c o l o r i m e t r y
DESCRIPTION
C O L O R I M E T R Y. 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. C O L O R I M E T R Y. - PowerPoint PPT PresentationTRANSCRIPT
COLORIMETRY
Prepared ByMichigan 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
COLORCOLORInteraction between
LIGHT and
MATTER
Matter“ORBIT”
Nucleus
Electrons
Energy Levelof
Electrons
Orbits = Energy Level
Leve
l 1
Leve
l 2
Leve
l 3
Each Electron Can Be In Only Certain Energy LevelsEach Electron Can Be In Only Certain Energy Levels
LIGHTLIGHT
Photon - “Energy Packet”
wavelength -
(time)
frequency - (gamma)
(lambda)
Wave
LIGHTLIGHT
E = h hc
=
The Energy (E) of the Photon is Related to the wavelength and the
frequency of the Wave
Where:h = Planck’s Constantc = Velocity of Light
E = hc
Constants
Every wavelength ()has a specificEnergy level.
LIGHTLIGHT
SummarySummary
Each Each ElectronElectron Can Be In Can Be In Only CertainOnly Certain Energy Energy LevelsLevels
Every Every wavelength wavelength has a has a specific specific EnergyEnergy Level. Level.
COLORIMETRY600 nm
700 nm
650 nm
COLORRESULTS WHEN
RADIATION IS ABSORBEDRADIATION IS ABSORBEDBY AN ELEMENTELEMENT OR
BY A COMPOUNDCOMPOUND FORMEDTHROUGH A REACTION
W H
I T
EW
H I
T E
RED
BLUE
YELLOW
YELLOW
BLUE
REDABSORBED
GR
EE
N
TRANSMITTANCE (T)
OF THE INTENSITY OF LIGHTLEAVING SOLUTION (I)
TO THE INTENSITY OF LIGHTENTERING SOLUTION (IO)
RATIO
TRANSMITTANCE
IT = IO
IO I
%T = T x 100
Units of Optical Path
Tra
nsm
itta
nce
0 1 2 3 4 5
1.0
.9
.8
.7
.6
.5
.4
.3
.2
.1
0
I0
I1
I2
I3I4
I5
Comparing Light Transmittance to Cell Length
In
LAMBERT’S LAWLAMBERT’S LAW
Relates the absorption of light to the depth or thickness of the colored liquid
Each layer of equal thickness will absorb the samefraction of light which passes through it
An arithmetic increase in thickness gives a geometric decrease in light intensity transmitted
Units of Concentration
Tra
nsm
itta
nce
0 1 2 3 4 5
1.0
.9
.8
.7
.6
.5
.4
.3
.2
.1
0
I0
I1
I2
I3I4
I5
Comparing Light Transmittance to Concentration
In
BEER’S LAWBEER’S LAW
Relates the absorption of light to the concentrationof 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
COLORIMETRYCOLORIMETRY
How Do We Use This Principle?How Do We Use This Principle?Perform a Chemical Reaction with the Perform a Chemical Reaction with the
Element to be Analyzed that Results in a Element to be Analyzed that Results in a Compound of that Element that Absorbs Compound of that Element that Absorbs
Light.Light.
Measure the AmountMeasure the Amountof Light Absorbed.of Light Absorbed.
COLORIMETRYCOLORIMETRY
1.1. The Chemistry Involved.The Chemistry Involved.2. The Length of Light Travel.2. The Length of Light Travel.
3. The Amount (Concentration) of 3. The Amount (Concentration) of Absorbing Material.Absorbing Material.
The Amount of Light AbsorbedThe Amount of Light AbsorbedIs Related To:Is Related To:
THE COMBINED LAMBERT’S LAWAND 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}
TRANSMITTANCE
T = 10 -abc
Absorbance = A = - log T
log T = log (10 -abc)
log T = -abc
-log T = -(-abc) = abc
A =
T = I Io
-log T = abc
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
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
COLORIMETRYCOLORIMETRY
Measurement of the amount of
LIGHT ABSORBED
by the
COLOR DEVELOPED
in a sample
CONCENTRATION CAN BE COLORIMETRICALLYCONCENTRATION CAN BE COLORIMETRICALLYDETERMINED IF: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 PreparationSample Preparation
DilutionDilution
Solids RemovalSolids Removal--- Coagulation--- Coagulation--- Centrifuge--- Centrifuge--- Filter--- Filter
pH AdjustmentpH Adjustment
DigestionDigestion
DIGESTION
Destroy Organics
Release Combined Constituent
Change Form of Constituent
ColorimetryColor Development
Color Must Be:
Stable Reproducible
Sensitive
Color DevelopmentMust Control :
pHTime
Ionic Strength
Temperature
COLORIMETRYCOLORIMETRY
Measurement of the amount of
LIGHT ABSORBED
by the
COLOR DEVELOPED
in a sample
Color MeasurementColor Measurement
“Color Comparators”
Compare Sample Color to Known Standards
O.K. For Control – Not For Reporting
Color MeasurementColor Measurement
Spectrophotometer
Compare Sample Color to Known Standards
“Calibration Curve” (verified)
Colorimetric InstrumentsColorimetric Instruments
Spectrophotometer
LightSource
Monochromator
SampleCell
Detector
Meter
Light SourceLight Source
ControllableConstant
Voltage Regulation
FatigueDiaphragm
Voltage Adjustment
WHITE
LIGHT
Color (wavelength) BandColor (wavelength) Band
Monochromator
PRISMOR
DIFFRACTIONGRATING
APERATUREOR
SLIT
Must be CAREFULLY Adjusted
Sample Cell
Cuvette
The Light Path is affected by the
Cuvette
Sample Cell
Cuvette
Must beCAREFULLY
Aligned
PHOTOELECTRIC TUBE“DETECTOR”
DifferingResponse
forVarious
WavelengthsBausch & Lomb33-29-71
340-600 nm33-29-72 (w / filter)
600-950 nm33-29-92 (w / filter)
400-700 nm
PHOTOELECTRIC TUBE“DETECTOR”
DifferingResponse
forVarious
WavelengthsMust Use the
Correct Combinationof Filter and Phototube
For WavelengthOf Analysis
INDICATING METERINDICATING METER
Gives the Readout inGives the Readout inTransmittance orTransmittance or
AbsorbanceAbsorbance
INDICATING METERINDICATING METER
Some Meters GiveSome Meters GiveReadout Directly inReadout Directly in
ConcentrationConcentrationUse Only those Readings Between the Lowest
and Highest Standard of Calibration
INDICATING METERINDICATING METER
Some Meters HaveSome Meters Have““Built-in” CalibrationBuilt-in” Calibration
These Calibrations Should Be Verified PeriodicallyUsing a Series of Standards and Only those Readings
Between the Lowest and Highest Standard of Calibration Should be Used
Optical SystemOptical System
LensesLensesMirrorsMirrorsAperturesAperturesOccludersOccluders
Optical SystemOptical System
The Instrument Must be Carefully Handled,The Instrument Must be Carefully Handled,Protected From Dust and Vapors, and Protected From Dust and Vapors, and
Serviced Only By Qualified TechniciansServiced Only By Qualified Technicians
Spectrophotometer
LightSource
SampleCell
Detector
Meter
Monochromator
COLORIMETRY
Instrument Operation:
Set Monochromator
Warm-up
Set Zero Absorbance w/Blank
Set ∞ Absorbance
Re-adjust as Needed
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:
COLORIMETER CALIBRATIONCOLORIMETER CALIBRATION
Calibration or StandardizedCalibration or StandardizedBy Measuring Absorbance ReadingsBy Measuring Absorbance Readings
of a Series of Known Standardsof 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
COLORIMETER CALIBRATIONCOLORIMETER CALIBRATION
Calibration or StandardizedCalibration or StandardizedBy Measuring Absorbance ReadingsBy Measuring Absorbance Readings
of a Series of Known Standardsof a Series of Known Standards
Verified FrequentlyAt Least One StandardIn Acceptable Range
Comparison of These Readings to the Reading for a Sample
Each Time Samples Are Analyzed
COLORIMETER CALIBRATIONCOLORIMETER 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:Repeat Calibration:
Calibration Steps: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”
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
Abs
orba
nce
Total Phosphorus Ascorbic Acid – Two Reagent MethodDD/MM/YY
650 nm ½ Inch CuvetteConc. Abs.0.2 0.1040.3 0.1530.4 0.2100.5 0.2580.6 0.312Calibration Calibration
CurveCurve(Using Phosphorus (Using Phosphorus Analysis Example)Analysis Example)
COLORIMETRY
Prepared ByMichigan Department of Environmental Quality
Operator Training and Certification Unit