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FPLC
All you need to know in the
available time!
FPLC
Fast Performance Liquid Chromatography- Liquid
chromatography is a term which refers to all
chromatographic methods in which the mobile phase is
liquid.
The stationary phase may be a liquid or a solid, in the form of a
matrix
A type of liquid chromatography where the solvent velocity
is controlled by pumps. The pumps control the constant
flow rate of the solvents
FPLC
The solvents are accessed through tubing from an outside
reservoir.
The flow rate of the solvent is set through computer input
and controlled by pumps.
There are various columns used in liquid chromatography
depending on the type of separation preferred. Each column
contains a small diameter packing material. The column is a
large (mm id) tube containing small (u) particles (gel beads)
known as stationary phase.
FPLC
The chromatographic bed is composed by the gel beads
alone when they are inside the column.
The sample is introduced into the injector and then carried
into the column by the flowing solvent.
Once in the column, the sample mixture separates as a result
of different components adhering to or diffusing into the
gel.
FPLC
As the solvents is forced into the chromatographic bed by
the flow rate, the sample separates into various zones of
sample components. These zones are referred to as bands.
Biomolecules have various characteristics such as molecular
weight, electric charge, and hydrophobicity.
As so, purification of the object is usually achieved by using
a combination of chromatographic methods
So, what can you do with a
FPLC?
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Answer- everything!!!!!!!
Only difference with FPLC and HPLC is pressure
HPLC allows for smaller column matrix due to the
increased pressure.
Remember the The Van Deemter Equation?
Allows solute to equilibrate between mobile and stationary phase
faster
HPLC is a better choice for analytical analysis.
Applications
Ion Exchange chromatography
Separates compounds based on their net charges. Negatively
or positively charged functional groups are covalently
bound to a solid support, yielding either a cation or anion
exchanger, respectively.
Hydroxyapatite chromatography
(Ca
5
(PO
4
)
3
(OH)
2
is a form of calcium phosphate that can be
used for the separation and purification of proteins,
enzymes, nucleic acids, viruses, and other macromolecules.
Applications
Affinity chromatography
A ligand is covalently bound to a solid support that is
packed into a chromatography column.
A mixture of components is then applied to the column. The
unbound contaminants, which have no affinity for the
ligand, are washed through the column, leaving the desired
component (protein, peptide, DNA fragment, etc.) bound to
the support.
Elution is accomplished by changing the pH and/or salt
concentration, or by applying organic solvents or a
molecule that competes for the bound ligand.
Applications
Reverse-Flow Chromatography for Affinity
Purifications
Especially useful when purifying antibodies using Protein A
or other affinity columns.
The protein is bound at the top of the column and
nonbinding material is washed out.
Flow to the column is then reversed and the bound proteins
elute from the top of the column in very concentrated form,
which helps prevent denaturation.
Applications
Size exclusion chromatography (SEC)
Also known as gel filtration chromatography
The gel medium consists of spherical beads containing
pores of a specific size distribution. Separation occurs when
molecules of different sizes are either included or excluded
from the pores within the gel support.
Small molecules diffuse into the gel pores and their flow
through the column is retarded, while large molecules do
not enter the pores and are rapidly eluted in the column's
void volume.
Applications
Size exclusion chromatography (SEC)
Consequently, molecules separate based on their size as
they pass through the column and are eluted in order of
decreasing molecular weight.
Common applications include fractionation and molecular
weight determination of proteins, nucleic acid separation,
plasmid purification, and polysaccharide fractionation.
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Applications
Size exclusion chromatography (SEC)
Resolution depends on particle size, pore size, flow rate,
column length and diameter, and sample volume.
Generally, the highest resolution is obtained with low flow
rates (210 cm/hr), long, narrow columns, small-particle-
size gels, small sample volumes (15% of the total bed
volume), and a sample viscosity that is the same as the
eluent.
Applications
Hydrophobic interaction chromatography (HIC)
Separates molecules based on their hydrophobicity.
Sample molecules containing hydrophobic and hydrophilic regions
are applied to an HIC column in a high-salt buffer.
The salt in the buffer reduces the solvation of sample solutes. As
solvation decreases, hydrophobic regions that become exposed are
adsorbed by the medium.
The more hydrophobic the molecule, the less salt needed to promote
binding.
Usually a decreasing salt gradient is used to elute samples from the column in
order of increasing hydrophobicity.
Applications
Immobilized metal ion affinity chromatography
(IMAC)
Most widely used nickel: also zinc and cobalt
Nickel binds molecules rich in electrons such as histidine
(from the His-tag fame!)
Consists of string of 6-10 histidine residues. Serve as a metal
binding site
IMAC uses the affinity of histidines imidazole side chains
for metal ions
Applications
Immobilized metal ion affinity chromatography
(IMAC)
The His-tag allows the protein to bind to the column beads
Note proteins with a naturally high histidine residues will also
bind
The elution buffer (containing imidazole) is then added to
the column
Competes with nickel for bound ligands and collection in
fractions
Applications
Immobilized metal ion affinity chromatography
(IMAC)
THIS IS WHAT YOU WILL BE
DOING!!!!!!!!!!!
Bio-Rad Biologic Duoflow
system
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Bio-Rad Biologic Duoflow system
1. UV Detector With
Conductivity Monitor
2. SV5-4 select valve
4. AVR9-8 switching valves
7. F40 workstation
8. BioFrac fraction collector
9. AVR 7-3 sample inject
valve
UV Detector With Conductivity Monitor
Standard 254 and 280 nm filters
Replaceable mercury lamp
214 nm expansion kit with zinc
lamp
365, 405, 436, and 546 nm
expansion filters for the mercury
lamp
Analytical 5 mm flow all UV
absorbance range from 0.0001 to
2.0 OD
Conductivity detection range
from 1 to 500 mS/cm
Conductivity
NaCl is an electrolyte
Becomes sodium ions (Na+) and chlorine ions (Cl-), each of
which is a corpuscle that conducts electricity.
IONS from the Greek word meaning wonderer
This means that the more Na+ and Cl- contained in water
the more electricity is carried, and the higher the
conductivity
Bio-Rad Biologic Duoflow system
1. UV Detector With
Conductivity Monitor
2. SV5-4 select valve
4. AVR9-8 switching valves
7. F10 workstation
8. BioFrac fraction collector
9. AVR 7-3 sample inject
valve
SV5-4 select valve
The SV5-4 valve is a low pressure,
5-port, 4-position valve used for
automatic buffer selection and
sample loading.
The SV5-4 valve may be used for:
Buffer and/or sample selection
when placed before the Workstation
pumps.
enables access to four separate
solutions
Purging or rinsing all tubing lines
for cleaning purposes
Bio-Rad Biologic Duoflow system
1. UV Detector With
Conductivity Monitor
2. SV5-4 select valve
4. AVR9-8 switching valves
7. F10 workstation
8. BioFrac fraction collector
9. AVR 7-3 sample inject
valve
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F10 workstation
The BioLogic DuoFlow workstation has a F10 pump,
Max flow rate of 10 ml/min
Pressure: 3,500 psi (233 bar, 23 MPa).
Bio-Rad Biologic Duoflow system
1. UV Detector With
Conductivity Monitor
2. SV5-4 select valve
4. AVR9-8 switching valves
7. F10 workstation
8. BioFrac fraction collector
9. AVR 7-3 sample inject
valve
AVR 7-3 sample inject valve
The Load position is for filling a
loop with sample or for running
buffer through a column
The Inject position is for
injecting contents of the sample
loop onto a column;
The Purge position is for
priming the DuoFlow
workstation gradient pump and
purging lines to waste without
removing the column from the
system.
The Mixer
The mixer ensures that the
buffers used are in the
correct proportion relative
to each other during the
course of the FPLC run
Comes into its own as the
gradient increases!
Other interesting components
not here at MSUM
Bio-Rad Biologic Duoflow system
1 QuadTec UV/Vis
detector
Monitor off-peak
wavelengths such as 245 nm
(instead of 280 nm, where
buffer components like
Triton X-100 absorb
strongly).
Can simultaneously monitor
at 224 nm for greater protein
sensitivity without
background interference.
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MAXIMIZER
5) The Maximizer enables buffer blending applications, doubles
pump flow rates, and doubles valving capacity to 6 low pressure
valves and 6 high pressure valves.
Proportioning valves on the Maximizer blend water, salt, and the
conjugate acid and base of a buffer to obtain a solution with a user-
defined pH and salt concentration.
MAXIMIZER
For example, after the user selects the desired pH for each
separation, the software determines the required acid/base ratio and
during the run compensates for changes in ionic strength and
temperature.
This provides tremendous convenience by enabling multiple
unattended runs, each at a different buffer pH and salt concentration.
What you need to do before
you load your sample
Before you load your sample
Degassing:
One major problem with pressurizing chromatography
systems using liquid solvents is that pressure reductions can
cause dissolved gases to come out of solution.
The two locations where this occurs are:
the suction side of the pump (which is not self-priming,
consequently a gas bubble can sit in the pump and flow is
reduced ).
the column outlet (where the bubbles then pass through
the detector causing spurious signals).
Before you load your sample
Degassing:
The problem is usually restricted to solvents that have
relatively high gas solubilities - usually involving an
aqueous component, especially if a gradient is involved
where the water/organic solvent ratio is changing.
As water usually has a higher dissolved gas content, then a
gradient programme may cause the gases to come out of
solution as the mobile phase components mix.
Before you load your sample
Degassing:
There are two traditional strategies used to remove problem
dissolved gases from chromatographic eluants. Often they
are used in combination to lower the dissolved gases.
Subject the solvent to vacuum for 5-10 mins. to remove the gases.
Subject the solvent to ultrasonics for 10-15 mins. to remove the
gases.
Note that most aqueous-based solvents usually have to be degassed
every 24 hours. Also remember that solubility of gases increases as
temperature decreases, so ensure eluants are at instrument
temperature prior to degassing.
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Degassing
The Backpressure Regulator
helps eliminate bubble information
within the detector.
As a solution is pumped through a
column, the column exerts a
backpressure that serves to keep any
air bubbles in solution.
Solution exiting the column returns to
atmospheric pressure and air bubbles
may form.
Degassing
As the bubbles pass through, or
lodge in the detector flow cell they
may cause artifacts on the baseline
chromatogram that appear as spikes.
This outgassing may be
minimized by thoroughly degassing
buffers and by placing a
backpressure regulator after the
Conductivity monitor.
The backpressure from the regulator
helps to keep the bubbles in solution
Degassing
The 40 psi backpressure regulator is
used with flow rates below 10
ml/min.
Plumb the backpressure regulator
following the direction of the arrow.
When using low pressure columns
such as an Econo-Pac cartridge,
plumb the 40 psi backpressure
regulator between the Workstation
pump outlet and the mixer. This aids
in seating the check valves,
preventing permanent damage to the
cartridge or column
Prime the FPLC pumps
In the manual mode, prime the FPLC pumps and wash the buffers
through the hoses.
Do this in the PURGE mode for the injection solenoid
Prime the FPLC pumps
Be careful not to let the last persons buffer contaminate yours. You
will want to flush the sample loop to remove water or old samples.
Look at the routing displayed on the sticker on the solenoid and
make sure you know what is going where.
What happens when you
load your sample
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AVR 7-3 sample inject valve
AVR7-3 in Load Position
(Figure A)
The column may be
equilibrated or eluted.
Sample is loaded into the
loop. If the loop is
overfilled, the sample runs
to waste.
Never remove the syringe
before the sample is
injected onto the column
AVR 7-3 sample inject valve
AVR7-3 in Inject
Position (Figure B)
The sample is injected
onto the column
AVR 7-3 sample inject valve
AVR7-3 in Purge
Position (Figure C)
Flow from the gradient
pump goes directly to
waste, bypassing the
column.
Basic AVR7-3 Sample Injection Valve
Operation using Load, Inject, and Purge
Positions
Load position is for filling a loop with sample or for
running buffer through a column.
Inject position is for injecting contents of the sample loop
onto a column.
Purge position is for priming the Duo-Flow workstation
gradient pump and purging lines to waste without removing
the column from the system.
Look at all the bloody
tubing!!!!!
The bloody machine looks like a Borg!
Yes, The FPLC
does look like a
Borg!!!!
However, do not panic!
Remember to look over
All of the instructions, follow
the lines and you will see
how it all works!
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Computer control Computer control
Flow Rate: The flow rate for washing
and eluting can be around 1.0 2.0
ml/min. Experiment with elution flow
rates. Equilibration with the
equilibration buffer can be performed at
the same flow rate
Fractions: Typically, I will collect
0.5-1.5 ml fractions for a 1 ml column
and a 1-2.5 ml fractions for a 2-5 ml
column. Collect when you want, the
whole thing or just specific fractions.
Computer control
SV5-4: Low pressure solenoid valve
used for preparative buffer selection.
The buffer name will appear in the
protocol screens isocratic flow,
linear flow, or change value dialog
box
SVT3-2: Low pressure solenoid valve
used as fraction collection diverter
1 Waste
2 Collect
AVR7-3: High pressure valve used to
inject sample
L load
I inject
P - purge
Making your method
Pick your available devices:
Fraction collector
Injection valve
Buffer blender for gradient
Detector
Ensure that valves and ports
match up!
Making your Protocol
You have to make your
protocol up by adding steps
1 turn lamp on
Isocratic flow to prime the
column
Zero baseline
Injection
Which loop or direct
inject?
What gradient?
Buffer %s?
Fraction collector!!!!!!
Type of injection
Static Loop: Standard fixed volume loop for sample
loading. Used with the AVR7-3 valve.
Sample can be loaded with a syringe through port 2,
Use Fill Before Inject and Rinse After Inject to select
loop fill volume and flow rate.
Refer to Chapter 8 for discussion of how to select volume
fill and rinse.
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Type of injection
Dynamic Loop: Uses Bio-Rads DynaLoop or other sliding-piston
sample loop. Both partial loop and full loop injections can be
programmed.
The dynamic loop can be filled manually through Inject valve port 2.
Use Fill Before Inject to select loop fill volume and flow rate. The
Rinse function is not available.
See Chapter 8 for DynaLoop loading
Type of injection
Direct Inject: Allows direct injection of sample through
either the Workstation pump or auxiliary load pump.
Pre-pump valves (such as the SVT3-2, SV5-4, or AVR9-8)
automate direct injection.
Refer to Chapter 8 for discussion of a direct inject
applications.
Have fun collecting your Data!