colorimetry-basics for analytical chemistry.ppt

Colorimetry

1. COLORIMETRY
Prepared By
Michigan Department of Environmental Quality
Operator Training and Certification Unit
Note: A printed description of colorimetry is available in the OTCU
Laboratory manual (Section 310) available on the OTCU website.

2. COLORIMETRY
Color Measurement

3. COLOR
Interaction
between
LIGHT
and
MATTER

4. Matter
“ORBIT”
Nucleus
Electrons
Energy Level
of
Electrons

5. Orbits = Energy Level
Each Electron Can Be In Only Certain Energy Levels

6. LIGHT
Photon -“Energy Packet”
wavelength -
(time)
frequency -(gamma)
(lambda)
Wave

7. LIGHT
E =h
hc

=
TheEnergy(E)of the Photonis
Related to thewavelength and the
frequency of the Wave()
()
Where:
h = Planck’s Constant
c = Velocity of Light

8. E =
hc

Constants
Every wavelength ()
has a specific
Energylevel.
LIGHT

9. Summary
Each ElectronCan Be In
Only CertainEnergy Levels
Every wavelength has a
specific EnergyLevel.

10. COLORIMETRY
600 nm
700 nm
650 nm

11. COLOR
RESULTS WHEN
RADIATION IS ABSORBED
BY AN ELEMENTOR
BY A COMPOUND FORMED
THROUGH A REACTION
W H I T E
RED
BLUE
YELLOW
YELLOW
BLUE
RED
ABSORBED
GREEN

12. TRANSMITTANCE (T)
OF THE INTENSITY OF LIGHT
LEAVING SOLUTION (I)
TO THE INTENSITY OF LIGHT
ENTERING SOLUTION (I
O)
RATIO

13. TRANSMITTANCE
I
T =
I
O
I
O I
%T = T x 100

14. Units of Optical Path
Transmittance
0 1 2 3 4 5
1.0
.9
.8
.7
.6
.5
.4
.3
.2
.1
0
I
0
I
1
I
2
I
3
I
4
I
5
Comparing Light Transmittance to Cell Length
I
n

15. LAMBERT’S LAW
Relates the absorption of light to the depth or
thicknessof the colored liquid
Each layer of equal thickness will absorb the same
fractionof light which passes through it
An arithmetic increasein thickness gives a
geometric decrease in light intensity transmitted

16. Units of Concentration
Transmittance
0 1 2 3 4 5
1.0
.9
.8
.7
.6
.5
.4
.3
.2
.1
0
I
0
I
1
I
2
I
3
I
4
I
5
Comparing Light Transmittance to Concentration
I
n

17. BEER’S LAW
Relates the absorption of light to the concentration
of the absorbing substance in the solution
The fractionof light absorbed is directly
proportional to the concentration of the absorbing
substance
An arithmetic increasein concentration gives a
geometric decreasein light intensity transmitted

18. COLORIMETRY
How Do We Use This Principle?
Perform a Chemical Reaction with the
Element to be Analyzed that Results in
a Compound of that Element that
Absorbs Light.
Measure the Amount
of Light Absorbed.

19. COLORIMETRY
1.The Chemistry Involved.
2. The Length of Light Travel.
3. The Amount (Concentration) of
Absorbing Material.
The Amount of Light Absorbed
Is Related To:

20. THE COMBINED LAMBERT’S LAW
AND BEER’S LAW
T = 10
-abc
Where:
a = constant for particular solution
b = length of absorbing layer (light path length)
c = concentration of absorbing substance
T =
I
I
o
{-Sign Indicates an Inverse Relation}

21. TRANSMITTANCE
T = 10
-abc
Absorbance = A = -log T
log T = log (10
-abc
)
log T = -abc
-log T = -(-abc) = abc
A =
T =
I
I
o
-log T= abc

22. ABSORBANCE (A)
Where:
a = constant for particular solution
b = length of absorbing layer (light path length)
c = concentration of absorbing substance
A = -log T
A = abc

23. ABSORBANCE (A)
If:
a = held constant by carefully performing the analysis
b = held constant by controlling the light path length
A is Directly Related to c (conc. of absorbing substance)
A = -log T
A = abc
Then:
If we can measure A, then we can determine c

24. COLORIMETRY
Measurement of the amount of
LIGHT ABSORBED
by the
COLOR DEVELOPED
in a sample

25. CONCENTRATION CAN BE COLORIMETRICALLY
DETERMINED IF:
1. Able to chemically develop a color with that substance and only that
substance
2. The developed color obeys (follows) Beer’s Law over a reasonable
range of concentrations
3. The developed color must be stablefor reasonable length of time,
reproducible,and sensitiveto small changes in concentration
4. All loss of transmitted light must be from absorbance by substance
measured (developed color)
5. All of substance present in sample must be available for reaction with
color developing agent
6. Able to measure amount of light absorbed

26. Sample Preparation
Dilution
Solids Removal
---Coagulation
---Centrifuge
---Filter
pH Adjustment
Digestion

27. DIGESTION
Destroy Organics
Release Combined Constituent
Change Form of Constituent

28. Colorimetry
Color Development
Color Must Be:

29. Color Development
Must Control :
pH
Time
Ionic Strength
Temperature

30. COLORIMETRY
Measurement of the amount of
LIGHT ABSORBED
by the
COLOR DEVELOPED
in a sample

31. Color Measurement
“Color Comparators”
Compare Sample Color to Known
Standards
O.K. For Control –Not For Reporting

32. Color Measurement
Spectrophotometer
Compare Sample Color to Known
Standards
“Calibration Curve”
(verified)

33. Colorimetric Instruments

34. Spectrophotometer
Light
Source
Monochromator
Sample
Cell
Detector
Meter

35. Light Source
Controllable
Constant
Voltage Regulation
Fatigue
Diaphragm
Voltage Adjustment

36. Color (wavelength) Band

37. Monochromator
PRISM
OR
DIFFRACTION
GRATING
APERATURE
OR
SLIT
Must be CAREFULLY Adjusted

38. Sample Cell
Cuvette
The Light Path is
affected by the
Cuvette

39. Sample Cell
Cuvette
Must be
CAREFULLY
Aligned

40. PHOTOELECTRIC TUBE
“DETECTOR”
Differing
Response
for
Various
Wavelengths
Bausch & Lomb
33-29-71
340-600 nm
33-29-72 (w / filter)
600-950 nm
33-29-92 (w / filter)
400-700 nm

41. PHOTOELECTRIC TUBE
“DETECTOR”
Differing
Response
for
Various
Wavelengths
Must Use the
Correct
Combination
of Filter and
Phototube
For Wavelength
Of Analysis

42. INDICATING METER
Gives the Readout in
Transmittance or
Absorbance

43. INDICATING METER
Some Meters Give
Readout Directly in
Concentration
Use Only those Readings Between the Lowest
and Highest Standard of Calibration

44. INDICATING METER
Some Meters Have
“Built-in” Calibration
These Calibrations Should Be Verified Periodically
Using a Series of Standards and Only those Readings
Between the Lowest and Highest Standard of Calibration
Should be Used

45. Optical System
Lenses
Mirrors
Apertures
Occluders

46. Optical System
The Instrument Must be Carefully Handled,
Protected From Dust and Vapors, and
Serviced Only By Qualified Technicians

47. Spectrophotometer
Light
Source
Sample
Cell
Detector
Meter
Monochromator

48. COLORIMETRY
Instrument Operation:
Set Monochromator
Warm-up
Set Zero Absorbance w/Blank
Set ∞Absorbance
Re-adjust as Needed

49. COLORIMETRY
General Rule –
Absorbance Between 0.100 and 0.700
Some Analyses More Restrictive
Best Readings –
Between Lowest and Highest Standards Used
In Calibration
Watch for Irregularities
Instrument Operation:

50. COLORIMETER CALIBRATION
Calibration or Standardized
By Measuring Absorbance Readings
of a Series of Known Standards
1. Computer Spreadsheet
2. Instrument with Internal Microprocessor
3. “Plotting” a Graph
Comparison of These Readings to
the Reading for a Sample

51. COLORIMETER CALIBRATION
Calibration or Standardized
By Measuring Absorbance Readings
of a Series of Known Standards
Verified Frequently
At Least One Standard
In Acceptable Range
Comparison of These Readings to
the Reading for a Sample
Each Time Samples Are Analyzed

52. COLORIMETER CALIBRATION
1. Significant Change In Procedure,
Equipment, or Reagents
2.Determined Length of Time
(Max. Six Months)
3. Verification Standard Not In Acceptable
Range
Repeat Calibration:

53. Calibration Steps:
1. Prepare Stock Solution
2. Prepare a Series of Dilutions
3. Same Preparation Steps as Sample
4. Develop Color
5. Measure Absorbance of Each
6. Prepare Calibration “Curve”

54. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
0.1
0.2
0.3
0.4
0.5
Concentration, mg/L
Absorbance
Total PhosphorusAscorbic Acid –Two Reagent Method
DD/MM/YY
650 nm ½Inch CuvetteConc.Abs.
0.2 0.104
0.3 0.153
0.4 0.210
0.5 0.258
0.6 0.312 Calibration
Curve
(Using Phosphorus
Analysis Example)

55. COLORIMETRY
Prepared By
Michigan Department of Environmental Quality
Operator Training and Certification Unit