HPLC Column Knowledge and Selection Factors
In the modern HPLC analysis, the selection of the column directly affects the separation effect. Choosing the right column can shorten the time required for method development and make the method more stable. However, there are many kinds of chromatographic columns on the market today, and different types of columns are suitable for different separation mission. Therefore, to make appropriate choices, the user must have a certain understanding of this.
Column Specifications: Physical properties
Column length and inner diameter, such as 250*4.6mm. The general HPLC column length is 2-250mm. The longer the column, the higher the degree of separation, but the higher the column pressure, the longer time required for separation. But the degree of separation is proportional to the square root of the number of theoretical plates, so simply increasing the column length is not the most effective separation method. Usually, 150mm and 5um filler can provide enough trays.
Particle size affects chromatographic separation. The smaller the particle size, the faster the separation and the higher the column efficiency. However, the higher the column pressure is, the easier the column is to be contaminated, results in a decrease of the column lifetime. Common analytical columns usually use 5um filler, complex multi-component sample separations typically use 3.5um, and larger diameter preparation columns typically use larger particle sizes.
Pore size, 60A, 120A, 300A and so on. With a small pore size, the porosity is high, the specific surface area is large, and the carbon load is high. The pore size of the chromatographic column filling must be matched with the molecular size to ensure that the molecules go through the pores smoothly and separate and distribute with the bonding phase in the inner surface of the pore. It is required that the pore diameter be more than 3 times of the molecular diameter. Generally, uses 80-120A for the small molecule and 300A for the macromolecule.
The shape of the particles is generally spherical and irregular. When a mobile phase with a large viscosity is used, the spherical particles can reduce the column pressure and extend the life of the column.
The specific surface area refers to the surface area per gram of filling, such as 180m2/g to 350m2/g, which is related to the particle size and the porosity. The large specific surface area will increase the reaction between the sample and the bonding phase and increase the retention and separation. The small specific surface area can shorten the analysis time and balance time. The size of the specific surface area doesn’t influence the performance of the column, chose the appropriate one.
Silicone substrate: the most common substrate, high strength, easy chemical modification, but with limited application range of pH (usually 2-8, special modification reaches 1-12)
Polymer substrate: mostly polystyrene-divinylbenzene or polymethacrylate, chemically stable, with a wide application pH range, strong hydrophobicity, good performance on separation of proteins and other samples, but less strength. Organic solvents can cause polymers to swell and suffer damage, batch reproducibility is poor.
Carbon loading: The proportion of bonded phase on the surface of the substrate. High carbon loading increases the retention, which makes it suitable for analysis of non-polar compounds.
Bonding phase: different bonding agents, different selectivity for compounds, generally long-chain alkyl bonding phase (C18 C8) is more stable than short-chain (C4 C3). Non-polar bonding is more stable compared to polar bonded phase (-NH2).
End-capping: Sealing the exposed silanols with a short chain to reduce the residual of silanol groups, and reducing the peak tailing caused by the reaction between the components to be tested and the acidic silanol groups. Especially for polar samples, uncapped columns have poor separation performance.