COLORIMETRY

&
SPECTROPHOTOMETR
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 0
vortex
0 5

Read
H O (ml) the 5Optical
2 4 3 Density
2 1 at 05550 nm
 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