Extraction, Chromatography & Spectrophotometry

Extraction Methods

General Purpose

• Separate analytes from complex matrices prior to identification/quantification.
• Major laboratory‐scale techniques:
  • Distillation
  • Solvent extraction (including Soxhlet & liquid–liquid)
  • Solid-phase extraction (SPE)

Distillation

• Definition: purification of liquids by boiling and condensing their vapour.
• Key physical principle: Vapour phase becomes enriched in the component with the lower boiling point (b.p.).
• Energy footprint: very high (heating + cooling).
• Applications
  • Volatile vs non-volatile separations (e.g.
    desalinating seawater).
  • Fractionating mixtures whose components differ in b.p. (e.g. crude-oil refining).

Types of Distillation

• Simple Distillation
  • Glassware: flask → condenser → receiver.
  • Best for a volatile liquid contaminated with non-volatile solids.
• Fractional Distillation
  • Extra fractionating column packed with beads/plates → repeated vapour–condensation cycles (= theoretical plates).
  • Allows separation of liquids with small b.p. differences.
  • Industrial scale example: petroleum tower.

Solvent Extraction

• Relies on differential solubility of components in two immiscible phases.

Soxhlet Extraction (Solid–Liquid)

• Setup: sample in paper thimble, solvent boiling in round-bottom flask, condenser on top.
• Cycle: vapour ⟶ condense ⟶ drip through sample ⟶ siphon back.
• Repeated wash concentrates analyte in boiling flask.

Liquid–Liquid Extraction

• Two immiscible liquids (e.g. water/hexane) shaken; solutes partition according to $K_D$.

Solid-Phase Extraction (SPE)

• Replaces bulk solvent with solid sorbent (powdered silica, C_18, etc.).
• Advantages:
  • Lower solvent usage
  • Higher enrichment & cleaner eluates
  • Compatible with automation.

Chromatography

Concept & Terminology

• Two phases:
  • Stationary phase (SP) – fixed (solid, liquid on solid, gel).
  • Mobile phase (MP) – moves (liquid or gas) and carries analyte.
• Separation arises from differential distribution (partitioning/adsorption/size exclusion) between MP & SP.
• Key vocabulary:
  • Analyte – substance of interest.
  • Chromatograph – instrument.
  • Chromatogram – detector output (peaks/spot pattern).
  • Retention factor $Rf = \frac{\text{distance analyte moved}}{\text{distance solvent front}}$ (PC/TLC).
  • Retention time $t_R$ – time between injection & peak maximum (column methods).

Essential Hardware

1. Stationary phase
2. Mobile phase
3. Sample introduction system/detector

Why Chromatography?

• Ultra-high sensitivity (µg–ng).
• Works for organic, inorganic, biological, environmental samples.
• Provides both qualitative (ID by $t_R$ or $Rf$) and quantitative (peak area) data.

Planar Chromatography

Paper Chromatography (PC)

• SP: cellulose paper fibres with bound water.
• MP: solvent mixture (e.g. $\text{H}_2\text{O}$ : ethanol).
• Procedure: spot sample near bottom ⟶ suspend paper in sealed jar with MP ⟶ solvent migrates by capillarity ⟶ develop chromatogram.
• Common classroom demo: separation of plant pigments (carotenes, xanthophylls, chlorophyll a/b).
• Quantification/ID by $Rf$ values, unique for a given solvent system.

Thin-Layer Chromatography (TLC)

• SP: thin layer (~250 µm) of silica gel $(\text{SiO}<em>2)</em>x$ (polar OH groups) on glass, aluminium, or plastic plate; often impregnated with fluorescent indicator.
• MP: organic solvent or mixture; chosen by trial & error for optimal resolution.

4 Stages of a TLC Experiment

1. Sample application – tiny spot using capillary on pencil guideline.
2. Development – plate placed upright in chamber; MP ascends by capillarity (≈20 min).
3. Visualization – under UV (quenched fluorescence) or iodine vapour; colourless compounds become visible.
4. Interpretation – measure spot/solvent distances; calculate $Rf$; compare with standards.

• Uses: monitor reaction progress, verify purity, preliminary solvent-system scouting for column/HPLC.

Column Chromatography Variants

High-Performance Liquid Chromatography (HPLC)

• MP: liquid pumped at high pressure (up to 600 bar) through tightly packed micro-particle column.
• Distinguishing features:
  • Sophisticated pump, injector, detector (UV-Vis, fluorescence, MS).
  • Small particle SP (≤5 µm) → high efficiency (many theoretical plates).
• Advantages: speed, resolution, reproducibility, ability to process preparative or trace-level samples.
• Choice when analytes are non-volatile or thermally unstable (unsuited to GC).

Gas Chromatography (GC)

• MP: inert gas (He, N_2, Ar).
• SP: viscous liquid coated onto inside of long capillary column or on solid support.
• Suitable for volatile, thermally stable compounds (≤400 °!C).
• Injection via heated port through septum; sample vapour swept onto column.
• Chromatogram provides:
  • Order of elution (linked to b.p. & polarity)
  • $t_R$ values
  • Relative peak areas (composition).
• Example b.p. data illustrate occasional polarity-induced reversal (toluene vs 4-methyl-2-pentanone).

Size-Exclusion Chromatography (SEC / Gel Filtration / Gel Permeation)

• SP: porous beads (Sephadex, Biogel, Sepharose).
• MP: buffer or solvent compatible with analyte (proteins, polymers).
• Separation purely by hydrodynamic volume (size):
  • Very large molecules excluded from pores → elute first.
  • Small molecules diffuse in/out → longer path → later elution.
• Critical parameters:
  • Column length (longer → higher resolution).
  • Buffer choice (avoid detergents/denaturants unless desired).
• Detected by UV at $280\,\text{nm}$ for proteins.

Ion-Exchange Chromatography (IEC)

• Exploits net charge of biomolecules at given pH (amphoteric nature).
• SP: resin with covalently attached ionic groups:
  • Cation exchanger – negatively charged matrix, binds $+$ proteins or metal cations; e.g. water softening (removal of $\text{Ca}^{2+}, \text{Mg}^{2+}$).
  • Anion exchanger – positively charged matrix, binds $-$ analytes (DNA, acidic proteins).
• Elution by changing pH or ionic strength (salt gradient) to weaken electrostatic attraction.

Affinity Chromatography

• Highest specificity: based on biospecific ligand–target binding.
• Preparation: immobilize ligand on solid matrix (e.g. agarose linked to bis-phosphothymidine).
• Workflow:
  1. Load sample → only target with affinity binds.
  2. Wash away non-binding components.
  3. Elute target by:
  • Competitive ligand (soluble)
  • High-salt / low-pH buffer
  • Denaturant (e.g. $8\,\text{M}$ urea) if acceptable.
  1. Dialyze to remove eluent reagents.
• Typical application: purification of antibodies, enzymes, or tagged recombinant proteins.

Spectrophotometry

Spectroscopy vs Spectrometry vs Spectrum

• Spectroscopy – study of spectra from matter–radiation interactions.
• Spectrometry – instrumental methods enabling spectroscopy.
• Spectrum – ordered array of signals by wavelength/frequency/mass.

Instrument Anatomy (UV-Visible Range)

• Radiation source – continuous white light (D_2, tungsten).
• Monochromator – prism/grating + slit selects narrow $\lambda$ band.
• Cuvette – typically 1 cm path length $b$.
• Detector – photodiode/PMT converts photons → electrical signal.
• Readout – displays %T or Absorbance $A$.

Operating Procedure (Good Laboratory Practice)

1. Turn on instrument, warm-up ≥15 min.
2. Clean cuvettes; handle frost-free sides only; rinse with DI water & small volume of sample.
3. Prepare blank (solvent only, identical colour/volume to samples).
4. Select wavelength where analyte shows strong absorbance & minimal interference (from prior scan or literature $\epsilon_{\lambda}$ values).
5. Insert blank, zero the instrument.
6. Measure samples; record %T and $A$ (optical density, OD).
7. Triplicate readings; average for accuracy.

Beer–Lambert Law

• Mathematical relation: $A = \epsilon b c$
  • $A$: absorbance (unitless)
  • $\epsilon$: molar absorptivity / extinction coefficient $\bigl(\text{L}\,\text{mol}^{-1}\,\text{cm}^{-1}\bigr)$, unique per compound & wavelength
  • $b$: path length (cm, usually $1.00$)
  • $c$: concentration (mol L$^{-1}$)
• Transmittance link: $T = \frac{P}{P_0}$, $A = -\log T = \log \left( \frac{P_0}{P} \right)$.
• Valid for monochromatic light, low–moderate absorbance (≈0.01–1.0).
• Generate calibration (Beer) plot (A vs c) → straight line; slope = $\epsilon b$; enables determination of unknown concentrations.

Biological/Analytical Applications

• Quantification of DNA (A_{260}), proteins (A_{280}, Bradford at $595\,\text{nm}$).
• Enzyme kinetics (change in A vs time).
• Plant metabolite analysis (vitamin C, anthocyanins).
• Semen cell counts (absorbance correlates with concentration).

Integrative & Practical Notes

• Choosing an extraction or chromatographic method depends on analyte volatility, polarity, size, charge, affinity, and concentration.
• Often multiple techniques are coupled (e.g. SPE cleanup → HPLC-UV quantification).
• Ethical/Environmental concerns:
  • High energy demand of large-scale distillation and solvent disposal from extractions.
  • Shift toward greener solvents, miniaturised SPE, and water-based MP in chromatography.
• Quality control: always run standards/controls; compare chromatograms (Fig. caffeine/aspirin example) to avoid mis-identification.
• Instrumental calibration (spectrophotometer blank, chromatograph retention markers) underpins data reliability.

Formulae & Key Equations (Quick Reference)

• $Rf = \frac{d_{\text{solute}}}{d_{\text{solvent front}}}$ (PC/TLC)
• $t_R = \frac{\text{distance (cm)}}{\text{chart speed (cm s}^{-1})}$ (chromatogram)
  – example: $6.5\,\text{cm} / 2\,\text{cm s}^{-1} = 3.25\,\text{s}$ ≈ $3\,\text{s}$.
• $A = \epsilon b c$ (Beer–Lambert)
• $T = \frac{P}{P_0},\quad A = -\log T$

End of Study Notes