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
Energy Level of Electrons
Nucleus
Electrons
5
Orbits Energy Level
Level 1
Level 2
Level 3
Each Electron Can Be In Only Certain Energy Levels
6
LIGHT
Photon -
Energy Packet
wavelength - ?
(lambda)
Wave
frequency - ?
(gamma)
7
LIGHT
hc ?
E
h?

Where h Plancks Constant
c Velocity of Light
8
LIGHT
Constants
Every wavelength (l) has a specific Energy level.
9
Summary
Each Electron Can Be In Only Certain Energy Levels
Every wavelength has a specific Energy Level.
10
COLORIMETRY
600 nm
650 nm
700 nm
11
COLOR
RESULTS WHEN RADIATION IS ABSORBED BY AN ELEMENT
OR BY A COMPOUND FORMED THROUGH A REACTION
RED
YELLOW
RED ABSORBED
YELLOW
W H I T E
GREEN
BLUE
BLUE
12
TRANSMITTANCE (T)
RATIO
OF THE INTENSITY OF LIGHT LEAVING SOLUTION
(I) TO THE INTENSITY OF LIGHT ENTERING SOLUTION
(IO)
13
TRANSMITTANCE
IO
I
I
T
IO
T T x 100
14
Comparing Light Transmittance to Cell Length
1.0 .9 .8 .7 .6 .5 .4 .3 .2 .1 0
I0
Transmittance
I1
I2
I3
I5
In
I4
0 1 2 3
4 5
Units of Optical Path
15
LAMBERTS LAW
Relates the absorption of light to the depth or
thickness of the colored liquid
Each layer of equal thickness will absorb the
same fraction of light which passes through it
An arithmetic increase in thickness gives a
geometric decrease in light intensity transmitted
16
Comparing Light Transmittance to Concentration
1.0 .9 .8 .7 .6 .5 .4 .3 .2 .1 0
I0
Transmittance
I1
I2
I3
I5
In
I4
0 1 2 3
4 5
Units of Concentration
17
BEERS LAW
Relates the absorption of light to the
concentration of the absorbing substance in the
solution
The fraction of light absorbed is directly
proportional to the concentration of the
absorbing substance
An arithmetic increase in concentration gives a
geometric decrease in light intensity transmitted
18
COLORIMETRY
Perform a Chemical Reaction with the Element to
be Analyzed that Results in a Compound of that
Element that Absorbs Light.
How Do We Use This Principle?
Measure the Amount of Light Absorbed.
19
COLORIMETRY
The Amount of Light Absorbed Is Related To

• The Chemistry Involved.
• 2. The Length of Light Travel.
• 3. The Amount (Concentration) of Absorbing
  Material.

20
THE COMBINED LAMBERTS LAW AND BEERS LAW
T 10 -abc
Where
a constant for particular solution
b length of absorbing layer (light path length)
c concentration of absorbing substance
- Sign Indicates an Inverse Relation
21
TRANSMITTANCE
Absorbance A - log T
T 10 -abc
log T log (10 -abc)
log T -abc
-log T -(-abc) abc
-log T
abc
A
22
ABSORBANCE (A)
A - log T
A abc
Where
a constant for particular solution
b length of absorbing layer (light path length)
c concentration of absorbing substance
23
ABSORBANCE (A)
A - log T
A abc
If
a held constant by carefully performing the
analysis
b held constant by controlling the light path
length
Then
A is Directly Related to c (conc. of absorbing
substance)
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) Beers
Law over a reasonable range of concentrations
3. The developed color must be stable for
reasonable length of time, reproducible, and
sensitive to 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
Stable
Reproducible
Sensitive
29
Color Development
Must Control
pH
Time
Temperature
Ionic Strength
30
COLORIMETRY
Measurement of the amount of
LIGHT ABSORBED
by the
COLOR DEVELOPED
in a sample
31
Color Measurement
Compare Sample Color to Known Standards
Color Comparators
O.K. For Control Not For Reporting
32
Color Measurement
Compare Sample Color to Known Standards
Spectrophotometer
Calibration Curve (verified)
33
Colorimetric Instruments
34
Spectrophotometer
Sample Cell
Detector
Monochromator
Light Source
Meter
35
Light Source
WHITE LIGHT
Constant
Controllable
Voltage Regulation
Diaphragm
Fatigue
Voltage Adjustment
36
Color (wavelength) Band
37
Monochromator
APERATURE OR SLIT
PRISM
OR DIFFRACTION GRATING
Must be CAREFULLY Adjusted
38
Sample Cell
The Light Path is affected by the Cuvette
Cuvette
39
Sample Cell
Must be CAREFULLY Aligned
Cuvette
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
Sample Cell
Detector
Monochromator
Light Source
Meter
48
COLORIMETRY
Instrument Operation
Warm-up
Set Monochromator
Set 8 Absorbance
Set Zero Absorbance w/Blank
Re-adjust as Needed
49
COLORIMETRY
Instrument Operation
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
50
COLORIMETER CALIBRATION
Calibration or Standardized By Measuring
Absorbance Readings of a Series of Known Standards
Comparison of These Readings to the Reading for a
Sample
1. Computer Spreadsheet
2. Instrument with Internal Microprocessor
3. Plotting a Graph
51
COLORIMETER CALIBRATION
Calibration or Standardized By Measuring
Absorbance Readings of a Series of Known Standards
Comparison of These Readings to the Reading for a
Sample
Verified Frequently
At Least One Standard
In Acceptable Range
Each Time Samples Are Analyzed
52
COLORIMETER CALIBRATION
Repeat Calibration
1. Significant Change In Procedure, Equipment,
or Reagents

• Determined Length of Time
• (Max. Six Months)

3. Verification Standard Not In Acceptable Range
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
Calibration Curve (Using Phosphorus Analysis
Example)
55
COLORIMETRY
Prepared By Michigan Department of Environmental
Quality Operator Training and Certification Unit