Chromatography Techniques

Chromatography Overview

  • Collective term for laboratory techniques to separate mixtures.

  • Involves mobile phase and stationary phase for isolation.

  • Types: Preparative (purification; isolation at a scale) and Analytical (measuring presence/amounts; very small amounts).

Key Terms in Chromatography

  • Analyte: Substance to be separated

  • Chromatogram: Visual output of chromatograph showing peaks for components.

  • Chromatograph: instrument used to carry out chromatographic separation

  • Retention time: Time for analyte to pass through system.

  • Mobile Phase: Phase that moves (liquid, gas, supercritical fluid).

    • Effluent: the mobile phase leaving the column.

  • Stationary Phase: Fixed phase in the chromatography setup.

    • Bonded Phase: is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing.

Thin Layer Chromatography (TLC)

  • Uses a flat carrier with a thin layer of adsorbent (e.g., silica: VERY POLAR, cellulose).

  • Mixture spotted on the plate and solvent rises by capillary action.

  • Components separate based on solubility and adsorption strength.

  • Retention Factor (Rf): Ratio of distance traveled by compound to solvent front.

    •    Constant under same conditions; higher Rf indicates less polarity. The most polar thing is the adsorbent.

    • The Elution profile.

    • Tailing means that other things are present, when you change the solvent, there is a possibility of the effluent changing your results.

CASE STUDY: TLC in Forensic Science—Food Adulteration

Food fraud: Finnish Customs seize 950g of tainted carcinogenic spices: Spices were incorrectly labeled and contained harmful substances. The purpose is for filler and color enhancement.

Synthetic Dyes and Palm Oils: The Palm nut has two distinct parts: the outer oil and the kernel oil.

Turmeric: Lead chromate pigments added to turmeric threaten public health.

Synthetic Dyes

  • Azo Dyes: Functional group R-N=R’. R and R’ are usually aryl

    • Many are non-toxic. However, different countries have different rules regarding the use of food dye. A dye may be approved in one country but not in other countries.

    • Tartrazine: Found on Nacho Cheese Doritos

    • Sudan Dyes: Very common in International Retail stores.

Gas Chromatography (GC)

  • Uses inert carrier gas (e.g., He, N2) and liquid stationary phase in a column.

  • Separates complex mixtures based on rate of passage influenced by interactions with stationary phase.

  • Detection based on retention times and order of elution.

GC/MS

  • GC/MS is a great test because it is both a category a (MS) and category b (GC). This is important for identifying and quantifying the components within a sample, providing valuable data for chemical analysis.

  • Three types of Mass Separators:

    • Quadrupole

    • Ion trap

    • Ion Detector

Separation Process in GC

  • Volatilization

    • Limitations: Too Heavy and too polar substances do not work.

  • Sample injected using micro-syringe; analytes move with carrier gas but also adsorb to stationary phase.

  • Separation efficiency increases with careful control of temperature and sample volume.

  • Carrier Gas must be chemically inert. Commonly N2 or He.

  • Injection: IMPORTANCE IS THERE ARE SPECIFIC PARAMETERS NOT THE PARAMETERS THEMSELVES.

  • Split/Splitless Injector; Cool on-column inlet; purge and trap system; solid-phase microextraction.

GC Column Types

  • Packed Columns: 1.5-10 m length; larger diameter; used with solid support material.

  • Capillary Columns: Smaller internal diameter (tenths of mm); more efficient; usually 25-60 m long.

Column Temperature Considerations

  • Temperature affects adsorption and sample flow rate; careful control is crucial.

  • Isothermal methods vs. temperature programming (ramping).

Detection in GC

  • Various detectors: selectivity based on response types (non-selective, selective, specific).

  • Common detectors include FID (destructive), TCD (universal), ECD (for specific compounds).

  • GC/MS: Combines GC with mass spectrometry for detailed analyte identification and quantification.