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.
COLORIMETRY
Color Measurement
COLOR
Interaction
between
LIGHT
and
MATTER
Matter
ORBIT
Nucleus Energy Level
of
Electrons
Electrons
Orbits = Energy Level
Each Electron Can Be In Only Certain Energy Levels
LIGHT
Photon - Energy Packet
wavelength - (lambda)
Wave
(time)
frequency - (gamma)
LIGHT
The Energy (E) of the Photon is
Related to the wavelength () and the
frequency () of the Wave
E = h = hc
Where:
h = Plancks Constant
c = Velocity of Light
LIGHT
Constants
E = hc
Every wavelength ()
has a specific
Energy level.
Summary
Each Electron Can Be In
Only Certain Energy Levels
Every wavelength has a
specific Energy Level.
COLORIMETRY
600 nm
650 nm
700 nm
COLOR
RESULTS WHEN
RADIATION IS ABSORBED
BY AN ELEMENT OR
BY A COMPOUND FORMED
THROUGH A REACTION
RED
WHITE
YELLOW
GREEN
RED
YELLOW ABSORBED
BLUE
BLUE
TRANSMITTANCE (T)
RATIO
OF THE INTENSITY OF LIGHT
LEAVING SOLUTION (I)
TO THE INTENSITY OF LIGHT
ENTERING SOLUTION (IO)
TRANSMITTANCE
IO I
I
T=
IO
%T = T x 100
Comparing Light Transmittance to Cell Length
I0 1.0
.9
.8
Transmittance
.7
.6
.5
I1
.4
.3
.2 I2
.1 I3 I5 In
I4
0
0 1 2 3 4 5
Units of Optical Path
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
Comparing Light Transmittance to Concentration
I0 1.0
.9
.8
Transmittance
.7
.6
.5
I1
.4
.3
.2 I2
.1 I3 I5 In
I4
0
0 1 2 3 4 5
Units of Concentration
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
COLORIMETRY
Perform a Chemical Reaction with the
How Do
Element to We Use This
be Analyzed Principle?
that Results in
a Compound of that Element that
Absorbs Light.
Measure the Amount
of Light Absorbed.
COLORIMETRY
The Amount of Light Absorbed
Is Related To:
1. The Chemistry Involved.
2. The Length of Light Travel.
3. The Amount (Concentration) of
Absorbing Material.
THE COMBINED LAMBERTS LAW
AND BEERS LAW
I
T =
Io
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}
TRANSMITTANCE
T = I
Io
Absorbance = A = - log T
T = 10 -abc
log T = log (10 -abc)
log T = -abc
-log T = -(-abc) = abc
A = -log T = abc
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
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
COLORIMETRY
Measurement of the amount of
LIGHT ABSORBED
by the
COLOR DEVELOPED
in a sample
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
Sample Preparation
Dilution
Solids Removal
--- Coagulation
--- Centrifuge
--- Filter
pH Adjustment
Digestion
DIGESTION
Destroy Organics
Release Combined Constituent
Change Form of Constituent
Colorimetry
Color Development
Color Must Be:
Color Development
Must Control :
pH
Time
Temperature
Ionic Strength
COLORIMETRY
Measurement of the amount of
LIGHT ABSORBED
by the
COLOR DEVELOPED
in a sample
Color Measurement
Compare Sample Color to Known
Standards
Color Comparators
O.K. For Control Not For Reporting
Color Measurement
Compare Sample Color to Known
Standards
Spectrophotometer
Calibration Curve
(verified)
Colorimetric Instruments
Spectrophotometer
Sample
Cell
Monochromator Detector
Light
Source
Meter
Light Source
Constant
Controllable
Diaphragm Voltage Regulation
Fatigue
Voltage Adjustment
Color (wavelength) Band
Monochromator
APERATURE
OR
SLIT
PRISM
OR
Must be CAREFULLY Adjusted
DIFFRACTION
GRATING
Sample Cell
The Light Path is
affected by the
Cuvette
Cuvette
Sample Cell
Must be
CAREFULLY
Aligned
Cuvette
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
PHOTOELECTRIC TUBE
DETECTOR
Differing
Response
for
Various
Wavelengths
Must Use the
Correct
Combination
of Filter and
Phototube
For Wavelength
Of Analysis
INDICATING METER
Gives the Readout in
Transmittance or
Absorbance
INDICATING METER
Some Meters Give
Readout Directly in
Concentration
Use Only those Readings Between the Lowest
and Highest Standard of Calibration
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
Optical System
Lenses
Mirrors
Apertures
Occluders
Optical System
The Instrument Must be Carefully Handled,
Protected From Dust and Vapors, and
Serviced Only By Qualified Technicians
Spectrophotometer
Sample
Cell
Monochromator Detector
Light
Source
Meter
COLORIMETRY
Instrument Operation:
Warm-up
Set Monochromator
Set Absorbance
Set Zero Absorbance w/Blank
Re-adjust as Needed
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
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
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
COLORIMETER CALIBRATION
Repeat Calibration:
1. Significant Change In Procedure,
Equipment, or Reagents
2. Determined Length of Time
(Max. Six Months)
3. Verification Standard Not In Acceptable
Range
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
Total Phosphorus Ascorbic Acid Two Reagent Method
DD/MM/YY
0.5
Conc. Abs. 650 nm Inch Cuvette
0.2 0.104
0.3 0.153
0.4 0.210
0.5 0.258
Calibration 0.4
0.6 0.312
Curve
(Using Phosphorus 0.3
Absorbance
Analysis Example)
0.2
0.1
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Concentration, mg/L
COLORIMETRY
Prepared By
Michigan Department of Environmental Quality
Operator Training and Certification Unit
