1. PUMPS USED IN HPLC, SIZE-
EXCLUSION, ION-EXCHANGE
CHROMATOGRAPHY
Presented by :-
Navneet Bhulli
M.Pharm 1st sem
PUMPS USED IN HPLC , SIZE-
EXCLUSION AND ION-
EXCHANGE
CHROMATOGRAPHY
Presented by :-
Navneet Bhulli
M.pharm 1st sem
2. CONTENTS
Introduction
HPLC
Pumps used in HPLC
Ion –exchange Chromatography
Pumps used in Ion-exchange
Size- exclusion Chromatography
Pumps used in Size-exclusion
3. INTRODUCTION
Chromatography is a technique by which mixture sample is
separated into its components by distribution into two or more
immiscible phases.
Chromatography was originally developed to isolate pigments
form plants, Hence, from Greek origin we get chromate, ‘color’ &
graph, ‘to record’.
It was invented by Mikhail Teswett in 1902 while doing a study of
the selective adsorption of leaf pigments on various adsorbents.
4. It consists of a stationary phase & a mobile phase. Components of
a mixture are carried through the stationary phase by the flow of a
mobile phase & the separations are based on differences in
migration rates of mobile phase components.
5. What are Pumps :-
Pump refers to the device that forces the mobile through a liquid
chromatography column at pressures up to 10,000 psi.
Pumps used in liquid chromatography are required to deliver very
constant liquid flows, free of pressure pulses.
Pressure pulses are one of the sources of detector noise.
The pressure pulses are reduced by employing carefully contoured
cams that drive the pistons
6. The cams are cut so that the liquid is delivered at a relatively
constant rate .
At the end of the piston delivery stroke, the cam is cut to allow a
relatively rapid piston return and refill.
Although this system reduces the pulsing effect, there remains
significant pressure pulses in the exit flow from the pump.
PUMP CARE :-
• Flush with water after running a buffer
• Replace seals in a timely manner.
• Maintain check valves.
• Do not allow solids in mobile phase.
7. Requirements of pumps :-
Capacity to withstand high load pressures.
Pulsations that accompany pressure fluctuations are small.
Flow rate does not fluctuate.
Solvent replacement should be easy.
The flow rate setting range should be wide and the flow
rate should be accurate.
8. Column chromatography rely on gravity or lower pressure pumping
systems for the supply of eluant to the column. It leads to lower flow
rates & high band broadening.
The use of faster flow rates is not possible as it can create pressure
sufficient enough to damage the matrix.
• HPLC is the most versatile & widely used type of elution
chromatography
• HPLC is a highly improved form of column chromatography. In this a
solvent is forced through under high pressures of up to 400
atmospheres. Makes it much faster.
HPLC :-
9. Criteria for HPLC Pumps :-
Constructed of materials inert toward solvents to be used.
Deliver high volumes (flow rates) of solvent (to 10 ml/min).
Deliver precise and accurate flow (<0.5% variation).
Deliver high pressure (to 10000 psi).
Deliver pulse free flow .
Have low pump-head volume .
Be reliable
11. Reciprocating piston pump :-
Currently used in 90% of commercially available HPLC systems
Usually consist of a small chamber in which the solvent is pumped by
the back/forth motion of a motor-driven piston.
Two ball check valves which open & close alternatively, control flow
of solvent into the and out of a cylinder.
The piston is driven by a stainless steel cam which forces the piston
into the chamber passing the solvent through the exit non-return valve.
Pistons are inert, generally made up of sapphire.
13. • Pumping capacity is : - 35 -400 µL per cycle.
• A wide range of flow rates are available by either varying the
stroke volume during each cycle or the stroke frequency.
• Since in this flow pulses are produced , some kind of pulse
dampers are required in the system.
Advantage: -
1. Small internal volume (35-400 L).
2. High output pressures(up to 10,000 psi).
3. Adaptability to gradient elution.
4. Constant flow rates .
Disadvantage:-
Pulse flow creates noise
--Forward Stroke ------ inlet check valve closes
and outlet check valve opens and M.P pump to the
flowing system
14. Syringe pumps
Large stainless steel motor driven hypodermic syringes.
They provide a very constant flow rate which was virtually
pulseless.
Pulse-less flow is achieved along with high pressure capability of
200 to 475 atm.
But required a frequent refilling process. Although the solvent
chamber has a finite capacity (250-500) ml before it must be
refilled.
15.
16. Piston driven motor forces the piston down into the reservoir
cylinder by means of a screw drive.
Mobile phase is forced out through a channel up the center of a
screw gear.
Advantages :-
1. Constant flow rate
2. Non – pulsating flow
Disadvantages :-
The separation is stopped while pump is re-filled.
17. Constant Pressure pumps
In these pumps the pressure from gas cylinder delivered through a
large piston drives the mobile phase.
Since the pressure of the solvent is proportional to the ratio of the
area of the two pistons (usually 30:1 or 50:1)
A low pressure gas source of 1-10 atm can be used to generate high
liquid pressure of 1-400 atm.
A valving arrangement permits the rapid refill of the solvent
chamber whose capacity is about 70 ml.
This system provides pulse-less & continuous pumping with high
flow rates . It is inconvenient for solvent gradient elution.
18. E.g. of HPLC Pumps :-
1. Waters 510 HPLC pump - Flow rate
0.1-9.9 ml/min,
Max pressure 6000 psi .
2. Waters 590 HPLC pump - Flow rate
0.5-45 ml/min,
Max pressure 5000 psi
19. Ion-Exchange Chromatography
In this the sample components are separated based upon attractive
ionic forces between molecules carrying charged groups of opposite
charge to those charges on the stationary phase.
Separations are made between a polar mobile liquid, usually water
containing salts or small amounts of alcohols, and a stationary phase
containing either acidic or basic fixed sites.
Ion exchange is probably the most frequently used technique for the
separation and purification of proteins, polypeptides, nucleic acids.
The reasons for success of ion- Exchange is widespread applicability,
high resolving power, high capacity and the simplicity of the method.
20. A pump delivers the mobile phase through the chromatographic
system. In general, Either single-piston or dual-piston pumps are
employed.
A pulse-free flow of the eluant is necessary for employing sensitive
UV/Vis and amperometeric detectors.
Therefore, pulse dampers are used with single-piston pumps and
a sophisticated electronic circuitry with dual-piston pumps
Mainly pump used in IEC is Single Piston High Pressure
Reciprocating Pump. Piston and cylinder are made from suitable inert
material
Pumps of IEC :-
21.
22. The piston is driven by a stainless steel cam which forces the piston
into the cylinder expressing the solvent through the exit non-return
valve.
After reaching the maximum movement, the piston returns as a
result of the pressure exerted by the return spring. During this
movement the cylinder is loaded with more solvent through the inlet
non-return valve.
The shape of the cam is cut to provide a linear movement of the
piston during movement of the solvent but a sudden return
movement on the refill stroke. In this way the pulse effect that results
from the refill action is reduced.
23. Size- exclusion Chromatography
The separation principle in size-exclusion chromatography (SEC) is
determined by the selective permeation of the polymers into and
out of the mobile-phase filled pores of the column packing.
The elution time of the polymer is governed by the time that it
expends in the pores; hence, larger molecules that expend less time
in the pores elute first, and smaller molecules elute later.
The principal factor affecting the size-exclusion chromatography of
a polymer is the hydrodynamic volume and not the molecular
weight.
24. Pumps used in SEC
• The most popular pumps used are the small volume, constant flow,
reciprocating pumps .
• It can provide accurately controlled flow rates of 1-15mL per min
against a column back pressure of up to 7250psi.
•The head of the pump contains a piston and a solvent chamber,
which can be of a very small volume having two check valves
mounted one on the other.
25.
26. • The main piston is withdrawn from the chamber, the valve to the
column open and allows mobile phase to enter the piston head.
• The inlet valve is now closed to prevent liquid from being drawn
out of the column.
• As the piston enters the chamber on the return stroke, valve to
the column opens and the pumps the mobile phase to the
column.
27. Comparison of Pumps :-
HPLC
SEC IEC
Pressure upto
10000 psi Upto 7200 psi Upto 1830 psi
Flow rate stability-
not more than 1%
Should be less
than 0.2%
Is 0.1 %
Flow rate range is
0.01-5ml/min It is 300cm/hr
0.1-4ml/min
