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