Unit 4: Visible Spectrophotometry & Colorimetry

- Detailed Notes
I. Introduction

 Spectrophotometry & Colorimetry: Quantitative Analytical Techniques

o Both methods quantify the concentration of a substance by
measuring its absorbance or transmission of light.
o Focus on the visible region (400-700 nm) for colorimetry and a
broader range (UV-Vis) for spectrophotometry.
o Key principle: Substances absorb light at specific wavelengths
depending on their chemical structure.
 Distinction between Spectrophotometry and Colorimetry:
o Historically, colorimetry used filters for wavelength
selection, while spectrophotometry uses monochromators.
o Modern instruments blur the line, with both offering
relatively narrow bandwidths.
o Spectrophotometry generally allows for more precise wavelength
selection and a broader spectral range.
 Applications:
o Pharmaceutical analysis: Drug purity, content uniformity,
dissolution studies.
o Clinical chemistry: Blood glucose, enzyme assays, electrolyte
determination.
o Environmental monitoring: Water quality analysis (pollutants),
air quality (particulates).
o Food chemistry: Food coloring, additives, vitamins.
o Research: Reaction kinetics, equilibrium studies, structural
elucidation.

II. Theory of Spectrophotometry & Colorimetry

 Light and Electromagnetic Spectrum:

o Light is a form of electromagnetic radiation characterized by
wavelength (λ) and frequency (ν).
o Visible region: 400 nm (violet) to 700 nm (red).
o Relationship: c = λν (where c is the speed of light).
 Interaction of Light with Matter:
o When light interacts with a substance, it can be:
 Transmitted (T): Passes through the substance.
 Absorbed (A): Energy is taken up by the substance.
 Reflected (R): Bounces off the surface.
o The sum of transmittance, absorbance, and reflectance equals
the incident light intensity (I₀). Usually, reflectance is
minimized.
 Absorbance (A) and Transmittance (T):
o Transmittance (T): The fraction of incident light that passes
through the solution. T = I/I₀ (where I is the transmitted
light intensity). Expressed as a percentage, %T = (I/I₀) * 100.
o Absorbance (A): The amount of light absorbed by the solution.
A = -log₁₀(T). Also called optical density (OD).
  Beer-Lambert Law (or Beer's Law): Foundation of Quantitative
Analysis
o Statement: The absorbance of a solution is directly
proportional to the concentration of the analyte and the path
length of the light beam through the solution.
o Equation: A = εbc
 A = Absorbance (unitless)
 ε = Molar absorptivity (or molar extinction coefficient)
(L mol⁻¹ cm⁻¹) - A measure of how strongly the substance
absorbs light at a given wavelength.
 b = Path length (cm) - The distance the light travels
through the solution. Usually 1 cm in standard cuvettes.
 c = Concentration (mol L⁻¹)
o Implications:
 Linear relationship between absorbance and concentration
at a fixed wavelength and path length.
 Allows determination of unknown concentrations by
comparing absorbance to known standards.
 Molar absorptivity (ε) is a characteristic constant for a
given substance at a specific wavelength and can be used
for identification.

III. Deviations from Beer's Law

 Real Deviations (Fundamental):

o Occur at high concentrations (>0.01 M) due to changes in the
refractive index of the solution. Interactions between analyte
molecules become significant.
o Effective concentration of the analyte may differ from the
actual concentration. Difficult to predict these deviations.
 Chemical Deviations:
o Result from chemical phenomena such as:
 Association: Analyte molecules aggregate in solution.
 Dissociation: Analyte molecules break apart in solution.
 Reaction with the solvent: Analyte reacts with the
solvent, forming a new species with different absorption
characteristics.
o These reactions shift the equilibrium and affect the
absorbance.
o Often pH-dependent, requiring careful pH control.
 Instrumental Deviations:
o Polychromatic Radiation: Beer's Law strictly applies to
monochromatic light. Using a wide bandwidth of light leads to
non-linearity. Monochromators minimize this.
o Stray Light: Unwanted light reaching the detector that is not
part of the selected wavelength range. Causes inaccurate
absorbance readings, especially at high absorbance values.
o Mismatch in Cuvettes: Variations in path length or optical
properties between cuvettes can lead to errors. Use matched
cuvettes or a single cuvette for all measurements.
o Detector Non-linearity: At very high or low light intensities,
the detector response may not be linear.
 Minimizing Deviations:
 o Work at concentrations where Beer's Law is obeyed (usually
dilute solutions).
o Use monochromatic light (narrow bandwidth).
o Minimize stray light.
o Use matched cuvettes or a single cuvette.
o Calibrate the instrument regularly.
o Control pH and ionic strength.
o Consider using standard addition methods to correct for matrix
effects.

IV. Instrumentation

 Basic Components of a Spectrophotometer/Colorimeter:

1. Light Source: Provides a stable and continuous spectrum of
light. Examples:
 Tungsten lamp: Visible region (320-2500 nm).
 Deuterium lamp: UV region (160-375 nm).
 Xenon arc lamp: UV-Vis region (200-1000 nm) – high
intensity across a broad spectrum.
2. Wavelength Selector (Monochromator/Filter): Isolates a
specific wavelength of light.
 Filters: Simple and inexpensive, but have a wider
bandwidth. Used in colorimeters.
 Monochromators: Use prisms or diffraction gratings to
disperse light and select a narrow band of wavelengths.
Found in spectrophotometers. Advantages: Higher
resolution (narrower bandwidth), wavelength scanning
capability.
3. Sample Holder (Cuvette): Holds the sample in the light beam.
 Material: Glass or plastic for visible region; quartz for
UV region (glass absorbs UV).
 Path Length: Typically 1 cm.
4. Detector: Measures the intensity of light transmitted through
the sample.
 Photomultiplier tube (PMT): Highly sensitive, used in
many spectrophotometers.
 Photodiode: Solid-state device, less sensitive than PMTs
but more stable and compact.
 Charge-Coupled Device (CCD): Array of photodiodes, allows
for simultaneous detection of multiple wavelengths.
5. Readout Device: Displays the absorbance or transmittance
reading. Computer, digital display, or meter.
 Types of Spectrophotometers:

o Single-Beam Spectrophotometer: Light passes through the

reference and sample separately. Requires blanking and
calibration before each measurement. Simpler design, less
expensive.
o Double-Beam Spectrophotometer: Light beam is split into two
paths: one through the reference and one through the sample.
Measures the reference and sample simultaneously, compensating
for fluctuations in the light source and detector drift. More
accurate.
 o Diode Array Spectrophotometer: Uses a diode array detector to
measure the entire spectrum simultaneously. Very fast
scanning. Suitable for kinetic studies and process monitoring.

V. Applications of Colorimetry & Spectrophotometry

 Quantitative Analysis:
o Determination of Concentration: Using Beer's Law (A = εbc) and
a calibration curve.
o Calibration Curve Preparation:
 Prepare a series of standard solutions of known
concentrations.
 Measure the absorbance of each standard at a specific
wavelength.
 Plot the absorbance values against the corresponding
concentrations.
 The resulting graph is the calibration curve. Ideally, it
should be linear.
 Measure the absorbance of the unknown sample.
 Use the calibration curve to determine the concentration
of the unknown.
o Standard Addition Method: Used to correct for matrix effects.
Known amounts of the analyte are added to the sample, and the
absorbance is measured. The concentration of the original
sample can be determined from the change in absorbance.
 Qualitative Analysis:
o Identification of Substances: By comparing the absorption
spectrum of an unknown substance to the spectra of known
compounds. The shape and position of the absorption peaks are
characteristic of the molecule.
o Purity Check: The presence of impurities can be detected by
changes in the absorption spectrum.
 Reaction Kinetics Studies:
o Monitoring the change in absorbance over time to determine the
rate of a chemical reaction.
o Enzyme assays are a common application.
 Molecular Weight Determination:
o By measuring the absorbance of a solution of known
concentration and using Beer's Law to calculate the molar
absorptivity. The molar absorptivity can be related to the
molecular weight.
 Pharmaceutical Applications:
o Drug Assays: Determining the concentration of active
ingredients in pharmaceutical formulations.
o Dissolution Testing: Monitoring the rate at which a drug
dissolves from a tablet or capsule.
o Stability Studies: Assessing the degradation of a drug over
time under different conditions.
 Clinical Chemistry Applications:
o Blood Glucose Determination: Measuring the concentration of
glucose in blood samples.
o Enzyme Assays: Measuring the activity of enzymes in blood
serum.
 o Electrolyte Analysis: Determining the concentration of
electrolytes (Na+, K+, Cl-) in blood samples.
 Environmental Monitoring Applications:
o Water Quality Analysis: Measuring the concentration of
pollutants (e.g., nitrates, phosphates, heavy metals) in water
samples.
o Air Quality Analysis: Measuring the concentration of
particulate matter and other pollutants in air samples.
 Food Chemistry Applications:
o Food Coloring Analysis: Determining the concentration and type
of food colorings in food products.
o Vitamin Analysis: Measuring the concentration of vitamins in
food products.