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 a c (Beer's
law) ------------- 1)
A 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.
No comments:
Post a Comment