Clinical Biochemistry

1. PHOTOMETRY
Dr. Mohammad Shiblee Zaman
MBBS, MD (Biochemistry)
Lecturer (Biochemistry)
Dhaka Medical College

2. Light

3. Light
Visible spectrum of
electromagnetic
radiation emitted in the
form of waves of
different wave lengths
ranging from 400nm-
700nm

5. Dispersion of light
 White light (stray light) is
the composite mixture of all
the wave length of visible
spectrum of electromagnetic
radiation
 When light is passed
through a dispersion
medium like prism, then we
will get a light of a
particular wave length.

6. Types of light
A. Stray light/ Polychromatic light
B. Monochromatic light

7. Common dispersion media
 Filter
 Prism
 Diffraction grating
 Interference filter

8. Light of different wave lengths
Violet
Blue
Green
Yellow
Orange
Red

9. How we see different color
 If light falls on a substance and totally absorbed, it will
be visualized as black.
 If the light is totally reflected, it will visualized as white.
 If some portion of light are absorbed and some are
reflected, the reflected wave length will determine the
color of substance.

10. Red
Wave length from 630nm – 750 nm

11. Wave length
• The distance between two peaks or two crests of two
adjacent waves is called wave length.

12. Frequency
It is the number of waves per second.
The more the wave length, the less the frequency.

15. Photometry
Technique used to measure the concentration of a
substance in a solution by adopting the property of
absorption or transmission or emission of light of a
particular wave length by that substance present in
the solution under controlled condition.

16. Principles of Photometry
 Substance to be measured by photometry must be colored to
begin with (like Hemoglobin) or can be made to produce color
derivatives by using certain reagents and reactions (e.g.
Glucose, Urea etc.).
 Intensity of produced color is proportional to the concentration
of the color producing substance present in the solution.
 Colored substance absorb light of a particular wave length and
the extent of color absorption depends on the concentration of
color producing substance in the solution.

18. Optical Density (OD)/ Absorbance/
Extinction
 The amount of light absorbed by the colored substance
is called OD.
 The OD may be defined as the logarithmic ratio of
incident light (I) to that of transmitted light (T)
So, OD= log10 (I/T)
Or, OD= log10 (100/T) [where incident light is taken as 100%]
Or, OD= log10 100 - log10 T
So, OD= 2 - log10 T

19. Transmittance
It is the ratio of intensity
of transmitted light (It) to
the intensity of incident
light (Io) across a solution.
Transmittance (T) = It/ Io

20. Laws of Photometry

21. Laws of Photometry
Beer’s law: This law states that, the intensity of
transmitted light decreases exponentially with the
increase in concentration of substance in the solution.
Lambert’s law: This law states that, the intensity of
transmitted light decreases exponentially with the
increase in the length of light pathway.

22. Using photometric laws in photometry
A∞ C × L Here, A= absorbance
A = K ×C ×L K= proportionate constant
K = A/ C × L C= conc. of substance
L= length of light pathway
For standard solution (s), For unknown solution (u),
we can write- we can write-
Ks = As/ Cs × Ls Ku =Au / Cu × Lu

23. K” is a photometric constant and is fixed for a particular substance
whatever may be the concentration. “L” is also kept constant by
using the cuvette having same diameter or length of light pathway.
So, we can write, Ks = Ku
So, As/ Cs × Ls = Au / Cu × Lu
Cu = Au × Cs / As [as Ls=Lu, because of using cuvette of same
length of light pathway]
Absorbance of Unknown X Concentration of Standard
Concentration of Unknown = ––––––––––––––––––––––––––––––––––––––––––––
Absorbance of standard

24. Colorimeter
It is an instrument by
which color is measured
or compared. Thus
colorimeter employs
color and color variation
to determine the
concentration of
substances in solution.

25. Parts of a Colorimeter
 A source of light
 The monochromatic light
and filter
 Cuvette
 A photosensitive element
 A sensitive galvanometer

26. Change of filter in colorimeter
 The filter should be changed
depending on the color of
solution.
 The chosen filter is opposite
from that of the color of
solution.
 If the solution is yellow, red,
orange then we have to
choose filter like violet,
green, blue.
Violet Blue Green
Yellow Orange Red

27. Spectrophotometer
In this instrument a
prism is used as a
dispersion media to
isolate the stray light
into UV or visible or
infrared light.

28. Colorimeter vs Spectrophotometer
Traits Colorimeter Spectrophotometer
Monochromator Filter Prism
Spectral bandwidth Broad band Narrow band
Spectral purity Less More
Spectral isolation Filter has to be changed with
different analysis
Desired wave length can be
adjusted
Stray light More Less
Accuracy Less More
Sample Larger volume needed Small volume needed
Cost Cheaper More costly
Range of electromagnetic
radiation
Only visible range of light can
be used
Beyond the visible range of
light can be used

29. Advantage of Spectrophotometer over Colorimeter
 Ensure high degree of spectral purity.
 Minimum stray light.
 Greater accuracy.
 More sensitive, specific and precision.

30. Prism, better than filter
 Prism gives narrow spectral bandwidth, so
gives better spectral purity.
 Choice of continuous wave length is available.
 Prism gives minimum stray light.

31. THANK YOU