How to select a column for HPLC method development

In high-performance liquid chromatography, to separate the components is our object and this occurs in the column (Stationary Phase), hence the column is the heart of the HPLC system. Changing the HPLC columns during method development will have the most impact on the resolution of the analytes. Typically, current reverse phase chromatography columns are made by packing with globular silica gel beads that are coated with the hydrophobic stationary phase. Typically the nature of the stationary phase has the most influence on the elution, capacity factor, selectivity, and efficiency. There are various types of matrices for stationary phase support, including polymers, silica, and alumina.

Silica is the most regular matrix for HPLC columns. Silica is chemically stable for low pH systems and most organic solvents. The drawback of silica solid support is that it will dissolve above pH 7. Nowadays HPLC columns are developed for use in high pH range. The particle size, nature, and shape of silica effect the separation of analytes. The use of small particle size of silica increases the separation efficiency or increases the number of theoretical plates. But, the use of small particles increases the backpressure of the system and the column becomes more easily plugged. The mobile phase in RP-HPLC is polar and the stationary phase is non-polar, whereby polar molecules are usually eluted earlier than non-polar molecules.

To form a stationary phase for RP-HPLC on silica supports, to introduce a non-polar surface free silanols are reacted with a chlorosilane with hydrophobic functionality. Because of static barriers, only about 1/3 of the silanols are derivatized. The remaining silanols may interact with the molecules, resulting in peak tailing. Typically, after column derivatization with the preferred stationary phase, chlorotrimethylsilane reacts with column STEM to eliminate the remaining free silanols and improves the efficiency of the column. C18 (octadecyl), C4 (butyl), Cs (octyl), phenyl (phenyl propyl) and nitrile (cyanopropyl) column are commonly used stationary phases. In common, higher carbon loads higher phase loadings, and longer alkyl chains gives better retention of non-polar components.

Here are listed some common bonded phases for HPLC columns.

SI, C1, C2, C3, C4, C5, C6, C8, C18, CN, NH2, NO2, OH, PHENYL, SCX, SAX, WCX, WAX.


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