Useful Terminology
Colorimetry
is the use of the human eye to determine the
concentration of colored species.
Spectrophotometry
is the use of instruments to make the same
measurements. It extends the range of possible
measurements beyond those that can be determined by
the eye alone.
Colorimetry
Visual Observations – Because colorimetry is based on
inspection of materials with the human eye, it is necessary to
review aspects of visible light.
Visible light is the narrow range of electromagnetic waves
with the wavelength of 400-700 nm.
ROYGBIV
= the mnemonic used to remember the colors of the visible spectrum.
COLOR
Interaction
between
LIGHT
and
MATTER
COLOR
RED
YELLOW
BLUE
RED
ABSORBED
YELLOW
BLUE
GREEN
WHITE
RESULTS WHEN
RADIATION IS ABSORBED
BY AN ELEMENT OR
BY A COMPOUND FORMED
THROUGH A REACTION
COLORIMETRY
Measurement of the amount of
LIGHT ABSORBED
by the
COLOR DEVELOPED
in a sample
Color Wheel
(ROYGBIV)
Complementary colors lie across the diameter on the color wheel
and combine to form “white light”, so the color of a compound
seen by the eye is the complement of the color of light absorbed
by a colored compound; thus it completes the color.
Observed Color of
Compound
Color of Light
Absorbed
Approximate
Wavelength of Light
Absorbed
Green
700 nm
Blue-green
600 nm
Violet
550 nm
Red-violet
530 nm
Red
500 nm
Orange
450 nm
Yellow
400 nm
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
Simple Spectrophotometer Schematic
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.
The amplifier increases the signal so that it is easier to read
against the background noise.
Spectrophotometer
Sample
Cell
Monochromator
Detector
Light
Source
Meter
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
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 Cell Length
I0
1.0
.9
Transmittance
.8
.7
.6
.5
I1
.4
.3
I2
.2
.1
I3
0
0
1
2
3
Units of Optical Path
I5
I4
4
In
5
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
Comparing Light Transmittance to Concentration
I0
1.0
.9
Transmittance
.8
.7
.6
.5
I1
.4
.3
I2
.2
.1
I3
0
0
1
I5
I4
2
3
Units of Concentration
4
In
5
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
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
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
Light Source
Controllable
Diaphragm
Voltage Adjustment
Constant
Voltage Regulation
Fatigue
Color (wavelength) Band
Monochromator
APERATURE
OR
SLIT
PRISM
OR
DIFFRACTION
GRATING
Must be CAREFULLY Adjusted
Sample Cell
Cuvette
The Light Path is
affected by the
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
Monochromator
Sample
Cell
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.
0.2
0.3
0.4
0.5
0.6
Calibration
Curve
0.4
Absorbance
(Using Phosphorus
Analysis Example)
650 nm ½ Inch Cuvette
Abs.
0.104
0.153
0.210
0.258
0.312
0.3
0.2
0.1
0
0.1
0.2
0.3
0.4
0.5
Concentration, mg/L
0.6
0.7
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.