•COLORIMETER
•Visible spectrum
•Colorimeter
Colorimeter is a
instrument used
for the
measurement of
coloured
substance in
solution.
The instrument is
ope...
•Photometry
Photometry means
measurement of light.
The colour of light is a function
of its wavelength.
As the wavelength ...
• Colorimeter - Principle
• When a white light passes through a coloured
solution, some specific wavelengths of light are
...
8/26/2013 12:23 AM
Components of the colorimeter
8/26/2013 12:23 AM
•COLORIMETER
•COLORIMETER
Relationship between absorbance and
transmittance
8/26/2013 12:23 AM
OD %T
• The measurement of colour intensity of a
coloured solution by photometry is governed
by two laws
Beer’s law Lambert’s la...
BEER’S LAW
• The concentration of a
substance is directly
proportional to the amount of
light absorbed or inversely
propor...
8/26/2013 12:23 AM
Beer’s law
Beer’s law
LAMBERT’S LAW
• When a ray of
monochromatic light passes
through an absorbing
medium its intensity
decreases exponentially...
•LAMBERT’S LAW
8/26/2013 12:23 AM
Preparation of solution for investigation
• In colorimetric estimation it is necessary to
prepare 3 solutions
BLANK(B)
STA...
8/26/2013 12:23 AM
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 concn of unknown
can be ...
TEST
Test solution is made by
treating a specific volume of
the test sample with reagents
As per procedure
8/26/2013 12:23...
•Derivation of the Formula
• Combining the two laws
• AαCxL
• OR A=KxCxL
• Let AT=absorbance of the test solution
• CT=con...
8/26/2013 12:23 AM
AT
AS
KxCTxL
KxCSxL
=
AT
AS
CT
CS
=
CT =
AT
AS
X CS
AS=KxCSxLAT=KxCTxL
8/26/2013 12:23 AM
CT =
AT
AS
X CS
Concentration
of TEST
solution
Absorbance of TEST
Absorbance of STANDARD
Concn of STAND...
8/26/2013 12:23 AM
Concentration
of TEST
/100ml
Absorbance of TEST
Absorbance of STANDARD
X
=
Xml
Concn of Std X 100
Conce...
•VERIFICATION OF BEER’S LAW
8/26/2013 12:23 AM
3% Cobalt
acetate in
1% HCl
1% HCl
REAGENTS
Colorimeter
Cuvettes
INSTRUMENT
8/26/2013 12:23 AM
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 m...
•By Graphical methood
15 30 60 90 120
O.D 0.02 0.08 0.14 0.2 0.26
0
0.05
0.1
0.15
0.2
0.25
0.3
O.D
Concentration in mg/6ml...
- -
THANK YOU
Colorimeter ppt BIOCHEMISTRY
Upcoming SlideShare
Loading in...5
×

Colorimeter ppt BIOCHEMISTRY

4,359
-1

Published on

2 Comments
4 Likes
Statistics
Notes
No Downloads
Views
Total Views
4,359
On Slideshare
0
From Embeds
0
Number of Embeds
2
Actions
Shares
0
Downloads
358
Comments
2
Likes
4
Embeds 0
No embeds

No notes for slide
  • Light source: tungsten filament lampSlit :It is adjustable which allows only a beam of light to pass through. It prevents unwanted or stray lightCondensing lens: Light after passing through a slit falls on a condenser which gives parallel beam of light.Filters : are usually made of colored glass. it is used for selecting narrow wavelength .they absorb light of unwanted wavelength and allow only monochromatic light to pass throughFor e.g.: , a green filter absorbs all colours,except green light which is allowed to pass through.light transmitted through a green filter has a wavelength from 500-560nm.Filters used is always complimentary to the colour of the solutionCuvette -may be square,rectangular or round shape with fixed diameter and having uniform surface-made up of plastic ,glass material-solution in the cuvette absorbs a part of the light and the remaining is allowed to fall on the detectorDetector (photocell): The detectors are photosensitive elements which converts light energy into electrical signal .the electrical signal is directly proportional to the intensity of light falling on the detectorOutput : the electrical signal generated in a photocell is measured by a galvanometer which displays transmission and optical density
  • Colorimeter ppt BIOCHEMISTRY

    1. 1. •COLORIMETER
    2. 2. •Visible spectrum
    3. 3. •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 8/26/2013 12:23 AM
    4. 4. •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. 8/26/2013 12:23 AM
    5. 5. • Colorimeter - Principle • When a white light passes through a coloured solution, some specific wavelengths of light are absorbed which is related to colour intensity. 8/26/2013 12:23 AM
    6. 6. 8/26/2013 12:23 AM
    7. 7. Components of the colorimeter 8/26/2013 12:23 AM
    8. 8. •COLORIMETER
    9. 9. •COLORIMETER
    10. 10. Relationship between absorbance and transmittance 8/26/2013 12:23 AM OD %T
    11. 11. • The measurement of colour intensity of a coloured solution by photometry is governed by two laws Beer’s law Lambert’s law 8/26/2013 12:23 AM
    12. 12. 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 8/26/2013 12:23 AM
    13. 13. 8/26/2013 12:23 AM Beer’s law Beer’s law
    14. 14. 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 8/26/2013 12:23 AM
    15. 15. •LAMBERT’S LAW 8/26/2013 12:23 AM
    16. 16. Preparation of solution for investigation • In colorimetric estimation it is necessary to prepare 3 solutions BLANK(B) STANDARD(S) TEST(T) 8/26/2013 12:23 AM
    17. 17. 8/26/2013 12:23 AM BLANK To eliminate the effect of light absorption by the reagent used Water BLANK Reagent BLANK
    18. 18. STANDARD Solution of known concentration of the substance Both O.D and concentration are known So concn of unknown can be calculated 8/26/2013 12:23 AM
    19. 19. TEST Test solution is made by treating a specific volume of the test sample with reagents As per procedure 8/26/2013 12:23 AM
    20. 20. •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 8/26/2013 12:23 AM
    21. 21. 8/26/2013 12:23 AM AT AS KxCTxL KxCSxL = AT AS CT CS = CT = AT AS X CS AS=KxCSxLAT=KxCTxL
    22. 22. 8/26/2013 12:23 AM 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
    23. 23. 8/26/2013 12:23 AM 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 - B X = Volume of ‘T’ Amount of ‘S’ X 100
    24. 24. •VERIFICATION OF BEER’S LAW 8/26/2013 12:23 AM 3% Cobalt acetate in 1% HCl 1% HCl REAGENTS Colorimeter Cuvettes INSTRUMENT
    25. 25. 8/26/2013 12:23 AM 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
    26. 26. •By Graphical methood 15 30 60 90 120 O.D 0.02 0.08 0.14 0.2 0.26 0 0.05 0.1 0.15 0.2 0.25 0.3 O.D Concentration in mg/6ml 8/26/2013 12:23 AM 0.17
    27. 27. - - THANK YOU
    1. A particular slide catching your eye?

      Clipping is a handy way to collect important slides you want to go back to later.

    ×