Instrumentation
HPLC systems
HPLC instrumentation includes a pump, injector, column, detector and recorder or data system, connected as shown in the Figure below. The heart of the system is the column where separation occurs. Since the stationary phase is composed of micrometer size porous particles, a high pressure pump is required to move the mobile phase through the column.
The chromatographic process begins by injecting the solute onto the top of the column. Separation of components occurs as the analytes and mobile phase are pumped through the column. Eventually, each component elutes from the column as a narrow band (or peak) on the recorder. Detection of the eluting components is important, and this can be either selective or universal, depending upon the detector used. The response of the detector to each component is displayed on a chart recorder or computer screen and is known as a chromatogram. To collect, store and analyze the chromatographic data, computers, integrators, and other data processing equipment are frequently used.
Functional schematic of a modern HPLC instrument.
In this introductory part, we should familiarize ourselves with the various parts of the LC system; more detailed information will be found in the later chapters. Figure above is a sensitive map, by clicking on any part of the instrument you will be addressed to the certain topic.
Syringe-Type Pumps
Syringe-type pumps generally consist of a cylinder that holds the mobile phase which is expelled by a piston. The piston is advanced by a motor connected through worm gears, to provide smooth pulseless flow.
Syringe pumps have a number of advantages. Pressure capability is generally quite high (up to 78,000 psi) and maintenance is infrequent since there are no fluctuating check valves; gears are simple and strong.
The disadvantages of the system are the limited possibilities to form gradients and the limited reservoir capacity. Widely used in the early stages of HPLC, syringe pumps are now mostly used for SFC and microcolumn chromatography where the flow rates are 1 - 100 µl/min. The 20 ml reservoir capacity is more than enough for the whole day operation.
Two of these pumps can be easily combined and driven through an electronic system which provides mixed mobile phase and gradient-programming operation, or drive can be arranged synchronously so that one pump can be refilled while the other is operating in order to obtain continuous elution.
Reciprocating Piston Pumps
The basic principle of the reciprocating single piston pump is shown below. The piston expels liquid through a one-way valve (check valve). The pumping rate is usually adjusted by controlling the distance the piston retracts, thus limiting the amount of liquid pushed out by each stroke, or by the cam rotating speed.
Schematic of the reciprocating single piston pump. CAM is pushing a sapphire piston back and force. When the piston is moving backwards it sucks the eluent through the inlet check valve (on the bottom). The sapphire ball is lifted and opens the path for the eluent. When the piston moves forward, the liquid pushes the inlet ball down and closes the path, but the outlet ball is lifted and opens the outlet valve (upper).
The main disadvantage of this type of pump is sinusoidal pressure pulsations which lead to the necessity of using pulse dampers.
Dual Piston Pumps
A more efficient way to provide a constant and almost pulse free flow is the use of dual-headed reciprocating pumps. Both pump chambers are driven by the same motor through a common eccentric cam; this common drive allows one piston to pump while the other is refilling. As a result, the two flow-profiles overlap each other significantly reducing the pulsation downstream of the pump; this is visualized below. Since the acceleration/deceleration profile is somewhat non-linear, the more efficient types of these pumps use eccentricity-shaped cams to obtain the best overlapping of the pressure curves and to obtain smooth flow.
Schematic of a dual-head reciprocating pumps.
The advantages of this pump are the unlimited solvent reservoir allowing long-term unattended use and quick changeover and clean out capability. However, unless special care has been exercised in manufacture, these pumps may have several disadvantages. There is a tendency for the incompletely compensated pulsations to be observable at high refractive index detector sensitivities, especially at low flow rates where piston cycles are widely spread. Furthermore, since each head has two check valves, pump reliability depends on the cleanliness of the mobile phase and continued sealing capability of four check valves on each cycle, with cycles normally occurring several times per minute.
Recent improvements to this popular pumping system include:
 | A computer-designed camshaft is used to achieve maximum overlap of pump strokes, resulting in virtually undetectable pulsation or ripple. |
| Staggered inlet/outlet lines are employed to allow complete flushing when liquids are changed or if air is inadvertently drawn through the pump. |
| Small-volume check valves are used to allow the pumps to function reliably at flow rates as low as 0.001 mL/min. This has the added benefit of providing excellent gradient reproducibility especially when programs start from extremely low concentrations. |
| There are fewer moving parts, with all maintenance-requiring components (pump seals, check valves) readily accessible from the front of the instrument. |
| A wide flow rate range (0.01 to 10 ml/min) is provided without gear change. |
Check valves on the reciprocating pump are the most weak part. It may be easily contaminated or clogged which leads to the pump misfunction. Most of the recent HPLC instruments use improved dual piston pumps which have three or even two check valves.
The schematic of this pump is shown above. The first piston, called low pressure, is sucks the liquid from the reservoir while the second (high pressure piston) is supplying the eluent to the system. Then the first piston refills the second piston very fast, during 1/100 of the whole pump cycle. This scheme allows the use of only 3 check valve, one of which is working under low pressure. There are no cavitation effects. Because the piston volumes are small (~100 µl), pressure pulastions are small and sharp and easy to damp.
Another type of dual piston pump uses only two check valves, but piston volumes are different.
While the smaller piston dispenses an eluent in the HPLC system, the bigger piston is sucking an eluent. When pistons change their direction, the bigger piston simultaneously refill the smaller chamber and dispenses eluent into the system.
This set-up allows only two check valves for the dual piston pump.
