Friday, July 26, 2013

Spectrophotometry - Applications of UV-Visible Spectrophotometry

In the previous posts, we have seen about the principles and the instrumentation of UV-visible spectrophotometry. By now, you might have understood that spectrophotometry is a highly versatile technique. So, it is difficult to jot down all the applications of the same. Here are summarized a few applications which are divided into two main groups as:
  1. Chemical Applications of UV-Visible Spectrophotometry
  2. Structural Applications of UV-Visible Spectrophotometry
We will discuss here one by one the applications. Starting first with the chemical applications of UV-visible spectrophotometry.

1. Chemical Applications of UV-visible spectrophotometry:

a. Quantitative Analysis (Identification of the concentration of the unknown substance):
UV-visible spectrophotometry is used to measure the concentration of an unknown sample. For this, first, the choice of the absorption band is to be made where the absorbance measurements will be taken (meaning in what range of wavelength will the sample absorb). The sample of interest’s absorption spectrum may be available in the literature if it has already been researched. If not, then double-beam spectrophotometry has to be performed to know where its absorption band will lie.
A suitable absorption band is now selected. Generally all the organic compounds will absorb in the UV-visible range of the spectrum and so a number of biological compounds may be measured using UV-visible spectrophotometer. Unknown concentration of nucleic acid and proteins are a good example. Nucleic acids absorb at 254nm (or 260nm) and proteins at 280nm. Nucleic acids absorption depends on the aromatic rings of purines and pyrimidines while that of proteins at 280nm depends on the number of amino acids - tyrosine and tryptophan content and a little due to phenylalanine content.


b. Qualitative Analysis (Identification of an unknown substance):
UV-visible spectrophotometry may be used to identify various classes of compounds in both pure state and as well as in biological preparations. This is done by plotting the absorption spectrum curves. These curves represent specific class of compounds and a knowledge of these curves will help in identification of any substance. For example, the substances which do not absorb in 220-280nm range are usually aliphatic or alicyclic hydrocarbons or their derivatives. Also, the complex systems will give rise to absorption curves with several maxima and each of them will have a characteristic shape and range indicating the presence of a particular functional group.
The graph on the right side shows the absorption spectrum of several plant pigments. As we can see each of the pigment has its own peculiar absorption spectrum which will help it identify in a mixture of compounds.

c. Enzyme Assay:
The enzyme activity can be easily, quickly and conveniently be calculated when the substrate or the product is colored or absorbs light in the UV range. In these cases. The rate of appearance or disappearance of light absorbing product or substrate can be measured with the help of spectrophotometer (which can also give the continuous record of the progress of reaction). We will take an example of the enzyme lactate dehydrogenase to understand how the enzyme assay is carried out or how enzyme activity is measured. Lactate dehydrogenase is an enzyme involved in the transfer of electrons from lactate to NAD+. The reaction is shown as follows:
So, as we can see here, the products are pyruvate, NADH and a proton. Here, one of the products, NADH absorbs radiation in the ultraviolet range at 340nm and its counterpart NAD+ does not. Neither any of the other substrate nor the product absorbs at 340nm. Thus, the progress of the reaction in the forward direction can be followed by measuring the increase in absorption at 340nm in spectrophotometer. Here, comes the role of optical assays which prompts their use in following the time course of an enzymatic reaction in which neither the substrate nor the product have a characteristic absorption spectrum.
Such reactions are then coupled to another enzymatic reaction (hence also called Coupled Assay) which can be measured easily optically. Example of such reaction is that of phosphoenopyruvate and ADP reacting to yield pyruvate and ATP catalyzed by pyruvate kinase.
Here, as neither any of the substrates nor the products absorb radiation, hence, this reaction can be coupled to the above mentioned first reaction. Here, if lactate dehydrogenase and NADH are added in excess, the system will be a little manipulated and we will get the coupled reaction as follows:
As we have added excess of NADH to the reaction, the system will now absorb at 340 nm. Thus, for each molecule of pyruvate formed in the first reaction, a molecule of NADH is oxidised to NAD+ in the second reaction where pyruvate in converted to lactate. As mentioned earlier, NAD does not absorb at 340nm, the absorbance goes on decreasing as  pyruvate gets converted to lactate.

2. Structural Applications of UV-Visible Spectrophotometry:

a. Control of Purification:
This is one of the most important application of UV-visible spectrophotometry. Impurities can be detected very easily by testing if the compound is not showing its characteristic absorption spectrum. Example: Benzene impurity in absolute alcohol can be detected by this method. This can be detected by measuring the absorbance at 280nm. As at 280nm, benzene will absorb, whereas alcohol (210nm) will not absorb. 

b. Study of Cis-Trans Isomerism:
The trans-isomer is more elongated as compared to its counterpart cis-isomer. Hence, this structural difference will be reflected in absorbance spectrum. The trans-isomer will have a higher wavelength of maximum absorption.
The graph at the adjacent shows the absorption spectrum of the azobenzene dye, 4-n-butyl-4'-methoxyazobenzene (BMAB) where both cis-BMAB and trans-BMAB have different absorption spectrum.

c. Molecular Weight Determination:
Suppose a compound forms a derivative with a reagent which has a characteristic absorption band. Suppose a compound forms a derivative with a reagent; now it will give the absorption band of a high intensity at a wavelength where the compound does not absorb, then the extinction coefficient of the derivative is usually the same as that of the reagent. Although the extinction coefficient will remain same for any of the derivatives formed, the optical density is different  for the compounds of different molecular weight. The molecular weight of the compound can then be calculated readily on the basis of absorption data.
                                                                   M = awb/OD
where  a – absorption coefficient
w – weight of the compound in g/l
b - path-length.

d. Turbidimetry:
Any particulate matter (or even bacteria) makes the solution look turbid. This is due to Tyndall effect which is because of the light scattering by the colloidal particles.
The particles in this solution will absorb at a particular wavelength and these particles will scatter the incident light. If this happens, then the radiation of a wavelength which is not absorbed by the solution is made to pass through the suspension and the apparent absorption will be solely because of the scattering by the particles. So, the transmitted light will have lower intensity as compared to that of the incident light.  As a result, if the intensity of the transmitted light is measured, it will give an idea of the number of particles in the suspension. This technique is turbidimetry. By using this technique, we can find out an approximate number of particles in a given suspension.