This document discusses different types of chromatography techniques and the pumps used in each. It covers high performance liquid chromatography (HPLC), ion-exchange chromatography, and size-exclusion chromatography. For HPLC, it describes reciprocating piston pumps that are able to deliver precise, pulse-free flow at high pressures up to 10,000 psi. For ion-exchange chromatography, it mentions pumps must provide pulse-free flow for sensitive detectors and single piston pumps are commonly used. Size-exclusion chromatography utilizes small volume reciprocating pumps for accurately controlled flow rates at pressures up to 7,250 psi.

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

10. Types of HPLC pumps :-
1. Reciprocating piston pump or
Constant flow pump
2. Syringe pump
3. Constant Pressure Pumps

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.

12. ---Backward Stroke--outlet check valve closes and
inlet check valves is refilled

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.

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 :-

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.

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

28. References
1. R.P.W. Scott , Liquid Chromatography
2. http://www.scribd.com/doc/7634222/HPLC
3. http://www.pdfchaser.com/pdf/hplc.html
4. http://media.wiley.com/product_data/excerpt/19
/35272870/3527287019.pdf
5. http://www.chromatography-online.org/ion-
chromatography