Cheatography

# UV & Fluorescence Spectroscopy Cheat Sheet (DRAFT) by eyeeyuu

UV & Fluorescence spectroscopy notes

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

### UV SPECTR­OSCOPY

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

### Beer-L­ambert Law

 Beer-L­ambert law= dependence of absorption on concen­tration and path length A=acl 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.

### FLUORE­SCENCE SPECTR­OSCOPY

 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 OH, OCH3, NH2, NHR, NR2 ↳ 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