Reverse and Normal Phase HPLC

Reverse and Normal Phase HPLC

A polar stationary phase and a non-polar mobile phase are used for normal phase HPLC. In normal phase, the most common R groups attached to the siloxane are: diol, amino, cyano, inorganic oxides, and dimethylamino. Normal phase is also a form of liquid-solid chromatography. The most non-polar compounds will elute first when doing normal phase HPLC.

Figure 2: Basic structure of a siloxane. The R groups can be varied depending on the type of column and analyte being analyzed. This figure was created with ChemBioDraw Ultra 12.0.

Reverse phase HPLC uses a polar mobile phase and a non-polar stationary phase. Reverse phase HPLC is the most common liquid chromatography method used. The R groups usually attached to the siloxane for reverse phase HPLC are: C₈, C₁₈,or any hydrocarbon. Reverse phase can also use water as the mobile phase, which is advantageous because water is cheap, nontoxic, and invisible in the UV region. The most polar compounds will elute first when performing reverse phase HPLC. Check the animation on the principle of reversed-phase chromatography to understand its principle.
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