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Chapter 3
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
DR ZIAD W JARADAT
PROTEIN BIOTECHNOLOGY BT 452
Types
Adsorption Chromatography; Liquid-solid
• This chromatographic method is considered one of the
most used methods in protein purification and analysis.
It is mainly based on the following concepts;
– The interactions of the solute or mobile phase molecules
at the surface of a solid particle forms the basis of the
adsorption mechanism.
– The separation happened due to the competition
between the analyte and the solvent for the binding site
on the stationary phase.
– A solvent that elutes compounds more rapidly and
therefore, competes better for the chromatographic sites
is called strong solvent while the one that can not
compete well is called weak solvent.
DR. ZIAD W JARADAT
DR. ZIAD W JARADAT
Example
• Water is a strong solvent for silica adsorbents while
hexane is a weak solvent for silica adsorbents. Thus,
solvent strength can be adjusted by using mixtures of
these solvents.
• There are two types of adsorbents;
– Polar adsorbents such as silica or alumina
– None-polar adsorbents such as hexane and heptane.
• The polar adsorbents are the most widely used stationary
phases in liquid-solid chromatography. Retention of solute
(substance to be separated) on these phases increases
with increasing polarity and requires displacement of
adsorbed solvent molecules from the stationary phase.
DR. ZIAD W JARADAT
• Partition and bonded phase chromatography; Liquid-Liquid
• This method is based on the separation of solutes by the use of
difference in their distribution between a liquid mobile phase and a
liquid stationary phase coated onto a solid support.
•
• Chemical forces that exist between molecules can be used in this
type of separation including
• hydrogen bonds,
• van der Waals,
•
ion-ion interactions
• In classical partition chromatography a polar liquid such as β, β’oxydipropionitrile (ODPN) was coated onto the support particles
and hexane was used as a mobile phase.
DR. ZIAD W JARADAT
• Polarity
• The role of polarity is central to the interaction of molecules in the
liquid or gaseous state and the polarity is a major determinant
property in the overall chromatographic process.
• The physicochemical basis for polarity is the interaction of attractive
forces that exist between molecules. These attractive forces are;
• Dispersive (van der Waals forces); these are important only when
other forces are lacking.
• Dipolar
• Hydrogen-bonding
• Dielectric interactions or electrostatic interactions
DR. ZIAD W JARADAT
• The more polar molecules have strong dipoles, an
ionic character, which is the ability to form strong
hydrogen bonds or a combination of the three
forces together.
• The less polar (non-polar) have dispersive forces
as the primary basis for interaction with a very
weak ability to interact through dipolar, H-bonding
or electrostatic forces.
DR. ZIAD W JARADAT
DR. ZIAD W JARADAT
Solvent polarity and solvent strength
• Solvent strength in liquid chromatography is a measure
of the ability of the mobile phase to compete with the
solute molecules for active sites (i.e interaction or
attraction sites) on the stationary phase.
• For instance when the stationary phase is silica gel, the
active sites are the highly polar hydroxyl groups (Si-OH).
• Therefore, in this case solvent strength increases with
the solvent or mobile phase polarity.
• When the stationary phase is non-polar (e.g polydivinylbenzene) then the solvent strength decreases with the
increase in solvent or mobile phase polarity.
• The strength of a solvent is directly related to its polarity.
DR. ZIAD W JARADAT
• The overall degree of interactive forces of the solvent
is quantitated in the polarity index P’. There are some
tables available showing polarity index of certain
materials.
• Criteria for selecting an elution solvent
– The first important step in solvent selection is to
maximize solute solubility in a given solvent i.e the
solvent to be a good mobile phase should dissolve the
sample to be eluted over a wide range of
concentrations. This is highly affected by the solvent
polarity.
– Polarity can be adjusted by mixing two solutions with
different polarities so that we can obtain a mobile
phase with the desired strength. Example; 50% of a 0.4 polar solution mixed with 50 of a 4.3 polar solution
would be a 1.95 P.
DR. ZIAD W JARADAT
DR. ZIAD W JARADAT
CHROMATIC PHASES
• Normal Phase; when the stationary phase is more polar than the
mobile phase
• Reverse Phase; when the mobile phase is more polar than the
stationary phase
• Reverse phase chromatography; The silica gel is polar and to be
used for the reverse phase separation, its polar surface has to be
changed. This can be done by attaching different functional groups
such as hydrocarbons mostly C-8 and C18 (none polar).
• As a result we create a none polar phase. This type is used more
than the Normal Phase, and the reason why it is more popular is
that its weak mobile phase is the high polar water, therefore, the
samples are applied in this weak mobile phase i.e applied in
aqueous status such as biological compounds.
• This makes it especially attractive in clinical chemistry for drug
confirmation, amino acid analysis and hormone separations.
DR. ZIAD W JARADAT
• Normal phase chromatography; although silica is polar, yet some
other groups are attached to it to strengthen its polarity these
groups are; CN- , NO2-, NH2- .
• In the above two phases, because the C8 or C18 or the use of CN, NO2-, NH2- are bonded to the silica gel this is called bonded
phase.
• How can we chose which phase to use for a certain solute?
• The material of choice should be the one that give high retention of
separated components.
• So if we know that the separated material are polar, we will chose
the normal phase because likes attract likes and vice versa.
•
Although the Normal phase was the first type to be used, it is not
used as the Reverse Phase because in the normal phase the polar
material is used on the stationary phase which is found to retain
the compounds most strongly.
DR. ZIAD W JARADAT
• Disadvantages of the partition chromatography;
• The liquid stationary phase will eventually bleed from
the column
• Due to this bleeding, reproducibility get affected
• And short column life
DR. ZIAD W JARADAT
• The mobile phase can be;
• Isocratic system; the use of a constant mobile
phase composition to elute solutes.
• Gradient system; eluting with gradient starting
from weakest mobile solution to the strongest
mobile phase. This system enhances the
resolution especially if the two components to
be resolved are close to each other.
DR. ZIAD W JARADAT
• Example Figure 5-14.
• In this example a mixture of mono, di and triglycerides
of lauric acid will be separated using partition
chromatography. The silica gel stationary phase has a
monolayer of water strongly held by hydrogen
bonding.
• The solute molecules are partitioned between liquid
mobile phase (chloroform; methanol) and the water
monolayer.
• The most polar component, the monoglycerides are
retained most by the polar stationary phase
• The intermediate polarity components, the
diglycerides are retained to a much lesser degree
• Non-polar components such as triglycerides are not
retained and pass quickly.
DR. ZIAD W JARADAT
DR. ZIAD W JARADAT
• Please note that if a none-polar material was
attached to the silica gel such as the
hydrocarbon octadecyl or octyl groups ( this will
be reversed phase). And in this case the
mobile phase will be a highly polar material
such as water, methanol or a cetonitrile.
• In this case the most polar elutes first while the
less polar retained and the none polar retained
the last.
DR. ZIAD W JARADAT
DR. ZIAD W JARADAT
FPLC
• It is an alternative chromatographic system to
the HPLC. It means Fast Protein Liquid
Chromatography. This system employes
operating pressure significantly lower than
those used in conventional HPLC systems.
• Lower pressure allows the use of matrix beads
based on polymers such as agarose. The
columns are constructed of inert glass while
those for HPLC are made of stainless steel.
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DR. ZIAD W JARADAT
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DR. ZIAD W JARADAT
End of Chapter
DR. ZIAD W JARADAT