Monday, December 21, 2015

Photometry: Principle, applications and types



Photometry

Fig. Principle of colorimetry
When light is passed through a coloured solution, certain wavelengths are selectively absorbed giving a plot of the absorption spectrum of the compound in solution. The wavelength at which maximum absorption is called the absorption maximum (λmax) of that compound. The light that is not absorbed is transmitted through the solution and gives the solution its colour.

Photometric instruments measure transmittance, which is defined as follows:



                               Intensity of the emergent (or transmitted) light         Ie
Transmittance (T)= ------------------------------------------------------------- = ------
                                               Intensity of the incident light                    Io

Transmittance is usually expressed on a range of 0 to 100%.

If the concentration of the substance in solution is increased linearly, or if the path length that the light beam has to traverse is increased, transmittance falls exponentially. So a term absorbance is defined so that it is directly proportional to the concentration of the substance.

                                                Ie
Absorbance (A)= log1/T = log  ----
                                                Io

Absorbance has no units. Photometric instruments electronically convert the measured transmittance to absorbance values.


Much of photometry is therefore based on two laws:


When a parallel beam of monochromatic light passes through a solution, the absorbance (A) of the solution is directly proportional to concentration(c)of the compound in the solution. This is Beer's law

Each successive layer of the solution absorbs a constant proportion of the light entering the solution, although the absolute amount entering each layer diminishes progressively. Therefore, absorbance is directly proportional to the thickness or length of the light path (l)  through the solution. This is Lambert's law.

a c (Beer's law)  ------------- 1)
a l (Lambert's law) -----------2)

By combining 1) and 2) ,  we get
                                                    A = εl c l
εl, the proportionality constant is termed the molar absorption coefficient.
It is specific for a given substance at a given wavelength. It is the absorbance of a one molar solution of a substance with a light path of one centimetre (if c is expressed in mol/L). But Beer's law applies to only dilute solutions and in practice the concentrations of the solutions that are used in photometry are usually in the mmol/L range. In colorimetry, the absorption coefficient is not usually used. Concentration of an unknown solution can be determined by using equation 1, which is derived as follows:

Absorbance of test sample (At) = ε x concentration of test (Ct) x l

Absorbance of standard sample (As) = ε x concentration of standard (Cs) x l

Hence,
                                 Absorbance of Test sample (At) 
Conc. of Test (Ct) = -------------------------------------------- X Conc. of standard (Cs)
                            Absorbance of Standard sample (As)  

The light path, l, is usually kept constant in photometric measurements at 1 cm. This is the diameter of the tube (called the cuvette) containing the solution.


A standard (or calibrator) is representative of the substance whose concentration is sought to be determined. The concentration of the compound in the test sample is obtained by comparing its absorbance with that of a known concentration of a standard solution. Ideally, a series of standards of known concentration are prepared to obtain a standard (calibration) curve. This helps to determine the range of concentrations over which Beer's law is obeyed.

Appropriate blanks to exclude the absorbance contributed by the solvents and reagents used- i.e. by anything other than the compound of interest- are also essential for any photometric measurement.

Colorimetry

Colorimetry uses the basic principles of photometry but the solutions have to be coloured, i.e. they must absorb light in the visible range. Colourless compounds are converted into coloured compounds using chemical reactions. Under defined reaction conditions, the quantity of colour formed is proportional to the quantity of the original colourless compound.

Photometric instruments



Colorimeter

It is used to measure the intensity of light transmitted through a coloured solution. It uses light only in the visible range. Ordinary light from a tungsten lamp is passed through a suitable filter to obtain light of a desired wavelength, which is then passed through the solution.

Transmitted light falls on the sensitive surface of selenium photocell which generates a current proportional to the light intensity. The cell is connected to a galvanometer, which is used to read out percentage transmission or absorbance.

Fig. Components of spectrophotometer


Spectrophotometer

A spectrophotometer works on the same principle as a colorimeter but it is more sensitive and sophisticated. There are light sources that emit light in the ultraviolet, visible and infrared regions of the spectrum. The wavelength is selected using a prism or diffraction grating and narrower bandwidths can be selected. Since light in the ultraviolet and infrared ranges is also emitted, the compound to be estimated does not necessarily has to be coloured and can be measured directly if they significantly absorb even at these wavelengths. This offers a significant advantage over the colorimeter, which is restricted only in the visible range.



Types of spectrophotometers:

Grating spectrophotometers: In these spectrophotometers, the monochromator is a diffraction grating, which disperses the white light into a continuous spectrum. By turning the wavelength adjustment knob, the grating is rotated and different parts of the spectrum are allowed to fall on the photocell. In this manner, the desired wavelength can be selected.

Prism spectrophotometers: The prisms may be made of glass or quartz. Light from a tungsten filament is focussed on the entrance slit and it passes through a glass prism that forms an extended spectrum. Only the light that falls on the exit slit can pass through the cuvet and illuminate the photocell. The monochromatic light obtained here is much more pure than that produced by light filters, i.e. a much narrower band of wavelength is present in the incident beam of light. Different photocells are used, one sensitive to the short wavelength range of the instrument (360-525nm), while the other is used for the longer wavelengths (600 to 1000nm). The total wavelength range at which measurements can be made includes the visible spectrum (400 to 750nm) and extends on each side into the near ultraviolet (360 to 450 nm) and the near infrared (750 to 1000nm). It is therefore possible to estimate substances that are more or less colourless in the visible region, but which absorb light in the ultraviolet or infrared regions.

Quartz prisms help in extending the wavelength range below 350nm down to about 190nm. A deuterium lamp with a quartz envelope is provided as an additional light source. This is a rich source of UV radiation. Quartz cuvets will also be needed if one has to work in the UV range.

Uses

A spectrophotometer can be used for routine analysis in clinical chemistry.  By using a narrower bandwidth than is available with ordinary filters, the absorbance is often higher and the relation between absorbance and concentration remains linear over a wide range. If very dilute colour is to be measured (e.g. in serum iron determinations), then a spectrophotometer is usually required.

Analytical methods depending on ultraviolet absorption are commonly used in clinical chemistry and research. Examples include serum enzyme assays, assays of glucose, urea, uric acid, etc, which take advantage of the UV absorption of the coenzymes NADH and NADPH at 340nm. For, such methods a quartz spectrophotometer is essential.

ELISA Reader

ELISA stands for "enzyme linked immuno-sorbent assay." The ELISA reader or microplate reader is modification of the spectrophotometer, which enables quantitation of upto 96 samples per ELISA microplate. Microplates have a 12 x 8 well format. A set of 8 lamps, 8 filters and 8 detectors enable the sequential determination of 96 samples.

Automated clinical chemistry analyzers:

These usually incorporate mechanized versions of basic manual laboratory techniques and procedures. They help in the processing, transport and testing of a large number of clinical specimens in an efficient manner. Work is simplified by using robotic and computer technology to undertake repetitive tasks like pipetting, dispensing and mixing. Advanced versions of the spectrophotometer help in increasing specimen throughput.


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