Semester 7
BP701T
IR Spectroscopy, Flame Photometry, AAS & Nepheloturbidometry
This unit covers four analytical techniques: (1) Infrared Spectroscopy — fundamental vibrational modes, sample handling techniques, instrumentation (sources, monochromators, specialized IR detectors), and applications for compound identification. (2) Flame Photometry — emission of characteristic wavelengths by metal ions in a flame for quantitative analysis. (3) Atomic Absorption Spectroscopy (AAS) — measurement of ground-state atomic absorption for elemental analysis. (4) Nepheloturbidometry — measurement of scattered/transmitted light in suspensions.
Syllabus & Topics
- 1IR Spectroscopy – Introduction: Infrared radiation (wavelength: 2.5-25 μm or wavenumber: 4000-400 cm⁻¹) causes vibrational transitions in molecules. Condition for IR absorption: (1) molecule must have a change in dipole moment during vibration. Homonuclear diatomics (O₂, N₂, H₂) → NO IR absorption (no dipole change). Heteronuclear diatomics (HCl, CO) → IR active. (2) Frequency of IR radiation must match natural vibrational frequency of bond. IR spectrum: plot of %Transmittance vs Wavenumber (cm⁻¹). Fingerprint region (1500-400 cm⁻¹): complex pattern unique to each molecule → molecular identification. Functional group region (4000-1500 cm⁻¹): characteristic absorptions of functional groups.
- 2Fundamental Vibrational Modes: Number of fundamental vibrations: Nonlinear molecule: 3N−6 (N = number of atoms). Linear molecule: 3N−5. Types: (1) Stretching vibrations (bond length changes): Symmetric stretching (both bonds extend/compress together) and Asymmetric stretching (one extends while other compresses — higher energy). (2) Bending/deformation vibrations (bond angle changes): In-plane: Scissoring (both atoms move toward each other), Rocking (both atoms move in same direction). Out-of-plane: Wagging (both atoms move above/below plane together), Twisting (one above, one below). Energy order: stretching > bending (stretching requires more energy → absorbs at higher wavenumber). Characteristic absorptions: O-H stretch (3200-3600 cm⁻¹, broad), N-H stretch (3300-3500), C-H stretch (2850-3300), C=O stretch (1650-1780, strong sharp), C=C stretch (1620-1680).
- 3IR – Sample Handling: Solids: (1) KBr pellet: sample ground with dry KBr powder (1:100 ratio) → pressed in die at high pressure → transparent pellet. Most common method. KBr is IR-transparent (no absorption). (2) Nujol mull: sample ground with mineral oil (Nujol — liquid paraffin) → paste applied between NaCl plates. Nujol absorbs at 2900, 1460, 1380 cm⁻¹ (C-H bands — must account for). (3) Thin film: polymer samples pressed into thin film. Liquids: placed between NaCl or KBr windows (salt plates) — hygroscopic, handle carefully. Neat liquid for pure samples. Solutions: dissolved in IR-transparent solvent (CCl₄, CS₂, CHCl₃). Gases: gas cells with long path length (cm to meters) and NaCl/KBr windows.
- 4IR – Factors Affecting Vibrations: (1) Mass of atoms: heavier atoms → lower vibrational frequency (lower wavenumber). C-H (3000) > C-D (2200) > C-Cl (800). (2) Bond strength: stronger bonds → higher frequency. C≡C (2100) > C=C (1650) > C-C (1200). Triple > Double > Single. (3) Hydrogen bonding: intramolecular/intermolecular H-bonding → broadens and shifts O-H, N-H absorptions to LOWER wavenumber. Free O-H: sharp at 3600 cm⁻¹. H-bonded O-H: broad at 3200-3400 cm⁻¹. (4) Conjugation: C=O in conjugation (with C=C or aryl) → absorption shifts to lower wavenumber (resonance weakens C=O bond). Isolated C=O: 1715 cm⁻¹ (ketone). Conjugated C=O: 1680 cm⁻¹. (5) Ring strain: small rings → C=O at higher wavenumber (cyclopentanone 1740 > cyclohexanone 1715). (6) Electronic effects: electron-withdrawing groups ↑C=O frequency; electron-donating groups ↓C=O frequency.
- 5IR – Instrumentation: Block diagram: Source → Sample → Monochromator → Detector → Recorder. (Dispersive IR: monochromator AFTER sample — to minimize thermal radiation from heated sample). Sources: (1) Nernst glower: rare earth oxide rod (ZrO₂, Y₂O₃, CeO₂), heated to ~1800°C. Emits continuous IR. Requires preheating (non-conducting when cold). (2) Globar: silicon carbide rod, heated to ~1200°C. More stable than Nernst, self-starting. (3) Incandescent wire: Nichrome coil, lowest intensity but simplest. Wavelength selectors: Diffraction grating monochromator (most common in dispersive IR). FTIR: uses Michelson interferometer (no monochromator) → measures all frequencies simultaneously → Fourier transform → spectrum. FTIR advantages: better S/N, faster, higher resolution, Fellgett’s (multiplex) advantage.
- 6IR – Detectors & Applications: Detectors (thermal — respond to heating by IR): (1) Golay cell (pneumatic detector): gas-filled cell → IR heats gas → gas expands → flexible membrane moves → detected optically. Sensitive but slow. (2) Bolometer: metal strip (Pt or Ni) or semiconductor → resistance changes with temperature. Slow but sensitive. (3) Thermocouple: junction of two dissimilar metals → IR heats junction → voltage generated (Seebeck effect). Robust, commonly used. (4) Thermistor: semiconductor whose resistance changes with temperature. Faster than thermocouple. (5) Pyroelectric detector (DTGS — deuterated triglycine sulfate): generates charge when temperature changes. Fast response → used in FTIR. Applications: (1) Identification of functional groups and molecular structure. (2) Pharmacopoeial identification test (IR fingerprinting). (3) Polymorphism studies (different crystal forms show different IR). (4) Studying hydrogen bonding, drug-excipient interactions.
- 7Flame Photometry: Principle: solution of metallic salt → aspirated into flame → atomization → thermal excitation → atoms excited to higher energy level → return to ground state → emit characteristic wavelength of light. Intensity of emission ∝ concentration (at low concentrations). Flame provides thermal energy for excitation. Suitable for: alkali metals (Li, Na, K, Rb, Cs) and alkaline earth metals (Ca, Ba) — low excitation energy → easily excited in flame. Characteristic emissions: Na (589 nm — yellow), K (766 nm — violet), Li (670.8 nm — red), Ca (622 nm — orange-red). Instrumentation: Burner (air-acetylene flame) → Atomizer/Nebulizer (aspirates solution into flame) → Optical filter (selects characteristic wavelength) → Detector (photocell/PMT) → Readout.
- 8Flame Photometry – Interferences & Applications: Interferences: (1) Spectral interference: emission lines of different elements overlap. Na (589 nm) and Ca may interfere if filters not selective enough. (2) Chemical interference: formation of refractory compounds in flame. Ca²⁺ + PO₄³⁻ → Ca₃(PO₄)₂ (resistant to atomization → ↓Ca signal). Solution: add releasing agent (LaCl₃ or SrCl₂) — preferentially binds PO₄³⁻. (3) Ionization interference: at high flame temperatures, atoms ionize → fewer ground/excited state atoms → ↓emission. Common for K, Cs. Solution: add ionization suppressor (excess Na or Cs to provide electrons). (4) Self-absorption: at high concentrations, ground-state atoms absorb emitted radiation → non-linear calibration. Applications: clinical analysis (serum Na⁺, K⁺), water analysis, cement/glass industry, soil/fertilizer analysis.
- 9Atomic Absorption Spectroscopy (AAS): Principle: ground-state atoms ABSORB radiation of specific wavelength from an external source → degree of absorption ∝ concentration (Beer-Lambert’s Law applied to atomic absorption). Different from flame photometry: FP measures EMISSION (excited→ground), AAS measures ABSORPTION (ground→excited). AAS advantage: more atoms remain in ground state than excited state → more sensitive, fewer interferences. Instrumentation: Hollow Cathode Lamp (HCL — element-specific source, emits sharp line spectrum of the element to be analyzed) → Chopper/modulator → Flame (atomizer: air-acetylene or nitrous oxide-acetylene for refractory elements) → Monochromator → Detector (PMT) → Readout. Hollow Cathode Lamp: cathode made of the element to be determined. Filled with inert gas (Ne/Ar). discharge → inert gas ions sputter cathode atoms → excited atoms emit characteristic radiation.
- 10AAS – Interferences & Nepheloturbidometry: AAS Interferences: (1) Chemical: incomplete atomization (refractory compounds) — same as FP. Solutions: hotter flame (N₂O-acetylene), releasing agents, chelating agents (EDTA). (2) Spectral: overlap of absorption lines (rare due to narrow HCL emission). Background absorption by molecular species → corrected by deuterium lamp background correction or Zeeman correction. (3) Ionization: same as FP — add ionization suppressor. (4) Matrix effects: viscosity/surface tension of sample solution affects nebulization. Solution: matrix matching or standard addition method. Applications: heavy metals in pharmaceuticals (Pb, Cd, Hg, As — ICH Q3D elemental impurities), water analysis, clinical (serum Ca, Mg, Fe, Zn), food analysis, environmental monitoring. Nepheloturbidometry: measurement of light scattered (nephelometry) or light transmitted through (turbidimetry) a SUSPENSION. Nephelometry: detector at 90° — measures SCATTERED light. More sensitive for dilute suspensions. Turbidimetry: detector at 0° — measures TRANSMITTED light (attenuation). For dense suspensions. Applications: bacterial suspension (McFarland standards), sulfate determination, immunoglobulin quantification (immunonephelometry).
Learning Objectives
Vibrational Modes: Explain stretching and bending vibrations with diagrams. Calculate the number of fundamental vibrations for H₂O and CO₂.
Sample Handling: Compare KBr pellet, Nujol mull, and solution methods for IR sample preparation.
IR Detectors: Describe the working principle of Golay cell, thermocouple, and pyroelectric detectors.
FP vs AAS: Compare flame photometry and AAS by principle, sensitivity, source, and applications.
AAS Interferences: List the types of interferences in AAS and methods to overcome each.
Exam Prep Questions
Q1. Why does AAS use a Hollow Cathode Lamp instead of a continuum source?
AAS requires a sharp line source (HCL) because atomic absorption lines are extremely narrow (~0.002–0.005 nm). A continuum source (like D₂ lamp) emits over a wide band — only a tiny fraction of its radiation would be absorbed by the narrow atomic line → very poor sensitivity. The HCL emits radiation at EXACTLY the same wavelengths that the analyte atoms absorb (same element in cathode and sample). This ensures maximum overlap between source emission and atomic absorption → high sensitivity.
Q2. Why is KBr used for IR sample preparation?
KBr is used because: (1) It is IR-transparent — it does NOT absorb in the mid-IR region (4000–400 cm⁻¹), so it won’t interfere with the sample spectrum. (2) It becomes plastic under pressure and can be pressed into a clear, thin pellet. (3) It is easily available and inexpensive. Important precaution: KBr is hygroscopic — it absorbs moisture from air, which would show O–H absorption in the spectrum. Therefore, KBr must be stored dry (desiccator) and pellets must be prepared in a dry environment.
Q3. What is the difference between Nephelometry and Turbidimetry?
Both measure light interacting with suspended particles, but from different angles: Nephelometry measures SCATTERED light at 90° to the incident beam → more sensitive for DILUTE suspensions (small amount of scattering easy to detect against dark background). Turbidimetry measures TRANSMITTED light (0°) — the decrease in intensity due to scattering → better for CONCENTRATED suspensions (significant light attenuation measurable). Think of it like fog: nephelometry = seeing the light scatter sideways through fog, turbidimetry = measuring how much dimmer a light appears through fog.