HPLC Detectors UV-Vis Fluorescence. Derek Jackson CHM410/1410 October 22, 2009 firstname.lastname@example.org. 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)
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October 22, 2009
Once a mixture of compounds has been separated by HPLC, how do we detect them?
Requirements for an HPLC detector
List of more common HPLC detectors
Why is our universe coloured?
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
Absorption occurs when light of a specific wavelength causes the electronic transition
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
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
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
Remember: Emission spectra are redshifted relative to absorption (excitation) spectra
π π* transitions
254 nm; ε = 200
270 nm; ε = 1450
280 nm; ε = 1450
269 nm; ε = 7800 (π π*)
330 nm; ε = 125 (n π*)
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
λMAX = 190 nm
λMAX = 360 nm
Top = 220 nm
Bottom = 280 nm
λ = 245 nm
UV and blue light and emit yellow-green
UV-Vis but the analyte must fluoresce!
What makes a good fluorophore?
Hence, fluorescence is more selective and sensitive due to noise reductions