Analytical technique used commonly in laboratories

2. Introduction
• Photometry is the most common analytical technique
used in the biochemical laboratory. It is designed to
measure the intensity of a beam of light.
• Photometric principles are applied to the several kinds
of analytical techniques:
(a) where absorbed or transmitted light is measured:
• Colorimetry
• Spectrophotometry
• Atomic absorption, and
• Turbidometry
(b) where emitted light is measured:
• Flame emission photometry

3. Introduction (cont.)
• The components of most photoelectric colorimeters are
basically the same and the basic method of operation is
also similar for all the instruments.
• In analytical chemistry, Colorimetry is a technique
“used to determine the concentration of colored
compounds (analytes) in sample solution” at visible
spectrum of light (400 – 800 nm).

4. Colorimetry
Principle:
Colored solutions have the property of absorbing certain
wavelength of light when a monochromatic light is passed
through them.
• The amount of light absorbed or transmitted by a
colored solution is in accordance with two laws:
– Beer’s law
– Lambert’s law

5. Beer’s law :
• When a monochromatic light passes through a colored
solution, amount of light transmitted decreases
exponentially with increase in concentration of colored
substance.
– i.e. the amount of light absorbed by a colored solution is
directly proportion to the conc. Of substance in the colored
solution.

6. Beer’s law
Beer’s law

7. Lambert’s law :
• The amount of light transmitted decreases
exponentially with increase in path length (diameter) of
the cuvette or thickness of colored solution through
which light passes.
– i.e. the amount of light absorbed by a colored solution
depends on path length of cuvette or thickness or depth of
the colored solution.

8. Relationship between absorbance and
transmittance
OD %T

9. Preparation of solution for investigation
• In colorimetric estimation it is necessary to
prepare 3 solutions:
BLANK(B)
STANDARD(S)
TEST(T)
3/18/2016 8:34 PM

10. BLANK
To eliminate the effect of light
absorption by the reagent used
Water BLANK
Reagent BLANK

11. STANDARD
Solution of known concentration of
the substance
Both O.D and
concentration are
known
So concentration of
unknown can be
calculated

12. TEST
Test solution is made by
treating a specific volume of
the test sample with reagents
As per
procedure

13. Combined Beer’s- Lambert’s law
• Combined Beer’s- Lambert’s law is thus expressed as
amount of light transmitted through a colored solution
decreases exponentially with increases in conc. Of
colored solution & increase in conc. of colored solution
& increase in the path length of cuvette or thickness of
the colored solution.
• Combining the two laws:
A α C x L
A = K x C x L
Let AT=absorbance of the test solution
CT=concentration of the test solution
AS=absorbance of the standard solution
CS=concentration of the standard solution

14. AT
AS
K x CT x L
K x CS x L
=
AT
AS
CT
CS
=
CT =
AT
AS
x CS
AS = K x CS x LAT = K x CT x L

15. CT =
AT
AS
xCS
Concentration
of TEST sol.
Absorbance of TEST
Absorbance of STANDARD
Con. of STANDARDx=
Concentration
of TEST/100ml
Absorbance of TEST
Absorbance of STANDARD
Concn of Std X 100
x=
X ml
=
ODT
ODS
x CS

16. Concentration of
TEST /100ml
O.D of ‘T’- O.D of ‘B’
O.D of ‘S’- O.D of ‘B’
x=
Volume of ‘T’
Amount of ‘S’
Concentration of
TEST /100ml
x=
Volume of ‘T’
Amount of ‘S’T - B
S - B
x 100
x 100

17. Standard curve (calibration curve)
• The standard curve is prepared to check whether the
method of assaying a particular substance follows
Beer’s Law, i.e. whether the absorbance of the
substance increases in a linear way with its
concentration.
• The standard curve is constructed by plotting a vertical
axis (y – axis, ordinate) for optical densities
(absorbance) and a horizontal axis (x – axis, abscissa)
the concentration of standard solution.
• The concentration of the test/unknown can be
measured from the graph (standard curve).

18. Verification of Beer’s Law
• Prepare 1% standard solution of glucose, i.e. 1gm/dl
1000mg/100ml.
• Make different dilutions of standard solution using the general
formula given as following for obtaining different concentrations
of a solution by dilution with diluent (DW):
Tube no. Conc. (mg%) Amount of mL needed, (V1)
C1 x V1 = C2 x V2
DW (mL) Total vol. V2 (ml)
1 50 1000 x V1 = 50 x 2, V1= 0.1 1.9 2
2 100 1000 x V1 = 100 x 2, V1= 0.2 1.8 2
3 150 1000 x V1 = 150 x 2, V1= 0.3 1.7 2
4 200 1000 x V1 = 200 x 2, V1= 0.4 1.6 2
5 250 1000 x V1 = 250 x 2, V1= 0.5 1.5 2
6 300 1000 x V1 = 300 x 2, V1= 0.6 1.4 2
7 350 1000 x V1 = 350 x 2, V1= 0.7 1.3 2
8 400 1000 x V1 = 400 x 2, V1= 0.8 1.2 2

19. 0.06 0.12 0.18 0.24 0.3 0.36 0.42 0.48
O.D2 50 100 150 200 250 300 350 400
0
50
100
150
200
250
300
350
400
450
CONC.OFGLUCOSE(mg/dL) OD of solution
Standard Curve / Calibration curve

22. Light spectrum and their wavelengths

23. Complimentary color
• Wavelength between 400nm to 760 nm form the visible
spectrum of light
• Light passed through a solution which selectivity absorbs
radiation at fixed wave lengths,then the color of the
transmitted light is complementary to that of the absored
light.

24. Colors and complimentary colors of visible
spectrum
Color of the
solution/ solution
color transmitted
Filter used/ color
absorbed
Wavelength (nm)
Yellow blue Violet 380 – 430
Yellow Blue 430 – 475
Orange Green blue 475 – 495
Red Blue green 495 – 505
Purple Green 505 – 555
Violet Yellow green 555 – 575
Blue yellow 575 – 600
Green blue Orange 600 – 650
Blue green Red 650 - 750

25. Colorimeter

26. Components of Colorimetry

27. Components of Colorimetry
1. Light source:
The light source is usually a tungten lamp, for
wavelength in the visible range (320 – 700nm) and a
deutarium or hydrogen lamps for ultraviolet light
(below 350nm).
a) Tungsten lamp  Visible range
b) Deutarium/hydrogen lamp (preferred)  UV Rays
c) Black body radiators (Nerst glower)  Infrared radiations

28. Light source
1. Tungsten lamp:
 filament mode of tungsten sealed in a glass envelope
 Filed with inert gas.
 Life time is limited due to gaseous tungsten formed by
sublimation.

29. Light source
Carbon arc lamp
• If sufficient intensity of light is not obtained
from tungsten lamp then carbon arc lamp can be
use as a source for color measurement.

30. Monochromators/Filters
• This is a means of selecting a sufficiently narrow wave
band
• Filter will absorb light of unwanted wavelength and
allow only monochromatic light to pass through.
– E.g.: a green filter absorbs all color, except green light which
is allowed to pass through.
Filter
Absorption filter Interference filter
Ex: Glass filter,Gelatin filter

31. Monochromators
• Early colorimeters used Absorption filters (i.e. glass
filter, Gelatin filter) that transmitted a wide segment of
spectrum (50nm or more).
• Newer instrument use Interface filters that consist of
thin layer of magnesium fluoride crystals with a
semitransparent coating of silver on each side.
• Monochromator consists of:
– Entrance slit
– Absorption/ interface filter and
– Prisms or diffraction grating for wavelength selection
– Exit slit

32. Sample Holder/ Cuvette
• Cuvettes are rectangular cell , square cell or
circular one.
• Made up of optical glass for visible wavelength
(quartz or fused silica for UV).
• Common one is square, rectangular to avoid
refraction artifacts.
• Optical path (length) of cuvette is always1cm.
• Capacity may be 3ml/2ml/1ml depending upon
the thickness of the wall of the cuvette.
• For accurate and precise reading cuvette must be
transparent, clean, devoid of any scratches and
there should be no bubble adhering to the inner
surface of the filled cuvette.

33. Photosensitive detectors
• Detectors are the transducers, which convert light
energy to electrical enagery. A detector should be
possess follwing characteristics:
1.Should be sensitive
2.Should have linear response
3.Its noise level Should be low
4.Should have short response time
5.Should stable.

34. Photosensitive detectors
• Different detectors used are:
1. Barrier layer cells (photocells) – simplest
2. Photoemmisive cells
3. Photomultiplier tube (for low intensity lights)
4. Photoconductive cells (photodiodes) – newest.

35. Read out devices
• The detector response can be measured by any
of the following devices:
a) Galvanometer
b) Ammeter
c) Recorder
d) Digital readout.
The signal may be transmitted to computer or print
out devices.

36. Criteria for satisfactory colorimetric
estimations
• Stability of color
Color may be fade of air oxidation,
photochemical decomposition, temperature.
• Intensity of color
The color of the solution should be intense in
order to detect small amount of constituents
and for making accurate result in low
concentration.

37. Criteria for satisfactory colorimetric
estimations
• Clarity of the solution
Substance under investigation should be completely
soluble in the solvent, since turbid solution,
suspension or colloidal solution absorb as well as
scatter light.
• Reproducibility
The intensity of the colored solution must be
reproducible. The effect of order of adding reagent,
pH and other variable should be clearly studied

38. Criteria for satisfactory colorimetric
estimations
• Specificity
Color produced should be specific for the desired
constituent. If other constituents interfere with color
reaction they be removed or prevented from or
prevented from functioning through appropriate
treatment like use of other coloring agent, altering
the oxidation state.
• Validity of Beer’s law
The intensity of color should be proportional to
concentration. It can be easily assessed by plotting
absorbance Vs concentration, where a straight line
passing through origin should be obtained.

39. Glass/gel filter is placed in the filter slot
3/4th of cuvette is filled with distilled water
and placed in the cuvette slot
Instrument is switched ‘on’ and allowed to
warm-up for 4-5 minutes

40. Button is adjusted using ‘coarse’ and ‘fine’ knobs to
give zero optical activity in the galvanometer
Blank solution is placed in an identical cuvette and
the OD is read (‘B’)
Blank solution is transferred to the original test tube

41. Test solution is taken in the same cuvette and O.D.
is read (‘T’)
Test solution is transferred back to the original test
tube
Standard solution is taken in same cuvette and O.D.
is read (‘S’)
Standard solution is transferred back to the test
tube
Cuvette is washed

43. Applications Of Colorimeter
• Estimation of biochemical compounds in blood, plasma,
serum, CSF, urine, etc.:
– Glucose
– Urea
– Creatinine
– Uric Acid
– Bilirubin
– Lipids
– Total Proteins
– Enzymes [e.g. ALT, AST, ALP]
– Minerals [Calcium, Phosphorus etc.] etc….