High performance liquid chromatography (HPLC) is a very common technique in analytical chemistry. It is used to separate different substances based on their different physical and chemical properties (polar/apolar, basic/acid pH, high/low molecular weight).
A small volume of the sample (a few µl) is injected in the flow of the mobile phase. The injected sample is transported into the analytical column where chemical and physical interactions take place with the stationary phase. The different compounds present in the sample remain in the column for different durations and are eluted at times which are specific for each compound (these times are called retention times).
HPLC relies on relatively fast flows (0.1 ml/min – 2-3 ml/min) and uses analytical columns packed with 2 –5 µm particles, leading to a loss of pressure of several dozens if not hundreds of bar. Under such conditions, linear velocity is high (2-10 mm/s) and diffusion is low. This, in turn, leads to increased resolution (separation) of all the compounds making up the mixture.
Solvents for HPLC analysis are mixtures of water and various organic liquids (methanol, acetonitrile, etc.). The water may include different salts and buffers.
The composition of the mobile phase may change during a run. Such a mode is called «gradient» or «graduated elution», in contrast to the «isocratic» mode where the composition of the mobile phase remains the same throughout the analysis. For example, in reversed HPLC, the stationary phase is apolar and the mobile phase is made up of water and methanol. In such as column, hydrophobic compounds are eluted at higher concentrations of methanol while hydrophilic substances are eluted at lower concentrations of methanol.
Main stationary phases
Normal phase columns contain a stationary phase that is hydrophilic and acid and an eluent that is apolar (such as hexane). This type of chromatography is historically the first one to have been used in HPLC.
The most common normal phase is made of a silica gel base with silanol (-OH) groups on its surface.
Normal phase HPLC is used to analyze polar compounds. At the same time, in the 1970s, normal phase started being replaced by reversed phase HPLC.
Normal phase has been upgraded recently to yield HILIC (hydrophilic interaction chromatography), a similar methods particularly well suited for analyzing strongly polar compounds.
The starting material of a reversed phase is a normal phase with alkyl chains bonded to the silanol groups (or other grafts, depending on the desired polarity). The stationary phase is usually composed of small silica particles with grafted chemical groups, commonly 8 or 18 carbon alkyl chains. This modification transforms the initial polar, hydrophilic phase (that lacks the «grafts») into an apolar and hydrophobic phase which is then called «reversed».
The pump is of major importance in HPLC analysis. Indeed, the pump allows to vary the composition of the mobile phase during a run. As many as 4 different solvents may be combined by a specific type of pump, called a quaternary pump.
The injector is usually equipped with a six-way valve allowing to introduce the sample into the flow of the mobile phase. The samples may be stored inside injector drawers under controlled temperatures (between 5 and 12 °C).
HPLC columns are much shorter than columns used in gas chromatography. At the same time, their specific format (length and diameter) depends strongly on the nature of the compounds that need to be analyzed.
See mass spectrometry.