Pharmaceutical Analysis · Short notes

Spectroscopy: Short Notes & Cheat Sheet

The electromagnetic spectrum, Beer–Lambert law, UV–visible, IR, fluorescence, atomic absorption, NMR and mass spectrometry.

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Short notes

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The electromagnetic spectrum used in analysis

increasing wavelength, lower energyX-ray< 10 nmCrystal structure(XRD)UV200–400 nmElectronic transitionsVisible400–800 nmColorimetryInfrared4000–400 cm⁻¹Bond vibrationsRadioMHzNMR: nuclear spin
Each region interacts with a different part of the molecule: electrons (UV–visible), bonds (IR) and nuclei (NMR).

UV–visible spectroscopy

  • UV is 200–400 nm and visible is 400–800 nm. Absorption promotes electrons to higher energy levels.
  • Beer–Lambert law: A = εbc, where A is absorbance, ε molar absorptivity, b path length and c concentration. A = log(I₀/I) = −log T.
  • Deviations occur at high concentrations, with stray light, with polychromatic light and when the analyte reacts chemically.
  • Chromophores absorb light (C=C, C=O, aromatic rings); auxochromes (–OH, –NH₂) shift or intensify absorption.
  • Shifts: bathochromic (red, to longer wavelength), hypsochromic (blue, to shorter), hyperchromic (more intense), hypochromic (less intense).
  • Instrument: deuterium lamp (UV) and tungsten–halogen lamp (visible), a grating monochromator, quartz cells for UV (glass absorbs UV), and a photomultiplier or diode-array detector.

IR and fluorescence

  • IR (4000–400 cm⁻¹) measures molecular vibrations. A vibration is IR-active only if it changes the dipole moment.
  • Samples are prepared as a KBr disc, a Nujol mull or by ATR. The fingerprint region below 1500 cm⁻¹ confirms identity.
  • Fluorimetry measures light emitted at a longer wavelength than that absorbed (Stokes shift). It is 10 to 1000 times more sensitive than UV, but suffers from quenching.
  • Fluorescence is measured at 90° to the exciting beam; quinine sulfate is a common standard.

Atomic and structural methods

  • Atomic absorption spectroscopy measures metals using a hollow-cathode lamp made of the element being measured, with a flame or graphite furnace.
  • Flame photometry (emission) measures sodium, potassium, lithium and calcium.
  • NMR uses radio waves on nuclei with spin (¹H, ¹³C). Chemical shift is in ppm relative to TMS (0 ppm). Signals split by the n + 1 rule.
  • Mass spectrometry separates ions by mass-to-charge ratio (m/z). The molecular ion gives the molecular mass; the base peak is the tallest peak (100%).
  • Nitrogen rule: an odd molecular mass means an odd number of nitrogen atoms.

Cheat sheet

High-yield facts in one place. Cover the right-hand column and test yourself.

Technique → what it measures

TechniqueBasis · use
UV–visibleElectronic transitions · assays
IRVibrations · identification
FluorimetryEmission · trace analysis
AASAtomic absorption · metals
Flame photometryAtomic emission · Na, K, Li
NMRNuclear spin · structure
Mass spectrometrym/z · molecular mass, structure

Components and materials

PartChoice
UV sourceDeuterium lamp
Visible sourceTungsten–halogen lamp
UV cellQuartz (silica)
IR sampleKBr disc, Nujol mull, ATR
AAS sourceHollow-cathode lamp
NMR referenceTetramethylsilane (0 ppm)

Memory aids

Bathochromic = Big wavelength (red)
A bathochromic shift moves absorption to longer wavelength; hypsochromic moves it shorter.

Exam traps

  • Glass cells absorb UV light; quartz must be used below about 340 nm.
  • Symmetrical vibrations that do not change dipole moment are IR-inactive.
  • Fluorescence is emitted at a longer wavelength, not a shorter one.
  • The base peak is the most intense peak, not necessarily the molecular ion.

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