HPLC Packing Material

HPLC Packing Material

HPLC columns are usually packed with pellicular, or porous particles. Pellicular particles are made from polymer, or glass beads. Pellicular particles are surrounded by a thin uniform layer of silica, polystyrene-divinyl-benzene synthetic resin, alumina, or other type of ion-exchange resin. The diameter of the pellicular beads is between 30 and 40 µm. Porous particles are more commonly used and have diameters between 3 to 10 µm. Porous particles are made up silica, polystyrene-divinyl-benzene synthetic resin, alumina, or other type of ion-exchange resin. Silica is the most common type of porous particle packing material.

Partition HPLC uses liquid bonded phase columns, where the liquid stationary phase is chemically bonded to the packing material. The packing material is usually hydrolyzed silica which reacts with the bond-phase coating. Common bond phase coatings are siloxanes. The relative structure of the siloxane is shown in Figure 2.

R group attached to siloxane	Chromatography method application
Alkyl	Reverse phase
Fluoroalkyl	Reverse phase
Cyano	Normal and reverse phase
Amide	Reverse phase
Amino	Normal and reverse phase
dimethylamine	Weak anion exchanger
Quaternary Amine	Strong anion exchanger
Sulfonic Acid	Strong cation exchanger
Carboxylic Acid	Weak cation exchanger
Diol	reverse phase
Phenyl	Reverse phase
Carbamate	Reverse Phase

 Table 4: This table shows the R groups that can be attached to the siloxane and what chromatographic method it is commonly applied to.

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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