Adsorption
Liquid Chromatography
Open Column Chromatography
Eluent
Silica gel
Glass Tube
Vial for fraction
collection
Open Column Chromatography
Open Column Chromatography
Advantages
Drawbacks
Simple
Very slow (hours)
Cheap
Bad reproducibility
(column is prepared by operator)
Recovery of the products
Limited quality of separation
No sample preparation
No detector
Exposure to silica gel and solvents
Flash Chromatography
Automated version of open column
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
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 columns
Capillaries
LC on adsorbents
Solid-Liquid chromatography
SP is a solid
Separation is due to a series of
adsorption / desorption steps
and polar interactions
Stationary phase
Silica gel, alumina, zirconia, titania
Mobile phase
Organic solvents (hydrocarbons to alcohols)
Mixtures of these solvents
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 particle
Pore size: from 60 to 500 Ǻ
determines the specific surface area
5 μm
The silica surface
H
O
H
H
H
H
O
H
H
O
Si
Free
silanol
group
O
H
H
O
O
Si O
Si
Bonded
silanol
groups
Hydrogen bonding
H
H
O
O
O
Si
-O
O
Hydrated
free
silanol
group
Si
O
Si
Ionised
silanol
group
H
O
Si
O
O
O
O
H
H
H
H
O
O
Si
Highly
hydrated
silica gel
O
H
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
Snyder’s model for adsorption chromatography
S
M
M
M
M
M
M
M
M
M
M
S
M
M
M
M
M
M
M
M
Stationary phase
M
S
Mobile phase molecule
The solute diplaces the solvent molecules
adsorbed on the stationary phase
Solute molecule
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
Solvent strength
Solvent strength is a measure of relative solvent polarity
(ability to displace a solute)
Scales are based on silica or alumina
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
Tocopherols and tocotrienols
α-tocopherol = Vitamin E
Natural anti-oxidant capabilities
Contained in most vegetable oils
and biological fluids
Compound
α
β
γ
δ
R1
CH3
CH3
H
H
R2
CH3
H
CH3
H
R3
CH3
CH3
CH3
CH3
Example: separation according to hydrocarbon volume
Tocopherols and tocotrienols
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.
Sanagi et al., Analytica Chimica Acta, 538 (2005) 71-76
Example: separation according to number of double bonds
Tocopherols and tocotrienols
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.
Sanagi et al., Analytica Chimica Acta, 538 (2005) 71-76
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
LiChroCart Alusphere Al 100 (250 x 4 mm, 5 μm)
Hexane – CH2Cl2-dioxane gradient elution
Diode-array UV-visible detection
Froescheis et al., J. Chromatogr. B, 739 (2000) 291-299
The silica surface
Interactions between the solute
and adsorbed solvent molecules
Competition for adsorption sites
between the solute and solvent
molecules
Interactions with the adsorbent
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.
Solvent strength