Semester 7
BP701T
Introduction to UV-Visible Spectroscopy & Fluorimetry
This unit covers the two most fundamental spectroscopic techniques in pharmaceutical analysis. UV-Visible spectroscopy — the principles of electronic transitions, chromophores and auxochromes, spectral shifts, solvent effects, Beer-Lambert’s law (derivation and deviations), detailed instrumentation (sources, monochromators, detectors), and applications (spectrophotometric titrations, single/multi-component analysis). Fluorimetry — theory of fluorescence (singlet/triplet states, Jablonski diagram), factors affecting fluorescence, quenching mechanisms, instrumentation, and applications.
Syllabus & Topics
- 1UV-Vis Spectroscopy – Principle: Based on absorption of UV (200-400 nm) or Visible (400-800 nm) radiation by molecules → electronic transitions occur (electrons promoted from ground state to excited state). The wavelength absorbed depends on the energy gap between electronic levels. Energy relationship: E = hν = hc/λ. Molecules with conjugated systems, aromatic rings, or heteroatoms absorb in UV-Vis range. UV-Vis spectrum: plot of Absorbance (A) vs Wavelength (λ). λmax = wavelength of maximum absorption — characteristic of the compound. Absorptivity (ε, molar absorptivity): intrinsic property of the compound at a given wavelength.
- 2Electronic Transitions: Types of molecular orbitals: σ (bonding), σ* (antibonding), π (bonding), π* (antibonding), n (non-bonding lone pairs). Transitions (in order of increasing energy/decreasing wavelength): σ→σ* (highest energy, <150 nm, vacuum UV — saturated hydrocarbons). n→σ* (150-250 nm — compounds with lone pairs: alcohols, amines, e.g., CH₃OH at 183 nm). π→π* (200-500 nm — compounds with C=C, C=O, aromatic rings. Intense: ε = 1,000-100,000). n→π* (250-500 nm — compounds with C=O, N=O, C=S. Weak: ε = 10-100, forbidden transition). Most useful for pharma: π→π* (conjugated systems) and n→π* (carbonyl compounds).
- 3Chromophores & Auxochromes: Chromophore: functional group responsible for UV-Vis absorption. Contains π or n electrons. Examples: C=C (ethylenic, λmax ~170 nm), C=O (carbonyl, n→π* ~270-280 nm), C=N, N=N (azo, ~340 nm), NO₂ (nitro, ~270 nm), C₆H₅ (phenyl, ~254 nm). Conjugation extends chromophore → ↑λmax, ↑ε (bathochromic shift). Auxochrome: group that doesn’t absorb itself but enhances absorption of a chromophore when attached. Contains n electrons for conjugation with chromophore. Examples: –OH, –NH₂, –OCH₃, –NR₂, –SH. Effect: when attached to chromophore → shifts λmax to longer wavelength (red/bathochromic shift) and ↑ε (hyperchromic effect).
- 4Spectral Shifts & Solvent Effects: Types of shifts: (1) Bathochromic (Red shift): shift to LONGER wavelength (lower energy). Caused by: extended conjugation, auxochrome attachment, polar solvents for π→π* transitions. (2) Hypsochromic (Blue shift): shift to SHORTER wavelength (higher energy). Caused by: loss of conjugation, polar solvents for n→π* transitions. (3) Hyperchromic: INCREASE in absorption intensity (↑ε). (4) Hypochromic: DECREASE in absorption intensity (↓ε). Solvent effects: Polar solvents (water, ethanol): stabilize π* more than n → ↓energy gap for π→π* → bathochromic shift. Stabilize n → ↑energy gap for n→π* → hypsochromic shift. Non-polar solvents (hexane): minimal interaction → spectra show fine structure (vibrational).
- 5Beer-Lambert’s Law – Derivation: Beer’s Law: A ∝ c (absorbance proportional to concentration). Lambert’s Law: A ∝ b (absorbance proportional to path length). Combined Beer-Lambert’s Law: A = εbc. Where: A = absorbance (log₁₀(I₀/I)), ε = molar absorptivity (L mol⁻¹ cm⁻¹), b = path length (cm), c = concentration (mol/L). Derivation: Consider monochromatic radiation of intensity I₀ entering solution of thickness b. Infinitesimal layer db absorbs: -dI/I = k·c·db. Integrate from 0 to b: ln(I₀/I) = kcb. Convert to log₁₀: log₁₀(I₀/I) = (k/2.303)·c·b = εcb. Therefore A = εbc. Transmittance: T = I/I₀; %T = (I/I₀) × 100; A = -log₁₀T = 2 – log₁₀(%T).
- 6Beer-Lambert’s Law – Deviations: (1) Real/Fundamental deviations: Beer’s Law valid only at LOW concentrations. At high concentrations (>0.01 M): intermolecular interactions change ε → non-linear A vs c plot. (2) Chemical deviations: analyte undergoes association, dissociation, or reaction (equilibrium shifts with concentration). Example: dichromate ↔ chromate equilibrium in K₂Cr₂O₇ solutions (pH-dependent). (3) Instrumental deviations: (a) Polychromatic radiation: Beer’s Law requires monochromatic light — if bandwidth too wide → negative deviation. Solution: use narrow slit width/better monochromator. (b) Stray light: radiation reaching detector without passing through sample → positive error at high A → negative deviation. (c) Mismatched cells: path length variations between reference and sample cells. (4) Personal errors: improper handling, fingerprints on cells, air bubbles.
- 7UV-Vis Instrumentation: Block diagram: Source → Wavelength selector → Sample cell → Detector → Readout. Sources: (1) Deuterium lamp (D₂): UV region (190-400 nm) — continuous spectrum from D₂ discharge. (2) Tungsten lamp: Visible region (350-800 nm) — incandescence. (3) Xenon arc lamp: UV + Vis (both regions, continuous). Wavelength selectors: (1) Filters: absorption filters (colored glass — low resolution) and interference filters (narrow bandwidth, ~10 nm). (2) Monochromators: prisms (glass for Vis, quartz for UV) and diffraction gratings (preferred — uniform dispersion, wider range, higher resolution). Components: entrance slit, dispersive element, exit slit. Slit width controls resolution and light intensity (narrow slit = better resolution but less light).
- 8UV-Vis Instrumentation – Cells & Detectors: Sample cells (cuvettes): Quartz/fused silica: for UV + Vis (transparent down to 190 nm). Glass: for Vis only (absorbs below ~320 nm). Path length: usually 1 cm. Must be optically matched (reference and sample cells identical). Detectors: (1) Phototube: light hits photocathode (alkali metal) → photoelectrons emitted → collected by anode → current. Simple, inexpensive. (2) Photomultiplier tube (PMT): phototube + electron multiplication. Photoelectrons accelerated through dynodes (each dynode multiplies electrons ~4-6×) → 10⁶-10⁷ amplification. Most sensitive detector for UV-Vis. (3) Photovoltaic cell (barrier layer cell): light on semiconductor (Se on Fe) → generates voltage proportional to intensity. No external power needed. Low sensitivity, used for filter photometers. (4) Silicon photodiode: semiconductor p-n junction → photocurrent. Used in photodiode array (PDA) detectors — all wavelengths measured simultaneously.
- 9UV-Vis Applications: (1) Spectrophotometric titrations: monitor absorbance at fixed λ during titration → plot A vs volume of titrant → endpoint from intersection of lines. Types: titrant absorbs (V-shaped), analyte absorbs (inverted V), both absorb, neither absorbs (product absorbs). (2) Single-component analysis: measure A at λmax → use Beer’s Law (A = εbc) to calculate concentration. Standard curve method: plot A vs c for known standards → determine concentration of unknown from graph. (3) Multi-component analysis: for mixture of components with overlapping spectra. Measure A at λmax of each component → solve simultaneous equations. For 2 components (X, Y): A_λ1 = ε_X1·b·c_X + ε_Y1·b·c_Y and A_λ2 = ε_X2·b·c_X + ε_Y2·b·c_Y → solve for c_X and c_Y. IP method: paracetamol + caffeine mixture analysis.
- 10Fluorimetry – Theory: Fluorescence: emission of light by a molecule after absorbing radiation → returning from excited SINGLET state to ground state. Jablonski diagram: Ground state (S₀) → Absorption (10⁻¹⁵ s) → Excited singlet state (S₁, S₂) → Internal conversion (S₂→S₁, non-radiative, 10⁻¹² s) → Vibrational relaxation → Fluorescence emission (S₁→S₀, 10⁻⁸ s, nanoseconds). Stokes shift: emitted light has LONGER wavelength (lower energy) than absorbed light (energy lost during vibrational relaxation). Electronic states: Singlet (S): all electrons paired (↑↓). Doublet: one unpaired electron. Triplet (T): two unpaired electrons (↑↑) — formed by intersystem crossing (S₁→T₁). Phosphorescence: emission from T₁→S₀ (slow, 10⁻³ to 10 s — forbidden transition).
- 11Fluorimetry – Factors, Quenching & Applications: Factors affecting fluorescence: (1) Structure: rigid, planar molecules with conjugated π systems → strong fluorescence (fluorescein, quinine, anthracene). Floppy molecules → internal energy dissipation → weak/no fluorescence. (2) Substituents: electron-donating groups (–OH, –NH₂) ↑fluorescence; electron-withdrawing groups (–NO₂, –COOH) ↓fluorescence. Heavy atoms (Br, I) ↑intersystem crossing → ↓fluorescence, ↑phosphorescence. (3) Solvent: polar solvents may ↑or ↓depending on compound. (4) Temperature: ↑T → ↓fluorescence (↑non-radiative decay). (5) pH: affects ionization state → changes fluorescence (fluorescein at pH 9-10). Quenching: any process that ↓fluorescence intensity. (1) Self-quenching (concentration quenching — at high conc.). (2) Collisional/dynamic quenching (O₂ is a common quencher — paramagnetic → ↑ISC). (3) Static quenching (complex formation). Instrumentation: Source (Xenon arc) → Excitation monochromator → Sample → Emission monochromator (90° angle to source) → Detector (PMT). 90° geometry: avoids transmitted exciting light reaching detector. Applications: quinine determination, drug analysis (riboflavin, thiamine), clinical (catecholamines), vitamins, amino acids (tryptophan).
Learning Objectives
Electronic Transitions: List all types of electronic transitions in order of energy and give examples of compounds for each.
Beer-Lambert’s Law: Derive Beer-Lambert’s law and explain all types of deviations.
UV-Vis Instrumentation: Draw the block diagram and describe each component including all detector types.
Multi-Component Analysis: Explain the principle of simultaneous multi-component UV analysis with equations.
Fluorimetry: Draw the Jablonski diagram and explain the difference between fluorescence and phosphorescence.
Exam Prep Questions
Q1. Why does Beer’s Law fail at high concentrations?
Beer’s Law assumes molecules absorb light independently (no interaction between absorbing species). At high concentrations (>0.01 M), solute-solute interactions occur — hydrogen bonding, complex formation, aggregation — which change the electronic environment and alter ε (molar absorptivity). Since ε is assumed constant in Beer’s Law, these changes cause deviations. Additionally, at high concentrations, the refractive index of the solution changes, further affecting absorption. This is why Beer’s Law is a limiting law valid only at dilute concentrations.
Q2. Why is the emission monochromator placed at 90° in a fluorimeter?
If the emission detector were placed in line with the excitation beam (0° or 180°), it would detect the TRANSMITTED exciting radiation along with the much weaker fluorescence → impossible to distinguish them. By placing the emission detector at 90°, the transmitted exciting light passes straight through and doesn’t reach the detector — only the fluorescence (emitted in all directions, including at 90°) is detected. This dramatically improves the signal-to-noise ratio and allows measurement of very low fluorescence intensities.
Q3. What is the Stokes Shift?
The Stokes shift is the difference in wavelength between the absorption maximum and the fluorescence emission maximum of the same molecule. Emission always occurs at a LONGER wavelength (lower energy) than absorption. Reason: after absorption (S₀→S₁), energy is lost through vibrational relaxation (within S₁) and internal conversion (S₂→S₁) before fluorescence occurs (S₁→S₀). The lost energy means the emitted photon has less energy → longer wavelength. A larger Stokes shift makes it easier to separate excitation and emission wavelengths.