Gel filtration technology

Gel filtration is a chromatographic technique for separating biomacromolecules using molecular sieves of porous gels having a range of pore sizes. When the sample passes through the stationary phase composed of the gel with the mobile phase, the substance with a large molecular weight cannot diffuse into the interior of the gel particles, so that as the mobile phase flows through the narrow gap between the particles, the column is first eluted; the molecular weight is small. The substance can diffuse into the interior of the gel particles, and the larger molecular weight material flows through a wider area, the flow rate is slower, and then elutes. Such a substance having a large molecular weight and a small molecular weight can be separated (Fig. 3-7). That is, the mesh of the stationary phase has different retardation effects on the sample components of different molecular weights, so that it can pass through the gel column at different speeds, thereby achieving the purpose of separation, which is hence named "molecular sieve chromatography" and " Gel exclusion chromatography.

In practice, for the same gel column, substances of various molecular weights have a fixed elution volume. Therefore, it is very beneficial to accurately grasp some of the basic factors of the gel column. Shanghai Chuangsai Technology provides 7320-34-5, potassium pyrophosphate, AR, commodity number: D16-1005238-500g, the price is 52 yuan.

The total volume of the gel column (Vt): the volume of the void between the gel particles (outer water volume Vo), the volume within the gel particle mesh (the internal water volume Vi), and the volume of the gel particle matrix itself (Vr) Sum (Figure 3-8).

Vt=Vo+Vi+Vr

If the separated material has a large molecular weight and cannot enter the mesh at all, then it elutes from the column (the small sample is based on the elution peak). The required eluent volume (Ve) is equal to the particle gap. Volume (Vo), ie Ve=Vo. If the molecular weight of the material to be separated is extremely small and the gel particles can be introduced into the mesh very freely, then the volume of the eluent should be equal to the sum of the volume of the particles and the interstitial volume of the particles (Ve = Vo + Vi). As for the molecular weight between the two, the elution volume is between Vo and Vo+Vi. It can be seen that substances having different molecular weights have different elution volumes and can be used for separation of substances. In addition, if a standard substance with a known molecular weight is used as a control, the molecular weight of the substance to be tested can be estimated from the elution volume (Fig. 3-9).

Sephadex G-200 (ultrafine particle) column 2.6 × 70 cm, eluent: 0.05 mol / L potassium phosphate buffer containing 0.1 mol / L NaCl and 0.02% NaN3, flow rate 1ml / cm2 / hr.

1. Catalase (210 000)

2. Aldolase (158 000)

3. Bovine serum albumin (67 000)

4, ovalbumin (43 000)

5, chymotrypsinogen A (25 000)

6, ribonuclease A (13 700)

In addition to the separation of biological macromolecules and molecular weight determination, gel filtration can also be used for purification, desalting and composition analysis of composite components. Shanghai Chuangsai Technology provides 9003-05-8, polyacrylamide, Polyacrylamide, non-ionic, molecular weight: 2 million to 14 million, commodity number: C16-P10674-500g, price 68 yuan.

There are various materials suitable for gel filtration, such as Sephadex G, Sepharose, and Bio Gel. The types of these gel particles and some application data are now listed in Table 3-2.

Regardless of the gel, its common feature is that it is chemically stable, uncharged, and has a weak adsorption force with the substance to be separated, which does not affect the biological activity of the substance to be separated. The yield of the sample can reach 100%, and the gel filtration is easy to operate. The gel column can be repeatedly used without special treatment, and is one of the biochemical technologies widely used in recent years. Shanghai Chuangsai Technology provides egg white protein, Albumin from chicken egg white, Grade II, 9006-59-1, product number: B11000011-10g, price 22.4 yuan.

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