Spectroscopy: UV, IR, NMR and mass

In short

Spectroscopy studies how a drug molecule interacts with electromagnetic radiation — UV-visible light excites electrons, infrared excites bond vibrations, radiofrequency in a magnetic field flips nuclear spins, and mass spectrometry weighs the ions the molecule breaks into. Together they give the four questions a chemist needs answered: how much is present (UV), which functional groups are present (IR), how the carbon-hydrogen skeleton is joined (NMR) and what the molecular mass and formula are (MS). The Indian Pharmacopoeia 2026 (10th edition) prescribes UV assays, specific absorbance limits and IR identity tests for a large number of monographs, and LC-MS/MS is the backbone of bioequivalence and impurity work in Indian industry. It is examined in Pharmaceutical Analysis and Pharmaceutical Chemistry papers, in GPAT and NIPER entrances, and in QC/QA/analytical-development interviews at Sun Pharma, Cipla, Dr Reddy's, Lupin, Zydus, Torrent, Aurobindo, Divi's, Syngene and the CROs.

Explained

Fundamentals common to all spectroscopy

• Electromagnetic radiation: E = hν = hc/λ; energy rises as wavelength falls. Order of increasing energy: radiofrequency (NMR) < microwave (rotational) < IR (vibrational) < UV-visible (electronic) < X-ray.
• Wavenumber ν̄ = 1/λ in cm⁻¹ is used in IR because it is directly proportional to energy.
• Absorption occurs only when the photon energy exactly matches a quantised energy gap (resonance condition) and the transition is allowed by a selection rule.
• Mass spectrometry is not absorption spectroscopy — it separates ions by mass-to-charge ratio, so it is destructive, while UV, IR and NMR are non-destructive.
• Qualitative use = identification (λmax, spectral fingerprint, shifts); quantitative use = assay, mainly UV-visible because absorbance is linear in concentration.

UV-visible spectroscopy

• Range: UV 200–400 nm (near UV), visible 400–800 nm; below 200 nm is vacuum UV (oxygen and nitrogen absorb).
• Electronic transitions in order of decreasing energy: σ→σ* (alkanes, below 150 nm, not seen), n→σ* (saturated halides, alcohols, amines, 150–250 nm), π→π* (alkenes, carbonyls, aromatics; high ε 10³–10⁵), n→π* (carbonyl, nitro; weak, ε 10–100, forbidden).
• Chromophore: the group responsible for absorption (C=C, C=O, NO₂, N=N, benzene ring). Auxochrome: a group with lone pairs (−OH, −NH₂, −OR, −Cl) that itself does not absorb above 200 nm but extends conjugation and shifts/intensifies the band.
• Shifts: bathochromic (red shift, to longer λ), hypsochromic (blue shift), hyperchromic (ε increases), hypochromic (ε decreases).
• Conjugation lowers the π→π* gap, so λmax rises: ethylene 171 nm → butadiene 217 nm → hexatriene 258 nm → β-carotene (11 double bonds) absorbs in the visible and looks orange.
• Solvent effect: polar solvents stabilise the more polar ground state of a carbonyl, so n→π* shows a blue shift with increasing polarity; π→π* shows a small red shift. Phenols and anilines are pH sensitive — phenoxide (alkali) is red-shifted, anilinium (acid) is blue-shifted. Hence IP assays fix the solvent (for example paracetamol in 0.1 M NaOH).
• Woodward–Fieser rules predict λmax of dienes and enones by adding substituent increments to a parent value (acyclic/heteroannular diene 217 nm, homoannular diene 253 nm, six-membered α,β-unsaturated ketone 215 nm; typically +5 nm per alkyl substituent, +30 nm per extended conjugation, +39 nm homoannular component).
• Beer–Lambert law and its deviations: real (concentration above about 0.01 M, refractive-index change), chemical (dissociation, association, complexation, pH change, solvolysis) and instrumental (polychromatic radiation, stray light, slit too wide, fluorescence, non-parallel beam).
• Instrumentation: source (deuterium/hydrogen discharge lamp 190–400 nm; tungsten-halogen 350–900 nm; xenon flash in diode-array instruments) → monochromator (holographic grating, formerly quartz prism) → sample cell → detector (photomultiplier, photodiode, diode array/CCD) → readout. Cells: quartz/silica below 350 nm, glass or plastic only in the visible; path length normally 1 cm.
• Single beam (one light path, blank measured separately, cheaper) versus double beam (beam split by chopper/mirror between sample and reference; corrects source drift, allows scanning) versus diode-array/photodiode-array (full spectrum in milliseconds, used as HPLC detector).
• Photometric accuracy is best when absorbance is roughly 0.2–0.8 (minimum relative error near A = 0.434); dilute or concentrate the sample to reach that window.
• Quantitative methods: direct A(1%, 1 cm) calculation (the IP route), calibration curve, standard-addition, simultaneous equation method for two-component mixtures, absorbance-ratio (Q-point) method, difference spectrophotometry, derivative spectrophotometry (removes matrix/excipient background).
• Calibration and control of the instrument: wavelength accuracy with a holmium oxide filter or solution and the deuterium lamp emission lines, absorbance accuracy with standard potassium dichromate in 0.005 M H₂SO₄, stray light with potassium chloride/sodium nitrite solutions, resolution with toluene in hexane — all described in the general chapters of IP 2026 (equivalent to USP ⟨857⟩).

Infrared spectroscopy

• Range: near IR 12,500–4,000 cm⁻¹, mid IR (analytically used) 4,000–400 cm⁻¹, far IR 400–50 cm⁻¹.
• Selection rule: a vibration is IR active only if it causes a change in dipole moment. Symmetrical stretches (N₂, O₂, the symmetric stretch of CO₂) are IR inactive but Raman active (mutual exclusion in centrosymmetric molecules).
• Number of fundamental modes: 3N − 6 for a non-linear molecule, 3N − 5 for a linear one (N = number of atoms).
• Vibration types: stretching (symmetric, asymmetric) and bending/deformation (scissoring, rocking, wagging, twisting). Stretching needs more energy than bending, so it appears at higher wavenumber.
• Hooke's-law consequences: wavenumber rises with bond strength (force constant k: C≡C > C=C > C−C) and falls with reduced mass (C−H ≈ 3000 > C−D ≈ 2200 > C−Cl ≈ 700).
• Regions: 4000–2500 (X−H stretch: O−H, N−H, C−H), 2500–2000 (triple bonds, C≡N, C≡C), 2000–1500 (double bonds C=O, C=C, C=N), 1500–400 cm⁻¹ = fingerprint region (single-bond stretches and bends; unique to the molecule, used for identity).
• Factors shifting group frequencies: hydrogen bonding (broadens and lowers O−H and C=O; free O−H sharp at 3600, H-bonded broad at 3300), conjugation (lowers C=O by about 20–30 cm⁻¹), ring strain (raises C=O: cyclohexanone 1715, cyclopentanone 1745, cyclobutanone 1780 cm⁻¹), inductive/electronic effects (acid chloride 1800 > ester 1735 > ketone 1715 > amide 1650 cm⁻¹), Fermi resonance, field effect and physical state (solid versus solution).
• Sampling: solids as KBr pellet (1–2% in dry, IR-grade KBr, pressed at high pressure; KBr is hygroscopic so wet KBr gives false O−H bands), Nujol mull (Nujol itself shows C−H bands around 2900, 1460, 1375 cm⁻¹), thin film/cast film, solutions in CCl₄ or CHCl₃ in NaCl/KBr cells, gases in long-path cells, and ATR (attenuated total reflectance — a diamond/ZnSe crystal; no sample preparation, now the routine QC method) or diffuse reflectance (DRIFT).
• FTIR uses a Michelson interferometer with a moving mirror; the interferogram is Fourier-transformed to a spectrum. Advantages over dispersive IR: all frequencies measured together (the Felgett or multiplex advantage) and high throughput with no slits (Jacquinot advantage), so it is much faster and far more sensitive, better wavenumber accuracy from the internal He–Ne laser, and spectra can be co-added to improve signal-to-noise.
• Pharmaceutical use: the IP identity test compares the sample spectrum with that of the reference standard or a reference spectrum; it also distinguishes polymorphs, detects pseudopolymorphs/hydrates, and supports NIR-based online blend-uniformity and PAT work.

NMR spectroscopy

• Requirement: a nucleus with non-zero spin, I ≠ 0. I = ½ for ¹H, ¹³C, ¹⁹F, ³¹P (sharp lines); ¹²C and ¹⁶O have I = 0 and are NMR silent; ¹⁴N (I = 1) is quadrupolar and broad.
• In a magnetic field B₀ a spin-½ nucleus has 2I + 1 = 2 orientations; absorption of radiofrequency flips it from the lower to the higher level at the Larmor frequency.
• Chemical shift (δ, ppm): electrons circulate and shield the nucleus. Electron-withdrawing neighbours deshield it → shift downfield (higher δ). δ is reported relative to TMS (tetramethylsilane) = 0; for aqueous work DSS or sodium 3-(trimethylsilyl)propanesulfonate is used. δ in ppm is field independent, which is why it is quoted instead of Hz.
• Typical ¹H shifts: TMS 0; alkane CH 0.9–1.7; allylic 1.6–2.6; CH next to C=O 2.0–2.6; CH−N 2.5–3.0; CH−O 3.3–4.5; vinylic 4.5–6.5; aromatic 6.5–8.5; aldehyde 9.5–10.5; carboxylic acid 10–13; phenol 4–12 (variable); alcohol/amine broad and variable.
• Anisotropy: the ring current of benzene deshields the ring protons (δ ~7.3) but shields anything above the ring; acetylene protons are shielded (δ ~2.5) despite sp carbon.
• Spin–spin coupling: neighbouring non-equivalent protons split a signal. n + 1 rule — n equivalent neighbours give n + 1 lines with intensities from Pascal's triangle. Coupling constant J is measured in Hz and does not change with field strength — the practical test to tell real coupling from two separate signals.
• Typical J: vicinal ³J 6–8 Hz (free rotation), axial–axial 8–12, axial–equatorial 2–5, geminal ²J 0–18, trans-alkene 12–18, cis-alkene 6–12, aromatic ortho 6–9, meta 1–3, para 0–1 Hz.
• Integration gives the relative number of protons (area under the peak), the basis of quantitative NMR (qNMR) and of counting protons in a structure.
• Exchangeable protons (OH, NH, SH, COOH) are broad, concentration and temperature dependent, and disappear on adding D₂O — a standard exam-level identification trick.
• ¹³C NMR: natural abundance only 1.1%, so sensitivity is low; FT and many scans are needed. Range 0–220 ppm; CH₃/CH₂ 0–50, C−O 50–90, alkene/aromatic 100–150, ester/acid 165–180, ketone/aldehyde 190–220. Routinely recorded proton-decoupled, so each carbon is a singlet and the number of lines counts the chemically distinct carbons. DEPT-135 distinguishes CH₃/CH (positive) from CH₂ (negative) and makes quaternary carbons disappear.
• Solvents: deuterated — CDCl₃ (residual peak δ 7.26 in ¹H, 77.0 in ¹³C), DMSO-d₆ (2.50 / 39.5), D₂O (4.79), methanol-d₄, acetone-d₆.
• Instrument: superconducting magnet (liquid helium) quoted by its ¹H frequency — 300, 400, 500, 600 MHz; higher field means better dispersion and sensitivity. 2D NMR: COSY (H−H through 2–3 bonds), HSQC/HETCOR (one-bond C−H), HMBC (2–3 bond C−H), NOESY (through-space proximity, stereochemistry and conformation).
• Uses: definitive structure elucidation, impurity and degradant identification, stereochemistry, and qNMR for reference-standard characterisation.

Mass spectrometry

• Sequence: sample inlet → ion source → mass analyser → detector → data system, with the analyser under high vacuum (about 10⁻⁵–10⁻⁷ torr) so ions do not collide.
• Ionisation — hard: EI (electron impact, 70 eV) gives rich fragmentation and library-matchable spectra, but the molecular ion may be weak or absent; suits volatile, thermally stable molecules (GC-MS).
• Ionisation — soft: CI (reagent gas, gives M+H⁺), ESI (electrospray; solution sprayed from a charged capillary; polar, ionic, thermolabile drugs, peptides, multiply charged proteins; the workhorse of LC-MS), APCI (less polar, moderately volatile analytes), MALDI (matrix-assisted laser desorption — proteins, polymers), FAB, field desorption.
• Analysers: magnetic sector (scanning by B), quadrupole (mass filter; cheap, robust, unit resolution), triple quadrupole QqQ (MS/MS; collision cell; MRM/SRM gives the highest selectivity and sensitivity for bioanalysis), time of flight (TOF) (high mass range, high speed, accurate mass), ion trap, Orbitrap/FT-ICR (very high resolution, accurate mass for formula assignment).
• Resolution R = m/Δm: unit-mass instruments R ≈ 10³; high-resolution MS R ≥ 10⁴–10⁵ gives mass accuracy of a few ppm, enough to assign an elemental formula.
• Key terms: molecular ion M⁺˙ (gives molecular mass); base peak (most intense peak, set to 100% relative abundance); fragment ions; metastable ions; isotope peaks M+1, M+2.
• Nitrogen rule: for a molecule of C, H, O, N, S and halogens, an even nominal molecular mass means zero or an even number of nitrogens; an odd mass means an odd number of nitrogens.
• Isotope patterns: M+1 ≈ 1.1 × (number of carbons) % of M; one Cl gives M : M+2 ≈ 3 : 1; one Br gives ≈ 1 : 1; S gives an M+2 of about 4.4%.
• Fragmentation pathways: α-cleavage (next to a heteroatom or carbonyl), benzylic and allylic cleavage (stable tropylium ion m/z 91 from alkyl benzenes), loss of small neutrals (H₂O 18, CO 28, ketene 42, CO₂ 44), McLafferty rearrangement (γ-H transfer in a carbonyl with a γ-hydrogen, loss of a neutral alkene), retro-Diels–Alder. Fragmentation favours the most stable carbocation and the most stable neutral.
• Pharmaceutical use: molecular-mass confirmation, impurity and degradation-product identification (ICH Q3A/Q3B), genotoxic impurity and nitrosamine testing at ppb level by LC-MS/MS, elemental impurities by ICP-MS (ICH Q3D), metabolite identification, and bioequivalence plasma assays.

Choosing and combining the techniques

• Practical structure elucidation runs: MS for molecular formula and mass → calculate double-bond equivalents → IR for functional groups → ¹H NMR for proton environments, counts and connectivity → ¹³C/DEPT for the carbon skeleton → 2D NMR to settle ambiguities.
• Regulatory frame in India: Drugs and Cosmetics Act 1940 and Rules 1945 (with the revised Schedule M good-manufacturing requirements), the Indian Pharmacopoeia 2026 monographs and general chapters, ICH Q2(R2) for analytical-procedure validation and Q14 for analytical development, and ICH Q3A/Q3B/Q3C/Q3D for impurities; CDSCO is the regulator and IPC Ghaziabad publishes the IP and reference standards. Analytical instruments must be qualified (DQ-IQ-OQ-PQ) with 21 CFR Part 11-style data-integrity controls (ALCOA+).

Key points

• Beer–Lambert law — absorbance is proportional to path length and concentration; the basis of every UV assay.
• A(1%, 1 cm) — the IP's specific absorbance: absorbance of a 1 g/100 mL solution in a 1 cm cell; ε = A(1%, 1 cm) × M / 10.
• Working absorbance window — 0.2 to 0.8 A, least relative photometric error near 0.434.
• Quartz cells below 350 nm — glass and plastic absorb in the UV.
• Chromophore absorbs, auxochrome modifies — conjugation and auxochromes cause a bathochromic (red) shift.
• n→π* blue-shifts in polar solvents; π→π* red-shifts — the standard solvent-effect question.
• IR activity needs a dipole-moment change — symmetric stretches of symmetrical molecules are IR inactive.
• 3N − 6 (non-linear) / 3N − 5 (linear) fundamental vibrational modes.
• Fingerprint region 1500–400 cm⁻¹ — molecule-specific; 4000–1500 cm⁻¹ is the functional-group region.
• FTIR beats dispersive IR on speed, sensitivity and wavenumber accuracy (Michelson interferometer + He-Ne laser reference).
• Wet KBr ruins a pellet — produces a spurious broad O−H band around 3400 cm⁻¹.
• δ in ppm is field independent; J in Hz is field independent too — but peak separation in Hz grows with field.
• n + 1 rule — n equivalent neighbouring protons split a signal into n + 1 lines, intensities by Pascal's triangle.
• Integration counts protons; ¹³C line count counts distinct carbons (proton-decoupled spectrum).
• D₂O shake removes OH, NH, COOH signals — confirms exchangeable protons.
• Nitrogen rule — odd nominal molecular mass implies an odd number of nitrogen atoms.
• Cl gives M:M+2 = 3:1, Br gives 1:1, M+1 ≈ 1.1 n% for n carbons — reading halogens and carbon count off the spectrum.
• ESI for polar/thermolabile drugs, EI for volatile ones; MRM on a triple quadrupole for trace bioanalysis.

Formulas and examples

Example 1 — UV assay of paracetamol tablets (IP method, A(1%, 1 cm) = 715 at 257 nm in 0.1 M NaOH). Powdered tablet equivalent to 100 mg (label claim) is dissolved and made up to 100 mL; 1.0 mL of this is diluted to 100 mL. Measured A = 0.701 in a 1 cm cell.

1. c (in the measured solution) = A / [A(1%, 1 cm) × b] = 0.701 / 715 = 9.804 × 10⁻⁴ g per 100 mL = 9.804 µg mL⁻¹.
2. Undo the 1 → 100 dilution: 9.804 × 100 = 980.4 µg mL⁻¹ = 0.9804 mg mL⁻¹.
3. Content in the 100 mL stock = 0.9804 × 100 = 98.04 mg.
4. Percentage of label claim = 98.04/100 × 100 = 98.0% — inside the IP limit of 95.0–105.0% for paracetamol tablets.

Note A = 0.701 lies in the 0.2–0.8 window, so the dilution scheme is sound.

Example 2 — converting specific absorbance to molar absorptivity, and the photon energy. Paracetamol, M = 151.16 g mol⁻¹, A(1%, 1 cm) = 715 at 257 nm.

• ε = 715 × 151.16 / 10 = 1.081 × 10⁴ L mol⁻¹ cm⁻¹ — large, so the band is an allowed π→π* transition of the substituted benzene ring, not an n→π*.
• Photon energy: E = 1240/257 = 4.82 eV, or 1.196 × 10⁵/257 = 465 kJ mol⁻¹.
• If the same solution gave %T = 19.95, then A = 2 − log₁₀(19.95) = 2 − 1.2996 = 0.700 — the same measurement expressed as transmittance.

Example 3 — IR: Hooke's law and DBE for paracetamol (C₈H₉NO₂, M = 151). Force constant of a C−H single bond k ≈ 500 N m⁻¹.

1. µ = (12 × 1)/(12 + 1) = 0.923 amu = 0.923 × 1.6605 × 10⁻²⁷ = 1.533 × 10⁻²⁷ kg.
2. √(k/µ) = √(500 / 1.533 × 10⁻²⁷) = √(3.262 × 10²⁹) = 5.711 × 10¹⁴ s⁻¹.
3. ν̄ = 5.711 × 10¹⁴ / (2π × 3 × 10¹⁰) = 5.711 × 10¹⁴ / 1.885 × 10¹¹ = 3029 cm⁻¹ — matching the observed aromatic/aliphatic C−H stretch near 3000 cm⁻¹.
4. DBE = (2×8 + 2 + 1 − 9)/2 = 10/2 = 5 → one benzene ring (4) plus one C=O (1).
5. Expected bands: 3330 (phenolic O−H, broad), 3160 (amide N−H), 1650 (amide C=O, lowered by conjugation and resonance), 1610 and 1510 (aromatic C=C), 1240 (C−O), 835 cm⁻¹ (para-disubstituted ring, two adjacent H).

Example 4 — NMR: field, frequency and reading a spectrum of benzocaine (ethyl 4-aminobenzoate, C₉H₁₁NO₂).

1. Field needed for a 400 MHz ¹H instrument: B₀ = 400 / 42.577 = 9.395 T. On the same magnet ¹³C resonates at 10.705 × 9.395 = 100.6 MHz.
2. Two aromatic signals at δ 7.85 and 6.64 are Δδ = 1.21 ppm apart = 1.21 × 400 = 484 Hz on this instrument, but only 1.21 × 100 = 121 Hz on a 100 MHz instrument — the shift in ppm is unchanged, the separation in Hz is not.
3. Spectrum: δ 7.85 (d, J = 8.6 Hz, 2H, ortho to C=O), 6.64 (d, J = 8.6 Hz, 2H, ortho to NH₂ — shielded by the electron-donating amine), 4.30 (q, J = 7.1 Hz, 2H, OCH₂), 4.05 (br s, 2H, NH₂ — vanishes on a D₂O shake), 1.36 (t, J = 7.1 Hz, 3H, CH₃).
4. Integration total = 2+2+2+2+3 = 11 H, exactly the hydrogen count of C₉H₁₁NO₂. DBE = (18 + 2 + 1 − 11)/2 = 5 → ring (4) + ester C=O (1).
5. The quartet/triplet pair with identical J = 7.1 Hz is the signature ethyl group; the two aromatic doublets with J ≈ 8.6 Hz confirm para substitution.

Example 5 — mass spectrum of paracetamol (EI).

No M+2 of about 33% or 100% is present, so no chlorine or bromine. If an unknown instead showed M at 1.0 and M+2 at 0.33 relative intensity, one chlorine is present; 1.0 : 1.0 means one bromine.

Where it is used

• QC laboratories of Sun Pharma, Cipla, Dr Reddy's, Lupin, Zydus, Torrent, Alkem, Mankind: UV assays and dissolution profiling, FTIR/ATR identity tests on every incoming raw material, and NIR for blend uniformity.
• Analytical development and R&D at Divi's, Aurobindo, Glenmark, Piramal and NIPER laboratories: structure elucidation of new molecules and impurities by NMR and high-resolution MS, forced-degradation studies, polymorph screening by IR with XRD and DSC.
• Bioanalytical CROs — Lambda Therapeutic, Veeda, Syngene, Accutest, Sai Life — run LC-MS/MS MRM assays for bioequivalence studies submitted to CDSCO and USFDA; job roles: bioanalytical scientist, method-validation chemist.
• Regulatory and compliance work: nitrosamine and genotoxic-impurity testing at ppb level by LC-MS/MS, elemental impurities by ICP-MS under ICH Q3D, residual solvents by GC-MS under ICH Q3C.
• Government and testing bodies: IPC Ghaziabad (reference-standard characterisation and monograph development), Central Drugs Laboratory Kolkata, state drug-testing laboratories, NABL-accredited private labs, forensic science laboratories (FTIR and GC-MS for drug-of-abuse and NDPS casework).
• Allied uses: herbal and nutraceutical fingerprinting, cosmetic and food-adulteration testing (FSSAI laboratories), biotech protein characterisation by MALDI-TOF and ESI-MS, and clinical biochemistry analysers based on visible-range colorimetry.

Interview questions

• Q: State the Beer–Lambert law and name two reasons a calibration curve bends at high concentration? A: Absorbance = ε·b·c. At high concentration, solute–solute interaction changes ε and the refractive index (a real deviation), and chemical effects like association or dissociation alter the absorbing species.
• Q: Why does an IP monograph specify the solvent for a UV assay? A: λmax and ε shift with solvent polarity and pH — for example phenolic drugs are red-shifted in alkali — so the stated A(1%, 1 cm) value is valid only in that solvent.
• Q: Why must a quartz cuvette be used below 350 nm? A: Glass and plastics absorb strongly in the UV, so they would add a large background absorbance; fused silica is transparent down to about 190 nm.
• Q: What absorbance range gives the most accurate result and why? A: About 0.2–0.8, with minimum relative photometric error near A = 0.434; outside this, transmittance readings are too close to 0% or 100%.
• Q: Which vibrations are IR inactive? A: Those that produce no change in dipole moment — for example the symmetric stretch of CO₂ or the stretching of N₂; they are Raman active instead.
• Q: Why is the amide carbonyl of paracetamol near 1650 cm⁻¹ when a ketone is near 1715? A: Nitrogen lone-pair donation and conjugation give the C=O partial single-bond character, lowering the force constant and hence the wavenumber.
• Q: Advantages of FTIR over a dispersive instrument? A: All wavenumbers are measured simultaneously, giving far better speed and signal-to-noise, plus high wavenumber accuracy from the internal laser and the ability to co-add scans.
• Q: Why is chemical shift quoted in ppm and not Hz? A: Because the shift in Hz is proportional to the magnet field, so ppm makes the value comparable across 300, 400 and 500 MHz instruments.
• Q: How would you confirm that a broad singlet is an −OH proton? A: Add a drop of D₂O and re-record; the exchangeable proton signal disappears and an HOD peak appears.
• Q: How does an ethyl group appear in ¹H NMR and why? A: A triplet (3H, CH₃, split by two CH₂ protons) and a quartet (2H, CH₂, split by three CH₃ protons) with the same J of about 7 Hz, by the n + 1 rule.
• Q: A compound shows M⁺ at 180 with M+2 at about one-third the intensity of M. What does that tell you? A: One chlorine atom is present (³⁵Cl:³⁷Cl ≈ 3:1), and the even mass suggests zero or an even number of nitrogens.
• Q: Why is ESI preferred over EI for most modern drug analysis? A: ESI ionises polar, ionic and thermolabile molecules directly from solution, so it couples to HPLC and preserves the molecular ion, whereas EI requires volatile, thermally stable samples and often destroys M⁺.
• Q: What is the McLafferty rearrangement? A: A six-membered-transition-state transfer of a γ-hydrogen to a carbonyl oxygen followed by β-cleavage, expelling a neutral alkene and giving a characteristic even-mass radical cation.

Common mistakes

• Mixing up the two concentration units: ε needs mol L⁻¹, A(1%, 1 cm) needs g per 100 mL — using one with the other shifts the answer by a factor of M/10.
• Forgetting to multiply back by the dilution factor in an assay, or ignoring the "equivalent to" weight of tablet powder, and then reporting an impossible percentage of label claim.
• Confusing wavelength and wavenumber, or quoting an IR band in nm; and reading the IR axis as increasing left-to-right.
• Using a damp KBr pellet or Nujol and then assigning the resulting broad 3400 cm⁻¹ band to the drug's O−H, or Nujol's own 2900/1460/1375 cm⁻¹ bands to the sample.
• Applying the n + 1 rule to equivalent protons (the three protons of a CH₃ do not split one another) or expecting splitting between protons that are chemically equivalent.
• Saying the coupling constant J increases with magnet strength — it does not; only the separation in Hz between different chemical shifts does.
• Assuming the highest-mass peak is always the molecular ion, forgetting isotope peaks, M+H⁺ adducts in ESI, or that EI may not show M⁺ at all; and ignoring the nitrogen rule when assigning a formula.
• Reporting a structure from one technique alone instead of cross-checking DBE, functional groups, proton count and molecular mass together.

Related topics

Chromatography: HPLC, GC and TLC · Pharmaceutical analysis: titrimetric and instrumental methods · Medicinal chemistry: drug classes and SAR · Analytical method validation and ICH Q2(R2) · Impurity profiling and stability studies · Good laboratory practice and data integrity.