It is well known that in modern high performance liquid chromatography, the choice of 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 columns on the market, and different types of columns are separated. Therefore, to make a suitable choice, you must have a certain understanding and understanding.
Column parameters
Physical properties
Column length, inner diameter, such as 250 * 4.6mm. Generally, the column length is between 2 and 250 mm. The longer the column, the higher the separation, but the higher the column pressure and the longer the separation time. However, the resolution is proportional to the square root of the number of theoretical plates, so the column length is increased. Not the most effective means of separation, in general, 150mm, 5um filler can provide enough number of plates.
Particle size affects chromatographic resolution. The smaller the particle size, the faster the separation and the higher the column efficiency, but the higher the column pressure, the more easily the column is contaminated, resulting in a decrease in column life. Common analytical columns typically use 5um packing, and complex multi-component sample separations typically use 3.5um particle sizes. Larger inner diameter preparative columns typically use larger particle sizes. If the stationary phase is chosen correctly, but the resolution is not sufficient, then a smaller particle size filler is useful. The column efficiency of the 3.5um packed column is nearly 30% higher than that of the 5um packing under the same conditions; however, the back pressure of the 3.5um column is twice that of 5um, so how to choose the particle size needs to be based on the actual situation. And set.
Aperture, 60A, 120A, 300A, etc. The pore size is small, the porosity is high, the specific surface area is large, and the carbon loading is high; the pore size of the column packing needs to match the molecular size to ensure that the molecules can freely enter and exit the packing pores and separate and distribute with the bonded phase of the inner surface of the pore, usually The pore diameter is required to be more than three times the molecular diameter, generally 80-120A for small molecules and 300A for macromolecules.
The shape of the particles is generally spherical and irregular. When a mobile phase with a high viscosity is used, the spherical particles can lower the column pressure and prolong the life of the column.
Specific surface area refers to the surface area per gram of filler, such as 180m2 / g - 350m2 / g, related to particle size and porosity; large specific surface area, will increase the reaction between the sample and the bonded phase, increase retention and resolution If the specific surface area is small, the analysis time and the equilibration time can be shortened, and it is not preferable that the specific surface area is large or small, and it is necessary to select a suitable specific surface area.
Chemical properties
Silica gel matrix: The most versatile matrix, high strength, easy to chemically modify, but the pH range used is limited (usually 2-8, special modification can reach 1-12).
Polymer matrix: mostly polystyrene-divinylbenzene or polymethacrylate, chemically stable, wide pH range, more hydrophobic, better separation of proteins and other samples; but less strength, Organic solvents may cause the polymer to swell and be damaged, the batch repeatability is poor, and there are not many commercial chromatography columns, which are generally more expensive.
Carbon loading: The proportion of the bonded phase on the surface of the substrate. If the carbon loading is high, the retention is increased, which is suitable for the analysis of non-polar compounds.
Bonded phase: different bonding reagents, different selectivity to compounds, generally long-chain alkyl bonded phase (C18 C8) is more stable than short-chain (C4 C3); non-polar bonding is more polar The bonded phase (-NH2) is stable.
End capping: The bare silicon hydroxy group is bonded with a short chain and sealed to reduce the residual silanol group and reduce the tailing of the chromatographic peak caused by the reaction of the component to be tested with the acidic silicic hydroxyl group. Especially for polar samples, the unblocked column has a poor separation effect.
Normal phase & reversed phase chromatography
At present, the main market is mainly reversed-phase chromatography, accounting for about 80%.
After understanding the basics of the column, the choice of column will be solved.
Column length and inner diameter selection
Length selection: The longer the column, the higher the total efficiency (the greater the value of n), the longer the column, the longer the analysis time. 250-300mm is the most common column length. More than half of the work in the laboratory uses this specification column, which is generally used to separate medium to complex mixtures of 10 to 50 components; 500-600mm, requiring higher resolution applications. , used to separate more than 50 components or complex samples containing difficult-to-separate substances for temperature-programmed analysis.
Inner diameter: The column efficiency is inversely proportional to the square of the column radius. The smaller the inner diameter, the higher the column efficiency, but the larger the inner diameter, the larger the column capacity, and the more the injection volume is allowed. When the injection volume exceeds the column capacity, the true balance cannot be established in each theoretical plate in the column, which will cause the chromatogram bee to be distorted, the column resolution is lowered, and the reproducibility is not good. Therefore, for complex samples requiring precise separation, a small inner diameter column must be used. On the other hand, if there are compounds with very different concentration components in the sample, in order to increase the sample capacity, it is necessary to use a column with a large inner diameter. Currently, the most common column inner diameter used in the laboratory is generally 4.6 mm.
General selection principle: analysis of large molecular weight compounds for large-aperture columns; for high pH or basic compounds, high-end or specially-capped columns are required to improve peak shape and extend column life.
Nowadays, there are a lot of commercial liquid chromatography columns. According to the parameters of the column, we can provide us with a preliminary choice. However, due to the differences in packing technology and bonding technology of various instrument manufacturers, even C18 columns, different series of the same brand. They all have different functions, such as being able to withstand low pH, high temperature resistance, suitable for alkaline samples, and the like. So study the column parameters before selecting the column. Read the column instructions carefully to find the right column and the appropriate separation method.
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