wavelength causes the electronic transition Molecular Orbital Theory

12. HOMO = highest occupied molecular orbital (σ, π, n) LUMO = lowest unoccupied molecular orbital (π*, σ * ) Most transitions we will be concerned with are from HOMO to LUMO The orbital types of HOMO/LUMO partially determine the energy required to make the transition Molecular Orbital Theory

13. Possible Transitions for Formaldehyde π  π * at 182 nm n  π * at 290 nm But do we see sharp peaks at those wavelengths? Why are electronic transitions broad? Answer: Vibrational transitions combined with condensed phase and solvent effects broaden UV-Vis peaks Formaldehyde UV-Vis

14. π  π * at 182 nm (ε = 10,000 L M -1 cm -1 ) n  π * at 290 nm (ε = 12 L M -1 cm -1 ) In formaldehyde, π  π * has strong absorption n  π * has very weak absorptions ε = Molar absorptivity Beer’s Law: A = ε c l Hence, UV-Vis can be used to quantify chromatography peaks linearly Absorption Intensities

15. After Absorption

16. Stokes Shift Remember: Emission spectra are redshifted relative to absorption (excitation) spectra

17. Aromatic Rings Benzene rings absorb “nominally” at about 254 nm but this can change depending on auxochromes Absorption bands are redshifted by:  Electron donating groups (OH, NH 2 ) redshift π  π * transitions  Extended conjugation (NO 2, C=O) which create n  π * transitions at longer wavelengths

18. Auxochromes 254 nm; ε = 200 270 nm; ε = 1450 280 nm; ε = 1450 269 nm; ε = 7800 (π  π * ) 330 nm; ε = 125 (n  π * )

19. Halogens Halogens redshift UV-Vis spectra in the order F << Cl < Br < I because of polarizability 1: 10 Br 2: 9 Br 3: 8 Br 4: 7 Br 5: 6 Br 6: 5 Br 7: 4 Br 8: Sunlight

20. Wavelength Selection For HPLC-UV, want to observe a chromatogram at the longest reasonable wavelength, why? Signal:noise is usually better at longer wavelengths due to reduction in noise from mobile phase and impurities Ex: DNPH derivitization of carbonyls

21. Wavelength Selection Acetone λ MAX = 190 nm DNPH-Acetone λ MAX = 360 nm

22. Solvent Cutoffs Acetonitrile190 nm Methanol205 nm Water200 nm Chloroform245 nm THF215 nm Ethyl acetate254 nm Toluene284 nm Acetone330 nm Pyridine330 nm

23. Chromatograms Top = 220 nm Bottom = 280 nm CBN CBD

24. Chromatograms λ = 245 nm

25. HPLC-UV-Vis Generally, UV-Vis HPLC detector not too different from a standalone UV-Vis (flow cell instead of a cuvette) Variable wavelength detector vs. Diode array detector

26. HPLC-UV-Vis Variable wavelength detector - monochromator PMT

27. HPLC-UV-Vis Diode array detector - polychromator

28. HPLC-UV-Vis Variable Wavelength detector  More sensitive due to photomultiplier tube or amplification circuitry  Requires more method development Diode array detector  Less sensitive due to photodiodes only  Very easy to develop a method

29. HPLC-UV-Vis

30. Fluorescence Example: Highlighter Pens absorb UV and blue light and emit yellow-green

31. Fluorescence Detectors

32. Greater sensitivity and selectivity over UV-Vis but the analyte must fluoresce! λ flu > λ abs What makes a good fluorophore? High absorbance, aromatic Fused rings, electron donating groups Quantum yield (Φ)

33. Fluorescence Detectors Need to select an excitation and an emission wavelength

34. Chromatogram Top: UV-Vis Bottom: Fluorescence Hence, fluorescence is more selective and sensitive due to noise reductions

35. Summary Refractive Index Detector  “Legacy” detector, insensitive, no gradients in mobile phase possible UV-Vis Detector  Detects absorption of chromophoric analytes based on molecular structure  Variable wavelength vs. Diode array detector Fluorescence Detector  Most sensitive and selective detector