Adsorption Liquid Chromatography

Open Column Chromatography Silica gel Glass Tube Eluent Vial for fraction collection

Open Column Chromatography

Advantages Simple Cheap Recovery of the products No sample preparation Drawbacks Very slow (hours) Bad reproducibility (column is prepared by operator) Limited quality of separation No detector Exposure to silica gel and solvents

Flash Chromatography Glass columns are replaced with pre-packed plastic cartridges safer and more reproducible Solvent is pumped through the cartridge, possible elution gradient safer, quicker and more reproducible Detectors and fraction collectors Automated version of open column chromatography Rapid purification or collection of fractions

Closed Column Chromatography Objective Increased speed through the use of a pressurized mobile phase High Pressure Liquid Chromatography High Performance Liquid Chromatography Limited contact with air for unstable solutes and limited evaporation of the MP Two types of columns: Packed columnsCapillaries

Stationary phaseSilica gel, alumina, zirconia, titania Solid-Liquid chromatography Mobile phaseOrganic solvents (hydrocarbons to alcohols) Mixtures of these solvents SP is a solid Separation is due to a series of adsorption / desorption steps and polar interactions LC on adsorbents

The stationary phase Analytical Column length: cm Column internal diameter: 2 – 5 mm Preparative Column length: cm Column internal diameter: 1 – 50 mm

The stationary phase Spherically shaped particles Irregularly shaped particles Porous silica particle Pore size: from 60 to 500 Ǻ determines the specific surface area 5 μm

O H H O H H O H H O H H Free silanol group Bonded silanol groups Hydrated free silanol group O H H Highly hydrated silica gel Si O O O O O O O O O H O H O H O H O H O O H O H - Ionised silanol group The silica surface Hydrogen bonding

The mobile phase The pressure drop along the column is due to flow resistance The more viscous the mobile phase, the larger the pressure drop

MMM MMM MMM S MMM MM M MMM S Stationary phase M Mobile phase molecule S Solute molecule The solute diplaces the solvent molecules adsorbed on the stationary phase No interactions are supposed to occur between solute and mobile phase Snyder’s model for adsorption chromatography

Retention is controlled by:  Specific surface area of the SP  Activation of the SP (amount of water adsorbed)  Cross-section area of the MP and solute molecules  Adsorption energies of the MP and solute molecules Solute retention Saturated unsaturated aromatic ethers nitro esters alcools amines amides hydrocarbons Snyder’s model for adsorption chromatography

Solvent strength is a measure of relative solvent polarity (ability to displace a solute) Scales are based on silica or alumina Solvent strength Solvent polarity = eluting strength heptane cyclohexaneTHFdioxaneACNiProHMeOH

Apparatus Unlike GC, many HPLC systems have a modular design can simply add a new « box » to change / extend capabilities (autosampler, fraction collector, derivatisation unit, multiple detection…)

Apparatus All solvents should be « HPLC grade » (filtered with a 0.2 um filter) to extend pump life by preventing scoring. Reduces the chances of a column plugging Solvents should be degassed prior to use. This reduces the chances of bubbles being formed in the column or detector Solvent is generally delivered at constant flow rate

Example: separation according to hydrocarbon volume CompoundR 1 R 2 R 3 αCH 3 CH 3 CH 3 βCH 3 HCH 3 γHCH 3 CH 3 δHHCH 3 Tocopherols and tocotrienols α-tocopherol = Vitamin E Natural anti-oxidant capabilities Contained in most vegetable oils and biological fluids

Tocopherols and tocotrienols Sanagi et al., Analytica Chimica Acta, 538 (2005) NPLC separations of palm oil extract obtained by Soxhlet extraction using n-hexane as extraction solvent Stationary phase: Hypersil silica (200 x 4.6 mm, 5 μm) Mobile phase, n-hexane:1,4-dioxane (96.0:4.0 v/v); flow rate, 1 ml min−1; temperature, 40 °C. Peaks: (I) α-tocopherol, (II) α-tocotrienol, (IV) γ-tocotrienol, (V) δ-tocotrienol and (III and VI) unknown. Example: separation according to hydrocarbon volume

Tocopherols and tocotrienols Sanagi et al., Analytica Chimica Acta, 538 (2005) NPLC separations of palm oil extract obtained by Soxhlet extraction using n-hexane as extraction solvent Stationary phase: Hypersil silica (200 x 4.6 mm, 5 μm) Mobile phase, n-hexane:1,4-dioxane (96.0:4.0 v/v); flow rate, 1 ml min−1; temperature, 40 °C. Peaks: (I) α-tocopherol, (II) α-tocotrienol, (IV) γ-tocotrienol, (V) δ-tocotrienol and (III and VI) unknown. Example: separation according to number of double bonds

Example: separation of isomers Lycopene Major carotenoid pigment present in tomatoes Associated with a decreased risk of various types of cancer (prostate, breast…) Existence of numerous (E,Z) isomers possibly displaying different bioactivity all-E isomer Z-isomers

Example: separation of isomers Lycopene Froescheis et al., J. Chromatogr. B, 739 (2000) LiChroCart Alusphere Al 100 (250 x 4 mm, 5 μm) Hexane – CH 2 Cl 2 -dioxane gradient elution Diode-array UV-visible detection

Competition for adsorption sites between the solute and solvent molecules Interactions with the adsorbent Interactions between the solute and adsorbed solvent molecules Both solute and solvent are attracted to the polar sites of the stationary phase If solutes have differing degrees of attraction to the phase, a separation is possible The silica surface

Mobile phases consisting of mixtures of polar and dispersive solvents frequently produce surface bi-layers when used with silica gel as a stationary phase and therefore a far more complicated set of interactive possibilities exist. The silica surface

Solvent strength