PAPER :

Biophysical Chemistry

Unit 3:

COLORIMETRY
Reference Books:

1) Biophysical Chemistry: Principles and Techniques.

By Upadhyay & Nath

2) Principles and Techniques of Biochemistry and Molecular

Biology .
B Keith
By K ith Wil
Wilson and
d JJohn
h WWalker
lk
• It is the most common analytical
y technique
q
used in biochemical estimation in clinical
laboratory.

• It involves the quantitative estimation of

color.
color

• A substa
substance
ce to be est
estimated
ated co
colorimetrically,
o et ca y,
must be colored or it should be capable of
forming chromogens (colored complexes)
through the addition of reagents.
• Colored substance absorb light in relation to
their color intensity.
• The color intensity will be proportional to the
conc. of colored substance.
• The instruments used in this method are
colorimeter or photometer or absorptiometers.
 Colorimeter - Principle
When a monochromatic light passes through a
coloured solution, some specific wavelengths of light
are absorbed which is related to colour intensity.

The amount of light absorbed or transmitted by a

colour solution is in accordance with two law i.e. Beer’s
& Lambert’s Law.
The measurement of colour intensity of a coloured
solution by photometry is governed by two laws
Beer’s law

When a monochromatic light passes through

a colored solution,
solution amount of light
transmitted decreases exponentially with
increase in concentration of colored
substance.

i.e. the amount of light absorbed by a colored

solution is directly proportion to the conc. of
substance
b t i the
in th colored
l d solution.
l ti
 Beer’s law

Beer’ss law
Beer
Lambert’ss law
Lambert

The amount of light transmitted decreases

exponentially with increase in pathlength
(diameter) of the cuvette or thickness of
colored solution through which light passes.

ii.e. the
th amountt off light
li ht absorbed
b b d by b a
colored solution depends on pathlength of
cuvette or thickness or dept of the colored
solution.
Combined beer’s- lambert’s law

Amount of light transmitted through a

colored solution decreases exponentially with
i
increases i conc. off colored
in l d solution
l ti &
increase in the path length of cuvette or
thickness of the colored solution
 Absorbance
Beer Lambert Law - the linear relationship between
Beer-Lambert
absorbance and concentration of an absorbing species.
A = abc
A is the absorbance
“a” is molar absorptivity in L/[(mole)(cm)]
Also called “extinction coefficient” or “”;
it is dependent on the material being studied.

“b” is the path length in cm

The diameter of the cuvette or sample holder which is the distance
the light travels through the absorbing sample. “b” is a constant
when the same size cuvette is used for all samples.
samples

“c” is the concentration of the sample in (mol/L)

Main use of Beer’s Law is to determine the concentration
of various solutions.
Transmittance is Related to Absorbance
Transmittance is given by the equation:
T = I/Io
where I is the intensity of the light after it has gone
through the sample & Io is the initial light intensity.

Absorbance is related to the %T:

A = -logT = -log(I/ Io)
 Equation Summary
T= (I/Io) = 10-A %T = (I/Io) x 100 A = -logT = log(1/T)

Sample Calculation
If %T = 95%,
95% then
h A = llog(100/95)
(100/95) = log(1/.95)
l (1/ 95) = -log(.95)
l ( 95)
A = 0.02227

Note the scale for Absorbance: 9/10th off th

the scale
l is
i from
f 0-1 d 1/10th is
0 1 and i from
f 1-2.
12
For this reason, the spectrometers have been calibrated in % Transmittance and
all readings will be taken in %Transmittance.
 Deviation in Beer and Lambert’s Law
• If a substance is following the beer
beer’ss law, we get a
straight line which is passing from the origin and the
slope is “ab”. But some time we do not get a straight
line.
line
• If we are getting much more value of absorbance than
desired, then deviation is said as +ve deviation. But if
we are getting much less value of absorbance than
desired, then deviation is said as -ve deviation.
 There are two reason for these deviations:
1. Instrumental Errors: The cause of instrumental errors
are as follows:
• Fluctuation in electricity
• Source of light is weak
eak or malfunction
• Arrangement of filter/monochromator is not proper
• Scattering of light is happening inside the
instrument
• Slit is not placed properly
• Outside knob are not working according to inner
instrumentation
• Sensitivity of detector is low or malfunction
2. Chemical Errors: The cause of chemical errors are as
follows:
• Presence of bacteria
• Solution is cloudy
• Pigmentation is there in the solution
• Acid-Base reaction is happening in the solution
• Association-Dissociation reaction is happening in the
solution
l i
• Polarization reaction is happening in the solution
g g with time
• If the color of the solution is changing
• If the absorption is happening due to solvent rather
than solute
• This law is only applicable to some extent of
concentration of the solution only. Below or above
that concentration these laws are not applicable.
Colorimetric analysis is two types
 Visual colorimetry
 Photo-electric colorimetry

Visual colorimetry is one of the oldest form of color

measuring technique which is not used now day,
natural or artificial light is used as light source
and determinations are made with a
colorimetry or color comparator where human
eye is used as detector.
Photoelectric colorimetry

Progress in the development of colorimetric

method has resulted largely due to the
application of photoelectric cell,
cell which eliminates
the difficulties of complicated visual comparison.

IIn this
hi method
h d human
h eye isi replaced
l d by b
suitable photoelectric cell, to afford a direct
measure of the light g intensity.
y Instruments
employing photoelectric cell measure the
light absorption and not color of substance
 Pre-requisite for a solution to be
analyzed by colorimeter:

• The solution must be colored.

• The solution must not be having any

contamination like Bacteria, Cloudy solution or
Pigmentation. The solution must be clear
(transparent).

• There must not be any reaction happening in the

solution like acid-base, association-dissociation or
polarization reaction.

• The solution must be having a particular

concentration. Because lambert and beer laws can
be applied only for particular range of concentration.
Components of the colorimeter
 Parts of the colorimeter

Light source: Tungsten filament lamp

Slit: It is adjustable which allows only a

g
beam of light to p
pass through.
g it p prevents
unwanted or stray light

Condensing
C d l
lenses: Light
i h after
f passing
i
through slit falls on condenser lense which
gives a parllel beam of light.
light
Filter:
M d off colored
Made l d glass.
l Filt
Filters are used
d for
f
selecting light of narrow wavelength.

Filters will absorb light of unwanted

wavelength
g and allow only
y monochromatic
light to pass through.

For ex: a green filter absorbs all color, except

green light which is allowed to pass through.
Light transmitted through a green filter has
a wavelength from 500-560 nm.
Filter used is always complimentary in color
to the color of solution.
Cuvette (sample holder):

The monochromatic light from the filter passes

through the colored solution placed in a cuvette.

It is made up of special glass/plastic/quartz

material.

It may be square/rectangular/round shape with

fixed diameter (usually 1 cm)& having uniform
surface. the colored solution in the cuvette
absorbs part of light & remaining is allowed to
fall on detector.

For ex: a solution of red color transmits red light

& absorbs the complimentary color green.
Detector (photocell):

Detector are photosensitive elements which

converts light energy into electrical energy.

The electrical signal generated is directly

proportional
ti l to
t intensity
i t it off light
li ht falling
f lli on the
th
detector.

Output: The electrical signal generated in

photocell is measured by
p yggalvanometer, which
displays percent transmission & optical
density.
 Wave length selection method:

1. Use other filter than the color of the solution.

Because the color of the solution is the color which is
not absorbed by the solution.
solution For eg.
eg In case of
CuSO4 solution, use red colored filter.
2. Find the highest absorption using different filters of
different colors.
3. The wavelength recommended for different solutions
is g
given in the S.O.P. ((standard operating
p gpprocedure))
manual of the instrument. So use that filter.
(ROYGBIV)
Relationship between the wavelength and colour

Color of the Filter used/ color Wavelength (nm)

solution/ solution absorbed
color transmitted
Yellow blue Violet 380 – 430
Yellow Blue 430 – 475
Orange Green blue 475 – 495
Rd
Red Bl green
Blue 495 – 505
Purple Green 505 – 555
Violet Yellow ggreen 555 – 575
Blue yellow 575 – 600
Green blue Orange 600 – 650
Blue green Red 650 - 750
Preparation of solution for investigation

In colorimetric estimation it is necessary to

prepare 3 solutions
Application of colorimeter
• It is widely used in hospital & laboratory for
estimation of biochemical samples, like plasma,
serum, cerebrospinal fluid ( CSF ), urine.

• It is also used to quantitative estimation of serum

components
co po e ts as we
well as g
glucose,
ucose, p
proteins
ote s aand
d ot
other
e
various biochemical compound.

• They are used by the food industry and by

manufacturers of paints and textiles.

• They are used to test for water quality, by

screening for chemicals such as chlorine, fluoride,
cyanide,
y , dissolved oxygen,
yg , iron,, molybdenum,
y , zinc
and hydrazine.
Advantage

• It is inexpensive

• V
Very wellll applicable
li bl for
f quantitative
i i analysis
l i off
colored compounds

• Easily transportable
Disadvantage
• Cannot be used for colorless compounds.

• It does not work in UV and IR regions.

regions

• We cannot set specific wavelength, as we have to

sett a range as a parameter.
t

• Similar colors from interfering substances can

produce errors in results.
SPECTROPHOTOMETRY
 What is spectrophotometry?
• A spectrophotometer is an instrument designed for
physical sample analysis via full spectrum color
measurement.
measurement

• By providing wavelength-by-wavelength spectral

analysis of a sample’s reflectance, absorbance,
or transmittance properties, it produces precise
data beyond
y that observable by
y the human eye.
y

• Spectrophotometry is the quantitative

measurement of the transmission properties of a
material as a function of wavelength.

• Spectrophotometry
S h d l
deals with
i h visible,
i ibl near-
ultraviolet, and near-infrared light.
 BASIC PRINCIPLES

• Light have dual characteristics.

characteristics corpuscular and
waveform.
• Beam of light: Known as an electromagnetic wave
wave-
form.
g
• The term electromagnetic is a p
precise description
p of
the radiation in that the radiation is made up of an
electrical and a magnetic wave which are in phase
and perpendicular to each other and to the direction of
propagation.
• Electromagnetic radiation is a type of energy that
is transmitted through space as a transverse wave at
huge velocity.
• I takes
It k numerous forms
f k
known as electromagnetic
l i
spectrum. The electromagnetic spectrum include
gamma ray,
g y, X-ray,
y, ultraviolet ((UV),
), visible,, infrared
(IR), microwave and radio-wave radiation.
Properties of Light
Particles and Waves
 Light waves consist of perpendicular, oscillating electric and
magnetic fields
 Parameters used to describe light

- Amplitude (A): height of wave’s electric vector

- Wavelength
W l h (l)
(l): distance
di ((nm, cm, m)) ffrom peak
k to peak
k

- Frequency (n): number of complete oscillations that the

waves makes each second
 Hertz (Hz): unit of frequency, second-1 (s-1)
 1 megahertz (MHz) = 106s-1 = 106Hz
Electromagnetic radiation is characterized by its
wavelength , Frequency  and energy E:
E = h= hc /  c=

Where h = Planck’s constant (6.626 x 10-34 J s)

c = speed
d off light
li h in
i a vacuum.

Energy
gy ((E):
) the energygy of one p
particle of light
g (p(photon))
is proportional to its frequency

(a) longer wavelength, lower energy;

(b) shorter wavelength, higher energy.
 Interaction of light with matter
 When a beam of light impinges upon a sample:
◦ a) some photons may have no interaction with a sample and be
transmitted
◦ b) some photons may be absorbed by a sample
◦ c) some photons may be scattered
◦ d) some photons may be reflected

 The extent of a) – d) depends on the material of the sample and

on the wavelength of the radiation
 In spectrophotometric measurements, c) and d) should by kept to
a minimum

absorption
off radiation
di ti The intensity of the transmitted
I0 transmitted radiation (I) is lower than
radiation
the intensity of the incident
I
radiation
di ti (I0)

scatter
 Electromagnetic spectrum

λ [m]

10-12
12 10-10
10 10-88 10-33 10-11
ABSORPTION SPECTRUM
The pattern off energy absorption
Th b i b a substance
by b
when light of varying wavelength passes through it
is uniquely
q y characteristic of the substance. This
pattern is known as an absorption spectrum.
 INSTRUMENTATION
• The essential components of a spectrophotometer
include:
(1) Energy source
(2) Monochromator
M h t
(3) Holder
(4) Photosensitive detector
 Source of light
light::
The light source must be fulfilling the following
requisitions:

• Source must be having proper intensity.

intensity

• Having all the frequencies of light

• Source must be stable.

 Sources of ultraviolet radiation
• Hydrogen lamp and the Deuterium lamp.
 Xenon, Mercury/Xenon lamp

• Xenon lamp may also be used for

ultraviolet radiation, but the radiation
produced is not as stable as the hydrogen
l
lamp.

Pure Xenon has very wide emission spectrum ~200 – 1200 nm

Sources of visible radiation
Tungsten filament lamp : source for visible
radiation.
 Carbon arc lamp
• If sufficient intensity
intensit of light is not obtained from
tungsten lamp then carbon arc lamp can be use as a
source for color measurement, which provides more
intense visible radiation
Sources of infrared radiation
Nernst Glower and Globar lamp

The Globar lamp consists of a

silicon
ili carbide
bid rodd which
hi h when
h heated
h d
to approximately 1200°C, emits
radiation in the 1-40 µ region.
g

Nernst Glower lamp employs a

hollow rod of zirconium and yttrium.

lt requires to be heated up to 1500°C

before it emits radiation in the range
g
of 0.4-20 µ.
Globar is more stable than the Nernst Glower
Wavelength Selectors
• Filter
Filt and
d Monochromators
M
Monochromators:
h t :

Filters:
Monochromators::
Monochromators

W
Wavelength
l th resolving
l i d
device
i
 Prism monochromators
The degree of dispersion by the
prism depends upon
(i) The apical angle of the prism
(usually 60
60°))
(ii) The material of which it is
made.
• Wavelengths at the red end of the spectrum are not fully
resolved
• Two types of prisms,
prisms
60° Cornu quartz prism
30° Littrow prism are usually used in commercial
instruments.

Silica, fused silica or quartz prism Ionic crystalline materials prism

e.g. NaCl,
N Cl KBr,
KB C B
CsBr, andd the
th
mixed crystalline material
Simple glass prisms commonly called KRS-5.
 Grating monochromators

The grating possesses

a highly aluminized surface

Grating resolves light Reflection followed by either constructive or

into its component destructive interferences
wavelength by virtue of
constructive
reinforcement and
destructive
interference of Foreprism
p
radiation reflected It preselects a portion of the
spectrum which is then
allowed to be diffracted by
the grating
Sample vessel (Cuvette
(Cuvette):
):
Cuvette are rectangular
g cell, square
q cell or circular one

1. For UV range: Quartz or fused silica cells

2. For visible range: Glass cuvette
3. For IR range: Glass tubes possess NaCl KBr or
CaF2 windows

Long pathlength 1 cm pathlength

cuvet

Short pathlength (b)

Detector:
Important requirements for a detector

(i) Hi Highh sensitivity

iti it to allow
ll the
h detection
d i off low
l l l
levels
of radiant energy.
((ii)) Short response
p time.
(iii) Long term stability
(iv) An electronic signal which is easily amplified for typical
readout apparatus
Mainly three type of detectors can be used:

1. Photovoltaic Cell
2. Photo tubes
3 Photo
3. Ph t multiplier
lti li tubes
t b

Recorder::
Recorder
Photovoltaic Cell
Photo Tubes
Photo multiplier tubes
 It is a very sensitive device in which electrons emitted
from the photosensitive cathode strike a second surface
called dynode which is positive with respect to the
original
g cathode.
 Electrons are thus accelerated and can knock out more
than one electrons from the dynode.
y
 If the above process is repeated several times, so more
than 106 electrons are finally collected for each photon
striking the first cathode.
There are two types of spectrophometer
available:
1. Single beam spectrophotometer
2. Double beam spectrophotometer
 The components of a single beam
spectrophotometer
Light source - white light of constant intensity

slits

G i
Grating
slits Separates white light
Phototube
into various colors
detects light &
measures intensity Rotating the grating
Sample
changes the wavelength going
When blank is the sample through the sample
Po is determined,
otherwise P is measured
67
 Double Beam Spectrophotometer

Beam Chopper Semi-transparent

Mirror
Sample
p

Tungsten
T t Sli
Slit Quartz
Q t Photo-
Grating
Lamp Cuvette multiplier
Reference
(Blank)
Mirror Mirror

Mirror 68
 Schematic diagram of a double beam scanning spectrophotometer

 In double beam arrangement, the light alternately passes through

the sample and reference (blank), directed by rotating half-sector
mirror (chopper) into and out of the light path.

 When light passes through the sample, the detector measures the P.
When the chopper diverts the beam through the blank solution, the
detector measures P0.

 The beam is chopped several times per second and the electronic
circuit automatically compares P and P0 to calculate absorbance
andd Transmittance.
T i

69
Advantages of double beam instruments over single beam instruments

Single beam spectrophotometer is inconvenient because

1. The sample and blank must be placed alternately in the light path.
2 For
2. F measurements at multiple
l i l wavelengths,
l h the
h bl
blankk must bbe run at eachh
wavelength.

In double beam instruments

1. The absorption in the sample is automatically corrected for the absorption
occurring in the blank, since the readout of the instrument is log the difference
between the sample beam and the blank beam.
2. Automatic correction for changes of the source intensity and changes in the
detector response with time or wavelength because the two beams are compared
and measured at the same time.
3 Automatic scanning and continuous recording of spectrum (absorbance versus
3.
wavelength).
70
 Applications of Spectrophotometer
• Primarily for
f quantitative analysis.

• Probably more widely used in chemical and clinical

l b
laboratories
i throughout
h h the
h world
ld than
h any other
h single
i l
method.

• Full
F ll spectrum analysis
l i also
l provides
id for
f greater specificity,
ifi i
potentially identifying color differences missed by
colorimeters.

• Spectrophotometric instruments are ideally suited for a

broad range of applications in the research and
development phase, including color formulation and color
system development, as well as color quality control
throughout production.

71
Applications of Spectrophotometer

72