Learn about laboratory chromatographic columns

The chromatographic column separation technique is based on the different partition coefficients between the fixed phase and the mobile phase in the mixture. The stationary phase is usually an adsorbent, such as silica gel, alumina, polyamide, etc., which is evenly filled in the chromatographic column. The mobile phase is the solvent that brings the mixture into the chromatographic column, also known as the eluent. As the mobile phase moves in the chromatographic column, each component stays on the fixed phase for different time due to the difference of partition coefficient, so as to achieve separation.

As an important separation tool, laboratory chromatographic column plays a vital role in chemical, biological, pharmaceutical and other fields. Based on the principle of chromatography, the separation of each component in the mixture can be realized effectively by using the difference of the partition coefficient, adsorption capacity and solubility of different substances between the fixed phase and the mobile phase. This paper will introduce the basic principle, type, selection method, operation procedure and application in various fields of laboratory chromatographic column in detail.

Laboratory chromatographic columns can be divided into many types according to their principle of action, filling material, shape and size.

◆ Classification by principle of action

1) Adsorption column chromatography: The adsorbent is used to separate the difference in adsorption capacity of each component of the mixture. The choice of adsorbent depends on the nature of the substance to be separated, such as silica gel is suitable for the separation of non-polar and polar compounds, and alumina is suitable for different types of compounds according to acid and alkali.

2) Partition column chromatography: The mixture is separated according to the difference in partition coefficient between the fixed and mobile phases. Partition column chromatography usually uses a solvent system as the mobile phase and adjusts the partition coefficient by changing the composition of the solvent.

3) Ion exchange column chromatography: The selective adsorption and release of ions in the mixture is separated by ion exchange resin. Ion exchange resins have specific ion exchange groups that can exchange ions in the separated substance.

◆ Sort by filling material

1) Silica gel column: Silica gel is a commonly used adsorbent, suitable for the separation of a variety of organic matter. Silicone columns have excellent thermal and chemical stability and can be used over a wide pH range.

2) Alumina column: Alumina column is divided into acidic, alkaline and neutral alumina column according to acid and alkali, suitable for different types of compound separation. Alumina column has strong adsorption capacity for polar substances.

3) Polyamide column: The polyamide column has the function of adsorption chromatography and partition chromatography, and is suitable for the separation of compounds that can form hydrogen bonds with polyamide, such as phenols, acids, quinones, etc.

◆ Sort by shape and size

1) Glass column: The type of chromatographic column commonly used in laboratories, with the advantages of transparency and easy observation. The size of the glass column is determined according to the amount of material to be separated and the degree of separation difficulty, and the diameter to height ratio is generally between 1:8 and 1:50.

2) Stainless steel column: Stainless steel column has the advantages of corrosion resistance and pressure resistance, which is suitable for high pressure chromatography systems. The use of stainless steel columns is relatively rare, but has advantages in certain conditions.

The selection of a suitable chromatographic column is crucial to achieve effective separation. When selecting chromatographic columns, the following factors need to be considered:

Laboratory chromatographic columns have a wide range of applications in a number of fields, including but not limited to the following:

◆ Protein separation and purification: Chromatographic column is one of the important tools for protein separation and purification. According to the difference of protein size, charge property and affinity, different chromatographic methods can be selected for separation and purification. For example, ion exchange column chromatography is suitable for proteins separated by charge properties; Affinity column chromatography uses specific binding properties to isolate and purify target proteins.

◆ Separation of nucleic acids: Chromatographic columns can also be used for separation of nucleic acids. Through specific chromatographic methods, nucleic acid molecules of different lengths and structures (such as DNA fragments and RNA molecules) can be effectively separated in the column to meet the needs of subsequent nucleic acid analysis, sequencing and other experiments.

◆ Separation of small molecule compounds: drug intermediates, small molecule components in natural products, etc., can be separated with the help of chromatographic columns. According to the difference of partition coefficient between fixed phase and mobile phase, suitable chromatographic method can be selected for separation and purification of small molecule compounds. For example, reversed-phase silica gel is suitable for the separation of lipophilic small molecules; The normal silica gel is suitable for the separation of hydrophilic small molecules.

◆ Fractional separation of polysaccharides: The chromatographic column can be used for fractional separation of polysaccharides. According to the size of polysaccharide molecules and branched chain structure, suitable chromatographic techniques can be selected to separate polysaccharide mixtures into different levels, which is helpful to further study the relationship between structure and function of polysaccharides.

◆ Other applications: Chromatographic columns are also widely used in the separation and extraction of intracellular products after cell breakage, the separation and purification of active components in plant extracts, the separation and purification of antibiotics, the separation and purification of enzymes, the purification of antibodies, and the separation and concentration of viruses. In addition, the chromatographic column also plays an important role in the separation and analysis of petrochemical products, the separation and detection of food additives, the separation and purification of cosmetic raw materials, the classification analysis of polymer materials and the separation and purification of natural spices.