High Performance Liquid Chromatographic Columns

High performance liquid chromatography (HPLC) is aslo known as liquid chromatography. HPLC uses a liquid moblie phase. The same basic principals from gas chromatography are applied to liquid chromatography. There are three basic types of liquid chromatographic columns: liquid-liquid, liquid-solid, and ion-exchange. Liquid-liquid chromatographic columns have the liquid stationary phase bonded or absorbed to the surface of the column, or packed material. liquid-liquid chromatographic columns are not as popular because they have limited stability and they are inconvenient. Partitioning occurs between the two different liquids of the mobile and stationary phases. In liquid-solid chromatographic columns the stationary phase is a solid and the analyte absorbs onto the stationary phase which separates the components of the mixture. In ion-exchange chromatographic columns the stationary phase is an ion-exchange resin and partitioning occurs with ion exchanges that occur between the analyte and stationary phase.

Usually HPLC has a guard column ahead of the analytical column to protect and extend the life of the analytical column. The guard column removes particulate matter, contaminants, and molecules that bind irreversibly to the column. The guard column has a stationary phase similar to the analytical column.

The most common HPLC columns are made from stainless steel, but they can be also made out of thick glass, polymers such as polyetherethelketone, a combination of stainless steel and glass, or a combination of stainless steel and polymers. Typical HPLC analytical columns are between 3 and 25 cm long and have a diameter of 1 to 5 mm. The columns are usually straight unlike GC columns. Particles that pack the columns have a typical diameter between 3 to 5 µm. Liquid chromatographic columns will increase in efficiency when the diameter of the packed particles inside the column decreases.

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.