Photometry broadly deals with the study of light absorption by molecules in solution. It is one of the most common analytical techniques used in clinical biochemistry laboratories to measure the intensity of a light beam. Most clinical chemistry reactions involve linking a chemical or enzymatic reaction to the development of a colored product, the intensity of which is then measured photometrically. The amount of light transmitted through a colored solution decreases exponentially with increases in the concentration and thickness of the colored substance, as governed by Beer's and Lambert's laws. Common photometers include colorimeters, which measure visible light, and spectrophotometers, which can measure ultraviolet and visible light.

1. PHOTOMETRY
Dr. Farhana Atia
Associate Professor, Dept. of Biochemistry
Nilphamari Medical College, Nilphamari
Email: farhana.atia@gmail.com

2. PHOTOMETRY
 Broadly deals with the study of the phenomenon of light
absorption by molecules in solution.
Photmeter
 Most common analytical techniques used in clinical
biochemistry laboratory.
 Designed to measure the intensity of a beam of light
 Most routine clinical chemistry reactions involve linking a
chemical or enzymic reaction to the development of a colored
product that is measured

3. PHOTOMETRIC PRINCIPLES
 When a monochromatic light with an original intensity ( )
passes through a solution, it may–
 Reflected
 Absorbed
 Transmitted ( )
Is < Io
O
I
S
I
S
I

4. PHOTOMETRIC PRINCIPLES
 The nature of light absorption
in a solution is governed by
Beer-Lambert law.
 Beer’s law: The amount of
light transmitted through a
colored solution decreases
exponentially with increase in
concentration of the colored
substance.

5. PHOTOMETRIC PRINCIPLES
 The nature of light absorption
in a solution is governed by
Beer-Lambert law.
 Lambert’s law: The amount
of transmitted light decreases
exponentially with increased
thickness of the layer of
solution through which the
light passes.

6. PHOTOMETRIC PRINCIPLES
 It is convenient to use absorbance (A) or optical density (OD)
which is directly proportional to concentration.
 OD & transmittance are related reciprocally.

7. PHOTOMETRIC PRINCIPLES
 Mathematical expression:
At a given wavelength, OD = A = kct
where, k = constant
c = concentration of colored substance
t = thickness of the layers
 Science the thickness is constant in the instrument,
OD is proportional to concentration.

8. TYPES OF INSTRUMENT
 No rigid classification
1. Where absorbed or transmitted light is measured.
 Colorimeter
 Spectrophotometer
2. Where emitted light is measured
 Flame emission photometer.

9. COLORIMETER
 Colorimeter is an instrument
used for the measurement of
colored substance in solution
 Simplest type
 Measure visible region of
spectrum (320-800nm)
 Specific wavelength of light is
isolated with interchangeable
filters

10. SPECTROPHOTOMETER
 More sophisticated.
 Measure ultra violate region (180 –
320nm) as well as the visible
range
 Glass prism or quartz prism
instead of filters
 Quartz cell in place of glass
cuvette
 Estimate the substance which are
more or less colorless in the visible

11. FLAME PHOTOMETER
 Deals with the quantitative
measurement of electrolytes
such as Na, K, Li.

12. PARTS OF PHOTOMETER
1. Light source.
2. Monochromators
3. Sample cuvettes
4. Optical path (Single or double beam)
5. Photosensitive detectors
6. Read out devices

13.  The instrument is set to zero (0) with the blank
 Concentration of test (T) =
𝑂𝐷 𝑜𝑓 𝑡𝑒𝑠𝑡
𝑂𝐷 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑
X Concentration of standard
CT =
AT
AS
HOW TO CALCULATE
XCS

14.  In colorimetric estimation it is necessary to
prepare 3 solutions
BLANK(B)
STANDARD(S)
TEST(T)
PREPARATION OF SOLUTION FOR INVESTIGATION

15. BLANK To eliminate the effect
of light absorption by
the reagent used
Water
BLANK
Reagent
BLANK
STANDARD Solution of known
concentration of the
substance
Both O.D and
concentration
are known
So conc. of
unknown can
be calculated
PREPARATION OF SOLUTION FOR INVESTIGATION

16. TEST
Test solution is made by
treating a specific volume of
the test sample with reagents
As per procedure
PREPARATION OF SOLUTION FOR INVESTIGATION

17. USES OF COLORIMETER
Estimation of-
 Blood glucose
 Blood urea
 Serum creatinine
 Serum proteins
 Serum cholesterol
 Serum biliburubin
 Urine creatinine
 Glucose in CSF

18. THANK YOU