Complete information on Frequently Used Spectroanalytical Methods

Spectrophotometry:

Absorption spectrophotometry in ultraviolet and visible region of electromagnetic spectrum is one of the oldest physical methods used for qualitative and quantitative analyses as well as structural determinations.

The wave length of ultraviolet rays starts at about 2000 A and end at 4000 A whereas wave length of visible region starts at about 4000 A and ends at about 7500 A. Wavelengths below 2000 A (called the vacuum ultraviolet) are also included among ultraviolet radiations. However, these high energy radiations find little use in the practice of analytical chemistry. Spectrophotometry can be divided into main sections:

(A) Visible Spectrophotometry:

Many substances are coloured or produce coloured products when they react with suitable reagents. When a beam of monochromatic light (light consisting of a narrow wave length band) is passed through a solution of such substance its intensity decreases. The decrease in its intensity is proportional to the concentration of the coloured substance in the solution if other factors like wave length of the radiation, the length of optical path through the solution and material of which the cell containing the solution, remain the same. It is this phenomenon which utilized in Absorption spectroscopy to determine the concentration of substance/substances in a sample.

The instruments used to measure intensity of electromagnetic radiations are: photometers, spectrophotometers and colorimeter. A Photometer is a cheap instrument which employs filters to isolate a relatively narrow wave length band of visible light and a photovoltaic or photoemissive cell to measure the intensity of radiations.

A Spectrophotometer is a instrument which employs a prism or grating to isolate a narrow band of electromagnetic radiations, instead filters. To -measure the intensity of radiations more sensitive photoemissive cell or a photomultiplier tube is used. With the help of this instrument a large region of electromagnetic spectrum can be scanned.

A very sensitive photomultiplier cell enables the instrument to measure very low light intensities. Any instrument which can be used for measuring the absorbance of a sample in the visible region of electromagnetic spectrum is called a Colorimeter. Thus, the name calorimeter can be applied to a photometer or a spectrophotometer working in the visible range of electromagnetic spectrum.

1. Description of Instruments used in Visible Spectrophotometry:

An instrument which measures the intensity of radiations in the visible and ultraviolet region of electromagnetic spectrum has following components:

a. A source of radiations.

b. Filters and monochromators.

c. A sample holder.

d. Detectors or the read-out devise.

(a) Source of Radiations:

To produce radiations or light with wave length ranging between 4000-7500 A the common house hold tungsten filament bulbs are mostly used. This lamp, however, suffers from the disadvantage that major part of radiant energy produced is in the red and infra-red region – only about 15% of it is of shorter wave lengths. The disadvantage can be over-come by raising the operating temperature of the lamp to about 2500°C.

At this temperature the total energy output shifts towards shorter wave lengths. Higher temperatures shorten life of the bulb. However, as these bulbs are quite cheap they can be replaced frequently. When higher intensities of light is required carbon arc is used which is a more intense source of visible light.

(b) Filters and Monochromators:

The source of relectromagnetic radiations usually produces radiations of mixed wave lengths spread over the entire visible range. For spectroscopic work we have to select light of a definite colour or a narrow band of wave length from the continuous spectrum. In simple instruments this is done by introducing filters of various colours in the optical path which isolates a narrow band of radiations of desired wave length. Coloured filters allow only a narrow band or radiations to be transmitted through while others are absorbed. This depends on the colour of the filter. Usually filters are made up of glass sheet which has been coloured by addition of some dye or colouring material.

In more sophisticated instruments Monochromators are used to isolate a narrow band from the radiations of mixed wave lengths. The essential parts of a monochromator are an entrance slit, a prism or grating which disperses incoming radiations into constituent wave length bands and an exit slit which allows only the radiations of desired wave length band to pass out of the monochromator.

The prism is made up of solid glass in the case of instruments designed to work with radiations of visible light. For ultra violet radiations quartz or fused silica prisms are used as glass is not completely transparent to the entire range of ultraviolet rays used in analytical work.

The prism is provided with devise to rotate it so that the required wave length band could be directed to pass through the exit slit. Many instruments employ a Grating to disperse bands of different wave lengths as they are cheaper. A grating consists of a large number of parallel lines (grooves) etched over a highly polished surface of some metal like Aluminium.

Usually 15,000-25,000 grooves per inch have to be etched for ultraviolet and visible region of electromagnetic spectrum. Light rays incident on the slopes of the grooves are spread out over a range of angles as they are reflected back. Bands of different wavelengths are reflected back at different angles and the cumulative effect of so many grooves effectively isolates narrow bands of different wave lengths at different angles.

By rotating the grating with the help of a knob provided for the purpose required wave lengths may be directed to pass out of the exit slit. The main advantage of a prism is those different waves lengths of radiations disperse by it do not overlap. However, gratings suffer from frequent overlapping of spectral bands. A system of filters has to be used in the instrument to obtain bands of clear monochromatic radiations when gratings are employed to disperse the radiations.

(c) Sample holders:

The beam of monochromatic radiations obtained from the monochromator is now directed to pass through the sample which is usually a solution kept in the sample cell placed in sample holder. In double beam instruments the beam of radiations from monochromator may be split up into two beams so as to pass alternatively through the sample holder and the reference cell provided to hold standard solutions or reference solutions.

This avoids the necessity of removing the sample and placing the reference solution each time we have to observe its absorbance. In single beam instruments light from monochromator enters straight into the sample holder. The sample and the reference cell which has to be placed in the sample holder have to be transparent to the wave lengths on which the absorbance is being recorded. For visible region of electromagnetic radiations colour corrected fused glass cells are usually used.

Both rectangular and cylindrical cells may be employed. The internal diameter of a cylindrical (or the width of the cell if rectangular) cell used is usually 1 cm. so that the thickness of solution traversed by light is 1 cm. In the case of ultraviolet radiations quartz or fused silica cells are used instead of glass cells.

(d) Detectors:

The intensity of the colour of sample solution or the colour produced after treatment of the sample with suitable reagents has to measure for the determination of concentration of substance in the sample. This may be done by preparing a graded series of solution of known concentration which is given exactly the same treatment as the sample and comparing the intensity of colour of the unknown solution with the known solution visibly. However, visual comparison – comparison with naked eyes – yields very crude results which are not so reliable.

A more accurate way to measure the intensity of colour of the sample solution is to convert the optical signal into an electric signal (electric current) which can be measured with precision and accuracy. The device which converts the optical signal into electrical signal is referred to as Detectors. Three main types of detectors are commonly employed for the purpose:

Barrier layer cell or Photovoltaic cells:

These cells are used in simple instruments and are not so sensitive as photoemissive or photomultiplier cells being described herein. It consists of a semiconductor such as Selenium which is deposited on a strong metal base. A very thin network of silver covers the surface of the semiconductor and acts as a collector electron which is connected to the terminals of a current measuring device.

The metal base acts as the other terminal. Radiations falling on the surface of semiconductor produce electrons which are collected by the silver net-work. A voltage difference between the silver net-work and the metal base is established causing a current to flow in the external circuit which is read by the meter. The deflection of the needle of the meter is proportional to the amount of radiations incident on the surface of the semiconductor.

Photoemissive ceil or Photocell:

A photoemissive cell consists of a light sensitive cathod which is shaped like a half cylinder of metal coated with Cesium or Potassium oxide and silver oxide. The anode is fixed towards the concave side of the cathod.

The entire device is enclosed in an evacuated bulb. As radiations fall on the tube cathod emits electrones which are captured by the anode. A potential difference is created between the anode and the cathod and current flows into the external circuit and is measured by a galvanometer provided for the purpose – the degree of deflection of the needle being proportional to the intensity of radiations incident on the photo cell. Photoemissive cells are more sensitive than photovoltaic cells discussed earlier.

Photomultiplierd cells:

Photomultiplier cells are more sensitive than either photovoltaic cells or photoemissive cells. It consists of a cathod covered with photoemissive material and a series of positively charged plates each of which is charged with successively higher potential (called dynodes).

These plates are also coated with material which emits several electrons for each electron collected on its surface. The electrons emitted by a series of charged plates are finally collected by an anode. The entire assembly of cathod, charged plates and anode are enclosed in an evacuated tube. When electromagnetic radiations hit the cathod through a transparent slit in the tube, electrons are discharged which strike the first plate resulting in release of many electrons.

These electrons strike the second positive charged plate releasing many more electrons and so on. In this way, a large number of electrons are dislodged which when finally collected at the anode cause a large potential difference to develop between the anode and the cathode. A strong current flows through the circuit – the deflection being proportional to the intensity of radiations incident on the cathode. Very low intensities of radiations which cannot be measured either by photovoltaic cell or by photoemissive cell can be measured by a photomultiplier cell.

2. Method adopted for measurement of concentration:

In practice spectrophometry involves measurement of the intensity of radiations after they have passed through a sample to determine its absorbance at the wave length which is absorbed most effectively by the substance in the sample. Subsequently the absorbance is compared with the absorbance of solutions of known concentration (standard solution) to determine the concentration of the substance present in the Sample. This usually involves following steps:

a. Determination of wave length at which the sample shows maximum absorbance. This is done by rotating the knob which turns the prism or grating to allow different wave lengths of electromagnetic radiations to pass through the sample one after the other. The instrument is finally set at the wave length at which maximum absorption or the absorption maxima of the substance occur.

b. Preparation of a graded series of solutions of the substance concerned and determination of their absorbance at the wave length determined above.

c. Preparation of a graph showing absorbance of known standard solutions on Y-axis against different concentrations on X-axis.

d. Determination of the concentration of unknown solution by drawing a line parallel to Y-axis from the point of absorbance of unknown to X-axis. The point where this line intersects the X-axis, is the point which denotes the concentration of the substance in the sample.