#10 -TLC and presumptive tests

Chromatography Theory

  • Chromatography: Method for separating mixtures with two phases—stationary and moving.

    • Stationary Phase: Remains fixed (e.g., silica gel).

    • Mobile Phase: Moves through the stationary phase (e.g., solvent).

  • Principle: Different solubilities and adsorption behavior of compounds leads to separation (adapted from Mohrig).

Thin Layer Chromatography (TLC)

  • Definition: A widely used method in drug identification.

  • Setup:

    • Utilizes a thin layer plate (plastic/silica coated).

    • Samples extracted and spotted on the plate.

  • Process:

    • The plate is placed in a solvent tank.

    • Solvent migrates upward by capillary action, separating sample components.

    • Visualization involves spraying with dye to reveal spots (e.g., THC in marijuana).

Applications of TLC

  • Suitable for identifying and comparing:

    • Drugs

    • Explosives

    • Inks

    • Biological samples (e.g., saliva, urine).

Solubility and TLC

  • Solubility: Amount of a solid substance dissolved in a liquid (solvent).

    • Common solvents for TLC: Organic liquids like alcohol/ether.

Adsorption Principles

  • Adsorption: Adherence of mixture components to the stationary phase.

    • Depends on molecule’s polar nature and localized charge.

Stationary Phase Characteristics

  • Typical materials: Silica gel and alumina, both polar.

    • Interactions:

      • Ion-dipole, dipole-dipole, hydrogen bonding, etc.

    • Polar Molecules: Strongly bind to adsorbents; weakly polar molecules move faster.

Mobile Phase Characteristics

  • Mobile Phase: Liquid containing sample for separation.

    • Crucial to select a suitable solvent; improper polarity results in lack of separation.

TLC Procedure Summary

  • Setup:

    • Prepare solvent, place TLC plates in the developing chamber.

    • Mark solvent front after it rises to near top.

  • Observation: Check under UV light to see separated compounds.

Retention Factor (Rf) Values

  • Rf Definition: Ratio of distance traveled by a substance to distance traveled by the solvent.

    • Rf Values range between 0.0 (no movement) to 1.0 (moved with solvent).

  • Example: If a compound travels 2.1 cm and solvent front travels 2.8 cm, Rf = 0.75.

Troubleshooting TLC

  • Common Issues:

    • Streaking: May indicate sample overload; dilute sample before re-running.

    • Smears: Strongly acidic/basic compounds may cause this; adjust eluting solvent.

    • Crooked solvent front: Ensure proper placement in developing jar.

    • No visible spots: Might indicate insufficient sample concentration or improper visualization method.

Presumptive Testing Overview

  • Color Spot Test: Mixing unknown substance with reagent produces color change.

    • Test Example: Cobalt Thiocyanate for cocaine results in blue change; Marquis test for heroin results in purple.

Presumptive Color Tests

  • Indicate presence of certain drug classes; classified as low discriminating power.

  • Portable test kits allow field use despite cost-effectiveness.

Presumptive Drug Tests

  • Initial screening tools to quickly identify drug involvement.

    • Less costly than definitive tests but can yield false positives/negatives.

Definitive Drug Tests

  • Highlight specific drug/metabolite presence.

    • High sensitivity and specificity through advanced lab technologies.

    • Useful for detecting various drugs that presumptive methods may miss, including new synthetic compounds.