Selection principle of chromatographic column

In the analysis of high performance liquid chromatography, the selection of chromatographic column directly affects the separation effect. Choosing the appropriate chromatographic column can shorten the development time of the method and make the method more stable.
1.jpg
At present, there are a wide range of commercial liquid chromatography columns. According to the parameters of chromatographic columns, we can provide a preliminary choice. However, due to the differences in packing technology and bonding technology of various instrument manufacturers. Even if they are all C18 columns, different brands, different series of the same brand have different functions, such as low pH resistance, high temperature resistance, and alkaline samples.
To make a proper choice, we must have a certain understanding of it.
Principle 1: look at physical properties
Column length, inner diameter, such as 250 * 4.6mm. Generally, the column length is 2-250mm. The longer the column is, the higher the separation degree is, but the higher the column pressure is, the longer the separation time is. However, the separation degree is directly proportional to the square root of the theoretical plate number, so increasing the column length is not the most effective separation method. Generally, 150 mm and 5 um packing can provide enough tray number.
The particle size affects the chromatographic resolution. The smaller the particle size is, the faster the separation is, and the higher the column efficiency is. However, the higher the column pressure is, the easier the column will be polluted and the column life will be reduced. 5 um packing is usually used in common analytical column, 3.5 um particle size is generally used for complex multicomponent sample separation, and larger particle size is usually used for preparation of chromatographic column with larger inner diameter. If the choice of stationary phase is correct, but the resolution is not enough, it is very useful to select a smaller size of filler. The column efficiency of 3.5um packing column is nearly 30% higher than that of 5um packing under the same conditions; however, the back pressure of 3.5um column is twice that of 5um, so how to select the packing particle size needs to be determined according to the actual situation.
Aperture, 60A, 100A, 120a, 300A, etc. When the pore size is small, the pore ratio is high, the specific surface area is large, and the carbon loading capacity is high; the pore size of chromatographic column packing should match with the molecular size to ensure that molecules can enter and leave the packing hole freely and separate and distribute with the bonding phase on the inner surface of the pore. Generally, the pore diameter is more than 3 times of the molecular diameter, 80-120a for small molecules and 300A for large molecules.
When the mobile phase with high viscosity is used, the spherical particles can reduce the column pressure and prolong the column life.
Specific surface area refers to the surface area per gram of filler, such as 180m2 / g-350m2 / g, which is related to the particle size and porosity; large specific surface area will increase the reaction between the sample and the bonding phase, and increase the retention and resolution; small specific surface area can shorten the analysis time and equilibrium time, not better if the specific surface area is large or small, so it is necessary to select the appropriate specific surface area.
Principle 2: chemical properties
Silica gel matrix: the most common matrix with high strength and easy chemical modification, but its pH range is limited (generally 2-8, and specially modified can reach 1-12).
Polymer matrix: mostly polystyrene divinylbenzene or polymethacrylate, chemically stable, with wide application pH range, stronger hydrophobicity, and good separation effect for protein and other samples; but its strength is small, organic solvent may cause polymer swelling and damage, batch repeatability is poor, there are not many commercial chromatographic columns, and the price is relatively high.
Carbon loading: the proportion of bonding phase on the surface of the matrix. If the carbon loading is high, the retention will increase, which is suitable for the analysis of non-polar compounds.
Bonding phase: different bonding reagents have different selectivity for compounds. Generally, long chain alkyl bonding phase (C18, C8) is more stable than short chain (C4, C3); nonpolar bonding phase is more stable than polar bonding phase (- NH2).
End capping: the exposed silicon hydroxyl group is bonded with short chain to reduce the residual silanol group and reduce the tailing phenomenon of chromatographic peak caused by the reaction between the component to be tested and the acid silicon hydroxyl group. Especially for polar samples, the separation effect of unsealed column is poor.
Principle 3: look at normal phase and reverse phase chromatography
At present, RP-HPLC is mainly used in the market, accounting for about 80%.
After understanding the basic knowledge of chromatographic column, the choice of chromatographic column is easy to solve
Principle 4: select according to column length and inner diameter
Length selection: the longer the column, the higher the total column efficiency (n value), the longer the column, the longer the analysis time. 250-300 mm is the most common column length. More than half of the laboratory work uses this specification column, which is generally used to separate 10 to 50 components of medium to complex mixtures; 500-600 mm, which requires higher resolution applications, is generally used to separate more than 50 components or complex samples containing difficult to separate substances.
Inner diameter: the column efficiency is inversely proportional to the square of column radius. The smaller the inner diameter is, the higher the column efficiency is. However, the larger the inner diameter is, the larger the column capacity is, and the more sample injection volume is allowed. When the injection volume exceeds the volume of the column, there is no real balance in each theoretical plate in the column, which will lead to distortion of chromatographic bees, decrease of column resolution and poor reproducibility. Therefore, it is necessary to use a small inner diameter column to separate complex samples accurately. On the other hand, if there are compounds with very different concentrations in the sample, in order to increase the sample capacity, columns with large inner diameter must be used. At present, the most common column inner diameter used in laboratory is 4.6mm.
General selection principle: select large aperture chromatographic column for analysis of high molecular weight compounds; select high capped or special capped chromatographic column for high pH or alkaline compounds, so as to improve the peak shape and prolong the service life of chromatographic column.
For more related technologies, please consult MS technologies technicians