To study the principles of colorimetry and

spectrophotometry and verification of Lambert-Beer’s

law.

Photometry: It is the most common analytical technique.

When a monochromatic light (light wave of a specific
wavelength) falls on a colored solution, the proportion of
incident light transmitted by the solution containing a light
absorbing material depends upon
1) Nature of substance
2) Wavelength of incident light
3) Amount of light absorbing material in the path of light and
this in turn is proportional to the concentration of substance
and depth of solution through which the light is passing.
When a monochromatic light with an original intensity Io
passes through a solution that can absorb radiant energy of
certain wavelength, the intensity of transmitted radiant energy
Is will be less than Io. Some of incident energy may be
reflected by the cell (containing the solution) or absorbed by
the cell as well as the solvent. To eliminate these factors and
to consider the absorption by the compound, a blank or
reference solution is used.

Blank solution or test tube: This contains all the reagents but
will not have the substance to be analyzed. Thus, the blank
test tube serves to set zero. This overcomes the absorption of
light by the solvent and the reagents. Distilled water can also
be used to set the zero but then the absorbance of a control
solution containing all the reagents has to be subtracted from
test and standard values.
Standard or Calibrator: It represents the substance whose
concentration is to be estimated. Concentration of standard is
known and ranges within limits found in the specimen. The
term calibration standard is used if it is necessary to avoid
confusion with other meanings of ‘Standard’. Various types of
standards used are:
 Arbitrary Standard: Calibrator standard containing an
unknown quantity of the specified substance. The content
is assigned by convention and expressed in arbitrary
units e.g. international biological standards,
immunological standards.
 Internal Standard: A known concentration of a
substance, which is not normally present in the sample
and clearly distinguishable from the substance to be
analysed, is added to sample or both standard and
sample, for the purpose of making results more accurate.
 Primary Standard: Substance of a known chemical
composition and sufficient purity is used for preparing
primary standard. The concentration is determined by
dissolving a weighed amount of primary standard
material in an appropriate solvent and making up to a
stated volume or weight.
 Secondary Standard: Solution used as a calibration
standard in which the concentration or other quantity has
been determined by an analytical method of stated
reliability.

The transmittance (T) is defined as

T = Is/ Io

The ratio of Is/Io is described as percentage,

So %T = Is/Io × 100 %

As the concentration of compound is increased, less light is

transmitted. %T varies inversely and logarithmically with the
concentration, so it is convenient to use Absorbance (A) as
optical density which is directly proportional to concentration.
A = - log Is / Io
= - log T
= log / T
= log 100 / %T
= log 100 – log %T
= 2 – log %T
So it is clear that Absorbance α concentration of substance
Absorbance α 1/ Transmittance

Beer’s law: Amount of light transmitted by the substance

decreases exponentially with increase in concentration of
substance i.e. amount of light absorbed by the coloured
substance is proportional to concentration of substance.
A α concentration C
A=k C
k is proportionality constant.

Lambert’s law: The amount of light absorbed by the

coloured substance is proportional to the thickness
(pathlength) of the solution through which light passes.
Aαl
A = kl
Combining two laws,
A=kCl
Also A = - log Is / Io
i.e. Is / Io = e kcl
If we have a standard solution, we can calculate concentration
of substance as
A=kCl
If l is constant
A=kC
 AT = k CT (T is unknown substance to be tested)
AS = k CS (S is standard)

and AT / AS = kCT / kCS

CT = A T × C S / A S

The concentration range over which substance follows Beer’s

law must be determined for each set of analytical conditions.
Deviation from Beer’s law occurs in the following conditions-
1) When concentrations are very high
2) When incident radiant energy is not monochromatic and
radiant energy reaches the detectors, without having passed
through the sample. So causing error in taking Absorbance.

Photocolorimeters

Components:
1) Light source:
In visible range, Tungsten lamp and
In UV range, hydrogen/ deuterium/ halogen/ quartz
lampsare used.
2) Filters:
It permits sufficiently narrow wavelength of light.
Coloured glass filters are used in simple instruments but in
sophisticated ones, prisms or diffraction gratings are used. For
each determination, a suitable wavelength should be selected
that can be attained by using a set of filters provided with the
instrument.
Different filters used depend on the colour of the resultant
solution as given in the table and it is because the absorbancy
is maximum when complementary filters are used.
Colour of solution Colour of filter Wavelength range
Bluish green Red 650-700
Green Blue Orange 600 – 650
Blue Yellow 575-600
Violet Yellow Green 555-575
Purple Green 505-555
Red Blue-green 495-505
Orange Green Blue 475-495
Yellow Blue 430-475
Yellowish Blue Violet 350– 430

Cuvettes: These are used to hold the solutions whose

absorbance is to be measured. A cuvette must be optically
transparent, thoroughly clean, devoid of any scratch and free
from any contamination. These can be square, rectangular or
circular in shape but square or rectangular ones with flat
surface are preferred. Optical path in cuvette is always 1 cm.
The capacity may vary. Glass Cuvettes are used in visible
range colorimetry while quartz or fused silica cuvettes are
used in UV range.
Photosensitive detectors: Either a photocell or a phototube
may be used to convert the light energy into electrical energy.
In photocell, a metal plate is coated with a thin layer of
photosensitive element such as selenium. This, in turn, is
coated with a thin transparent layer of a metal such as gold or
copper. When the light strikes the selenium layer, electrons
are liberated which pass on to the transparent metal layer
making it electronegative. A potential difference is created
between the transparent metal layer and the metal plate.
 The phototube contains a cathode, consisting of a
metal plate coated with a photosensitive element, and an
anode made up of a metal wire. When light strikes the
photosensitive element, electrons move from the cathode to
anode generating a potential gradient. Different types of
detectors are:
 Barrier layer cells
 Photoemissive tubes
 Photomultiplier tubes
 Photoconductor cells
Measuring devices: The detector response can be measured
by anyone of the following readout devices:
 Galvanometers
 Ammeters
 Recorder.
 Digital readout
The signal may be transmitted to computer or printout device.
Most modern instruments are of direct reading type where the
amplified detector signal operates a galvanometer.
Spectrophotometer: A Spectrophotometer works on the
same principle as a colorimeter but it is more sensitive and
sophisticated. There are light sources (quartz, deuterium. etc.)
that emit light in the UV, visible and infrared regions of
spectrum. The wavelength is selected using a prism or
diffraction grating and narrower bandwidth can be selected.
Since the 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.
The reliability criteria of an analytical method are defined as:

Accuracy: Agreement between the best estimate of a quantity

and its true value. It has no numerical value but is expressed
by the term ‘inaccuracy’ i.e. the numerical difference between
the mean of a set of replicate measurements and the true
value. Bias and systematic error are synonymous with
inaccuracy.
Precision: It is the agreement between replicate
measurements i.e. reproducibility of an analysis.
It has no numerical value but is expressed by the term
‘imprecision’, the standard deviation (SD) or coefficient of
variation (CV) of the results in a set of replicate
measurements. The phrase ‘random error’ is one times used
for imprecision.
Specificity: It is the ability of an analytical method to
determine solely the component(s) it is supposed to measure.
It has no numerical value.
Sensitivity: The ability of an analytical method to detect
small quantities of the measured component. It has no
numerical value but is expressed by the term ‘detection limit’
i.e. the smallest single result which, with a stated probablity
(commonly 95%), can be distinguished from a suitable blank.
For the purpose of quality control (QC), control samples or
specimens are used. These are usually inserted into an
analytical run, a set of consecutive assays performed without
interruption. The results re usually calculated from same set of
calibration standard readings.
QC has been divided into:
Internal QC: It is the procedure of utilising the results of
only one laboratory for QC puposes i.e. by replicate
measrement of a sample over a period of time.
External QC: In this type of program, results of several
laboratories, which analyse the same specimen are utilised.

`
Verification of Lambert-Beer’s law:
Take six test tubes and label them as B (blank), T1, T2, T3, T4
and T5. Dilute Protein standard (2.8mg/ml) as under-

Protein Standard (ml) Distilled water (ml) Biuret reagent (ml)

Blank - 2.5 3.5

T1 0.5 2.0 3.5
T2 1.0 1.5 3.5
T3 1.5 1.0 3.5
T4 2.0 0.5 3.5
T5 2.5 - 3.5

Wait for 10 minutes and measure OD of each solution at

wavelength 540 nm after setting zero with blank. Plot a graph
taking concentration on X-axis and OD on Y-axis.
Result: Linear graph verifies the Lambert-Beer’s law.
Precautions:
1) Wait for 10 minutes at room temperature before taking the
OD.
2) All test tubes should be clean and dry.