UV/Vis Interpretation

  1. UV Spectrogram
    1. Full Spectrum Analysis
    2. Calibration Curve
  2. Energy Levels
  3. Auxochromes
  4. Absorbance Shifts
    1. Bathochromic (Red Shift)
    2. Hypsochromic Shift (Blue Shift)
  5. Application
    1. Concentration

1. UV Spectrograms:

1.1 Full Spectrum Analysis

Before, we start talking about effect of various factors, on UV analysis and interpretation, it is best to start learning the different spectrograms and their relationships to each other. The figure below shows a spectrogram we might get by running a whole spectrum analysis on a molecule (e.g Drug molecule).

As mentioned in Theory, UV/Vis wavelength ranges from 10 nm to 800 nm. If we use the instrument to see whether the drug absorbs at every single single wavelength, we will achieve the graph above. Here we can simply see that our Drug has an absorption at 250 nm.

Now if we try to run a full spectrum analysis, (like what we did above) using different concentrations of our Drug, we get a spectrogram like the one below at different concentrations.

By looking at the spectrogram above, one can easily realise that we can use it to our advantage to find the unknown concentration of that specific drug by creating a CALIBRATION CURVE because the intensity of the graph (A11) is always the same, even at different concentrations.

1.2 Calibration Curve:

Calibration curve is simply graph of Absorbance against the concentration which gives us a valuable opportunity to find an unknown concentration of the drug in question.
In order to create a calibration curve, we need to have minimum of 5 data points (known concentrations), and obtain their absorbances using the UV instrument. Plotting absorbance against the concentration, will give us a linear graph, from which we can calculate our sample with unknown concentrations using its line equation (Look at the figure below).

Some important notes:

  1. The plot is line of best fit and the close your R2 to 1, the more reliable your graph is for finding the concentration.
  2. The gradient (415) represents the A11
  3. The concentrations used to obtain absorbances (data points), should be such that absorbance readings WOULD BE between 0-1 otherwise the absorbance values are neither accurate, nor you will get a straight line after 1.

2. Energy Levels:

When the UV/Vis is radiated to a molecule, we see an absorption. The wavelength at which the radiation is absorbed, provides an energy for an electrical transition. This electrical transition is from the stable highest occupied molecular orbital (HOMO) to lowest unoccupied molecular orbital (LUMO) which is the unstable antibonding orbital. The UV radiation simply provides the energy required for this transition. Now considering the transition of π το π*, the electrons move from the stable regions of orbital to unstable regions of orbital which does not happen under normal condition. Two important small contributions to the unstable regions of the orbitals are as it follows:

  1. electrons spinning in orbitals in such that both electron spin in orbital of one atom making it negative and the other one positive. This is the HOMOLYTIC FISSION taught in chemistry books when chain reaction happens and ozone gets destroyed... and so on.
  2. The other minor probability is the HETEROLYTIC FISSION where both electrons in π orbital move to the unstable regions of the atomic orbitals in each atom separately, leaving both atoms involved neutral.

Look at the illustration below, nothing explains these stuff better than a nice diagram:

As we can see it is obvious that if ethene is dissolved in a polar solvent (H2O) the energy required for transition from the ground state (HOMO) to unoccupied state (LUMO) is decreased, because the excited state is stabilised by the polar solvent. This is really important because it means the molecule will absorb at low energy higher wavelength and UV absorbance shifts will happen.

3. Auxochromes:

Auxochrome is just a functional group bound to the chromophore with lone pair of electrons (unshared electrons). Now why is it important?
The existence of these groups affect the absorption of UV by chromophore by shifting their wavelength. There are two common ways they can do it:

  1. These groups are usually polar (oxygen in ketone) and so if we put our molecule in polar solvent, the ground state will be stabilised and BLUE SHIFT (see below) happens because we need more energy for the transition (n to π*).
  2. If we have an acidic group ( -OH in phenol), placing the molecule in a basic solvent will de-protonate the Oxygen and the increase in resonance effect will stabilise the transition and as a result RED SHIFT happens (see below).

4. Absorbance Shifts

2.1 Bathochromic (Red Shift):

If we have a sample such as phenol, when we add a base to the system, phenol becomes deprotonated. This will lead to induction of the pair of electrons on oxygen, into the benzene ring, creating a conjugated system (resonance structures) which decreases the energy difference between ground state and excited state of the electrons. As a result, we need less energy for the electronic transition of the compound. Less energy means, absorption at higher wavelengths and therefore an increase in our λmax. Because λmax gets closer to the infrared wavelength, this effect is called red shift or the fancy name of bathochromic

2.2 Hypsochromic Shift (Blue Shift)

The blue shift also follows the same principle as the red shift. This kind of shift happens when the energy of transition is increased. An example can be the stabilisation of the ground state such that it would be harder to excite the electrons to the higher energy levels. As explained above, imagine having auxochrome interacting with a polar solvent; this way we need some energy to break the interaction between the molecule and solvent before being able to excite the electron.
The smaller the wavelength the more energy is provided by the radiation, and as the absorption curve moves towards to ultraviolet wavelength (see diagram above), it is called the blue shift or hypsochromic shift.

As explained in this section, you can now appreciate the importance of solvent selection and pH regulation when carrying out UV analysis.

3. Application of Knowledge

Now that you have all the information you need, I think it is a good idea to wrap this section up with a nice real life example!

3.1 Concentration Calculation:

You are working in a formulation department in a prestigious pharmaceutical company. Your boss comes and gives you white powder code named wonderD15x. You are asked to find its intrinsic solubility using UV analysis. Note your boss was kind enough to work out the specific absorption of the molecule (A11 = 415). Before reading the answer, just think how you would do this...

Answer:

  1. You prepare a saturated solution of your sample.
  2. Now using the beer lambert law and A11, calculate, at what concentrations you will get an absorption of (A = 1), because as explained above, if you do, you will lose accuracy. This will give you an idea of to what extent to dilute your saturated solution for the analysis.

    So at what concentration do you think we get an absorbance of 1 ? (go back to Theory if you have forgotten the beer lambert law!)