What is high pressure pump used in HPLC?
High-performance liquid chromatography (HPLC) is generally composed of an infusion system (pump), injection system, separation system (column), detection system, and data processing system. A pump is the most important equipment to ensure the normal operation of the HPLC instrument. The amount of separation is highly dependent on the ability of the infusion system to achieve a precise and stable flow rate. Accurate flow rates mean accurate retention times. Without reproducible flow and composition of the mobile phase, quantitative results cannot be given. The columns used in HPLC are very thin (1-6 mm) and the size of the stationary phase used is also very small (a few μm to tens of μm), so the mobile phase is subject to high resistance to flow in the column. At atmospheric pressure, the mobile phase flow rate is very slow, and the column efficiency is low and time-consuming. In order to achieve fast and efficient separation, a large pressure must be applied to the mobile phase to accelerate its flow rate in the column. For this purpose, a high-pressure pump must be used for high-pressure infusion.
High-pressure pump used in HPLC is to applies high pressure to the mobile phase to speed up its flow in the column. High pressure and high speed are one of the characteristics of HPLC.
High pressure pumps used in HPLC should meet the following conditions.
a. Constant flow rate, no pulsation, and a large adjustment range (generally 1~10 mL/min).
b. Resistant to solvent corrosion.
c. Having a high infusion pressure.
d. For general separation, a pressure of 60×10^5Pa is sufficient, while for high-efficiency separation, a pressure of 150~300×10^5Pa is required.
Working principle of high pressure pump used in HPLC
The
high pressure pump used in HPLC is one of the key components of the instrument and is divided into two types according to the principle. One is a constant flow pump, and the other is a constant pressure pump.
Regardless of how the resistance of the column changes, regardless of external influences, the flow rate of the mobile phase output from the constant-flow pump can remain basically unchanged, which is exactly the basic requirement for the work of the HPLC system.
The output of the constant pressure pump is basically a constant pressure. Under normal conditions the resistance of the HPLC system does not change, so constant pressure can achieve constant flow. However, as the column and temperature may vary, it may cause the flow rate to change.
Type of high pressure pump used in HPLC
| Pump used in HPLC | Description |
| Intermittent pump | Intermittent pumps can continuously deliver a certain volume of solvent until the solvent is exhausted, so they are also known as syringe pumps.
This type of pump is usually used when a high precision flow rate needs to be maintained. Intermittent pumps include syringe pumps and start replacement pumps. |
| Continuous pump | Continuous pumps are most commonly used and can continuously deliver solvents. Reciprocating piston pumps and hydraulic diaphragm pumps belong to this type of pump. |
| Double plunger parallel pump | A two-plunger parallel pump feeds the mobile phase continuously into the column. The pump operates with the two plungers 180 degrees out of phase, and the pulsation generated by the interaction of the two plungers reduces the total pulsation. While one plunger is working to feed the mobile phase into the column, the other plunger is working to draw the mobile phase into and fill the pump chamber. |
| Double plunger tandem pump | A dual plunger tandem pump has a 2:1 solvent in the primary and secondary pump cavities. When the secondary pump feeds the mobile phase into the column, the primary pump sucks liquid to fill the primary pump cavity. When the secondary pump cavity is emptied, the main pump not only infuses liquid to fill the secondary pump cavity but also feeds the mobile phase into the column. |
| Reciprocating piston pump/diaphragm pump | The reciprocating piston pump/diaphragm pump has the advantage of keeping the plunger out of contact with the harmful mobile phase during use and better plunger lubrication. One side of the stainless-steel membrane plating stores oil, while the other side is in contact with the mobile phase. When the reciprocating plunger drives the oil against the diaphragm, the mobile phase is fed into the column. When the plunger is retracted, the pressure disappears and the mobile phase enters the pump chamber on the west side of the diaphragm.
There is a ball valve in the reciprocating piston pump. The purpose of using a ball valve or check valve is to ensure that the mobile phase flows in one direction. The components of the ball valve include a sapphire gasket, a ruby ball, and a spring that provides tension. As the plunger moves to draw solvent from the mobile phase reservoir, the ruby ball at the inlet is pushed up by the spring, which allows the solvent to fill the solvent chamber. The ruby ball at the outlet is held against the gasket, which prevents the backflow of solvent that previously entered the column. As the plunger moves forward, the mobile phase pushes the ruby ball at the outlet of the gasket. At the same time, the mobile phase pushes the ruby ball at the inlet against the gasket to prevent the mobile phase from flowing back into the reservoir. |
The plunger in the pump is usually made of synthetic sapphire, which must be checked periodically for scratches. To maintain retention time and peak area reproducibility, the plunger seal must be replaced periodically.
Most infusion systems (
high pressure pumps used in HPLC) are equipped with an in-line filter or filter plug before the injector. This filter or screen takes into account particles generated by the wear of the pump seal, which prevents particles from damaging the injection valve or other components. It needs to be replaced periodically.
The purpose of the damper is to reduce the pressure pulsation caused by the pump operation. The damper consists of a diaphragm that separates the mobile phase from the compressible liquid and is located between the primary and secondary pump chambers in a dual plunger compensated pump.
Feature of high pressure pump used in HPLC
HPLC pump pressure features
A. High pressure resistance
High pressure resistance is one of the basic requirements of the HPLC system for high pressure pumps. Generally, the maximum working pressure of the infusion pump is 34.47-41.36Mpa (5000-6000psi) when the mobile phase flow rate is 1-3mL/min using a column with an average particle size of about 4µm and an inner diameter of about 5mm.
B. Good stability and small pulsation
The stability and pulsation size of the infusion high-pressure pump will directly affect the stability of the HPLC system (drift and repeatability), affect the noise of the system, and reduce the sensitivity. Maintaining stability and small pulsation is more important for HPLC systems using detectors such as electrochemical detectors and differentials, or for gradient separation. Therefore, the pressure pulsation of many current high-pressure pumps can be controlled to within 1%.
C. Continuous adjustable flow rate and wide flow range
At present, the flow range of the high-pressure pump of HPLC can reach 0.001-10.0mL/min, and the flow rate of preparative chromatography is even larger, sometimes requiring thousands of mL/min, which can simultaneously meet the analytical work requirements of various columns with different internal diameters.
D. Good flow rate repeatability
The flow repeatability of the high-pressure pump used in HPLC can reach 0.07% (RSD), but the repeatability is related to the test method.
a. Weight method
The outflow fluid per unit time of the pump is collected and weighed directly on the balance to judge the repeatability.
b. Retention time method
The repeatability of the flow rate is judged according to the length of the retention time.
In the analytical work of HPLC, its qualitative analysis is generally based on the retention time. The repeatability of the high-pressure pump (RSD) is one of the most important indicators, it is also a very important performance indicator that affects the repeatability of the entire HPLC system.
E. Small dead volume
The dead volume of the high-pressure pump affects the analytical precision and accuracy of HPLC, especially the effect of gradient elution. A small dead volume of the high-pressure pump is conducive to the rapid replacement of solvents and to the timely discharge of air bubbles from the system. Therefore, the smaller the dead volume, the better.
F. High flow rate accuracy
HPLC high-pressure pump flow rate accuracy is important because it directly affects the repeatability of analytical test data (reliability). The current domestic and foreign HPLC pump flow repeatability is generally around 1% (related to the test method), the best can reach 0.06%.
G. With gradient elution function
In the case of analytical work that cannot be solved by using isocratic elution, it is possible to solve the problem well by using the gradient elution method. Therefore, users of HPLC often need the instrument's high-pressure pump to have a flow rate program and a gradient elution program.
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