Hi I'm Gerard Sharp. Welcome to the February 2015 issue of the monthly newsletter. This month the Tech Tip is on How a HPLC Pump Works.

Dr Gerard Sharp...

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While the diagrams shown in this article are static, the online eLearning content (HPLC Harware) incorporates these diagrams as interactive animations with voiceovers explaining their function. Some of these interactive animations can be viewed for free by visiting this link.The HPLC Hardware' module is suitable for new users as well as anyone wanting to update their fundamental knowledge of HPLC.

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Tech Tip - How HPLC Pumps Work
 

Introduction

The aim of a modern HPLC pump is:

  • to produce a constant flow of solvent through the column regardless of the resistance encountered by the mobile phase
     
  • to maintain this constant flow regardless of variation in resistance (backpressure)
     
  • to change the proportion of solvents flowing to the column
     

If the pump consisted of a constant head pressure system then the flow would decrease as the resistance in the system increased. Resistance increase can be due to buildup of:

  • particulates produced by pump seal wear and tear which can block filters and connecting tubing
     
  • particulates in the mobile phase or sample due to inadequate filtering
     
  • breakdown of column material to smaller particles which can block frits or connecting tubing


A constant flow rate in HPLC is very important because there is much reliance on retention time matching between peaks in a sample and peaks in a standard. A change in the column flow rate will change retention times. Therefore the system has to be robust enough to withstand flow changes due to an increase in resistance in the system. The measure of this increase in resistance is the backpressure. This is the pressure which is generated behind (back) of the point of resistance when the pump is trying to maintain constant flow. Backpressure is one of the main diagnostics used in HPLC for system performance. The main type of pump used in HPLC is a reciprocating piston pump.

 

Single Piston Pump
 

A single piston reciprocating pump is shown here. Either a cam / cam shaft or a ball screw drive causes the piston to move back and forth and thereby either withdrawing solvent from a container or delivering solvent to the system. Two valves are needed on either side of the piston. The oulet check valve shown here is present to ensure that once the solvent is inside the chamber and the piston is returning to pump this solvent towards the column, there is no piston solvent which is returned to the mobile phase container. Additionally, the inlet check valve is present to ensure that once the solvent has been delivered to the column, this valve is closed to ensure no downstream solvent is pulled back into the system.

For single piston pumps, once the piston is pulling back solvent into the piston, there is no delivery to the column. Single piston pumps handle this deficiency in two ways. Firstly, by minimizing the time between dispensing flow to the system and refilling the piston. This is often simply achieved by very large springs which pull back the piston very quickly and thereby reduce the time between piston strokes. The second technique is to dampen the flow after the pump has dispensed the solvent and thereby the amount of ripple is minimized. Single piston pumps however are not suitable for pumping at low flows.

 

Dual Piston Pump - in parallel

 

A better, albeit a more expensive, solution is to introduce a second piston which is 180º out of phase with the first piston. As shown in this diagram, the second piston is dispensing solvent to the system while the first piston is pulling solvent into the pump. This results in a continuous flow to the system compared with a single piston pump. The relative amount of flow variation resulting from a difference in flow from the pump is known as the percentage ripple. Modern pumps usually have a specification of 2% maximum for this value i.e. 0.2mL variation in output flow in 1.0mL/min flow rate.

  

Dual Piston Pump - In Series

 

It is also common in modern dual piston HPLC pumps to have the pistons in series rather than in parallel. In this example shown here, the first piston (with the two valves attached) is twice the volume of the second piston. Thus, as the first piston is pushing solvent out, half of this solvent is dispensed to the system (through the purge valve) and the other half is dispensed to the second piston. Once the second piston chamber is full, this piston delivers the solvent to the system while the first piston has to fill the contents of its chamber by pulling back at twice the stroke speed of the second piston.

The advantage of the piston-in-series design is that only two check valves are needed, rather than four in a piston-in-parallel design. A dampener may be located between the two pistons.

 

Quaternary Pumps

 

It is also common in HPLC to have the pumps do the mixing of solvents as this is more reproducible than mixing off-line. Of the dual piston pumps, there are essentially two types available in HPLC - the binary pump and the quaternary pump. A quaternary pump consists of only one dual piston pump. Solvents from different bottles can be drawn into the pump through a proportioning valve system (shown here) before the pistons. Because the mixing occurs before the pump, this style is referred to as a low pressure mixing pump.

There are usually four valves able to choose from four different solvents hence the name. For the example described above, to achieve 50 / 50 mix of water and methanol, the valve for bottle A is opened 50% of time and then closed followed by the valve for bottle B opening. The combined valve opening time is defined by the time for one piston stroke.

 

Binary Pumps

 

The binary pump consists of two pumps each with two pistons. Each pump is capable of delivering a solvent to the system. If a HPLC method requires a mobile phase mix of 50% water and 50% methanol, this can be achieved by having one bottle of water on pump 1 and one bottle of methanol on pump 2. If each pump is running at the same speed, there will be equal flow delivered to the system i.e. a 50 / 50 mix. Because the mixing occurs after the pump, this style is referred to as a high pressure mixing pump.

Good mixing of the water and methanol needs to be carried out before delivery to the column. The ratio of the water and methanol can be changed during the analysis by changing the speed of the pistons from each pump. For example, in this case, changing the pistons speed on pump 1 to twice the speed of pump two will result in a 66:33 mix of water : methanol. The mixing ratio is precisely controlled through the software. The binary pump can be retrofitted with a selection valve on each pump which converts the binary solvent selection to quaternary solvent selection.


eLearning

 

Chromatography eLearning is yet another way to enhance your chromatography knowledge. While not ever being able to replace a classroom course, eLearning does offer advantages on cost and rich interactive content.  

Also, unlike a classroom course you can learn at your own pace and return to the content time after time.

Ardent Scientific offers varying levels of membership for viewing eLearning content. The Single Membership starts at $49 / year and corporate memberships are also available. View here for full details.

 

Sent by Ardent Scientific
P.O. Box 549, Box Hill Vic 3128, Australia ph 0422 872 212
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