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COLORIMETRY

&
SPECTROPHOTOMETR
COLORIMETRY & SPECTROPHOTOMETR

• Many biochemical
experiments involve
the measurements of
compound or group
of compounds
present in a complex
mixture.
COLORIMETRY & SPECTROPHOTOMETR

• The most widely used


method for determining
the concentration of
biochemical compounds is
colorimetry, which makes
use of the property that
when white light passes
through a colored
solution, some wavelength
are absorbed more than
others.
COLORIMETRY & SPECTROPHOTOMETR

• Many compounds
are not themselves
colored but can be
made to absorb light
in visible region by
reaction with suitable
reagents.
COLORIMETRY & SPECTROPHOTOMETR

• These reactions are


fairly specific and in
most cases very
sensitive, so that
quantities of material in
the region of mM / L
concentrations can be
measured.
COLORIMETRY & SPECTROPHOTOMETR

• The big advantage of is


that complete isolation
of compound is not
necessary and the
constituents of a
complex mixture such
as blood can be
determined after little
treatment.
COLORIMETRY & SPECTROPHOTOMETR

• The depth of the color


is proportional to the
concentration of the
compound being
measured, while the
amount of light is
proportional to the
intensity of the color
and hence the
concentration.
Measurement of Extinction:
• The earliest colorimeters relied on the human
eye to match the color of a solution with that
of one of a series of colored discs. The results
obtained were too subjective and not
particularly accurate.
The Colorimeter:
• Colorimeter is generally any tool that characterizes
colour samples to provide an objective measure of
colour characteristics.
• In chemistry, the colorimeter is an apparatus that
allows the absorbance of a solution at a particular
frequency (colour) of visual light to be determined.
Colorimeters hence make it possible to determine the
concentration of a known solute, since it is
proportional to the absorbance.
The Colorimeter:
• Different chemical substances absorb varying
frequencies of the visible spectrum.
• Colorimeters rely on the principle that the
absorbance of a substance is proportional to its
concentration i.e., a more concentrated
solution gives a higher absorbance reading.
The Colorimeter:
• Filter in the colorimeter is used to select the color of
light which the solute absorbs the most, in order to
maximize the accuracy of the experiment.
• Note that the colour of the absorbed light is the
'opposite' of the colour of the specimen, so a blue
filter would be appropriate for an orange substance.
Sensors measure the amount of light which has
passed through the solution, compared to the amount
entering, and a display reads the amount absorbed.
The Colorimeter:
• A quantitative reading for the concentration of
a substance can be found by making up a
series of solutions of known concentration of
the chemical under study, and plotting a graph
of absorbance against concentration. By
reading off the absorbance of the specimen
substance on the graph, a value for its
concentration is found.
How colorimeter works?
1- White light from a tungsten lamp passes through a
slit, then a condenser lens, to give a parallel beam
which falls on the solution under investigation
contained in an absorption cell or cuvette. The cell is
made of glass with the sides facing the beam cut
parallel to each other.

Light source slit condenser cuvette filter photocell galvanometer


lens
How colorimeter works?
2- Beyond the absorption cell is the filter, which is
selected to allow maximum transmission of the color
absorbed. If a blue solution is under examination,
then red is absorbed and a red filter is selected.
• NOTE: The color of the filter is complementary to the
solution.

Light source slit condenser cuvette filter photocell galvanometer


lens
How colorimeter works?
3- The light then falls on to a photocell which
generates an electrical current in direct
proportion to the intensity of light falling on it.

Light source slit condenser cuvette filter photocell galvanometer


lens
How colorimeter works?
4- This small electrical signal is increased by the
amplifier which passes to a galvanometer of
digital readout to give absorbance reading
directly.

Light source slit condenser cuvette filter photocell galvanometer


lens
How colorimeter works?
• Among the simplest and most common
colorimeters are the Spectronic 20 and
Spectronic 21. They are commonly called the
Spec 20 and Spec 21.
The Spectrophotometer:
• Is a sophisticated type of
colorimeter where
monochromatic light is provided
by prism.
• The band with of the light passed
by a filter is quite board, so that it
may be difficult to distinguish
between two components of
closely related absorption with a
colorimeter. A spectrophotometer
is then needed.
The Spectrophotometer:
• All types require a Blank:
which is a solution that contains the entire
reagents except the substance to be measured.
• It is used to adjust the device to zero.
Here is a summary of the steps of operation of
a Spec 20 & spectrophotometer:
1- Power
Turn on power.
2-Warm up
Allow about 5 minutes when first turned on.
3-Wavelength
Select appropriate wavelength.
4- Zero
With sample holder empty and closed, adjust meter
needle to 0%T (or infinite A) using zero control knob.
Here is a summary of the steps of operation of
a Spec 20 & spectrophotometer:
5- Blank
Fill tube half full with water.
Place in sample holder and close cover.
Adjust meter needle to 100%T (or 0 A) using light
control knob.
Here is a summary of the steps of operation of
a Spec 20 & spectrophotometer:
6- Standard (Measure absorbance (or %T) of known solution).
Fill tube half full with sample of known
concentration.
Place in sample holder and close cover.
Read absorbance value (or %T) from meter.
Repeat this step if making a calibration curve or
verifying proportionality (Beer's Law).
Here is a summary of the steps of operation of
a Spec 20 & spectrophotometer:
6- Standard (Measure absorbance (or %T) of known solution).
Fill tube half full with sample of known
concentration.
Place in sample holder and close cover.
Read absorbance value (or %T) from meter.
Repeat this step if making a calibration curve or
verifying proportionality (Beer's Law).
7- Sample
Measure absorbance (or %T) of solution with
unknown concentration as in previous step.
Types of spectrophotometer:
1- Visible spectrophotometer.

2- Ultraviolet (UV)
spectrophotometer.
COLORIMETRIC
DETERMINATION OF
VITAMIN B-12
Vitamin B12
- Vitamin B12 is water soluble vitamin.
- RDA= 2.4 µg /day for adult.
- It is found only in animal sources.
Functions:
1- Aids folic acid in synthesis of heme.
2- It prevents anemia.
3- Required for protein digestion and absorption.
B 1 2 3 4 5 Unk.

St. Viatamin (ml) 0 1 2 3 4 5 0

Shake
Unk. Vitamin (ml)
0 0
Well
0
with
0
vortex
0 0 5

Read
H O (ml)the Optical
2 5 4 Density
3 2 1at 555
0 nm
0
Results
0.6

0.5

0.4
Abs.

0.3

0.2

0.1

0
0 1 2 3 4 5 6
Conc.
* Calculations
Absorbance of Unknown Absorbance of Standard
=
Concentration of Unknown Concentration of Standard

CUn. = A Un. × C St.


A St.

Samar A. Damiati