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HPLC Detectors UV-Vis Fluorescence - PowerPoint PPT Presentation

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HPLC Detectors UV-Vis Fluorescence. Derek Jackson CHM410/1410 October 22, 2009 djackson@chem.utoronto.ca. 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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Hplc detectors uv vis fluorescence

HPLC DetectorsUV-VisFluorescence

Derek Jackson


October 22, 2009


Hplc detectors
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

Hplc detectors1
HPLC Detectors

List of more common HPLC detectors

  • Refractive Index

  • UV-Visible

  • Fluorescence

  • Conductivity (for ion chromatography)

  • Mass Spectrometry

Refractive index detector
Refractive Index Detector

  • “Legacy” bulk property detector

  • Almost universal, rugged

  • Low sensitivity (high ppm), no gradient programs possible

Uv visible detector
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

Molecules and light
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)

Molecular orbital theory
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

Molecular orbital theory1
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

Molecular orbital theory2
Molecular Orbital Theory


σπ n

Molecular orbital theory4
Molecular Orbital Theory

Absorption occurs when light of a specific wavelength causes the electronic transition

Molecular orbital theory5
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

Formaldehyde uv vis
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

Absorption intensities
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

Stokes shift
Stokes Shift

Remember: Emission spectra are redshifted relative to absorption (excitation) spectra

Aromatic rings
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


254 nm; ε = 200

270 nm; ε = 1450

280 nm; ε = 1450

269 nm; ε = 7800 (π π*)

330 nm; ε = 125 (n  π*)


  • 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

Wavelength selection
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

Wavelength selection1
Wavelength Selection


λMAX = 190 nm


λMAX = 360 nm


Top = 220 nm

Bottom = 280 nm




λ = 245 nm

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

Hplc uv vis1

  • Variable wavelength detector - monochromator


Hplc uv vis2

  • Diode array detector - polychromator

Hplc uv vis3

  • 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


  • Example: Highlighter Pens absorb

    UV and blue light and emit yellow-green

Fluorescence detectors1
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)

Fluorescence detectors2
Fluorescence Detectors

  • Need to select an excitation and an emission wavelength


Top: UV-Vis

Bottom: Fluorescence

Hence, fluorescence is more selective and sensitive due to noise reductions


  • 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