Unit 5 – Extraction, Chromatography & Spectrophotometry

5.1 EXTRACTION METHODS

Core Idea

• Goal: isolate target analytes from complex matrices before analysis.

Main Techniques

• Distillation – exploits boiling-point differences.

• Solvent Extraction – exploits differential solubility.

• Solid-Phase Extraction (SPE) – replaces bulk solvent with functionalised solid surfaces.

Distillation

• Definition: purification by boiling a liquid and condensing its vapour.

• Applications

  • Remove non-volatile impurities.

  • Separate two volatile liquids with differing b.p.

• Principle

  • Vapour above a boiling mixture is richer in low-b.p. components → condensate becomes the distillate.

  • Energy intensive (heating + cooling).

• Variants

  1. Simple Distillation

  • Apparatus: distillation flask, condenser (with water-in / water-out), thermometer, collection flask.

  • Best for volatile + non-volatile system (e.g. salt water → pure water).

  1. Fractional Distillation

  • Adds a fractionating column packed with beads to provide repeated vapour–liquid equilibria → higher resolution.

  • Used industrially in crude-oil “fractionation”.

Solvent Extraction

• Concept: partitioning of solutes between two immiscible phases.

• Two major sub-methods:

  1. Soxhlet (solid–liquid)

  • Sample in paper thimble, located in Soxhlet chamber.

  • Solvent boils, vapour condenses, drips onto sample, leaches analyte, siphons back once level reached.

  • Continuous, exhaustive extraction with minimal solvent replacement.

  1. Liquid–Liquid Extraction (LLE)

  • Shake immiscible solvents in separatory funnel → solute partitions according to affinity.

  • Denser phase drains first.

  • Key for inorganic/organic clean-up.

Solid-Phase Extraction (SPE)

• Replaces bulk solvent with powdered silica or other functionalised particles (hydrophobic, ion-exchange, etc.).

• Advantages: lower solvent use, higher efficiency, amenable to automation.

5.2 CHROMATOGRAPHY – PRINCIPLES & TECHNIQUES

Big Picture

• Versatile separation strategy: components distribute between a stationary phase (SP) and a mobile phase (MP).

• Outcomes

  • Determine purity, isolate components, identify unknowns.

• Name origin: Greek chromos (colour) + graphy (writing).

Essential Components

• Stationary phase – solid, liquid, or liquid on solid support; "stays put".

• Mobile phase – gas or liquid that carries sample; "motivating force".

• Sample mixture – applied at column/plate origin.

Key Interactions

• Surface adsorption, partitioning/solubility, charge, size exclusion, biospecific affinity.

Fundamental Terms

• Analyte – substance being separated.

• Chromatograph – instrument; chromatogram – visual/printed output.

• Retention factor (Rf) for planar methods:
  $R_f = \frac{\text{distance travelled by solute}}{\text{distance travelled by solvent front}}$

• Retention time (t_R) for column methods: time between injection and peak apex.

General Analogy

• "Bees vs. hornets in a flower bed": species with affinity (bees) delayed, others exit first.

Paper Chromatography (PC)

• SP: cellulose paper; MP: solvent (e.g. water–ethanol).

• Spot mixture near bottom; solvent rises via capillarity → separates pigments (chlorophyll a, b, xanthophylls, carotenes).

• Identify components via distinct $R<em>f$ values (e.g. yellow pigment $R</em>f = 0.72$, cyan $R_f = 0.36$).

• Imperatives: keep origin above solvent; mark solvent front quickly.

Thin-Layer Chromatography (TLC)

• SP: thin silica-gel or alumina layer on glass/plastic.

• MP: organic solvent.

• Workflow

  1. Sample Application – capillary spot on start line.

  2. Development – plate stands in closed jar; solvent ascends.

  3. Visualization – UV lamp (fluorescent indicator) or iodine vapour.

  4. Interpretation – compute $R_f$; compare to standards.

• Pros vs. Paper:

  • Adsorption vs. partition mechanism.

  • Faster (15–45 min), sharper spots, heat-resistant, UV-detectable.

Liquid Chromatography (LC)

• MP: liquid; SP: liquid on solid support/solid/ion exchanger.

• Versatile for ions & organics.

High-Performance Liquid Chromatography (HPLC)

• Pressurised LC (pump-driven).

• Hardware: solvent reservoir, pump, injector, high-efficiency column, detector, data station.

• Advantages: speed, resolution, sensitivity, recovery, reproducibility.

• Suited for non-volatile or thermolabile analytes; preparative or trace work.

Gas Chromatography (GC)

• MP: inert gas (He, N₂, Ar).

• SP: liquid coated on inert solid inside column (WCOT/SCOT) or porous solid.

• Injection port with rubber septum; oven-controlled column; detector outputs chromatogram.

• Separation based on volatility & polarity – order generally follows increasing boiling point, but polarity can override (e.g. toluene vs. 4-methyl-2-pentanone case).

Size-Exclusion Chromatography (SEC) / Gel Filtration

• Bead gel with defined pores (e.g. Sephadex, agarose, polyacrylamide).

• Large molecules excluded from pores → elute fastest; smaller molecules delayed.

• Non-denaturing; ideal for proteins, antibodies, enzymes.

• Monitor effluent at $280\,\text{nm}$ (protein UV absorbance).

• Critical choices: column length (↑ length = ↑ resolution), compatible buffer (avoid detergents that disrupt proteins).

Ion-Exchange Chromatography (IEC)

• SP: resin with fixed charges; separation by electrostatic attraction dependent on pH.

  • Cation exchanger – negatively charged groups (e.g. –SO₃⁻) bind cations / positive proteins.

  • Anion exchanger – positively charged groups (e.g. –NH₃⁺) bind anions / negative proteins.

• Elution by changing pH or ionic strength (salt gradient).

• Real-world: household water softeners remove $\text{Ca}^{2+},\text{Mg}^{2+}$.

Affinity Chromatography

• Relies on biospecific interactions (enzyme–substrate, antibody–antigen, receptor–ligand).

• Column contains immobilised ligand.

• Only target binds; others wash through.

• Elution strategies: excess soluble ligand, high salt, low pH, or denaturant (e.g. $8\,\text{M}$ urea).

• Example: purification of staphylococcal nuclease on bis-phosphothymidine agarose.

• Post-elution dialysis removes disruptive agents.

Summary Table – Chromatography Types

• Paper → partition; qualitative TLC alternative.

• TLC → adsorption; quick monitoring.

• LC/HPLC → pressure-driven liquid columns.

• GC → gas phase; volatile organics.

• SEC → size; desalting & MW estimation.

• IEC → charge; protein purification, water softening.

• Affinity → highly selective lock-and-key separations.

5.3 SPECTROPHOTOMETRY

Spectroscopy vs. Spectrometry

• Spectroscopy – study of spectra produced by matter–radiation interactions.

• Spectrometry – measurement techniques to perform spectroscopy.

• Spectrum – ordered array by wavelength, frequency or mass.

Spectrophotometer – Purpose & Components

• Splits polychromatic light → monochromatic beam.

• Measures absorbance → determines solute concentration via Beer’s Law.

• Anatomy

  • Continuous light source (e.g. tungsten filament).

  • Monochromator (prism/diffraction grating + slit).

  • Cuvette holder (fixed path length).

  • Detector → converts light to electrical signal.

  • Display/data system.

Operating Procedure (Step-by-Step)

1. Warm-up (≈15 min).

2. Prepare & rinse cuvettes (avoid fingerprints; use kimwipes).

3. Load $0.5{-}1\,\text{mL}$ sample; new pipette tip per sample.

4. Blank/Control – solvent only (same dye, if any) in identical cuvette.

5. Wipe exterior, select analysis wavelength (one that analyte absorbs strongly).

6. Calibrate-zero with blank (set transmission to $100\%$ or absorbance $0$).

7. Insert sample; wait ~10 s for stable reading; record %T or $A$.

8. Run each sample ≥3× and average.

Beer’s (Beer–Lambert) Law

• Quantifies absorbance–concentration relationship: $A = \varepsilon b c$ where

  • $A$ – absorbance (unitless).

  • $\varepsilon$ – molar absorptivity (L mol⁻¹ cm⁻¹).

  • $b$ – path length (cm).

  • $c$ – concentration (mol L⁻¹).

• Transmittance relation:
  $T = \frac{P}{P<em>0}, \qquad A = -\log T = \log\left(\frac{P</em>0}{P}\right)$

• Beer’s Law holds for monochromatic light and dilute solutions (typically $A = 0.01{-}1.0$).

Beer’s Law Plot (Calibration Curve)

• Plot $A$ vs. $c$ at fixed $\lambda$.

• Linear region enables concentration determination of unknowns.

Biological / Analytical Applications

• Sperm counting, DNA/Protein quantification, vitamin C or anthocyanin assays in plants.

Ethical / Practical Implications

• Energy cost of distillation → incentive for greener separations.

• Selectivity of affinity methods yields high-purity biopharmaceuticals, benefiting medicine.

• Accurate spectroscopic quantification underpins quality control, clinical diagnostics.

Connections & Reinforcement

• Extraction often precedes chromatography to pre-clean samples.

• Chromatographic fractions frequently quantified via spectrophotometry (same cuvette path length $b$ appears in Beer’s equation and in chromatographic UV detectors).

• Ion-exchange & affinity columns are also used as SPE cartridges, blurring technique boundaries.

Key Equations (LaTeX-formatted)

• Distillation energy consideration qualitatively proportional to $Q = m c \Delta T$.

• Retention factor $R<em>f$: $R</em>f = \frac{d<em>{solute}}{d</em>{solvent\ front}}$.

• Beer’s Law: $A = \varepsilon b c$.

• Transmittance: $T = \frac{P}{P_0}, \quad A = -\log T$.