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 = Planck’s 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
 LAMBERT’S 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
 BEER’S 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 LAMBERT’S LAW
AND BEER’S 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) Beer’s 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 ½ 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