principal and application of calorimeter, beers lambarts law, how to major lambda max, and how to use calorimeter,

1. COLORIMETRY

3. Objectives-
1. To explain the principle and application of colorimetry
2. To explain the λmax of KMnO4 by colorimetry
3. To determine the concentration of given unknown solution by
using λmax

4. To explain the principle and application of
colorimetry

5. Photometry
 Photometry literally means measurement of light.
 Colorimetry refers to measurement of light in the visible
spectrum (400 – 700 nm).
 Spectrophotometry refers to the measurement of light in
the visible spectrum (400 – 700 nm), UV (190 nm – 400 nm)
and the IR (700 – 1000 nm) regions.

6. When white light passes through a solution, certain energies (colors or
wavelengths) of the light are absorbed while other color(s) are allowed to
pass through (i.e. transmitted) by the solution.
Color of solution is due to the light which is not absorbed, i.e. wavelengths
which are transmitted.
Photometers measure this transmitted light (transmittance %) and convert
it into absorbance values
.
If all the wavelengths are transmitted, the solution appears colorless or
transparent
Colors of Solutions

7. A green solution appears green because of the following
reason:
• It is absorbing light only in the red wavelength (complementary colour of
green) and transmitting all other wavelengths.
• These transmitted wavelengths combine together to appear as green colour.
WHITE LIGHT

8. Observed Color of
Compound
Color of Light Absorbed Approximate Wavelength of
Light Absorbed
Green Red 700 nm
Blue-green Orange-red 600 nm
Violet Yellow 550 nm
Red-violet Yellow-green 530 nm
Red Green 500 nm
Orange Blue 450 nm
Yellow Violet 400 nm

9. Transmittance is related to Absorbance
Transmittance is given by the equation:
T = I/Io
Where, I is the intensity of the emergent light &
Io is the initial light intensity.
Absorbance is related to the %T:
A = -log %T = -log(I/ Io)

10. Absorbance v/s Transmittance
• Absorbance is directly proportional to the concentration.
• Transmittance is inversely & logarithmically proportional to the
concentration.
• Hence, A  log (1/T) or A  - log T

11. Beer's law: When a parallel beam of monochromatic light passes through a
solution, the absorbance (A) of the solution is directly proportional to
concentration (C) of the compound in the solution.
A  C
Lambert’s law: Absorbance is directly proportional to the thickness or length
of the light path (l) through the solution
A  l
or A = ε C l
ε , the proportionality constant is termed the molar absorption coefficient
Defined as the absorbance of a one molar solution of a substance with a light
path of one centimeter (if C is expressed in mol/L)
Specific for a given substance at a given wavelength.
Beer's & Lambert’s law

12. Now let us pass this red light through various concentrations of the
green solution
T = 50 %
T = 25 %
T = 10 %
T = 1 %
A = 1
A = 2
A = 0.6
A = 0.3
•CONCLUSION : as the concentration of the substance increases, the
absorbance also increases. (OR)
A  c ----- Beer’s law.

13. • Absorbance of test sample (At) = ε x concentration of test (Ct) x l
• Absorbance of standard sample (As) = ε x concentration of standard (Cs) x l
• Conc. of test (Ct) = x Conc. of standard (Cs)
Absorbance of test (At)
Absorbance of standard (As)
Determination of Concentration of Unknown Sample

14. • Blank contains all substances except the analyte of interest.
• Absorbance of the blank must be subtracted from the absorbance values
of test & standard.
• Aim is to remove any absorption of light due to the solvent & other
reagents
• All the measured absorbance is only due to the analyte.
Conc. of test (Ct) =
Abs. of test (At) – Abs. of blank (Ab)
Abs. of standard (As) – Abs. of blank (Ab)
x Conc. of standard (Cs)
What is Blank ?

15. The lamp emits all colors of light (i.e., white light).
The monochromator selects one wavelength and that wavelength is sent
through the sample.
The detector detects the wavelength of light that has passed through the
sample.
Colorimeter and its Parts

16. Clinical Application
We can measure the concentration & strength of various biological fluid like
plasma, serum, CSF, urine.
E.g. Determination of –
• Blood glucose
• Blood urea
• Serum protein
• Serum creatinine
• Serum cholesterol
• Serum inorganic phosphate
• Urine creatinine
• Glucose in CSF etc.

17. REAGENT (A) SAMPLE (B) COLORED PRODUCT (A+B C)

18. Colorimeter
• Light emitted in visible range
(400-700 nm).
• Solution has to be colored.
• Wavelength selector – filter.
• Moderate sensitivity
Spectrophotometer
• Light emitted in UV (200-400
nm), visible & IR range (700-
900nm)
• Solution need not be colored.
•Wavelength selector – prism or
diffraction grating.
• High sensitivity.
Difference between Colorimeter &
Spectrophotometer

19. Colorimeter
Spectrophotometer

20. To explain the λmax of KMnO4 by colorimetry

21. •You will be provided a 12 mg/dl solution of KMnO4
• Measure the absorbance of this solution at various
wavelengths from 400 nm to 700 nm.
• Plot the absorption spectrum from the observed values.
• Find out the λmax.
Today, you will perform a similar experiment to find out
the λmax for KMnO4 solution

22. • Set the filter at the desired wavelength.
• Take DW (distilled water) as blank and adjust the
absorbance of the instrument to zero
• Record the absorbance of the test solution
• Repeat the same procedure for different wavelengths
• Plot the graph of wavelength vs absorbance
Wavelength that gives maximum absorbance is λmax of the
test solution.
Protocol for Determination of λmax

23. Observation table for calculating the λmax of KMnO4 solution
S.No. Filters (Wavelengths)
(nm)
Blank O.D.
(Water )
O.D./Absorbance
1. 400 0.00 0.20
2. 420 0.00 0.24
3. 480 0.00 0.34
4. 500 0.00 0.50
5. 520 0.00 0.69
6. 540 0.00 0.71
7. 620 0.00 0.37
8. 680 0.00 0.12

24. To determine the concentration of given
unknown solution by using λmax

25. S.No.
Required
concentrations
Volume of KMNO4 Volume of DW Total volume
1 1.5 mg/dl 1.5 ml 12 ml 12 ml
2 3 mg/dl 3 ml 9 ml 12ml
3 6 mg/dl 6 ml 6 ml 12 ml
4 9 mg/dl 9 ml 3 ml 12 ml
5 12 mg/dl 12 ml 0 ml 12 ml
Protocol for Making a standard Curve
• The working solution of KMnO4 provided has a concentration of 12 mg/dl
• Plot a standard curve using different concentrations of KMnO4 by making
concentrations of 1.5, 3, 6, 9 and 12% w/v (12 ml each)
• Measure the absorbance at λmax

26. Concentration Absorbance / O.D.
1 1.5 mg/dl
2 3 mg/dl
3 6 mg/dl
4 9 mg/dl
5 12 mg/dl

27. • Set the colorimeter at the λmax of the test solution
• Take DW (distilled water) as blank and set the absorbance of the
instrument at zero.
• Record the absorbance of the test solution at different concentrations.
• Plot the graph of concentration vs. absorbance and obtain the
standard curve
Protocol for Making a standard Curve (Cont.)

28. Concentration (mg/dl)
Optical
Density Standard Curve
12
10
8
6
4
2
0
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
0.10
0.20
1.20
1.30
Straight Line Graph