Liquid-phase preparation is a high-precision separation technology widely used in various fields. It can effectively separate, collect and purify mixtures. This article will focus on the influencing factors of separation and purification, extract the essence from chaos, and provide more precise and reliable support for separation and purification experiments.

Key points of preparative liquid chromatography research

To achieve separation of a mixture, separation factor is commonly used as an overall indicator of the separation efficiency of the chromatographic column to assess the separation status of the substances to be separated during the chromatographic process. Separation factor, also known as resolution, is denoted by R and is defined as twice the difference in the retention times of adjacent peaks divided by the sum of their peak widths.


The separation factor of the substance to be separated can also be expressed by the following equation:

In the equation, a represents the separation factor, N represents the number of theoretical plates, which represents the column efficiency, and k represents the capacity factor, which reflects the elution time of the peak and is also commonly known as the retention factor. The larger the R value, the more separated the two peaks are.

Separation factor: It represents the degree of separation between adjacent peaks, that is, the ratio of the retention times of a pair of adjacent peaks. The larger the separation factor, the better the separation effect between two peaks, and conversely, the smaller the separation factor, the worse the separation effect between two peaks.

Column efficiency: refers to the separation ability of the column itself. The higher the column efficiency, the better the ability of the chromatographic column to effectively separate two or more components. For example, compounds that cannot be separated on a short column may be well separated on a long column. Therefore, the impact of column efficiency on the chromatographic separation effect is very significant.

Capacity factor: It is the degree to which a compound is distributed between a selective stationary phase (such as a chromatographic column) and a mobile phase, that is, the relative retention time of a pair of components. The size of the capacity factor determines the retention time and separation degree of the substance in the column. The larger the capacity factor, the better the separation effect, which allows substances with greater differences to be better separated. The size of the capacity factor is also affected by factors such as the polarity of the mobile phase, temperature, pH value, etc. Therefore, the three factors of separation factor, column efficiency, and capacity factor have a significant impact on the accuracy and sensitivity of chromatographic separation.

Next, we will try to adjust these three factors to achieve the best chromatographic separation effect.

Separation factor a

How to increase the separation factor? Under the premise of ensuring the properties of the sample remain unchanged, the following two aspects need to be considered primarily:

01 Selection of Stationary Phase

In general, the stationary phase material should interact with the sample components to achieve effective separation. For hydrophobic samples, a reverse phase chromatography stationary phase can be chosen, while for hydrophilic samples, a normal phase chromatography stationary phase can be chosen. For biomacromolecules, an ion exchange chromatography stationary phase can be selected. For carbohydrates, a hydrophobic interaction chromatography stationary phase can be used. For inorganic ions, an ion chromatography stationary phase is best used, while for synthetic polymers, a gel chromatography stationary phase is preferred.

02 Choice of Mobile Phase

The choice of mobile phase is first determined by the type of stationary phase. In normal phase chromatography, a mixture of n-hexane and isopropanol is the most basic solvent system. Adjusting the ratio of the two solvents can change the polarity of the solvent system. If the basic solvent system cannot meet the separation needs, other solvents such as methyl tert-butyl ether, dichloromethane, acetonitrile, or their mixtures can be added to the basic solvent system to change their selectivity. If it is reverse phase chromatography, a water+organic solvent system should be used. Common organic solvents are methanol, acetonitrile, and tetrahydrofuran. Adjusting the ratio between them and water can change the polarity of the solvent.

Column Efficiency (N)

In the preparation of liquid chromatography, in addition to trying to make the distance between two chromatographic peaks as large as possible, it is also necessary to avoid excessive distribution of peak widths to ensure column efficiency. The main factors that affect column efficiency include the following aspects:

  1. Sample

In order to improve the efficiency of preparing the target compound, the sample is usually slightly overloaded during liquid phase injection. However, if the sample is excessively overloaded, it can lead to reduced column efficiency and decreased separation ability. In addition, the chemical properties, concentration, purity, and distribution ratio of the sample in the two phases can also affect column efficiency.

2. Fixed phase

The influence of the fixed phase mainly includes the particle size and distribution range of the filler, the shape of the filler, the chemical properties of the surface, the size of the surface area, whether the filler is evenly filled, whether it is tightly packed, and the size of the chromatographic column.

3. Column Length and Inner Diameter

Short columns (15-100mm): have shorter run time and lower column pressure, and are commonly used in high-throughput analysis and situations with higher sample throughput.

Long columns (150-250mm): have higher resolution and longer run time, and are suitable for some research-oriented analysis situations.

4. Mobile phase

The factors affecting the mobile phase mainly include the composition, chemical properties, viscosity, diffusivity, and linear velocity of the mobile phase.

5. Flow rate

A flow rate that is too fast can have a negative impact on chromatographic separation because the rapidly flowing solvent can easily wash away chemical substances on the surface of the packing material. When the flow rate is low, the residence time of substances on the packing material surface increases, which may result in impurities in the final product.

6. The effect of temperature

Increasing temperature can reduce the viscosity of the solvent and increase the solubility of the sample.

Capacity factor (k):
Refers to the quantitative distribution ratio of the target molecule between the mobile phase and the stationary phase in the separation column. Proper increase in the capacity factor can improve the separation effect. As shown in the figure below, when the capacity factor reaches a certain value, its influence on the separation degree is not significant. Increasing the capacity factor will consume a lot of time and solvent, so we should avoid increasing the value of the capacity factor too much. Unless the target material is very valuable, it is not recommended to use the method of increasing the capacity factor.

By establishing appropriate separation conditions, we can help to obtain the target substance from the mixture. It is hoped that this article can provide useful references for the development and application of liquid phase separation technology.

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