colorimeter ppt biochemistry
TRANSCRIPT
Colorimetery
•COLORIMETER
•Visible spectrum
•Colorimeter
Colorimeter is a instrument used
for the measurement of
coloured substance in
solution.
The instrument is operative in the visible range of
the electromagnetic
spectrum
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•Photometry
Photometry means measurement of light.
The colour of light is a function of its wavelength.
As the wavelength changes alteration in colour is detected.
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• Colorimeter - Principle
•When a white light passes through a coloured solution, some specific wavelengths of light are absorbed which is related to colour intensity.
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Components of the colorimeter
Tungsten lamp
Slit
Condensing lens
Filter
Cuvette
Photocell
Galvanometer
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•COLORIMETER
•COLORIMETER
Relationship between absorbance and transmittance
IOIE
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OD %T
• The measurement of colour intensity of a coloured solution by photometry is governed by two laws
Beer’s law Lambert’s law
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BEER’S LAW• The concentration of a
substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light
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AαC
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Beer’s law
Beer’s law
LAMBERT’S LAW• When a ray of
monochromatic light passes through an absorbing medium its intensity decreases exponentially as the length of the light path through light absorbing material increases
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AαL
•LAMBERT’S LAW
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Preparation of solution for investigation• In colorimetric estimation it is necessary to prepare 3 solutions
BLANK(B)
STANDARD(S)
TEST(T)
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BLANKTo eliminate the effect of light absorption by the reagent used
Water BLANK
Reagent BLANK
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STANDARDSolution of known concentration of the substance
Both O.D and concentration are known
So concn of unknown can be calculated
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TESTTest solution is made by treating a specific volume of the test sample with reagents
As per procedure
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•Derivation of the Formula• Combining the two laws• AαCxL• OR A=KxCxL• 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
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AT
AS
KxCTxL
KxCSxL=
AT
AS
CT
CS=
CT =AT
AS
X CS
AS=KxCSxL
AT=KxCTxL
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CT =AT
AS
X CS
Concentration of TEST solution
Absorbance of TEST
Absorbance of STANDARD
Concn of STANDARDX=
Concentration of TEST /100ml
Absorbance of TEST
Absorbance of STANDARD
Concn of Std X 100X=
Xml
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Concentration of TEST /100ml
Absorbance of TEST
Absorbance of STANDARD
X=
Xml
Concn of Std X 100
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’ X 100
Concentration of TEST /100ml
T - B
S - BX=
Volume of ‘T’
Amount of ‘S’ X 100
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•VERIFICATION OF BEER’S LAW
3% Cobalt acetate in 1% HCl
1% HCl
REAGENTS
Colorimeter
Cuvettes
INSTRUMENT
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B S1 S2 S3 S4 S5 S6 S7 T
Volume of 3% cobalt acetate[in ml]
- 0.5 1 2 3 4 5 6 -
Volume of 1% HCl [in ml] 6 5.5 5 4 3 2 1 - -
Conc. Of cobalt acetate in mg/6ml
- 15 30 60 90 120 150 180
O.D at 490nm 0.01 0.03 0.06 0.12 0.18 0.24 0.30 0.36 0.21
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•By Graphical methood
15 30 60 90 120
O.D 0.02 0.08 0.14 0.2 0.26 NaN
0.025
0.075
0.125
0.175
0.225
0.275
Concentration in mg/6ml
O.D
0.17
