How to choose the HPLC column for analysis & preparative tests?
The selection of the HPLC column is a common topic for laboratory personnel and production managers. The selection criteria are complicated. Here we are going to talk about it.
Here is some basic principle for HPLC column choice.
- Matrices / Substrate
- Stationary phase / Ligand
- Particle diameter
- Pore size
- Column size
- Guard columns
Matrices / Substrate
Silica gel is an ideal chromatography packing material for bonded-phase HPLC columns. Silica gel is a rigid particle that can withstand the compression of liquid streams. It is widely used in analysis and preparative HPLC.
PSDVB (polystyrene divinylbenzene) packing is ideal for small-scale chromatographic analysis. It is chemically stable, rigid, and does not show soluble substances or precipitate. PSDVB particles can be used as reversed-phase chromatography packing material that doesn't need a bonding phase. After coating and derivatization, it can be made into cation and anion exchange resins.
Core-shell resin is widely used in UPLC columns. It is relatively new material. Compared with silica gel, its particle diameter can be less than 3um.
Stationary phase / Ligand
The traditional ligands are C18(octadecyl silane, ODS), C8, C4, NH2, CN, phenyl, diol, PFP, AQ. Other ligands are double-phenyl, biphenylyl, naphthyl.
The bonded phase can provide a significantly different selectivity than the stationary phase to achieve successful separation, or increased selectivity for certain mobile phases, or other bonded phases.
For the ion-exchange column, the ligands are classified into strong cation, weak cation, strong anion, and weak anion. The selection is based on the sample properties.
The common particle diameter for analysis HPLC columns is 3um, 3.5um, 5um, 8um, 10um. For preparative columns, there are particles of 10um, 20um, 30um, 40um, 50um, 75um.
Particles are less than 3um, like 2.7um, 1.8um, 1.2um, which are used in UPLC analysis.
The back pressure of the column is higher with smaller packing materials. There is a pressure limit in the HPLC system, making sure that the column pressure didn't over your system limit.
The most popular pore size of HPLC columns is 100A, 120A, 300A. Some suppliers also provide particles with 80A, 200A, 500A.
For PSDVB particles, there are particles with a pore size of 1000A, 2000A, 5000A.
The choice of pore size is according to the size of your samples.
The inner-diameter of HPLC columns is 2.0/2.1mm, 3.0mm, 4.0mm, 4.6mm, 7.8mm, 10mm, 20mm, 21.2mm, 30mm, and 50mm. It is international convention in HPLC development.
The length of columns is customized from 25mm to 500mm. Some customers even use a column length of 1000mm.
For small particles like 3um packing materials, we highly recommend using thin and short columns.
If the particle diameter of the packing material is halved, the number of plates will be doubled (the column length remains the same), and the column pressure is increased by a factor of 4. If the column length is doubled, the number of plates and analysis time will be double. The longer the column length, the more the pressure increases linearly.
Whether or not to use a guard column depends on the sample and experimental conditions. The column size of our guard column is 4.6mm*10mm. We also provide tiny guard columns from the USA with nearly zero dead volume.
Manager & Engineer in GALAK Chromatography. Master of Chemical Engineering.
During my college study, I found liquid chromatography to be a profound subject. I know the painful struggle a novice needs to go through to get started. I share this article to help you solve your problems quickly.