1. What is UV/Vis Spectroscopy:
It is an analytical technique commonly used to find the quantity or concentration of the sample using the calibration curve. It mainly works on conjugated systems (double bonds and stuff...). Now lets see how we get the spectra, the rationale and the way in which we calculate the quantity of sample dissolved.
2. UV/Vis Radiation
UV/Vis have high radiation energy with wavelength ranging from 10 to 800 nm. When UV is beamed at structures electrons in σ and π bonds are transmitted from stable electronic ground state to unstable electronic excited state.
If the σ bond breaks, the whole molecule will collapse as these are the bonds that hold the molecules together. These bonds are also strong and require a UV radiation of < 150 nm. As a result the radiations less than 150 nm is useless for us.
However if we excite the weak π bond to [π *] in unsaturated systems, there would be no harm and we can get a reading without damaging our molecule. Having said that, the reading will be useless because the absorption would be similar to that of the solvent and the air! (Oxygen absorbs UV at about 200 nm)
So in order to have a meaningful reading, we need to have CHROMOPHOREs, which is explained in next section...
3. What is Chromophore:
Chromophore part of the molecules containing electrons involve in electronic transition. Extended system of double bond in unsaturated polyenes that absorb a longer wavelength UV. Resonance structures as well as aromatic compounds such as benzene are also chromophores. Take a look at the pic below to learn some examples. Molecules with lone pairs of electrons are also able to absorb the UV/Vis to have an electron transition, therefore are also a chromophore.
Conjugated systems such as compound B, requires less Energy for transition of electrons between energy levels, from highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO), as a result low energy LONGER wavelengths are required to provide the energy for these transition. As an example if the compound A absorbs at 100 nm wavelength, compound B would absorb at longer wavelength 200 nm because its excited state is more stable and the ground state requires less energy for the transition.
4. Practical & Useful Absorptions:
The absorption of UV by chromophores, are practically useful for us for the following reasons:
- the conjugated systems are unique for each compounds, meaning we have a degree of specifity.
- These systems of two or more double bonds, absorb the UV at higher wavelength and in a higher intensity.
5. Absorption (A):
Before we jump into the beer lambert equation, lets start talking about what is absorption.
The equation is obvious because when the radiation is emitted (I0), some off the radiation will be absorbed and therefore less radiation (It) will reach the instrument detector. "A" is then simply amount of radiation absorbed and has NO UNIT (it is arbitrary). I0 is usually calculated by just beaming UV through the solvent ONLY (calibration), look up instrumentation for more on these two!
6. Beer Lambert Law:
This is the most important equation of UV theory for scientists such as pharmacist who just need to apply the theory not caring about concepts as much as analytical scientists.
The equation simply relates ABSORBANCE to CONCENTRATION. As a result it is a very useful equation in order to find an unknown concentration of a known chemical in a medium (pharmaceutical industry).
- "ε" is absorption coefficient, which is absorption of UV by 1 mole/dm3 of sample, this is constant for each chemical. [In pharmaceuticals is mostly replaced by A(1%, 1cm). look below.]
- "l" is the length the cell which the the radiation has to go through. Look at the instrumentation for more details.
- "c" is the concentration which is always the unknown in usual lab practical.
We get the absorbance using the UV/Vis instrument, we know the cell length and extinction coefficient. so can easily calculate the concentration
7. A(1%, 1cm) or A11:
Extinction coefficient (ε) is not that much of a use in pharmaceutical industry as most of the times we are dealing with an unknown compounds. As a results using specific absorbance A(1%, 1cm) is a gold standard, and even in British Pharmacopeia, Clarke's Analysis of Drugs and Poisons and many more, A(1%, 1cm) values are only used for analytical purposes.
A11 is the absorbance value 1% w/v (1g in 100 mL). It is important to note that the concentration calculated from this equation will also be in (g in 100 mL).
8. UV Spectrum:
The the image below represents a general UV spectrum you get in the labs. All terminology you need to learn are explained in the graph, so spend a bit of time reading it before you go to the interpretation.
The intensity of the the peak is related to the extinction coefficient and will be explained in interpretation. The λmax is commonly used for analysis and finding the unknown concentration. As an example British Pharmacopeia tells you to measure your sample of paracetamol at 275 nm and calculate your concentration using 715 as your A11.
Now carry on to instrumentation or jump to interpretation.
