Adsorption Liquid Chromatography
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AdsorptionLiquid Chromatography
Open Column Chromatography
Silica gel
Glass Tube
Eluent
Vial for fraction collection
Open Column Chromatography
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 ChromatographyHigh Performance Liquid Chromatography
Limited contact with air for unstable solutes and limited evaporation of the MP
Two types of columns:
Packed columns Capillaries
Stationary phase Silica gel, alumina, zirconia, titania
Solid-Liquid chromatography
Mobile phase Organic 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: 10 - 25 cm
Column internal diameter: 2 – 5 mm
Preparative
Column length: 2 - 50 cm
Column internal diameter: 1 – 50 mm
The stationary phase
Spherically shaped particles
Irregularly shaped particles
Porous silica particlePore 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
Bondedsilanol groups
Hydratedfree
silanol group
O
H
H
Highly hydrated silica gel
Si O Si Si OO Si O Si O Si O Si O Si O
O
H
O
H
O
H
O
H
O
H
O O
H
O
H-
Ionisedsilanol group
The silica surface
Hydrogen bonding
The mobile phase
The pressure drop along the column is due to flow resistanceThe more viscous the mobile phase, the larger the pressure drop
M M M
M M M
MMM
S
M M M
M M
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 cyclohexane THF dioxane ACN iProH MeOH
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
Compound R1 R2 R3
α CH3 CH3 CH3
β CH3 H CH3
γ H CH3 CH3
δ H H CH3
Tocopherols and tocotrienols
α-tocopherol = Vitamin E
Natural anti-oxidant capabilities
Contained in most vegetable oilsand biological fluids
Tocopherols and tocotrienols
Sanagi et al., Analytica Chimica Acta, 538 (2005) 71-76
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) 71-76
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) 291-299
LiChroCart Alusphere Al 100 (250 x 4 mm, 5 μm)Hexane – CH2Cl2-dioxane gradient elutionDiode-array UV-visible detection
Competition for adsorption sites between the solute and solvent moleculesInteractions 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
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