A selection method of liquid chromatographic column

 

1. Determine the purpose of separation to determine whether your application requires high separation, short analysis time, high sensitivity, long column life, low experimental cost, etc.

 

2. Evaluate the chemical properties of analytes evaluate the chemical properties of analytes, such as chemical structure, solubility, stability, etc.

 

3. Select suitable column to understand the physical and chemical properties of chromatographic packing.

 

Selection method of two liquid chromatographic columns

 

Packing matrix

 

1. Silica gel matrix: high purity, low cost, high strength, easy chemical modification, but limited pH range. Most silica based fillers are stable between ph2-8, but the specially modified silica bonded phase can be stable at ph1.5-10.

 

2. Polymer matrix: wide range of pH value, stable temperature (high temperature can reach more than 80 ℃), small mechanical strength.

 

Particle shape

 

Most modern HPLC fillers are spherical particles, but sometimes they are irregular particles. Spherical particles provide lower column pressure, higher column efficiency and stability, and longer column life when using high viscosity mobile phase; irregular particles have large specific surface area and relatively low price.

 

Particle size

 

The smaller the particle size, the higher the column efficiency and the higher the resolution, but at the same time, the higher the column pressure drop. The packing of 1.5-3 μ m is selected to solve some complex samples. The packing of 1.5 μ M can be used in UPLC, and the packing of 10 μ m or larger can be used as semi preparation or preparation column.

 

Carbon content

 

Carbon content refers to the proportion of bonded phases on the surface of silica gel, which is related to specific surface area and bonding coverage. High carbon content improves the column capacity, resolution and analysis time, which are used for complex samples requiring high separation; low carbon content analysis time is short and shows different selectivity, which is used for rapid analysis of simple samples and samples requiring high water content active phase conditions. Generally, the carbon content of C18 column varies from 7% to 19%.

 

Aperture and specific surface area

 

HPLC adsorption medium is porous particles, and most of the reaction surfaces are in the pores. Therefore, molecules must enter into the pore to be adsorbed and separated.

 

Pore size and specific surface area are two complementary concepts. The aperture is small, the specific surface area is large, and vice versa. Large specific surface area increases the reaction between the sample and the bonding phase, increases the preservation, sample loading and the separation of complex components; small specific surface area, fast equilibrium time, suitable for gradient analysis.

 

Pore volume and mechanical strength

 

Pore volume, also known as "pore volume". It refers to the volume of void per unit particle. It can reflect the mechanical strength of packing very well. The mechanical strength of packing with large pore volume is slightly weaker than that of packing with small pore volume. The packing with pore volume of 1.5ml/g or smaller is mostly used for HPLC separation, while the packing with pore volume larger than 1.5ml/g is used for size exclusion chromatography and low-pressure chromatography.

 

End sealing

 

The end capping can reduce the tailing peak of polar alkalescent compounds due to the interaction with the exposed silanol group. The selectivity of the unsealed phase is different from that of the capped phase, especially for the polar samples. ,