Colorimetry & Spectrophotometry

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 RESULTS WHEN RADIATION IS ABSORBED BY AN ELEMENT OR BY A COMPOUND FORMED THROUGH A REACTION RED YELLOW RED ABSORBED W H I T E YELLOW GREEN BLUE BLUE

Measurement of the amount of 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   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   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 500 nm Orange Blue 450 nm 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 Detector Monochromator Light Source

RATIO TRANSMITTANCE (T) 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

Transmittance Comparing Light Transmittance to Cell Length I0 I1 I2 I3 1.0 .9 .8 .7 .6 .5 .4 .3 .2 .1 Transmittance I1 I2 I3 I5 In I4 0 1 2 3 4 5 Units of Optical Path

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

Transmittance Comparing Light Transmittance to Concentration I0 I1 I2 1.0 .9 .8 .7 .6 .5 .4 .3 .2 .1 Transmittance I1 I2 I3 I5 In I4 0 1 2 3 4 5 Units of Concentration

COLORIMETRY The Amount of Light Absorbed Is Related To: 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 T = I I o 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}

Absorbance = A = - log T A = TRANSMITTANCE T = I Io Absorbance = A = - log T T = 10 -abc log T = log (10 -abc) log T = -abc -log T = -(-abc) = abc -log T = abc A =

ABSORBANCE (A) A = abc A = - log T Where: a = constant for particular solution b = length of absorbing layer (light path length) c = concentration of absorbing substance

ABSORBANCE (A) A = abc A = - log T If: Then: 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 --- Filter pH Adjustment --- Coagulation --- Centrifuge --- Filter pH Adjustment Digestion

Release Combined Constituent Change Form of Constituent DIGESTION Destroy Organics Release Combined Constituent Change Form of Constituent

Colorimetry Sensitive Color Development Stable Reproducible Color Must Be: Stable Reproducible Sensitive

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

Compare Sample Color to Known Standards “Calibration Curve” (verified) Color Measurement Compare Sample Color to Known Standards Spectrophotometer “Calibration Curve” (verified)

Colorimetric Instruments

Light Source Constant Controllable WHITE LIGHT Voltage Regulation Diaphragm Fatigue Voltage Adjustment

Color (wavelength) Band

Monochromator Must be CAREFULLY Adjusted APERATURE OR SLIT PRISM OR DIFFRACTION GRATING Must be CAREFULLY Adjusted

The Light Path is affected by the Cuvette Sample Cell The Light Path is affected by the Cuvette Cuvette

Sample Cell Must be CAREFULLY Aligned Cuvette

PHOTOELECTRIC TUBE Differing Response for Various Wavelengths “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

of Filter and Phototube PHOTOELECTRIC TUBE “DETECTOR” Differing Response for Various Wavelengths Must Use the Correct Combination of Filter and Phototube For Wavelength Of Analysis

Gives the Readout in Transmittance or Absorbance INDICATING METER Gives the Readout in Transmittance or Absorbance

Some Meters Give Readout Directly in Concentration INDICATING METER Some Meters Give Readout Directly in Concentration Use Only those Readings Between the Lowest and Highest Standard of Calibration

Some Meters Have “Built-in” 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

The Instrument Must be Carefully Handled, 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

Instrument Operation: Set Zero Absorbance w/Blank COLORIMETRY Instrument Operation: Warm-up Set Monochromator Set ∞ Absorbance Set Zero Absorbance w/Blank Re-adjust as Needed

Instrument Operation: Watch for Irregularities 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 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”

(Using Phosphorus Analysis Example) Calibration Curve (Using Phosphorus Analysis Example)

How Do We Use This Principle? 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.