High-Performance Liquid Chromatography Plumbing

HPLC Plumbing

An important feature of HPLC instrumentation (see Figure 12.26) is the presence of several solvent reservoirs. As discussed in the previous section, controlling the mo- bile phase’s polarity plays an important role in improving a liquid chromatographic separation. The availability of several solvent reservoirs allows the mobile phase’s composition to be quickly and easily varied. This is essential when using a gradient elution, for which the mobile-phase composition is systematically changed from a weaker solvent to a stronger solvent.

Before they are used, mobile-phase solvents must be treated to remove dis- solved gases, such as N₂ and O₂, and small particulate matter, such as dust. Dis- solved gases often lead to the formation of gas bubbles when the mobile phase en- ters the detector, resulting in a distortion of the detector’s signal. Degassing is accomplished in several ways, but the most common are the use of a vacuum pump or sparging with an inert gas, such as He, which has a low solubility in the mobile phase. Particulate material capable of clogging the HPLC tubing or column is re- moved by filtering. If the instrument is not designed to do so, degassing and filter- ing can be completed before the solvents are placed in their reservoirs.

The mobile-phase solvents are pulled from their reservoirs by the action of a pump. Most HPLC instruments use a reciprocating pump consisting of a piston whose back-and-forth movement is capable both of maintaining a constant flow rate of up to several milliliters per minute and of obtaining the high output pressure needed to push the mobile phase through the chromatographic column. 

A solvent proportioning valve controls the mobile phase’s composition, making possible the necessary change in the mobile phase’s composition when using a gradient elution. The back and forth movement of a reciprocating pump results in a pulsed flow that contributes noise to the chromatogram. To eliminate this problem a pulse damper is placed at the outlet of the pump.