Intro to Chromatography

Sports Drug Testing

  • Used in sports drug testing to detect banned substances

  • Involves:

    • Collection and storage of biological samples

    • Separation of analytes (drugs/metabolites) from the matrix

    • Detection of substances

      • Selectivity

      • Sensitivity

      • Confidence in the method(s)

Sample Preparation

  • Biological samples often complex and limited in volume/concentration

  • Urine preferred over blood:

    • Non-invasive

    • No trained staff required

    • Fewer health & safety issues

Stability & Storage Considerations

  • Sensitive to:

    • Light

    • Oxygen

    • Moisture

    • Temperature

Matrix Interference

  • Other compounds can interfere:

    • Salts

    • Proteins

    • Naturally occurring steroids

  • Requires sample preparation:

    • Liquid-liquid extraction (LLE)

    • Solid phase extraction (SPE)

    • Must assess recovery

Analysis

  • Chosen based on:

    • Nature of the sample

    • Type of required information

Chromatography - General Principles

  • Biological samples = mixtures → require separation

  • Common techniques:

    • Gas Chromatography (GC)

    • High-Performance Liquid Chromatography (HPLC)

  • Often coupled with detection methods:

    • GC-FID, GC-MS

    • HPLC-UV, HPLC-MS, HPLC-NMR

Basic Components

  • Stationary phase: Fixed medium

  • Mobile phase: Moves the analyte

  • Separation depends on interactions between:

    • The analyte

    • Stationary phase

    • Mobile phase

  • Interaction depends on:

    • Chemistry of molecule

    • Stationary phase chemistry

    • Mobile phase properties

Types of Chromatography

  • HPLC: Separates by hydrophobicity

  • Ion exchange chromatography: Separates by ionic charge

Thin Layer Chromatography (TLC)

  • Simplest chromatography method

  • Stationary phase: Powder on a plate (silica, alumina)

  • Mobile phase: Solvent moves by capillary action

  • Requires TLC tank with lid to saturate atmosphere

  • Components separate based on interaction strength:

    • Stronger interaction = travels less far

  • Retention factor (Rf) used for identification

    • Constant under standardised conditions

Stationary Phases

  • Silica (~90%) – acidic

    • Acidic compounds travel further (less retained)

  • Alumina – basic

    • Acidic compounds retained more strongly

Spot Visualization

  • Fluorescence under UV (254 nm)

  • Spray reagents:

    • Ninhydrin: Amines, amino acids, sugars

    • Sulfuric acid: Black carbon deposit

    • Marquis reagent: Alkaloids (e.g., morphine)

    • FPN: Phenothiazine drugs

TLC Limitations

  • Transfer losses

  • Contamination

  • Hard to integrate into on-line analysis

Other Chromatography Techniques

  • Offer better:

    • Control

    • Reproducibility

    • Sensitivity

    • Analytical information (depends on detector)

  • Chromatogram: Graph of instrument response vs. time