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Chapter 17: Fundamentals of Spectrophotometry
Major Concepts About Light
Wavelength (λ):
- Definition: The distance between wave peaks, measured in meters (m).
Frequency (ν):
- Definition: The number of oscillations in a second, measured in Hertz (Hz).
Key Relationship:
- The speed of light (c) is expressed as:
- Where:
- c: Speed of light (constant, approximately m/s)
- λ: Wavelength
- ν: Frequency
- The speed of light (c) is expressed as:
Photon Energy (E):
- Formula:
- Where:
- E: Energy per photon
- h: Planck's constant, approximately J·s
- ν: Frequency
- Formula:
Wavenumber (v):
- Definition: The number of waves per unit distance, given by the formula:
- Definition: The number of waves per unit distance, given by the formula:
Speed (c):
- Constant, independent of the frequency and wavelength.
Electromagnetic Spectrum
Energy Levels: Energy associated with different wavelengths expressed in kJ/mol.
- Y-rays: kJ/mol
- X-rays: 12,000 kJ/mol
- Ultraviolet: 310 kJ/mol
- Electronic excitation: 150 kJ/mol
- Bond breaking and ionization: 0.080 - 150 kJ/mol
Frequency Levels in Hz:
- Ranges from:
- Hz (Y-rays) to Hz (Microwave)
Visible Spectrum:
- Wavelength (m):
- to
- Represented in nanometers (nm):
- 400 nm (violet) to 800 nm (red)
Beer’s Law (Beer-Lambert Law)
Absorbance (A):
- Formula:
- Where:
- P₀: Incident light intensity
- P: Transmitted light intensity
- T: Transmittance
- Formula:
Components of Beer’s Law:
- Where:
- ε: Molar absorptivity (extinction coefficient)
- b: Path length (cm)
- c: Concentration (Molarity)
Application of Beer’s Law
Example 1: Quantifying a component in a mixture.
- A 250.0 mL solution contains 25.8 mg benzene (C₆H₆, FM: 78.11) in hexane with:
- Absorption peak at 256 nm.
- Absorbance: 0.266 in a 1.000 cm cell.
- Calculation Required: Find the molar absorptivity of benzene.
- A 250.0 mL solution contains 25.8 mg benzene (C₆H₆, FM: 78.11) in hexane with:
Example 2: Hexane contaminated with benzene.
- Absorbance: 0.070 at 256 nm in a 5.000 cm cell.
- Calculation Required: Find the concentration of benzene (mg/L).
- Key Advantage: Non-destructive evaluation of samples.
Example 3: Potassium permanganate (KMnO₄).
- Given:
- Concentration:
- Transmittance: 44.1% in a 2.10 cm cell at wavelength 525 nm.
- Calculation Required: Find the absorbance of this solution and the molar absorptivity of KMnO₄.
- Given:
Additivity of Absorbance in Mixtures
- Total Absorbance Formula:
- Where:
- A: Absorbance at a single wavelength.
- No interaction between species affecting absorbance.
Spectrophotometric Titration
- Purpose: Monitoring absorbance during a titration to determine the equivalence point.
- Advantage: Corrects for physical dilution of solutions without altering the dissolved material concentration.
Example of Absorbance Correction
- Given:
- Observed absorbance after adding 75 µL of ferric nitrilotriacetate to 1.500 mL of apotransferrin is 0.222.
- Final volume: 1.5 mL + 0.075 mL = 1.575 mL
- Corrected Absorbance Formula:
What Happens When a Molecule Absorbs Light?
Processes Involved:
- Electronic excitation transitioning the molecule from ground state to excited states.
- States:
- Ground state
- Excited states
- Energy levels from to
Basic Vibrational Excitation Modes:
- Modes such as scissoring, rocking, wagging, in-plane bending, and twisting.
Electronic States: Singlet and Triplet
- Definitions:
- Singlet State: All electrons are paired.
- Triplet State: Two unpaired electrons, resulting in different spins.
- Electronic Excitation: Each state may contain energy levels leading to diverse molecular behaviors.
Vibrational Modes for Formaldehyde
- Number of Modes:
- Formula: where n = number of atoms in the molecule.
- Example Calculation for formaldehyde (4 atoms):
modes.
- Wavenumber (cm⁻¹):
- Stretching modes have higher frequencies than bending modes.
Intersystem Crossing and Energy Conversion
- States Involved:
- : Ground electronic state
- : Lowest excited singlet state
- : Lowest excited triplet state
- Processes:
- Absorption, fluorescence, and phosphorescence processes identifiable in time scales.
- Fluorescence: Typically on the order of to seconds.
- Phosphorescence: Can last from seconds to hours.
Comparison of Phosphorescence vs. Fluorescence
- Characteristics:
- Intensity: Phosphorescence is less intense, often around 10 times lower than fluorescence.
- Duration: Phosphorescence persists significantly longer than fluorescence.
- Energy Levels: Phosphorescence generally occurs at lower energies with longer wavelengths compared to fluorescence.
Spectra Characteristics
- Absorption and Emission Spectra:
- Typically mirror images of each other but can vary based on energy transitions and molecular geometry.
- Example: Absorption (black line) vs. Emission (colored line) of bis(benzylimido)-perylene in dichloromethane solution.
Correlation Between Absorption and Emission Spectra
- Observed Patterns:
- Absorption and emission spectra are correlated but show differences due to relaxation of geometrical configurations in ground and excited states after transitions.
Typical Luminescence Experiment
- Setup and Measurement:
- Instruments used include light source and monochromators to separate emitted light based on wavelength.
Emission and Excitation Spectra
- Emission Spectrum:
- Distribution of light emitted versus wavelength.
- Excitation Spectrum:
- Emission intensity versus different excitation wavelengths.
Example of Fluorescence Intensity at Low Concentration
- Expression for Intensity:
- Where:
- c: Concentration
- P_o: Irradiance
- Fluorescence Intensity Chart:
- Demonstrates intensity changes with concentration.
Chemically Selective Luminescence Examples
- Examples:
- Fluorescein, Calcein, reactive groups selective for specific ionic species (e.g., Ca²+).
- Fluorescein structure and its related functionalities in luminescence.