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
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 resin
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
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 3μm.
Stationary phase / Ligand #
The traditional ligands are C18(octadecyl silane, ODS), C8, C4, $\small{\textrm{NH}}_2$, 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.
Particle diameter #
The common particle diameter for analysis HPLC columns is 3μm, 3.5μm, 5μm, 8μm, 10μm. For preparative columns, there are particles of 10μm, 20μm, 30μm, 40μm, 50μm, 75μm.
Particles are less than 3μm, like 2.7μm, 1.8μm, 1.2μm, 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.
Pore size #
The most popular pore size of HPLC columns is 100$\mathring{A}$, 120$\mathring{A}$, 300$\mathring{A}$. Some suppliers also provide particles with 80$\mathring{A}$, 200$\mathring{A}$, 500$\mathring{A}$.
For PSDVB particles, there are particles with a pore size of 1000$\mathring{A}$, 2000$\mathring{A}$, 5000$\mathring{A}$.
The choice of pore size is according to the size of your samples.
Column size #
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 3μm packing materials, we highly recommend using thin and short columns.
If the particle diameter of the packing material is halved, the nμmber 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 nμmber of plates and analysis time will be double. The longer the column length, the more the pressure increases linearly.
Guard columns #
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.