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UV & Fluorescence Spectroscopy Cheat Sheet (DRAFT) by

UV & Fluorescence spectroscopy notes

This is a draft cheat sheet. It is a work in progress and is not finished yet.


Chromo­phore = portion of a molecule that absorbs light

Conjugated structure = 2+ adjacent C=C double bonds
More pi-ele­ctrons = more conjugated double bonds
↳ compound will absorb more light and have a longer wavelength and chromo­phore

Analytical purposes

When a beam of radiation is passed through a sample,
- some radiation is absorbed by the sample
- some radiation is reflected or scattered
- some radiation passes straight through
For analytical purposes, we are interested in the amount ABSORBED by the sample and so we want to eliminate reflection and scatte­ring.
This is done by:
- taking I0 as the intensity of the light passing through a cell when filled with a blank solution (every­thing except substance being measured)
taking I as the intensity passing through the cell when filled with the sample

Calcul­ating transm­ittance


Beer-L­ambert Law

Beer-L­ambert law= dependence of absorption on concen­tration and path length
a = absorption coeffi­cient
Represents the absorbance of a solution of unit concen­tration when measured in a cell of unit path length

Quanti­tative analysis

Two methods are used to convert the measured absorbance into a concen­tra­tion:
1. Calibr­ation curve
2. Using the Beer-L­ambert law with a given 'a' value
Calibr­ation curve
5 or more standards are prepared from a sample of pure material to be determ­ined. Their absorbance is measured and their values are used to construct a calibr­ation curve.
The sample absorbance is measured and its concen­tration is read off the curve.
A pure analyte sample must be available
Beer-L­ambert law
Use of A=acl will be needed with additional steps to work out the dilution factor

Relati­onship between path length and concen­tration

The value of T (or I) depends on the cell's path length, concen­tration of absorbing substances and the nature of the substance.
- Increased path length = decreased intensity → light passes MORE through solution and interacts MORE with molecule

Wavelength selection

The wavelength for the assay should be chosen so that only the substance of interest absorbs, with no impurities

Analysis of mixtures

If we assume that the components of the mixture do not react with one another, then the absorbance at some wavelength = the sum of absorbance for each component

Mixture of 2 substances

For a mixture of 2 substances with different chromo­phores, each will have different powers of light absorption at some wavele­ngth(s) in the spectrum.
If measur­ements are made on the mixture at 1 and 2, then a pair of simult­aneous equations can be set up to find the unknown concen­tra­tions.


What is fluore­scence?
When molecules absorb UV-visible radiation, they absorbed energy is converted into kinetic energy due to collis­ions.
A few excited molecules get rid of excess energy by emitting the absorbed energy as light = FLUORE­SCENCE

Why use fluore­scence?

Fluore­scence is more selective than absorption of energy
↳ The chances of finding 2 substances which absorb and emit at the same wavelength is LESS than finding 2 substances that absorb at the same wavelength
Not all substances fluoresce
↳ Advantage: it makes fluori­metry more selective for compounds that fluoresce
Disadv­antage: not all compounds fluoresce
It's more selective than absorption spectr­oph­oto­metry

Process of fluore­scence

- The emitted radiation is of lower energy than the absorbed radiation
- Molecules in the ground state absorb light and are excited to a vibrat­ional level in the 1st excited state
- Excess vibration is then lost by collisions with other molecules until they are in the V=0 level of the 1st excited state
- The molecules get rid of the remaining energy by emitting it as radiation and dropping to a vibrat­ional level in the ground state

Types of fluore­scence

1. Chemi-­lum­ine­scence
When the product molecules are left in an excited state, light is emitted when the molecules return to the ground state
2. Bio-lu­min­escence
Bioche­mical reactions that produce light

Relati­onship between structure and fluore­scence

e.g. Fluore­scein → fluore­scent and rigid structure (prevents loss of energy)
e.g. Phenol­pht­halein → non-fl­uor­escent and non-rigid structure (can twist which converts its absorbed energy into rotational and vibrat­ional energy)

Fluore­scence enhancers and inhibitors

Fluore­scence enhancers
Fluore­scence inhibitors
↳ Increase the no. of deloca­lised electrons
COOH, NO2, NO, F, Cl, Br, I

Factors affecting fluore­scence intensity

More energy absorbed = more energy emitted
Intensity of fluore­scence is PROPOR­TIONAL to amount of light absorbed

Relati­onship between I0 and I given by the Beer-L­ambert law
- Intensity of emitted radiation is directly propor­tional to conc
- Fluore­scence is directly propor­tional to intensity of excitation state
↳ brighter lamp = greater fluore­scence
- Fluore­scence depends on molar absorption coeffi­cient (E)
↳ stronger absorbance = greater fluore­scence

Fluore­scence intensity vs. concen­tration

At high concen­tra­tions, the fluore­scence emission becomes concen­trated near the sides of the sample cell to which the exciting radiation enters.
As the exciting radiation passes through the sample, its intensity falls and the fluore­scence produced also decreases.

Under these condit­ions, the equation for fluore­scence intensity no longer holds (as Ecl is no longer small

Experi­mental measur­ement of fluore­scence

1. Light source
- As intense as possible at wavelength of max absorption
- Continuous source
- Wavelength region of 200-500nm

2. Excitor and detector monoch­romator
- Used to select excita­tio­n/e­mission wavele­ngths
- Low dimini­shing and high light gathering powers
- Filters can be used but are less versatile

3. Sample cell
- Usually silica
- 1x1cm
- 4 clear, optically worked surfaces

4. Detector
- Must be sensitive as possible (varies)
- Photom­ult­iplier tube

Factors affecting fluore­scence measur­ements

1. Lower limit of detection
Lowest concen­tration of substance that can be determined
2. Fluore­scent impurities
Usually from the reagents or from the cell
Ensure the cell is clean and is always placed in the same way (as the 4 sides have different background intens­ities)
3. Photod­eco­mpo­sition
The sample can undergo decomp­osition due to the high intensity of the exciting radiation samples, and standards should NOT be exposed to the radiation longer than necessary
4. Quenching
Substance fluore­scence is affected more by its enviro­nment then the absorption of the sample
Quenching = when fluore­scence is decreased by the presence of another sample