C O L O R I M E T R Y

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