UV-Visible and fluorescence spectroscopy
Learning objectives:
Describe the physical principle behind UV-Vis spectroscopy
Draw the basic elements of a spectrometer
Define absorbance and transmission of light
Apply the Beer-Lambert law to measure concentration
Calculate the extinction coefficient of a protein using its primary sequence
Review the applications of UV-Vis spectroscopy in monitoring reactions
Review the applications of UV-Vis spectroscopy in studying metallo-proteins
UV-Visible spectroscopy (electron absorption spectroscopy)
Principle:
- Measure the absorption of specific wavelengths of UV and Visible light. Due to transitions of electrons from a
ground state to an excited state
- This is an energy difference between the two electron energy levels (or orbitals).
- Energy difference = Planck's constant * speed of light /wavelength of approaching light
- To cause a particular electronic transition only light with the same energy (wave length) will be absorbed by the
molecule
- Examples:
groups that absorb visible (400-800 nm) and UV (185-400 nm) light have unsaturated bonds, lone pair
electrons or D orbitals
- Light source from a light bulb
- Light is only absorbed if it equals the energy gap if not it will pass through the other side
- Water does not absorb visible light and therefore can see through it
- The wavelength of light absorbed gives us information about these energy gaps
- Every molecule has different electrical orbital
The colours of light
Aromatics absorb at 280nm
Carbonyl stretch absorbs at 1600nm
Red is at the long end hence infrared and UV is strong radiation
Violet at the short end
Copper sulphate is blue in solution as absorbs red and yellow light
, Haemoglobin
Haemoglobin is red because it has a porphine ring and a metal ion
The heme in haemoglobin absorbs light at 440nm, which is not the red colour but blue
UV Visible spectrometer
Range between £5,000-£25,000
Quarts cuvets are £100
Plastic cuvets are cheaper
Path length is 1 cm
Width is 1 cm
UV-Vis Spectrometer instrument
Light source: typically, 2 lamps a deuterium for UV and tungsten for visible
Monochromator:
- diffraction grating to separate wavelength of light.
- Physically turns to catch the different wavelengths of light.
- Differaction gradient records each wavelength of light
Sample holder:
- typical cell is 1 cm
Detector:
- photo multiplier
- Measures light.
Plotter:
- computer
These are optics inside the instrument
Absorbance and Transmittance
Learning objectives:
Describe the physical principle behind UV-Vis spectroscopy
Draw the basic elements of a spectrometer
Define absorbance and transmission of light
Apply the Beer-Lambert law to measure concentration
Calculate the extinction coefficient of a protein using its primary sequence
Review the applications of UV-Vis spectroscopy in monitoring reactions
Review the applications of UV-Vis spectroscopy in studying metallo-proteins
UV-Visible spectroscopy (electron absorption spectroscopy)
Principle:
- Measure the absorption of specific wavelengths of UV and Visible light. Due to transitions of electrons from a
ground state to an excited state
- This is an energy difference between the two electron energy levels (or orbitals).
- Energy difference = Planck's constant * speed of light /wavelength of approaching light
- To cause a particular electronic transition only light with the same energy (wave length) will be absorbed by the
molecule
- Examples:
groups that absorb visible (400-800 nm) and UV (185-400 nm) light have unsaturated bonds, lone pair
electrons or D orbitals
- Light source from a light bulb
- Light is only absorbed if it equals the energy gap if not it will pass through the other side
- Water does not absorb visible light and therefore can see through it
- The wavelength of light absorbed gives us information about these energy gaps
- Every molecule has different electrical orbital
The colours of light
Aromatics absorb at 280nm
Carbonyl stretch absorbs at 1600nm
Red is at the long end hence infrared and UV is strong radiation
Violet at the short end
Copper sulphate is blue in solution as absorbs red and yellow light
, Haemoglobin
Haemoglobin is red because it has a porphine ring and a metal ion
The heme in haemoglobin absorbs light at 440nm, which is not the red colour but blue
UV Visible spectrometer
Range between £5,000-£25,000
Quarts cuvets are £100
Plastic cuvets are cheaper
Path length is 1 cm
Width is 1 cm
UV-Vis Spectrometer instrument
Light source: typically, 2 lamps a deuterium for UV and tungsten for visible
Monochromator:
- diffraction grating to separate wavelength of light.
- Physically turns to catch the different wavelengths of light.
- Differaction gradient records each wavelength of light
Sample holder:
- typical cell is 1 cm
Detector:
- photo multiplier
- Measures light.
Plotter:
- computer
These are optics inside the instrument
Absorbance and Transmittance
