COLORIMETER &

SPECTROPHOTOMETER

Dr. Marwa Ibrahim Khedr

Lecturer of Medical Biochemistry and Molecular Biology, MD
Clinical pathology specialist, MS
Dr. Marwa Ibrahim Khedr, MD
COLORIMETRY & SPECTROPHOTOMETR

• Many biochemical
tests involve the
measurements of
compound present in
a complex mixture.

Dr. Marwa Ibrahim Khedr, MD

 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.

Dr. Marwa Ibrahim Khedr, MD

COLORIMETRY & SPECTROPHOTOMETR

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

Dr. Marwa Ibrahim Khedr, MD

 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.

Dr. Marwa Ibrahim Khedr, MD

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.

Dr. Marwa Ibrahim Khedr, MD

Beer-Lambert’s law is a linear relationship
between the absorbance and the
concentration of an absorbing species.
Dr. Marwa Ibrahim Khedr, MD
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.

Dr. Marwa Ibrahim Khedr, MD

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.

Dr. Marwa Ibrahim Khedr, MD

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.

Dr. Marwa Ibrahim Khedr, MD

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.

Dr. Marwa Ibrahim Khedr, MD

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.

Dr. Marwa Ibrahim Khedr, MD

 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.

Dr. Marwa Ibrahim Khedr, MD

 How colorimeter works?
2- before 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

Dr. Marwa Ibrahim Khedr, MD

 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
Dr. Marwa Ibrahim Khedr, MD
 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

Dr. Marwa Ibrahim Khedr, MD

Dr. Marwa Ibrahim Khedr, MD
 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.

Dr. Marwa Ibrahim Khedr, MD

 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.
Dr. Marwa Ibrahim Khedr, MD
Spectrophotometer is based on the
photometric technique which states that :
When a beam of incident light of intensity I0
passes through a solution, a part of the
incident light is reflected (Ir), a part is
absorbed (Ia) and rest of the light is
transmitted (It)
Thus, I0 = Ir + Ia + It

Dr. Marwa Ibrahim Khedr, MD

In photometers
(colorimeter & spectrophotometer)
• the amount of light reflected (Ir) is kept
constant by using cells that have identical
properties.

• (I0 ) & (It ) is then measured

Dr. Marwa Ibrahim Khedr, MD

Dr. Marwa Ibrahim Khedr, MD
 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.

Dr. Marwa Ibrahim Khedr, MD

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.

Dr. Marwa Ibrahim Khedr, MD

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.

Dr. Marwa Ibrahim Khedr, MD

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).

Dr. Marwa Ibrahim Khedr, MD

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.

Dr. Marwa Ibrahim Khedr, MD

Types of spectrophotometer:
1- Visible spectrophotometer.

2- Ultraviolet (UV)
spectrophotometer.

Dr. Marwa Ibrahim Khedr, MD

Dr. Marwa Ibrahim Khedr, MD
Dr. Marwa Ibrahim Khedr, MD
Dr. Marwa Ibrahim Khedr, MD
Parts of Spectrophotometer
1. Light source – To generate light
• Tungsten Halogen lamp for visible light (400- 800 nm)
• Hydrogen or deuterium lamp for Ultraviolet light (200-400
nm)
• Xenon flash lamp for both visible and UV radiations (190-
1000 nm)
2. Monochromator – To select the particular wavelength
Accept polychromatic input light and outputs monochromatic
light
• Entrance slit
• Exit slit
• Dispersion device
 Prizm
 Absorption filter (Glass)
 Interference filter (Metal or thin film)
Dr. Marwa Ibrahim Khedr, MD
Dr. Marwa Ibrahim Khedr, MD
Dr. Marwa Ibrahim Khedr, MD
 COLORIMETRIC
DETERMINATION OF
Biochemical compounds (for
ex. Uric acid, cholesterol

Dr. Marwa Ibrahim Khedr, MD

 Results
0.6

0.5

0.4
Abs.

0.3

0.2

0.1

0
0 1 2 3 4 5 6
Conc.

Dr. Marwa Ibrahim Khedr, MD

* Calculations
Absorbance of Unknown Absorbance of Standard
=
Concentration of Unknown Concentration of Standard

CUn. = A Un. × C St.

A St.

Dr. Marwa Ibrahim Khedr, MD

Dr. Marwa Ibrahim Khedr, MD