How to select a column for HPLC method development? 👩‍🔬

Select a column for HPLC method development

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Select a column for HPLC method development
1. Below are some common bonded phases for HPLC columns

In high performance liquid chromatography, our goal is to separate components, and this happens in the column (stationary phase), so the column is the heart of the HPLC system. Replacing HPLC columns during method development has the greatest impact on analyte resolution.

select a column for HPLC method development

Typically, current reversed-phase columns are made by packing them with spherical silica beads coated with a hydrophobic stationary phase. In general, the type of stationary phase has the greatest impact on elution, capacity factor, selectivity, and efficiency. There are several types of stationary phase support matrices, including polymers, silica, and alumina. Silica is the most regular matrix for HPLC columns. Silica is chemically stable to low pH systems and most organic solvents. A disadvantage of the silica solid support is that it dissolves above pH 7.
Today, HPLC columns have been developed for use in the high pH range. The particle size, type and form of silica can affect the separation of analytes.

The use of small particle size silica improves separation efficiency or increases the number of theoretical separation stages. But using small particles increases back pressure in the system and the column is more prone to plugging. The mobile phase in RP-HPLC is polar, the stationary phase is non-polar, and polar molecules typically elute earlier than non-polar molecules.

To form the stationary phase for RP-HPLC on silica supports, free silanols are reacted with hydrophobic functional chlorosilanes to introduce non-polar surfaces. Due to the static barrier, only about 1/3 of the silanols were derivatized. The remaining silanols can interact with the molecule, resulting in peak tailing.
Typically, after column derivatization, trimethylchlorosilane is reacted with the preferred stationary phase for column STEM to eliminate residual free silanols and improve column efficiency. C18 (octadecyl), C4 (butyl), Cs (octyl), phenyl (phenylpropyl), and nitrile (cyanopropyl) columns are commonly used stationary phases

In general, higher carbon loading, higher phase loading, and longer alkyl chains lead to better retention of non-polar components.