Methods and suggestions for selection of HPLC columns
A simple method for selection of HPLC column1. Determine the separation purpose and determine whether your application requires high resolution, short analysis time, high sensitivity, long column life, and low experimental cost.
2. Evaluate the chemical properties of analytes, such as chemical structure, solubility, stability, etc.
3. Select appropriate chromatographic column to understand the physical and chemical properties of chromatographic packing.
Selection methods of two liquid chromatographic columns | conditions for selection of liquid chromatographic columns
Filler matrix
1. Silica gel matrix: high purity, low cost, high strength, easy chemical modification, but limited pH range. Most of the silica matrix fillers are stable between ph2-8, but the specially modified silica bonded phase can be stabilized at ph1.5-10.
2. Polymer matrix: wide range of pH value, stable temperature (high temperature can reach more than 80 degrees), low mechanical strength.
Particle shape
Most modern HPLC fillers are spherical particles, but sometimes irregular particles. Spherical particles provide lower column pressure, higher column efficiency and stability, and longer column life when using high viscosity active phase; irregular particles have larger specific surface area and relatively low price.
Particle size
The smaller the particle size, the higher the column efficiency and the higher the separation, but at the same time, it will lead to higher column pressure drop. In order to solve some complex samples, 1.5 μ m packing can be used in UPLC, and 10 μ m or larger filler can be used as semi preparative or preparative column.
Carbon content
Carbon content refers to the proportion of bonded phase on silica gel surface, which is related to specific surface area and bonding coverage. High carbon content progressive column capacity, resolution and analysis time are used for complex samples requiring high resolution; low carbon content analysis time is short, showing 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 varies from 7% to 19%.
Pore size and specific surface area
HPLC adsorption medium is porous particles, most of the reaction surface is in the pore. Therefore, molecules must enter the pore to be adsorbed and separated.
Pore size and specific surface area are two complementary concepts. The pore size is small and the specific surface area is large, and vice versa. Large specific surface area can increase the reaction between the sample and the bonding phase, increase the storage, loading amount and the resolution of complex components; the specific surface area is small and the equilibrium time is fast, which is suitable for gradient analysis.
Pore volume and mechanical strength
Pore volume, also known as "pore volume". Refers to the void volume per unit particle. It can well reflect the mechanical strength of the filler. The mechanical strength of the filler with large pore volume is slightly weaker than that of the filler with small pore volume. The packing with pore volume of 1.5ml/g or less is mostly used for HPLC separation, while the packing with pore volume greater than 1.5ml/g is used for size exclusion chromatography and low pressure chromatography.
End capping
The end capping can reduce the tailing peak of polar basic compounds due to the interaction with exposed silanol groups. The open end bonding phase has different selectivity to the capped phase, especially for the polar sample.
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