1. Colorimetry
&
Spectrophotometry

2. 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.

3. 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 nm.
                                                   
ROYGBIV
= the mnemonic used to remember the colors of the visible spectrum.

4. COLOR
Interaction between
LIGHT
and
MATTER

5. 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

6. Measurement of the amount of
COLORIMETRY
Measurement of the amount of
LIGHT ABSORBED
by the
COLOR DEVELOPED
in a sample

7. 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.

8. 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

9. 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

10. 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.

11. Spectrophotometer Sample Cell Detector Monochromator Light Source

12. RATIO TRANSMITTANCE (T) OF THE INTENSITY OF LIGHT LEAVING SOLUTION (I)
TO THE INTENSITY OF LIGHT
ENTERING SOLUTION (IO)

13. TRANSMITTANCE IO I I
T = IO
%T = T x 100

14. 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

15. 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
Units of Optical Path

16. 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

17. 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
Units of Concentration

18. 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.

19. THE COMBINED LAMBERT’S LAW
AND BEER’S LAW
T = I
I o
T = 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}

20. 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 =

21. 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

22. 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

23. 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

24. Sample Preparation Dilution Solids Removal --- Filter pH Adjustment
--- Coagulation
--- Centrifuge
--- Filter
pH Adjustment
Digestion

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

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

27. Color Development
Must Control : pH
Time
Temperature
Ionic Strength

28. Color Measurement Compare Sample Color to Known Standards
“Color Comparators”
O.K. For Control – Not For Reporting

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

30. Colorimetric Instruments

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

32. Color (wavelength) Band

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

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

35. Sample Cell
Must be
CAREFULLY
Aligned
Cuvette

36. PHOTOELECTRIC TUBE Differing Response for Various Wavelengths
“DETECTOR”
Differing
Response
for
Various
Wavelengths
Bausch & Lomb nm
(w / filter) nm
(w / filter) nm

37. 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

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

39. 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

40. 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

41. Optical System
Lenses
Mirrors
Apertures
Occluders

42. 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

43. Spectrophotometer Sample Cell Monochromator Detector Light Source

44. 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

45. Instrument Operation: Watch for Irregularities
COLORIMETRY
Instrument Operation:
General Rule –
Absorbance Between and 0.700
Some Analyses More Restrictive
Best Readings –
Between Lowest and Highest Standards Used In Calibration
Watch for Irregularities

46. 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

47. 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

48. 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

49. 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”

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

51. 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.