HPLC Column Selection Guide

1. Basic knowledge of column 

Chromatography is a laboratory technique commonly used for the separation of a mixture. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. Column chromatography is a separation technique in which the stationary bed is within a tube.  Thus, the column used in this analytical method is the heart of the chromatographic system.  Common requirements for columns include high column efficiency, good selectivity and fast analysis speed etc.  Packing materials for HPLC are mainly all porous spherical silica gel, organic polymer matrix or core-shell silica gel. Their particle sizes, for instance, 1.8 µm, 3 µm, 5 µm, 7 µm and 10 µm are available. The selection of columns,  and classification and main features of packaging materials are described herein. 

2. Selection of column dimensions and specifications of packing materials 

2.1 Length of column

2.2 Inner diameter of column

Generally, 4.6 mm is the most widely used for standard HPLC instruments. The 2.1 mm and 1.0 mm columns have result in higher sensitivity but can be affected by the dead volume outside the column. We recommend using users to use  these columns with low dead volume in the system. 

2.3 Particle size of packing materials 

Column with smaller particle size packing materials yields better resolution than that of larger particles. On the other hand, column of larger particles has lower back pressure and better tolerance of contamination.  Particle <2 um is generally used for UPLC which requires purer mobile phase.   

2.4 Pore size of packing materials

The selection of pore size depends on the size of the molecules to be analyzed.  The larger the molecules, the larger the pores should be chosen. Generally, compounds with molecular weight less than 2000 can use  a pore size of 120 Å, while compounds with molecular weight greater than 2000 are recommended to use a pore size of 200 Å and above. Pore size also affects the resolution: , the smaller the pore, the more surface and interaction with the molecules to be analyzed. 

To decide which method and column is suitable, the molecule to be analyzed should be considered in the first step.  Below process Selection I describes a step by step guides to identify the right analysis modes.    Once the analysis mode was has been determined, one should consider the column phase.  C18 column is most used for many analyses.  But if one wants to analyze a very polar molecule, it may not interact with the C18 material. In this case, C4, CN, diol or phenyl phases are more suitable. In another case if the analyte is too unpolar it can irreversibly retain on the column and cannot elute from the column. In this case a silica, CN or NH2 phase can be tried.  Once the column phase is decided, the focus will be on the selection of dimensions and specifications for the column, such as particle size, pore size, internal diameter, and carbon loading. In addition, the composition and pH for the composition also needs to be optimized to achieve best separation, Selection 2 provides a guideline for how to select the model of columns based on the mobile phase conditions.  

If you have any questions on selecting a HPLC column or require further information, please feel free to contact us at sales@auroraprosci.com.