What are the applications of gel permeation column chromatography?

Gel permeation chromatography is a chromatography technique that separates solute molecules according to their size. When sample solutions with different solute sizes pass through the gel column, the solutes with different molecular sizes will be subject to different retardation effects due to the molecular sieve effect of the network structure inside the gel particles. The large molecular weight is not easy to penetrate into the network and is excluded from the particles, so the blocking effect is small

Gel granules 2Molecule 3Small molecule 4

The macromolecules flow out of the chromatography bed first, and the small molecular weight can penetrate into the network Figure 1, the schematic diagram of the gel permeation chromatography principle The internal elution process is long, so the retardation effect is large, and then it flows out of the chromatography bed, so that the purpose of separation can be achieved.

The key to gel chromatography is to establish liquid-solid equilibrium and then separate the different substances out at the appropriate elution rate. The high temperature facilitates the rapid establishment of liquid-solid equilibrium. However, high temperatures also cause solutes to diffuse too quickly in the liquid, resulting in reduced separation efficiency.

The purpose is to separate mixtures and obtain a certain number of pure components, which includes the purification of organic synthesis products, the separation and purification of natural products.

1) As an analytical tool: mixtures with large molecular weight differences can be analyzed by gel chromatography. On the basis of gel chromatography, paper chromatography or thin layer chromatography can be used in combination to identify the individual dissociating components.

2) As a desalting tool: the salt impurities in the polymer (such as protein nucleic acid, polysaccharides, etc.) solution can be removed with the help of gel chromatography technology, which is called desalting. Decanan gel SephadexG-25 is often used for desalting protein solutions due to its small flow resistance and suitable cross-linking.

3) Concentration of polymer solution: (4) For the removal of pyrogen substances: pyrogen substances are certain substances produced by microorganisms that make human skin annihilated, such as some polysaccharide protein complexes, etc., deionized water can be treated with gel to obtain pyrogen-free water suitable for the preparation of injections.

5) For the determination of the molecular weight of polymer substances, a series of standard samples with known molecular weight are put into the same Burning Beat Gel chromatography column, so that they can be separated by gel chromatography under the same conditions, the elution volume of each component is recorded, and the logarithm of the molecular weight is plotted by the elution volume, and the straight line can be obtained within a certain molecular weight range, which is the standard curve of molecular weight. (6) It is used for the separation and purification of substances.

The principle of gel permeation chromatography is as follows:

Gel chromatography, also known as sterically exclusion chromatography. It is a method of separating and analyzing the components of the mixture by using some gels due to their different molecular weights and different blocking effects. The separation catechism of gel chromatography is different from other chromatography methods, it is similar to the function of molecular sieves, but the pore size of gels is much larger than molecular sieves, generally a few hundred to several thousand angstroms.

The column is filled with a gel with pores of a certain size. When the sample enters the chromatographic column, sample molecules of different sizes flow along the external crevices of the gel particles and the pores of the gel with the mobile phase, and the molecules in the volume are excluded because they cannot penetrate into the gel pores, so they pass through the gel column relatively smoothly and are washed out by the mobile phase earlier. Medium volumes of molecules produce partial osmosis.

Small molecules can penetrate into the pores of the gel and are retarded, and are washed out of the mobile phase later due to an equilibration process. In this way, the specimen components flow out of the column sequentially according to the molecular size of the molecule that are hindered, so as to achieve the purpose of separation.

Photogel chromatography uses an aqueous solution as the mobile phase, which is called filtered gel chromatography (HFC), while when an organic solvent is used as the mobile phase, it is called gel permeation chromatography (GPC). The separation of GPC is based on the volume exclusion mechanism, which is filled with porous gels or particles, and the pore size is similar to that of the polymer molecules to be separated, and the large volume of high-fraction stove compounds cannot enter the gel pores.

It flows out of the gel particles first, the leaching volume (time) is the smallest, and the polymers are leached out in order from large to small according to their molecular weight. In this way, by making a standard curve of polymers of known molecular weight, it is possible to analyze the polymer compounds of unknown homologies to be tested.

Desalting: Low molecular weight impurities in polymer (such as proteins, nucleic acids, polysaccharides, etc.) solutions can be removed by gel chromatography, which is called desalination. The desalination operation of this method is simple and fast, and proteins and enzymes are not easy to denature during the desalination process.

Suitable gels are Sephadexg or Bio-Gel-P. The ratio of column length to diameter is 5-15, and the sample volume can reach 25%-30% of the column bed volume, in order to prevent the formation of precipitate adsorption on the column after the solubility of protein desalting, the chromatography column is generally balanced with volatile salt buffers such as ammonium acetate, and then the sample is added, and then eluted with the same buffer, and the volatile salts are removed by freeze-drying of the collected eluent.

For separation and purification: Gel chromatography has been widely used for the separation and purification of enzymes, proteins, amino acids, polysaccharides, hormones, alkaloids and other substances. The gel has a strong adsorption force on pyrogen, which can be used to remove pyrogens in ion-free water to prepare water for injection.

Determination of the molecular weight of polymer substances: a series of standards with known molecular weight are placed in the same gel column, chromatography under the same conditions, the elution volume of each component (standard of known molecular weight) is recorded, and the logarithm of molecular weight is plotted by elution volume, and a straight line, that is, the standard curve of molecular weight, can be obtained within a certain molecular weight range. When determining the molecular weight of an unknown substance, the sample can be eluted on a gel column with a measured standard curve, and its molecular weight can be found on the standard curve according to the elution volume of the substance.

Concentration of polymer solution: Usually sephadexg-25 or 50 dry rubber is put into the dilute polymer solution, then the water and low molecular weight substances will enter the pores inside the gel particles, and the polymer substances are excluded outside the gel particles, and then centrifuged or filtered to separate the swollen gel to obtain a concentrated polymer solution.

Choose different gel types according to the purpose of the experiment. If the purpose of the experiment is to separate the large and small molecules in the sample, due to their significant differences in partition coefficients, this separation is also known as group separation, Sephadex G-25 and G-50 are generally used, and Sephadex G-10, G-15 and Bio-Gel-P-2 or 4 can be used for the desalting of small peptides and low molecular weight substances (1000-5000). If the purpose of the experiment is to separate some substances with similar molecular weights in the sample, this separation is also called fractionation.

Generally, gels with exclusion limits slightly greater than the highest molecular weight substances in the sample are selected, and these substances can penetrate into the gel to varying degrees during the chromatography process, and finally be separated due to different KDs. According to experience, when separating groups, most of the chromatography columns with a length of 2-30cm are used, and when fractionating and separation, a chromatography column about 100cm long is generally required, with a diameter of 1-5cm, less than 1cm produces a wall effect, and more than 5cm is a serious dilution. The ratio of length l to diameter d l d should generally be between 7-10, but it should be between 30-40 for slow-moving substances.

Gel chromatography can not only be used to separate and determine the relative molecular weight and relative molecular weight distribution of polymers, but also to separate fat-soluble and water-soluble substances according to the gel fillers used, and the relative molecular mass of the separation can range from several million to less than 100. In recent years, gel chromatography has also been widely used for small molecule compounds. Substances with similar molecular mass but different chemical structures cannot be completely separated and purified by gel permeation chromatography.

Gel chromatography cannot distinguish compounds of similar molecular size, and the relative molecular weight difference needs to be more than 10% to be separated.

(1) All components are eluted before the elution of solvent molecules, and the separation time is short.

2) Can be elution time, can be continuous injection.

3) The separation process of gel chromatography does not rely on intermolecular forces, and in general, there are no strongly retained molecules accumulated in the column, so the sample components will not be lost during separation, and the service life of the column will be extended.

4) The retention time is short, the chromatographic peak is narrow, and it is easy to detect.