HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

1
Chapter 3

• HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

2
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.

3
(No Transcript)
4
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.

5

• 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 ß, ß- oxydipropionitrile (ODPN)
  was coated onto the support particles and hexane
  was used as a mobile phase.

6

• 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

7

• 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.

8
(No Transcript)
9
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
  polydivinyl-benzene) 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.

10

• 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.

11
(No Transcript)
12
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.

13

• 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.

14

• 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

15

• 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.

16

• 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.

17
(No Transcript)
18

• 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.

19
(No Transcript)
20
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.

21
(No Transcript)
22
(No Transcript)
23
(No Transcript)
24
(No Transcript)
25
(No Transcript)
26
(No Transcript)
27
(No Transcript)
28
(No Transcript)
29
(No Transcript)
30
(No Transcript)
31
(No Transcript)
32
(No Transcript)
33
(No Transcript)
34
(No Transcript)
35
End of Chapter