image of solvent reservoirs

Solvent choice:

The mobile phase is used to transport the sample components through the column over the surface of the stationary phase. In normal phase hplc, it would typically be a non-polar solvent or mixture of solvents, such as hexane, cyclohexane, toluene, or dichloromethane. In reverse phase hplc, it is typically either one of, or a mixture of two or more of, the following: water, an aqueous buffer, methanol, acetonitrile, or tetrahydrofuran (THF).

The exact choice of mobile phase depends on the nature of both the sample components to be separated and the column stationary phase. Ion chromatography, for example, often uses an aqueous buffer, while organic compounds are commonly separated using water-methanol or water-acetonitrile mixtures.

Sparging:

It is important to remove any dissolved gas from the mobile phase before it is pumped into the system. This prevents bubble formation that might damage the column and degrade system performance. Most systems provide a means to pass helium through the solvent reservoirs in a process called “sparging”. This strips out any dissolved gas, but helium’s low solubility and inert nature prevent it from being a problem itself. The image above shows this procedure being performed on all four reservoirs.

Continue the tour:

HPLC:   solvents →  pump →  injector →  column →  detector