HPLC Detectors UV-Vis Fluorescence

1. HPLC DetectorsUV-VisFluorescence 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. Molecular Orbital Theory FORMALDEHYDE σπ n

10. Molecular Orbital Theory BENZENE ππ*

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

12. Molecular Orbital Theory 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

13. Formaldehyde UV-Vis Possible Transitions for Formaldehyde π π* at182 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

14. Absorption Intensities π π* at182 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

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, NH2) redshift π π* transitions • Extended conjugation (NO2, 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 λMAX = 190 nm DNPH-Acetone λMAX = 360 nm

22. Solvent Cutoffs

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. Fluorescence Detectors • Greater sensitivity and selectivity over UV-Vis but the analyte must fluoresce! • λflu > λabs 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