Spectrophotometry

Spectrophotometry

The present aim of the clinical chemists is the development of micro and ultramicro –methodology for the analysis of all the constituents of blood and body fluids. The study of functions of the body in both health and diseases critically requires the quantitative analysis of blood and body fluids for their various constituents.

Because so much of the quantitative methodology of biological chemistry is based on colour or light measurement, consideration must be given to the physical properties involved and to the fundamentals of the instrumental procedures. Many methods for the quantitative analysis of blood , tissue, urine, and other biological material are used on the separation of the substance in question and its chemical conversion to a compound which is capable of absorbing radiant energy. If the reaction product in solution absorbs light in the visible region of the spectrum then the solution will be coloured. The intensity or depth of colour of such a solution can be used as a measure of concentration of the dissolved material. Determinations involving quantitative estimation of colour are known as colorimetric analyses.

Many biochemical experiments involve the measurement of the compound or group of compounds present in a complex. Probably, the most widely used method for determining the concentration of biochemical compounds is colorimetry which makes use of the property that when white light passes through a coloured solution, some wave lengths are absorbed more than others. Many compounds are not themselves coloured, but can be made to absorb light in the visible region by reaction with suitable reagents.These reactions are fairly specific and in most cases very sensitive, so that quantities of materials in millimolar quantities can be measured.

A knowledge of the physical nature of colour indicates that it is produced when specific regions or wavelengths of the visible spectrum are absorbed. To take a simple example , a solution has blue colour because , it absorbs a lesser proportion of the blue components of the mixed white light passing through it than any other coloured components. Thus the white light entering the solution will emerge in diminished intensity and have a preponderance of the blue wave lengths so the solution appears to be blue. The proportion of the various wave lengths of light absorbed is directly related to the concentration of light absorbing material. The intensity of the remaining transmitted colour is also a measure of the concentration of the material present in the solution .

Analytical procedures based upon the direct measurement of light absorption at specific wavelengths or regions of the spectrum are known as photometric procedures and the instruments used are photometers and spectrophotometers. In addition , there are methods which are dependent on the ability of insoluble particles to scatter light, called turbidometric methods and methods which are dependent on the ability of materials to emit light under specified conditions, called fluorimetric methods.

Principle: Spectrophotometric technique is based on the basic laws of light absorption. For uniform absorbing medium the proportion of the light radiation passing through it is called the transmittance, T, where T=I/I₀. I₀ = Intensity of the incident radiation, I= Intensity of the transmitted radiation. The extent of radiation absorption is more commonly referred to as the absorbance (A) or extinction (E) which are equal to the logarithm of the reciprocal of the transmittance,

i.e., A = E = log 1/T = logI₀/I

Transmittance is generally expressed on a range of 0-100% and used in certain type of turbidity measurement. Absorbance or extinction varies from 0 to ∞.

The Beer –Lambert Law

When a monochromatic light of initial intensity I₀ passes through a solution in a transparent vessel, some of the light is absorbed so that the intensity of the transmitted light I is less than I₀ .There is some loss of light intensity from scattering by particles in the solution and reflection at the interfaces, but mainly from absorption by the solution. The relationship between I and I_o depends on the path length of the absorbing medium, l, and the concentration of the absorbing solution, c. These factors are related in the laws of Lambert and Beer (Fig 10.9).

Lambert’s law: When a ray of monochromatic light passes through an absorbing medium its intensity decreases exponentially as the length of the absorbing medium increases.

I = I₀ e^- k₁l

Beer’s law : When a monochromatic light passes through an absorbing medium its intensity decreases exponentially as the concentration of the absorbing medium increases.

I = I₀ e^- k₂ c

These two laws are combined together in the Beer- Lambert law:

I = I₀ e^- k₃ cl

Transmittance: The ratio of intensities is known as the transmittance (T) and this is usually expressed as percentage

Percent T = I/I₀ 100 = e^{- k}₃^cl

Extinction: If logarithms are taken of the equation instead of a ratio then

log_e Io/ I = k₃cl

log₁₀ Io/I= k₃cl / 2.303

log₁₀ Io/I= kcl

The expression log₁₀ Io/I is known as the extinction (E) or absorbance(A). The extinction is some times referred as optical density.

Therefore

A (or) E = k cl

where k is molar extinction co-efficient for the absorbing material at wave length λ, c = molar concentration of the absorbing solution, l = path length in the absorbing material in cm. If the Beer- Lambert law is obeyed correctly and l is kept constant, then a plot of extinction against concentration gives a straight line passing through the origin (Fig 10.10)

Some colorimeters and spectrophotometers have two scales, a linear one of percent transmission and a logarithmic one of extinction (Fig 10.11). The extinction scale is related linearly to the concentration and this scale is used in the construction of a standard curve. With the aid of such a curve the concentration of an unknown solution can easily be determined from its molar extinction.

Molar extinction coefficient : If l is 1 cm and c is 1 mol/ litre then the absorbance is equal to k, the molar extinction coefficient, which is characteristics for a compound. The extinction coefficient k is thus the extinction given by 1 mol / litre in a light path of 1 cm and usually written E_1CM , it has the dimention of mol^-1 cm^-1. The instruments used for the measurement of extinction by the molecules to be quantified are spectrophotometer and photoelectric colorimeters.

The photoelectric colorimeter:

A diagram of the basic arrangement of a typical colorimeter is given in Fig 10.12.

White light from a tungsten lamp passes through a slit then a condenser lense, to give a parallel beam which falls on the solution under investigation contained in absorption cell or cuvette. The cell is made of glass with the sides facing the beam cut parallel to each other. In most of the colorimeters, the cells are 1 cm square and will hold 5 ml of solution .

Beyond the absorption cell is the filter, which is selected to allow maximum transmission of the colour absorbed. If a blue solution is to be measured, a red filter should be selected.The colour of the filter is, therefore, complementary to the colour of the solution under investigation (Table 10.1). In some instruments the filter is located before the absorption cell.

The light then falls on to a photocell which generates an electrical current in direct proportion to the intensity of light falling on it. This small electrical signal is increased in strength by the amplifier , and the amplified signal passes to a galvanometer, or digital readout, which is calibrated with logarithmic scale and the extinction can be read directly. The blank solution (which does not contain the material under investigation) is first taken in the cuvette and reading adjusted to zero extinction and this is followed by the test solution and the extinction is recorded directly.

A better method is to split the light beam , pass one part through the sample and the other through the blank, and balance the two circuits to give zero. The extinction is determined from the potentiometer reading which balances the circuit..

Photometric analysis: There are four general steps in carrying out a photometric analysis:

·           separation of the substance from the complex mixture- for e.g., estimation of blood glucose requires the precipitation of lipids and proteins by using deproteinising agents which otherwise interfere with the colour reaction of glucose

·           quantitative conversion to a coloured or light absorbing substance-for e.g., after deproteinisation as mentioned above for glucose estimation, the supernatant is made to react with orthotoluidine reagent to give a greenish blue coloured complex

·           measurement of light absorption- for e.g., the colour intensity of the above mentioned complex is measured by using a red filter.

·           calculation of the concentration of the substance - for e.g., by comparing the extinction with that of the standard solution of the same substance of known concentration.

UV Absorption Spectrophotometry

A spectrophotometer is a sophisticated type of colorimeter where monochromatic light is provided by a grating or prism in the place of filter in ordinary colorimeter. The band width of the light passed by a filter is quit broad, so that it may be difficult to distinguish between two compounds of closely related absorption with a colorimeter. Some compounds absorb strongly in the ultra violet region and their concentration can be determined by using a more expensive type of spectrophotometer which operates down to 190 nm. For e.g.,

·           The activity of enzymes requiring NAD as coenzymes can be determined by treating the enzyme source with the relevant substrate and measuring the NADH formed (colourless) which gives strong absorption at 340 nm. The increase in absorbance is proportional to the concentration of the enzyme.

·           the concentration of uric acid can be estimated by measuring the extinction of the solution at 293 nm before and after treatment with an excess of the enzyme uricase. At pH 9.0, uric acid which absorbs at 293 nm, is oxidized by uricase to allantoin, which has no absorption at this wave length. The decrease in absorbance at 293 nm is a measure of uric acid level.

The main components of a simple spectrophotometer are shown in Fig 10.13

Absorption spectra

Many compounds have characteristic absorption spectra in the ultra violet and visible regions so that identification of those materials in a mixture is possible.

Proteins: Proteins absorb strongly at 280 nm according to their content of the amino acids tyrosine and tryptophan, and this provides a sensitive and non-destructive form of assay.

Nucleic acids: Nucleic acids and their component bases show maximum absorption in the region of 260nm. The extent of absorption of nucleic acid is a measure of their integrity, since the partial degraded acids absorb more strongly than the native materials.

Haem proteins: These conjugated proteins absorb in the visible region as well as in the UV region of the spectrum due to haem group. The visible spectra of the oxidized and reduced forms of cytochrome C are sufficiently different so that the relative amounts of these forms can be determined in a mixture.

Things to remember: The detailed operation of a particular instrument must be obtained by carefully reading the instruction manual. Few important points concerning the use and care of calorimeters and spectrophotometers are given below.

·           Cleaning the cuvette:The cuvette should be cleaned by soaking in 50 per cent v/v nitric acid and then thoroughly rinsed in distilled water.

·           Using the cuvette: First of all, fill the cuvette with distilled water and check them against each other to correct for any small difference in optical properties. Always wipe the outside of the cuvette with soft tissue paper before placing in the cell holder. When all the measurement have been taken, wash them with distilled water and leave in the inverted position to dry.

·           Absorption of radiation by cuvettes: All cuvettes absorb radiation and the wave length at which significant absorption occurs depend on the material from which the cuvette is made. Silica cuvettes are the most transparent to U/V light but they are expensive. Glass cuvettes are much cheaper than silica, and so they are used whenever possible and invariably in the visible region of the spectrum.

·           Light source : A tungsten lamp produces a broad range of radient energy down to about 360 nm. To obtain the ultra violet region of the spectrum a deuterium lamp is used as the light source.

·           Blanks: The extinction of a solution is read against a reagent blank which contains all the reagents except the compound to be measured. The blank is first placed in the instrument and the scale adjusted to zero extinction before reading any solution . Alternatively, the extinction can be read against distilled water and the blank reading can be subtracted from that of the test solution

·           Duplicates: It is essential to prepare all blanks, standard solutions and unknown solutions in duplicates so that the accurate standard curve can be obtained.

·           Construction of standard curve : A series of concentrations of standard solution are taken in different test tubes and made to react with colouring agents. The blank tube is also treated similarly but by replacing standard solution with water. The absorbance are measured at the corresponding wavelength and a graph is plotted as concentration of the standard versus the absorbance.

Applications of spectrophotometry

Colorimetry and spectrophotometry have widest application in biological sciences. These techniques are used for the determination of

·           glucose, proteins, lipids, nucleic acid etc

·           turbidity of solutions( bacterial cell mass)

·           absorption spectrum of a compound

·           purity of compound by knowing the molar extinction coefficient which is maximum for a pure compound.