Pumps

1. PUMPS USED IN HPLC , SIZE-EXCLUSION AND ION-EXCHANGE CHROMATOGRAPHY Presented by :- Navneet Bhulli M.pharm 1 st sem

2. 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 :- P ump 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 . A t 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 :- Reciprocating piston pump or Constant flow pump Syringe pump Constant Pressure Pumps

11. Reciprocating piston pump :- C urrently used in 90% of commercially available HPLC systems U sually 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: - Small internal volume (35-400 L). High output pressures(up to 10,000 psi). Adaptability to gradient elution. 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. 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 :- Constant flow rate Non – pulsating flow Disadvantages :- The separation is stopped while pump is re-filled.

16. 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.

17. E.g. of HPLC Pumps :- Waters 510 HPLC pump - Flow rate 0.1-9.9 ml/min, Max pressure 6000 psi . Waters 590 HPLC pump - Fl ow rate 0.5-45 ml/min,  Max pressure 5000 psi 

18. 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 acid s. The reasons for success of ion- Exchange is widespread applicability, high resolving power, high capacity and the simplicity of the method.  

19. 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 M ainly pump used in IEC is Single Piston High Pressure Reciprocating Pump. Piston and cylinder are made from suitable inert material   Pumps of IEC :-

20. 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.

21. 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.

22. 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.

23. 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.

24. 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

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