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.

Spectrophotometry - UV-Visible Spectrophotometry

We have seen the basic instrumentation of spectrophotometer in the last post. Here, we will discuss the instrumentation in more details. In this post, we will discuss one of the types of spectrophotometry - UV-Visible spectrophotometry.

Components of UV-Visible Spectrophotometer:

We will discuss the following as the components of UV-visible spectrophotometer.

• Source
• Monochromator
• Prism
• Diffraction Grating
• Cuvettes
• Detectors
• Photomultiplier
• Silicon Diode
• Diode Array 

1. Source
The source should be stable during the measurement period, i.e.; the intensity of the radiation that is emitted should not fluctuate. Also, the radiation source should not change abruptly over its wavelength range.

Source of UV radiation:
Ultra-violet (UV) light is generally derived from a deuterium arc that provides emission of high intensity and adequate continuity in the range of wavelength 180-380nm. The mechanism for this involves the formation of an excited molecular species, which breaks up to give two atomic species and an UV photon. A quartz or silica envelope must be used in these lamps and the cuvettes should also be of the same material because of the heat generated and also to transmit the shorter wavelengths of UV radiation (glass absorbs radiation of wavelengths less than 350nm). However, the limiting factor is the lower limit of the atmospheric transmission at about 190nm.
Source of visible radiation:
The main source of visible radiation is tungsten filament lamp. This type of lamp uses the wavelength 350nm-2500nm. Recently, in modern instruments, the source is tungsten-halogen (also described as quartz iodine) lamp. In this type, lamps contain a small amount of iodine in a quartz ‘envelope’ which also contains tungsten filament. Here, iodine reacts with tungsten (gaseous state formed by sublimation) thereby producing the compound WI2 which is volatile. When the WI2 molecules hit the filament they decompose, redepositing tungsten back on the filament. The lifetime of a tungsten/halogen lamp is approximately double than that of an ordinary tungsten filament lamp. These lamps are very efficient. 

2. Monochromator:

Coming to the next component of UV visible spectrophotometry. The function of a monochromator is to produce a beam of 'monochromatic radiation' meaning 'single wavelength radiation' that can be selected from a wide range of wavelengths.
The essential components of the monochromator are:

• Entrance Slit
• Collimating device (which produces parallel light)
• Dispersing system (prism or dispersion grating)
• Focusing lens or mirror
• Exit slit

Polychromatic radiation (radiation of more than one wavelength) enters the monochromator through the entrance slit. The beam is collimated with the help of collimating device and then strikes the dispersing device (which can be prism or grating which is explained below) at an angle. The beam is split into its component wavelengths by the grating prism. By moving the dispersing element or the exit slit, radiation of only a particular wavelength leaves the monochromator through the exit slit. Below are explained two types of dispersing system as prisms and diffraction gratings.

a. Prisms:
A prism is a material that provides a continuous spectrum in which the component wavelengths are separated in space. It improves the definition of light entering the source through the prism by using an entrance slit which defines the incident beam. This beam then strikes the collimator which is used to produce a parallel beam at the prism as can be seen in the diagram showing a typical prism monochromator. Once the dispersion occurs, the spectrum is focused at the exit slit which scans the beam to isolate the required wavelength. Mirrors are used instead of lenses in UV-visible systems for the sake of efficiency and also is cheaper.

b. Diffraction Grating:
Diffraction grating is another means for producing monochromatic light. It consists of a series of parallel grooves (or lines) on a reflecting surface. These grooves are considered as separate mirrors from which the reflected light interacts with light reflected from neighbouring grooves to produce interference, and so to select preferentially the wavelength that is reflected when the angle of grating in the incident beam is changed.

Advantages of gratings over prisms:
There is better resolution and linear dispersion. Hence, the constant bandwidth and simpler mechanical design for wavelength selection. 

3. Cuvettes:
The container in which the sample or the reference solution is placed is called the cuvette. It must be transparent to the radiation which will pass through it. The material of which the cuvette is made is generally quartz or fused silica for spectroscopy in UV region. Silicate glasses can also be used for the manufacture of cuvettes for its use between 350 and 2000nm. 

4. Detectors:
There are four principal detectors as the photoconductive cell, the photomultiplier, the silicon diode, and the diode array. Of these four, the photoconductive cell is so severely restricted in both wavelength response and sensitivity and hence is never found in instruments these days. The most commonly encountered detectors are the last three which we will very briefly discuss here one by one.
a. Photomultiplier:
Light causes emission of electrons from a photocathode which accelerate past a series of dynodes. Electrons striking the first dynode release a secondary emission that is stronger than the original beam and so on through the series of the dynodes to produce a cascade effect. The electron density released by the final dynode to the anode can be many orders of magnitude greater than that from the cathode. The photomultipliers have an internal amplification that gives them great sensitivity and a wide spectral range.
b. Silicon Diode:
These are mechanically robust (as it is a solid-state device) and electronic benefits include reduced power supply and control circuit requirements.
c. Diode Array:
Diode array is an assembly of individual detector elements arranged in a linear manner or matrix-form which can be mounted so that the complete spectrum is focused onto an array of appropriate size. 

This was about the UV-visible spectrophotometry.

Spectrophotometry - Principles

We will be discussing some of the biophysical techniques in some posts following from now. In this post, we will have a look at some of the basic principles of spectrophotometry.

Electromagnetic Radiation (EM Radiation)

We will have to brush-up a little knowledge about Physics and Chemistry before going into the details of spectrophotometry to make things easy. Recall what you studied regarding light in your graduation. Briefly, we will revise here. The beam of light consists of a stream of photons or electromagnetic wave-form disturbance. Basically, the term 'electromagnetic' is a precise description of radiation such that the radiation is made up of an electrical and magnetic wave which are in phase and perpendicular to each other and to the direction of propagation as can be seen in the diagram above.

If matter is exposed to electromagnetic radiations (for example, infra-red rays), as can be seen in the adjacent figure, the radiation can be absorbed, transmitted, reflected, scattered or undergo photoluminescence (which can include a number of effects like fluorescence, phosphorescence etc. about which we will discuss in our future posts). Electromagnetic radiation is produced by events at molecular, atomic or nuclear level. A little understanding of Chemistry is needed here to understand the events that give rise to electromagnetic radiations such as the oscillations of nuclei and electrons in electrical or magnetic fields, molecular bending and vibration, excitation of orbital electrons, ejection of an inner orbital electron and rearrangement of the other electrons and nuclear break-up. Each of these events differ in terms of energy that is involved and thus, the radiation that they will emit will have different wavelengths. Thus, a complete spectrum of electromagnetic radiation is produced. Such a spectrum is shown in the form of the table below.

Spectrophotometry:

Now, coming to what is spectrophotometry. The spectrophotometry takes the advantage of dual nature of light namely:
              A particle nature which gives rise to photoelectric effect.
              A wave nature which gives rise to visible spectrum of light.

A spectrophotometer consists of two words as ‘spectrometer’ for producing light of selected wavelength and ‘photometer’ for measuring the intensity of light. Thus, a spectrophotometer is an instrument which is used to measure the amount of light (electromagnetic radiation) that a sample absorbs. The instruments are so arranged such that the sample can be placed between the spectrometer beam and photometer thereby measuring an unknown analyte concentration. In other words, it operates by passing a beam of light through a sample and measuring the intensity of the light reaching the detector.

Points to remember:

• The distance of one cycle is the wavelength (λ).
• The frequency, ν, is the number of cycles passing a fixed point per unit time.
• λ = c/ν (where c – velocity of light, 3x10⁸ms^-1)
• The shorter the wavelength, the higher the energy, E=hν

Now, the question arises, how does spectrophotometer know the concentration of unknown sample just by measuring the amount of light it absorbs. Here, the "laws of absorption" will play a crucial role and will clear all the doubts.

Laws of absorption:

Beer-Lambert Law (or Lambert-Beer Law or Beer’s law) states that there is a linear relationship between the absorbance and concentration of a sample. For this reason, Beer's law can only be applied when there is a linear relationship. The simple equation is:

                               A=εbc

where,

A - Absorbance (no unit)
ε - Molar absorptivity (Unit: Lmol^-1cm^-1) 

b - Path length of the sample (i.e.; the path length of the cuvette in which the sample is contained; unit: cm)
c - The concentration of the compound in the solution (Unit: mol L^-1)

Experimental measurements are usually made in  terms of transmittance which is:

                             T = I/Io                 

where,
I – Intensity of light after it passes though the sample.
Io – Initial intensity of the light.

The relation between A and T is:

 A = -log T = -log(I/Io)

The spectrophotometer displays either the % transmission or absorbance. Thus, unknown concentration of an analyte (sample) can be determined by measuring the amount of light the sample absorbs by applying Beer’s law. If the absorptivity coefficient is not known, then the unknown concentration can be determined by using a working curve of absorbance versus the concentrations derived from the standards. The graph on the left will make it clear.

Instrumentation and Mechanism:

Here, we will just see the outline of the instrumentation of the spectrophotometer. The basic structure of spectrophotometer is illustrated in the figure below. 

As we have seen above, the working of spectrophotometer is described as two instruments namely, 'spectrometer' and 'photometer'. Hence, here I will describe the role of each to make things easy to understand.
Spectrometer: It consists of a light source which produces a desired range of wavelength of light. Then there is a collimator which is a lens that will transmit a straight beam of light (photons). This beam of light will then pass through a monochromator which can be a prism or a grating that will spilt the light into several component wavelengths which will give rise to a spectrum. Then the slit will transmit only the desired wavelength (as in the figure, the desired wavelength being transmitted is in the range of yellow color).

Photometer: After the desired range of wavelength of light passes through the solution of a sample in cuvette, the detector or photocell detects the amount of photons that is absorbed and then sends a signal to galvanometer or a digital display.

I hope the basics and principles of spectrophotometer are clear to you.

In the next few posts, we will have a look at the different types of spectrophotometry like that of UV-visible spectrophotometry, IR spectrophotometry, fluorimetry.
Any doubts are welcome!