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Basic Principals of
Chromatography
Concepts and Definitions
Chromatography is the most important separation method
for biomolecules.The outcome of a chromatography
experiment is a CHROMATOGRAM
Column chromatography
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Concepts and Definitions
Chromatography is the most important separation method
for biomolecules.The outcome of a chromatography
experiment is a CHROMATOGRAM
Planar chromatography
Why Chromatography?
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Why Chromatography?
Adaptable to wide range of compounds, because
variety of :separation principles (retention
mechanisms), and types of experimental setup
(planar or column - gas and liquid phase elution).
Why Chromatography?
Adaptable to wide range of compounds, because
variety of :separation principles (retention
mechanisms), and types of experimental setup
(planar or column - gas and liquid phase elution).
Separated analyte is immediately available for
identification or quantification Can be scaled
up for preparative use.
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Why Chromatography?
Adaptable to wide range of compounds, because
variety of :separation principles (retention
mechanisms), and types of experimental setup
(planar or column - gas and liquid phase elution).
Separated analyte is immediately available for
identification or quantification Can be scaled
up for preparative use.
WHY NOT? Some instrumentation expensive and
not easily portable Often need preliminary bench
"work-up" of sample to avoid column contamination
Examples of analytical applications Drug analysis -
therapeutic monitoring or abuse detection
Vitamins, hormones, specific peptides and proteins
Environmental pollution, pesticide residues etc
Basic Chromatographic
Principles
All chromatographic systems contain: A
stationary phase A mobile
phaseSample molecules (mixture for
separation)
- Movement of molecules determined by
the balance between two forces :
Impelling force of mobile phase carries
with it molecules for which it has affinity -
favoured by solubility (LC), volatility (GC)
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Factors which influence
chromatography
Theoretical Plates the number of
equilibrations that a compound makes
with the stationary phase.
Chromatography columns are
considered to consist of a number of
adjacent plates or zones where there
is enough space for a compound to
achieve complete equilibrium
between the mobile and stationary
phase. Each zone is called a
theoretical plate and the length of the
column the plate height. The more
theoretical plates the better the
resolution of protein. Consider three
columns each with a different number
of theoretical plates
Relative distribution on the column
Factors which influence
chromatography
Peak resolution- due to how you
elute the protein. Step vs gradient
(Shallow vs steep).
peak broadening - Due to pulsing
or size of matrix. Small beads =
sharp peaks (T Plates) but high
pressure.
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Factors which influence
chromatography
Column velocity
A slow flow rate will
increase the time a protein
is in the mobile phase and
diffusion occurs resulting in
peak broadening.
A high flow rate will
decrease the interaction of
the protein with the
stationary phase and
decrease the number of
possible equilibration,
resulting in a reduction of
theoretical plates possible.
A Van Deemter plot is a plot of plate
height vs. average linear velocity
of mobile phase. Such plots are of
considerable use in determining
the optimum mobile phase flow
rate
Retention Mechanisms
Overview of
five common
retention
mechanisms
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Partition Chromatography
Stationary phase = sorbed solvent held on the
surface, or within the grains or fibers of an inert solid
supporting matrix
Sample molecules equilibrate (PARTITION) between liquid stationary phase
and mobile phase. (Mobile phase is liquid in LC and HPLC systems and
gaseous in GLC systems). Retention depends on a sample molecule's
escaping tendency into the mobile phase versus its solubility in the
stationary phase.
K
D
=
Quantitatively given by the PARTITION
COEFFICIENT, KD, the ratio of solubilities in the
two phases
Solute in mobile phase
Solute in stationary
phase
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Examples of Partition
Chromatography Systems
Liquid chromatography systems based on partitioning include
paper chromatography, and thin-layer chromatography
(TLC) on polar matrixes such as silica gel and alumina.
The mobile phase is always a solvent mixture- one
component is polar (eg H2O) --> taken up by polar matrix to
form the stationary phase. Sample molecules soluble in this
solvent will be retarded - this component of solvent is the
retarder. - one component is non-polar (eg butanol) --->
travels around stationary phase.
Sample molecules soluble in this solvent don't spend time in
stationary phase -this component of mixture is the mover. -
sometimes a third component to maintain miscibility and
adjust pH (eg acetic acid).
Adsorption
Chromatography
Solute in liquid (or gas) phase interacts with
adsorption sites on solid surface (finely
divided particles for maximum surface area).
Suitable solids include HYDROXYAPATITE
(Ca3(PO4)2.Ca(OH)2), ALUMINA (Al2O3),
MAGNESIUM CARBONATE. Polar groups on
solid form dipolar interactions (eg hydrogen
bonds) with sample dissolved (usually) in organic
solvent. Elute by increasing polarity of the solvent
(eg if using acetonitrile CH3CN, add methanol
(CH3OH)) --> competing bonds with adsorption
sites.Gradient elution useful (also for ion-
exchange and partition chromatography)
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Ion-Exchange Chromatography
Retention by attraction between groups on stationary phase
with opposite charge to sample molecules. Stationary phase
= insoluble, but solvent permeable polymer matrix (eg
cellulose) chemically modified to introduce ionizable groups
(eg -COOH).
Elute by Change of pH to
neutralise charged group on either
solute or stationary phase.
Increase [salts] (especially
polyvalent) in eluant buffer -->
Displace by competing ions. pH
or salt gradient to enhance
separation. Ion-exchange media
are classified according to
whether the attached ionizable
group is strongly or weakly
acidic or basic --> determines
the usable pH range
Affinity Chromatography
Retention due to biospecific interaction using a ligand
molecule chemically coupled to a dextran or cellulose
matrix - Hence may be able to isolate analyte from
complex mixture.
Elution can be by
displacement with ligand
molecules in free solution.
But analyte then eluted as
complex with ligand.
Better to elute by change
of pH to weaken
binding.Chemistry of
ligand coupling to matrix
using cyanogen bromide.
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Affinity Chromatography
HPLC
High Performance (Pressure) Liquid Chromatography
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HPLC vs. FPLC
Components and Uses
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Std Routing
Gradient pump inlet
valves may be used to
select a different
buffer, to add
sanitation or storage
buffer at the end of a
run, or to load sample
directly through the
gradient pump. Any
valve may be
attached to gradient
pump inlets A or B.
Two examples are
shown. Use the
Manual screen valve
control to switch
between valve ports
and to prime each
valve position using a
syringe before
running a method
Sequential Binding and Elution
A sample is loaded
onto one column,
nonbinding proteins
are washed away,
and then a fraction
of eluted proteins is
diverted to a
second column.
This column is
washed, eluted,
and fractions
diverted to a third
column, which in
turn is washed and
eluted. Gradient
pump inlet valves
are used if different
buffers are needed
for equilibration
and elution of each
column.
Step 1
With valve 1 in
Purge position and
valves 2 and 3 in
Load position, the
gradient pump may
be primed with the
buffers from the
gradient pump inlet
valves.
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Sequential Binding and Elution
Step 2A (not shown)
Valve 1 is placed in Load position to load sample into the
loop. Valve 2 is in Inject position and valve 3 is in Load
position. Columns 2 and 3 are now bypassed.
Step 2B
Valves 1 changes to Inject position and sample is loaded
onto column 1.
Step 3
Valve 1 changes to Load position to wash column 1. The
flow goes to the fraction collector or to waste via the
fraction collector diverter valve.
Step 4
Valve 2 changes to Load position. The flow from column 1
passes through column 2 and then to the fraction collector.
The flow path is shown.
Step 5
Valve 1 changes to Purge position to bypass column 1.
With valves 2 and 3 in Inject position, the flow passes only
through column 2 and then to the fraction collector (or
waste via the fraction collector, as shown).
Step 6
Valve 3 changes to Load position.The flow passes through
column 2 and column 3. The flow path is shown.
Step 7
Valve 2 changes to Load position. Column 2 is now
bypassed.
Valve 3 changes to Inject position and column 3 is now
washed and eluted.
Tandem Chromatography
Tandem chromatography is used to automatically collect
selected fractions from one column and inject them onto a
second column. Eluted fractions from the first column are
collected into a DynaLoop or other sample loop. After injection
of the collected sample, it is eluted from the second column.
Gradient pump inlet valves are used if different buffers are
needed for equilibration and elution of each column. An example
of this application is the purification of monoclonal antibodies,
where column 1 is a cation exchanger and column 2 is ceramic
hydroxyapatite. This automated procedure can be performed at
neutral pH, as an alternative to affinity chromatography, which
requires low-pH buffers to elute the antibodies.
Step 1A (not shown)
The sample is loaded into the loop of valve 1 and injected onto
column 1. With the appropriate buffer selected from the
gradient pump inlet valves, column 1 is eluted. Valve 4 directs
the flow to the fraction collector for collection or diversion to
waste.
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Step 1B
At a predetermined time
or volume, valve 4
moves to position 2 and
a portion of the column
eluate is captured in the
loop/DynaLoop attached
to valve 2.
Step 2
Valve 1 moves to
Purge position to put
valve 2 and column 2
into the flow path.
Valves 3 and 4 change
position to connect
column 2 to the
detectors and fraction
collector. With the
appropriate buffer
selected from the
gradient pump inlet
valves, the sample
stored in the
loop/Dynaloop is
injected onto column 2
when valve 2 moves to
Inject position.
Step 3 (not shown)
Valve 2 reverts to
Load position and
column 2 is eluted.