L12 - Lecture 12 HPLC Detectors UV-Vis Fluorescence HPLC...

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Lecture 12 HPLC Detectors UV-Vis Fluorescence
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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
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HPLC Detectors List of more common HPLC detectors UV-Visible Fluorescence Refractive index detector Conductivity (for ion chromatography) Mass Spectrometry
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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
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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)
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Molecular Orbital Theory Molecular Orbital: the probability of finding an electron within a given region of space in a molecule which is similar to atomic orbitals Each molecular orbital has a specific energy- level and a specific shape, and each of them can be occupied by a maximum of 2 electrons with opposite spins
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Molecular Orbital Theory Molecular orbitals exist at different energy levels; 2 bonding orbitals (sigma/pi), 1 non bonding orbitals and 2 anti-bonding orbitals (sigma*/pi*) Molecular absorption occurs when photonic energy causes promotion of an electron to a higher energy orbital, different types of transitions possible
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Hydrogen gas H H Each H atom has 1s 1 to give sigma overlap H H In phase Out of phase
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Carbon Atoms have enough electrons to occupy p- orbitals and have pi overlapping In phase Out of phase C C C C
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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
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FORMALDEHYDE σ π n Molecular Orbital Theory
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BENZENE π π* Molecular Orbital Theory
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wavelength causes the electronic transition Molecular Orbital Theory 6 transitions between 3 types of occupied (i.e., sigma, pi, and n) and 2 types of unoccupied MO (sigma* and pi*)
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This note was uploaded on 01/05/2012 for the course CHM 410 taught by Professor - during the Fall '11 term at University of Toronto.

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L12 - Lecture 12 HPLC Detectors UV-Vis Fluorescence HPLC...

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