Photometry

• Photometry is based on the laws governing the radiant energy of light. It

involves the quantitative measurement of light absorbed by a coloured
solution formed from the substance under investigation.
• The extent of light absorption is directly related to the concentration of the
analyte.
• Photometric methods are broadly classified according to the nature of light
measured:
[Link] of absorbed or transmitted light:
• Colorimetry, spectrophotometry, atomic absorption spectrometry,
turbidometry
[Link] of emitted light:
• Flame emission photometry, fluorimetry
 Colorimetry
Introduction
• Colorimetry is an analytical technique used for the
quantitative estimation of substances that form
coloured solutions. It is based on the measurement
of light absorbed by a coloured solution, where the
intensity of colour is proportional to the
concentration of the substance.
Principle
• Colorimetry follows the Lambert–Beer law, which
states that the absorbance of a coloured solution is
directly proportional to its concentration and the
path length of light passing through it.

• where A = absorbance, c = concentration, and l = path length.

 Laws of Absorption of Light
Lambert–Beer’s Law
• The functioning of colourimeters and spectrophotometers is based on Lambert–Beer’s
law, which is derived from two fundamental principles:
[Link]’s law:
• The optical density (absorbance) of a solution is directly proportional to the
concentration of the absorbing substance.
[Link]’s law:
• The optical density of a coloured solution is directly proportional to the path length of
light passing through the solution.
• Mathematically:

• where A = absorbance, c = concentration, and l = path length.

 Expression

• The extent to which light is absorbed by a solution can be

expressed in the following ways:

i. Percentage transmittance (%T )

ii. Optical density (OD)
iii. Absorbance (A) or Extinction (E)
 Components of a Colorimeter
1)Light source (tungsten lamp)
2)Filter (wavelength selector)
3)Cuvette (holds the solution; path length = 1 cm)
4)Photodetector
5)Readout system (meter/digital display)
1. Light Source
• A tungsten lamp is commonly used for the visible range (420–760 nm). For
ultraviolet (UV) measurements, hydrogen or deuterium lamps are employed.
2. Entrance Slit
• Focuses the light beam into the monochromator.
3. Monochromator / Filters
• In colourimeters, coloured filters are used to isolate light of a specific
wavelength.
• Complementary filters are selected to maximize sensitivity.
• In spectrophotometers, diffraction gratings are used to produce a narrow
and precise wavelength band.
 Selection of Filters
Peak Transmission
Colour of Solution Filter Used
Range (nm)
Bluish-green Red 580–680
Blue Yellow 520–580
Purple Green 490–520
Red Blue-green 470–490
Yellow Blue 430–470
Yellowish-green Violet 400–430
4. Exit Slit
• Ensures that only the selected wavelength reaches the sample.
5. Cuvette
• Holds the test solution. The optical path length is usually 1 cm.
• [Link] cuvettes: used for visible range measurements
• [Link]/silica cuvettes: used for UV range measurements
6. Detector (Photosensitive Element)
• Converts transmitted light into an electrical signal.
7. Galvanometer / Meter
• Measures and displays the electrical output corresponding to light intensity.
Modern instruments use digital readout systems.
 Basis of Measurement

1) When white light passes through a coloured solution, a

specific wavelength is absorbed.
2) The remaining light is transmitted and measured.
3) Complementary coloured filters are used to select the
appropriate wavelength for maximum absorption.
 Procedure
Steps :
• The operation of a colorimeter follows a systematic sequence to ensure
accurate and reproducible results:
1. Switching On and Warm-up
• Switch on the colorimeter and allow it to warm up for 5–10 minutes.
• This stabilizes the light source and electronic components.
2. Selection of Filter
• Select the appropriate complementary filter corresponding to the colour of
the solution to be measured.
• This ensures maximum absorbance and sensitivity.
3. Preparation of Solutions
• Prepare the blank, standard, and test solutions as per the analytical method.
• Ensure solutions are clear and free from turbidity or air bubbles.
4. Setting the Blank
• Fill a clean cuvette with the blank solution.
• Wipe the cuvette with tissue paper to remove fingerprints.
• Place it in the cuvette holder.
• Adjust the instrument to zero absorbance (0 OD) or 100% transmittance
(%T) using the blank.
5. Measurement of Standard
• Replace the blank with the standard solution.
• Note the absorbance (or %T) of the standard.
6. Measurement of Test Sample
• Place the test solution in the cuvette holder.
• Measure and record the absorbance under the same conditions.
• 7. Calculation of Concentration
• Calculate the concentration of the test sample by comparing its absorbance with that
of the standard using Beer–Lambert law:

8. Switching Off
• After measurements, switch off the instrument and cover it to protect from dust.
Flow Summary:
Warm-up → Filter selection → Blank setting → Standard reading → Test reading →
Calculation
 Terminology
Blank, Standard, and Test
• In photometric estimations, measurements are performed using three types of solutions:
Blank, Standard, and Test. Each has a specific role in ensuring accuracy and reliability of
results.
1. Blank :
• The blank contains all reagents except the substance to be estimated.
• It is used to set the instrument to zero absorbance (or 100% transmittance).
• The blank corrects for:
Ø Absorbance due to reagents
Ø Solvent effects
Ø Cuvette imperfections
• It eliminates background interference .
Example:
• For blood glucose estimation, the blank contains reagent + distilled water (no glucose).
CUVETTES
 2. Standard
• The standard contains a known concentration of the substance being
estimated.
• It is used to calibrate the instrument and to compare the test sample.
• The absorbance of the standard serves as a reference for calculating the
concentration of the test.
Purpose:
• Ensures accuracy of measurement
• Allows quantitative estimation using proportionality (Beer–Lambert law)
 3. Test (Sample)

• The test contains the unknown concentration of the substance (patient

sample or experimental sample).
• Its absorbance is measured under the same conditions as the standard.
• The concentration of the test is calculated by comparing its absorbance
with that of the standard.
Calculation Formula
 Summary Table
Component Contents Purpose

Reagents + solvent (no Zero setting &

Blank
analyte) background correction

Reagents + known analyte

Standard Calibration & reference
concentration

Reagents + unknown Estimation of analyte

Test
sample concentration
 Applications of a Colorimeter
2. Medical Diagnostics
1. Clinical Biochemistry Ø Assessment of liver, kidney, and
• U s e d ro u t i n e l y i n h o s p i ta l a n d metabolic disorders
diagnostic laboratories for estimation Ø Monitoring disease progression and
of: treatment response
• Blood glucose 3. Biochemical and Research
• Serum urea Laboratories
Ø Estimation of enzymes and metabolites
• Creatinine
Ø Analysis of vitamins and hormones
• Cholesterol (after colour development)
• Bilirubin Ø Kinetic studies involving coloured
• Total protein and albumin reactions
• Hemoglobin
4. Pharmaceutical Industry 6. Food and Beverage Industry
ØMeasurement of food colours and
• Quality control of drugs additives
• Estimation of active ingredients ØDetermination of sugar, protein, and
• Stability testing of formulations preservative content
ØQuality control and standardization
5. Environmental Analysis
7. Teaching and Training
• Estimation of pollutants in water ØDemonstration of Beer–Lambert
(nitrates, phosphates, iron, fluoride) law
• Monitoring water quality in ØPractical training for students in
environmental studies biochemistry and laboratory
techniques
 Advantages of a Colorimeter

1)Simple to operate and user-friendly

2)Economical and affordable
3)Rapid analysis with minimal training
4)Suitable for routine clinical and teaching laboratories
5)Requires small volume of sample
6)Portable models available for field use
 Limitations of a Colorimeter
1. Works only in the visible light range (≈400–700 nm)
2. Less sensitive and less accurate than spectrophotometer
3. Uses filters, which give a broad wavelength band
4. Not suitable for very dilute solutions
5. Interference occurs with turbid or cloudy samples
6. Limited applications in advanced research
 Spectrophotometer
A spectrophotometer is an instrument used to measure the intensity
of light absorbed or transmitted by a solution at a specific wavelength.

Basic components of a spectrophotometer include:

1)A stable source of radiant energy

2)An entrance slit
3)A wavelength selector (filter or grating)
4)An exit slit
5)A cuvette holder
6)A radiant energy detector
7)A readout device for electrical signals
 Types of spectrophotometers
[Link]-beam spectrophotometer
[Link]-beam spectrophotometer – all components are duplicated except
the light source.
This design allows simultaneous correction for fluctuations in light intensity,
grating efficiency, and slit-width variations, improving accuracy and stability.
 Comparison: Colorimeter vs Spectrophotometer
Feature Colorimeter Spectrophotometer
Wavelength
Visible region only UV and visible regions
range
Wavelength
Coloured filters Monochromator (prism/grating)
selection
Sensitivity Moderate High
Accuracy Lower Higher
Type of light Broad band Narrow band
Routine clinical
Suitable for Research & precise analysis
tests
Cost Low High
Sample
Relatively high Can detect very low concentrations
concentration
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