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M.Pharm 2nd Semester - Advanced Spectral Analysis (MPC201T)

Complete Study Notes

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COURSE OVERVIEW

• Interpret NMR, Mass, and IR spectra of organic compounds
• Develop theoretical and practical skills for hyphenated instruments
• Identify and characterize organic/drug molecules from spectral data

1. Spectrometric Identification of Organic Compounds - Silverstein, 6th Ed., John Wiley & Sons
2. Principles of Instrumental Analysis - Skoog, Holler & Nieman, 5th Ed.
3. Instrumental Methods of Analysis - Willard's, 7th Ed., CBS Publishers
4. Organic Spectroscopy - William Kemp, 3rd Ed., ELBS

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UNIT I - UV and IR Spectroscopy (8-12 Hours)

A. UV-Visible Spectroscopy - Woodward-Fieser Rules

1. Rules for Conjugated Dienes (1,3-Butadienes)

2. Rules for Cyclic Dienes

• Homoannular (both double bonds in same ring): Base = 253 nm
• Heteroannular (double bonds in different rings): Base = 217 nm
• Same increments as above apply

3. Woodward-Fieser Rules for α,β-Unsaturated Carbonyl Compounds (Enones)

• Acyclic enone or 6-membered ring enone: 215 nm
• 5-membered ring enone: 202 nm
• α,β-unsaturated aldehyde: 208 nm
• Carboxylic acid/ester: 195 nm

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B. IR Spectroscopy

FTIR (Fourier Transform IR)

• Uses Michelson interferometer + Fourier transformation
• Advantages over dispersive IR: faster, higher sensitivity, better resolution, signal averaging possible
• Wavenumber range: 4000-400 cm⁻¹

ATR-IR (Attenuated Total Reflectance IR)

• Sample placed on a high-refractive-index crystal (ZnSe, diamond, Ge)
• Evanescent wave penetrates sample ~0.5-2 μm
• No sample preparation needed; suitable for solids, liquids, pastes
• Applications: pharmaceutical raw material ID, tablet coating analysis

Key IR Absorption Bands

IR Interpretation Strategy

1. Check 3600-2500 cm⁻¹ region (O-H, N-H, C-H stretches)
2. Check 2000-1500 cm⁻¹ region (C=O, C=C, C=N)
3. Fingerprint region 1500-600 cm⁻¹ (unique to each molecule)
4. Confirm with supporting evidence

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UNIT II - NMR Spectroscopy (10-12 Hours)

Principles of NMR

• NMR-active nuclei: ¹H, ¹³C, ¹⁵N, ³¹P, ¹⁹F
• Larmor frequency: ν₀ = γB₀/2π (γ = gyromagnetic ratio)
• Chemical shift (δ): Position of resonance relative to TMS (tetramethylsilane), measured in ppm

1D NMR

¹H NMR - Chemical Shift Ranges

¹³C NMR - Chemical Shift Ranges

Spin-Spin Coupling (J-coupling)

• n+1 rule: A proton with n equivalent neighboring H appears as n+1 multiplet
• Coupling constant J measured in Hz (not ppm - field independent)
• ³J (vicinal): 6-8 Hz; ²J (geminal): 10-15 Hz
• Coupling patterns: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m)

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2D NMR Techniques

COSY (Correlation Spectroscopy)

• Shows H-H coupling through bonds (³J)
• Cross-peaks indicate which protons are neighbors
• Used to identify connected spin systems
• Application: Trace connectivity along a carbon chain

NOESY (Nuclear Overhauser Effect Spectroscopy)

• Shows H-H correlations through space (not bonds)
• NOE is inversely proportional to r⁶ (r = inter-proton distance)
• Cross-peaks between protons within ~5Å
• Application: Determine stereochemistry, 3D structure, cis/trans configuration

HSQC / HMQC (Heteronuclear Single/Multiple Quantum Coherence)

• Shows one-bond C-H correlations (¹J_CH)
• Maps each H to its directly attached C
• Differentiates CH, CH₂, CH₃ groups

HMBC (Heteronuclear Multiple Bond Correlation) - "HECTOR" variant

• Shows 2-3 bond C-H correlations (²J and ³J)
• Bridges quaternary carbons with neighboring H
• Essential for connecting fragments across heteroatoms

INADEQUATE (Incredible Natural Abundance Double Quantum Transfer Experiment)

• Shows C-C connectivity directly
• Detects ¹³C-¹³C coupling (natural abundance ~1.1%)
• Very insensitive - requires large sample amounts
• Provides complete carbon skeleton connectivity map

2D NMR Summary Table

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UNIT III - Mass Spectrometry (8-12 Hours)

Principles

Ionization Techniques

Mass Fragmentation Rules

General Rules

1. Molecular ion (M⁺): Highest m/z peak; must be odd-electron ion
2. Base peak: Most abundant peak (100% relative intensity)
3. Nitrogen rule: If compound has odd number of nitrogen atoms, M⁺ will be odd
4. Ring + double bond rule (Degree of unsaturation): DBE = (2C + 2 + N - H - X) / 2

McLafferty Rearrangement

• Occurs in carbonyl compounds (aldehydes, ketones, esters, carboxylic acids)
• Requires a γ-hydrogen and a C=O group
• Mechanism: 6-membered cyclic transition state → γ-H migrates to C=O oxygen → β-cleavage occurs
• Produces an enol ion + neutral alkene
• Example: Pentan-2-one → m/z 58 (McLafferty product)

Fragmentation of Functional Groups

• Loss of water (M-18) - especially primary and secondary
• Loss of H (M-1)
• α-cleavage adjacent to oxygen
• Characteristic peaks at m/z = 31 (CH₂=OH⁺), 45, 59...

• α-cleavage is dominant (most favorable)
• Loss of small alkyl groups
• Iminium ion: [CH₂=NH₂]⁺ at m/z = 30 (primary aliphatic amines)
• Even-numbered M⁺ (if no N) → odd number N gives odd M⁺

• Ketones: α-cleavage on both sides of C=O; acylium ion (RCO⁺)
• Aldehydes: Loss of H (M-1); McLafferty rearrangement; m/z = 29 (CHO⁺)
• Esters: McLafferty rearrangement; loss of OR (loss of alkoxy group)

• CnH₂n+1⁺ ion series (m/z = 15, 29, 43, 57...) - differ by 14 mass units
• Branched alkanes fragment at branch points preferentially
• Little or no M⁺ for long chain alkanes

• Strong M⁺ (aromatic ring stabilizes)
• Benzyl cleavage → tropylium ion C₇H₇⁺ (m/z = 91) - most stable carbocation
• Loss of 28 (CO) from phenols (M-28)

Metastable Ions

• Appear at non-integer m/z values as broad humps
• Confirm fragmentation pathway: m* = (m₂)²/m₁
• Where m₁ = precursor ion, m₂ = product ion
• If m* matches a peak position, transition m₁ → m₂ is confirmed

Isotope Peaks

• M+1 peak: Due to ¹³C (1.1% natural abundance); height ≈ 1.1 × number of carbons
• M+2 peak (Chlorine pattern): ³⁵Cl:³⁷Cl = 3:1 → M:M+2 = 3:1
• Bromine pattern: ⁷⁹Br:⁸¹Br = 1:1 → M:M+2 = 1:1
• Sulfur: M+2 peak ~4.4% (due to ³⁴S)

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UNIT IV - Raman Spectroscopy (2-3 Hours)

Principle

• Based on inelastic scattering of monochromatic light (Raman scattering)
• When photons interact with molecules, most scatter elastically (Rayleigh) but ~1 in 10⁷ scatter inelastically with energy shift
• Stokes lines: Scattered photon has lower energy (molecule gains energy)
• Anti-Stokes lines: Scattered photon has higher energy (molecule loses energy)

Selection Rule

• IR active: Change in dipole moment (μ) required
• Raman active: Change in polarizability (α) required
• Mutual exclusion rule: In centrosymmetric molecules, a mode cannot be both IR and Raman active

IR vs Raman Complementarity

Instrumentation

• Source: Laser (Nd:YAG 1064 nm, Ar ion 514 nm, diode lasers)
• Sample holder: Cuvette, fiber optic probe, or direct contact
• Detector: CCD detector or photomultiplier

Applications in Pharmacy

• Polymorphism identification (different crystal forms of same drug)
• Counterfeit drug detection (non-destructive)
• Process analytical technology (PAT) - in-line monitoring
• Identification of active pharmaceutical ingredients through packaging

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UNIT V - Structural Characterization of Natural Compounds

Approach for Structure Elucidation

1. Determine molecular formula from HRMS (High Resolution MS) → calculate DBE
2. UV: identify chromophoric system (conjugation pattern)
3. IR: identify functional groups
4. ¹H NMR: number of protons, chemical shifts, multiplicity
5. ¹³C NMR / DEPT: type and number of carbons
6. 2D NMR (COSY, HMBC, HSQC): connectivity
7. Confirm by comparison with authentic standard or literature

Examples

Penicillin

• IR: β-lactam C=O at ~1770 cm⁻¹ (strained ring); amide C=O at ~1650 cm⁻¹; S-C stretching
• ¹H NMR: Characteristic H-5 (thiazolidine ring), H-6 (β-lactam), gem-dimethyl singlets
• MS: M⁺, loss of CO₂ (M-44), loss of side chain

Morphine

• UV: λmax ~285 nm (phenanthrene-type aromatic chromophore)
• IR: OH (3400 cm⁻¹), C=C aromatic (1600 cm⁻¹), C-O-C ether (1100 cm⁻¹)
• ¹H NMR: Aromatic H (δ 6.4-6.6), N-CH₃ (δ 2.4), vinyl protons (double bond in ring)
• MS: M⁺ = 285; characteristic fragments at m/z 229, 215, 162

Camphor

• IR: C=O stretch at ~1740 cm⁻¹ (bridged ring ketone, slightly elevated)
• ¹H NMR: 3 methyl groups (gem-dimethyl + bridge methyl); complex CH₂ patterns
• MS: M⁺ = 152; McLafferty product; loss of CH₃ (M-15)

Vitamin D (Calciferols)

• UV: λmax ~265 nm (conjugated triene system - characteristic!)
• IR: OH stretch (3400 cm⁻¹), C=C (1600-1650 cm⁻¹)
• MS: M⁺ = 384 (Vit D₂) or 384 (Vit D₃); loss of side chain fragments

Quercetin (Flavonoid)

• UV: Two bands - Band I (~370 nm, B-ring cinnamoyl), Band II (~255 nm, A-ring benzoyl)
• IR: OH phenolic (3200-3400 broad), C=O (1650 cm⁻¹), C=C aromatic
• ¹H NMR: H-3' and H-4' (B-ring ortho coupling), H-6 and H-8 (A-ring meta coupling), OH signals
• MS: M⁺ = 302; retro-Diels-Alder fragmentation characteristic of flavonoids

Digitalis Glycosides (e.g., Digitoxin)

• UV: ~220 nm (α,β-unsaturated lactone of aglycone)
• IR: Lactone C=O (~1770 cm⁻¹ for 5-membered butenolide), OH stretches
• MS: Sequential loss of sugar residues (deoxy sugars, -148 Da each)

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UNIT IV/VI - Chromatographic Hyphenated Techniques (12 Hours)

A. GC-MS (Gas Chromatography - Mass Spectrometry)

• Interface: Direct coupling (capillary column flows directly into MS ion source)
• Ionization: Usually EI (70 eV) - allows NIST library matching
• Applications:
  • Volatile drug analysis (anesthetics, solvents)
  • Residual solvent testing in pharmaceuticals (ICH Q3C)
  • Drug metabolite identification in urine/plasma
  • Forensic drug analysis - matches fragmentation pattern to library

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B. GC-AAS (Gas Chromatography - Atomic Absorption Spectroscopy)

• Combines GC separation with element-specific detection by AAS
• Used for speciation of organometallic compounds (e.g., organo-mercury, organo-lead)
• Very sensitive for trace metal analysis

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C. LC-MS (Liquid Chromatography - Mass Spectrometry)

• Interface challenge: LC uses liquid mobile phase; MS requires gas-phase ions - solved by ESI or APCI
• ESI (Electrospray Ionization): Solution sprayed through high-voltage needle → charged droplets → solvent evaporates → multiply charged ions formed
• APCI (Atmospheric Pressure Chemical Ionization): For less polar compounds
• Applications:
  • Drug and metabolite profiling in plasma
  • Bioavailability/bioequivalence studies
  • Impurity profiling of APIs
  • Protein/peptide analysis (with ESI)

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D. LC-FTIR (Liquid Chromatography - FTIR)

• Hyphenation of LC with FTIR for functional group identification of LC fractions
• Interface problem: Common HPLC solvents (water, acetonitrile) absorb IR strongly
• Solutions: Solvent elimination interface (flow cell dried), or deuterated solvents
• Applications: Polymer identification, pharmaceutical impurity structure determination

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E. LC-NMR

• Combines LC separation with online NMR detection
• Challenges: NMR is insensitive; deuterated solvents required (expensive); peak dilution
• Modes: Stop-flow (pause LC when peak of interest elutes), on-flow continuous
• Applications: Natural product structure determination without isolation, metabolite ID

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F. CE-MS (Capillary Electrophoresis - Mass Spectrometry)

• CE separates charged molecules by charge/size ratio in capillary
• MS provides molecular weight and structural information
• Interface: ESI or MALDI
• Advantages: Very high efficiency separation; tiny sample volumes (nL)
• Applications: Peptide/protein analysis, chiral drug analysis, ionic pharmaceuticals

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G. HPTLC (High Performance Thin Layer Chromatography)

• Upgraded version of TLC with pre-coated HPTLC plates (silica 5-7 μm)
• Equipment: Automated sample applicator (CAMAG), horizontal developing chamber, densitometer/video documentation
• Advantages over TLC: Better resolution, quantitative, reproducible
• Applications:
  • Fingerprinting of herbal drugs
  • Quantitative analysis of pharmaceutical formulations (P.D. Sethi method)
  • Stability testing and impurity profiling
  • Authentication of raw materials

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H. Supercritical Fluid Chromatography (SFC)

• Mobile phase: Supercritical CO₂ (+ small % organic modifier)
• Above critical point (31°C, 73 atm for CO₂)
• Properties: Density like liquid, viscosity like gas → fast, efficient
• Advantages: Faster than HPLC; easy removal of mobile phase; green chemistry
• Applications: Chiral separations, lipid analysis, drug impurity profiling

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I. Ion Chromatography (IC)

• Separates ionic species (anions and cations) by ion-exchange
• Suppressor column reduces background conductance
• Detection: Conductivity (most common), UV-Vis
• Applications: Counter-ion analysis in drug salts, inorganic impurities, preservative analysis

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J. Ion Exclusion Chromatography (IEC)

• Separates weak acids from strong acids and neutral compounds
• Based on Donnan exclusion (strongly ionized species excluded from resin pores)
• Applications: Organic acid analysis in pharmaceutical fermentation broths

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K. Flash Chromatography

• Rapid preparative LC using positive pressure (compressed air/nitrogen)
• Silica or reverse-phase cartridges; automated systems available
• Used for rapid isolation/purification of compounds during synthesis

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UNIT V - Thermal Methods and Biological Assays

A. Thermal Analysis

DSC (Differential Scanning Calorimetry)

• Endothermic events: Melting, glass transition (Tg), desolvation/dehydration
• Exothermic events: Crystallization, decomposition, oxidation

• Heat-flux DSC: Single furnace; temperature difference measured
• Power-compensation DSC: Two separate furnaces; power difference measured

• Melting point determination and purity assessment
• Polymorphism detection and form characterization
• Excipient compatibility studies
• Lyophilization (freeze-drying) development - Tg determination
• Characterization of amorphous vs crystalline forms

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DTA (Differential Thermal Analysis)

• Similar information to DSC but measures ΔT rather than heat flow
• Less quantitative than DSC (can't directly give enthalpy)
• Can operate at higher temperatures than DSC
• Often coupled with TGA

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TGA (Thermogravimetric Analysis)

• Uses precise microbalance
• Weight loss events: dehydration, desolvation, decomposition, oxidation

• Moisture and solvent content determination
• Thermal stability profiles
• Decomposition temperature
• Distinction between monohydrate, dihydrate, anhydrous forms
• Composition of blends

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B. Biological Standardization

Bioassay

• Quantal assay: All-or-none response (e.g., convulsions, death) - ED₅₀, LD₅₀
• Graded assay: Graduated dose-response (e.g., blood pressure change, blood sugar)

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ELISA (Enzyme-Linked Immunosorbent Assay)

1. Direct ELISA: Antigen coated on plate → enzyme-labeled antibody → substrate → color
2. Indirect ELISA: Antigen → primary antibody → enzyme-labeled secondary antibody
3. Sandwich ELISA: Capture antibody → antigen → detection antibody → enzyme → color (most sensitive)
4. Competitive ELISA: Sample antigen competes with labeled antigen for antibody binding

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RIA (Radioimmunoassay)

• Label: ¹²⁵I (gamma emitter), ³H, ¹⁴C
• Curve: As unlabeled antigen increases → less labeled antigen bound → lower counts
• Standard curve: Plot of %bound vs. concentration of standard

• Digoxin measured by RIA in therapeutic drug monitoring
• Antibody raised against digoxin-protein conjugate
• Critical because therapeutic range is narrow (0.8-2.0 ng/mL)

• Developed by Yalow & Berson (Nobel Prize 1977)
• Quantifies plasma insulin, important for diabetes diagnosis and research

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QUICK REVISION - IMPORTANT EXAM POINTS

Must-Remember Numbers

2D NMR - One Line Summary Each

• COSY - "neighbors through bonds" (H-H, 3 bonds)
• NOESY - "neighbors in space" (H-H, within 5Å) - stereochemistry
• HSQC - "which H is on which C" (one bond C-H)
• HMBC - "connecting across the molecule" (2-3 bond C-H, quaternary C)
• INADEQUATE - "C-C skeleton map" (insensitive, low abundance)

Hyphenated Techniques - Interface Summary

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1. Master Woodward-Fieser rules with numerical examples (calculation-type questions)
2. Practice mass fragmentation of alcohols, amines, ketones, aromatics
3. Understand each 2D NMR technique - what it shows, one example application
4. Know DSC applications in pharma (polymorphism, purity - very frequently asked)
5. ELISA sandwich method and RIA principle are common short-answer topics