Gel filtration chromatography

Alternative name for exclusion chromatography

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synonym Molecular sieve chromatography (Gel filtration chromatography or volume exclusion chromatography) generally refers to gel filtration chromatography

Gel filtration chromatography is based on the use of porous Reticular structure Granular molecular sieve Function, according to the components in the separated sample relative molecular mass It is a technology for elution and separation based on the difference of size.^[1]

gel filtration Chromatography (gel filtration chromatography) method, also known as exclusion chromatography or molecular sieve method, is mainly based on protein The size and shape, that is, the quality of the protein, are separated and purified. Chromatography column The filler in is some inert porous network structure material, mostly cross-linked Glycan (e.g glucan or agarose ）Class substances, which make the substances in the protein mixture Molecular size To be separated. It is also called molecular exclusion chromatography. It uses porous gel beads as matrix to separate proteins or other molecular mixtures according to molecular size Chromatography technique 。 Generally macromolecule First flow out, Small molecule Then it flows out.

Chinese name: Gel filtration chromatography
Foreign name: gel filtration chromatography
Alias: Exclusion chromatography or molecular sieve method

Classification: A technology of elution and separation
Classification by: Size and shape of protein
Application: be used for Separation and purification , desalination, etc

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Fundamentals

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Gel filtration chromatography is also called Molecular sieve chromatography , exclusion chromatography. Is to use Reticular structure Gelatinous molecular sieve Action, according to the Molecular size Different to separate. The filler in the chromatographic column is some inert porous network structure material, mostly cross-linked Glycan (e.g glucan or agarose ）Substance like, Small molecule Matter can enter its interior, and the distance is longer when it flows down Macromolecular substances But they are excluded from the outside. The journey down is short solution When the gel filtration chromatography column is passed, the substances in the solution are separated by different molecular weight sieves.

advantage

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Its outstanding advantages are that the gel used for chromatography is an inert carrier, without charge, and has weak adsorption force, Operating conditions It is relatively mild, and can be carried out in a relatively wide temperature range Organic solvent , and for separated components Physical and chemical properties The maintenance of is unique. It has good separation effect for high molecular substances.

usage method

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1. The choice of gel depends on the purpose of the experiment. If the purpose of the experiment is to Macromolecular substances and Small molecule Matter is separated because they are partition coefficient There are significant difference , which is also called group separation, generally SephadexG-25 and G-50 can be selected Small peptide And low molecular weight substances (1000-5000) desalination SephadexG can be used -10 G-15 and Bio-Gel-p-2 or 4. If the purpose of the experiment is to separate some substances with similar molecular weight in the sample, this separation is also called Fractionation 。 General selection Exclusion limit The gel is slightly larger than the most high molecular weight substance in the sample, and these substances can penetrate into the gel in varying degrees during chromatography. Due to different Kd, they are finally separated.

2. The diameter and length of the column are based on experience. When groups are separated, 2-30cm long Chromatography column , generally, a chromatographic column with a length of about 100cm is required for graded separation, and its diameter is 1-5cm, less than 1cm Tube wall effect If it is greater than 5cm, the dilution is serious. The ratio L/D of length L to diameter D should generally be 7-10, but it should be 30-40 for slow moving materials.

3. Preparation of gel column After the gel model is selected, suspend the dry gel particles in 5-10 times distilled water or Eluate Moderate sufficiency swelling After swelling, the extremely fine particles are poured out. Natural swelling takes a long time, and heating can accelerate the swelling, that is, after boiling water bath The wet gel slurry is gradually heated to nearly boiling, and the gel can be fully expanded and dissolved within 1-2 hours. Heating method It can save time and sterilize.

Filling of gel: vertically fix the chromatographic column on the shelf with the ground Clip Clamping, one with Mixing device The column is filled with eluent, and the gel is mixed into thinner slurry Sheng in In the container at the top of the column, and then slightly stir to make the gel sink into the column, so that the gel particles rise horizontally until the required height, remove the device at the top of the column, and gently cover the surface of the gel bed with corresponding filter paper. Let it stand for a while, and then start flow balance. The flow rate should be lower than the flow rate required for chromatography. stay Equilibrium process The flow rate gradually increased to the chromatography flow rate should not exceed the final flow rate. Balance the gel bed overnight. Before use, check whether the chromatography bed is even and whether there is“ lines ”Or bubbles, Or plus Some colored substances are used to observe the movement of the color band. If the band is narrow, even and flat, it indicates that the performance of the chromatographic column is good. If the color band is distorted, scattered, or widened, the column must be reinstalled.

4. After the balance of the sample adding and eluting gel bed, leave several milliliters of eluent at the top of the bed to saturate the gel bed, and then Dropper Add samples. Generally, the sample volume is not greater than the total gel volume Bed volume 5% - 10% of. The sample concentration has nothing to do with the distribution coefficient, so the sample concentration can be increased, but for the substance with larger molecular weight, the viscosity of the solution will change with Concentration increase And increase so that Molecular motion Limited, so the sample and eluent Relative viscosity It shall not exceed 1.5-2. After adding the sample, open the outlet to allow the sample to penetrate into the gel bed. When the sample liquid level is just at the same level with the surface of the gel bed, add several milliliters of eluent to wash the pipe wall, so that all of it enters the gel bed collector Connect, design the flow rate in advance, then collect the eluent separately, and conduct qualitative and quantitative determination for each fraction.

5. Reuse of gel column, gel recovery and storage can be used repeatedly after being loaded once, without special treatment, and will not affect the separation effect. To prevent gel Infection , 0.02% Sodium azide , before the next chromatography Bacteriostatic agent Remove, so as not to interfere with the determination of eluent.

Recovery method

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If it is no longer used, it can be recovered. The general method is to wash the gel with water and filter it dry, and then use 70%, 90%, 95% Ethanol dehydration Balance to ethanol When the concentration reaches above 90%, filter it dry and then reuse it Ether Wash off ethanol, filter dry, dry and store. The wet storage method is to add bacteriostatic agent or water in the gel slurry to wash it to neutral, and then seal it Autoclave preservation.

Application of gel chromatography

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⑴ desalination : Polymer (such as protein 、 nucleic acid , polysaccharide, etc.) can be removed by gel chromatography, which is called desalination. The desalting method is simple and fast, and the protein and enzyme are Desalting process It is not easy to change. Applicable gel is Sephadex G-10 , 15, 25 or Bio Gel-p-2, 4, 6. The ratio of column length to diameter is 5-15, and the sample volume can reach Column bed volume 25% - 30%, in order to prevent protein desalting solubility It will form sediment and adsorb on the column when it is reduced. It is generally used Ammonium acetate etc. volatility salt Buffer Balance the chromatographic column, then add the sample, elute with the same buffer, and use Freeze drying method Remove volatile salts.

(2) For Separation and purification : Gel chromatography has been widely used in enzyme, protein amino acid 、 Polysaccharide 、 hormone 、 alkaloid Separation and purification of other substances. The gel has strong adsorption power to pyrogen, and can be used to remove Pyrogen preparation Water for injection 。

⑶ Determination of molecular weight of high molecular substances: use a series of known molecular weight Standard Put into the same gel column under the same conditions Chromatography , record the Elution volume And plot the logarithm of elution volume to molecular weight to get a straight line within a certain molecular weight range, that is standard curve 。 When determining the molecular weight of an unknown substance, the sample can be added to the gel column where the standard curve has been determined for elution, and its molecular weight can be found on the standard curve according to the elution volume of the substance.

⑷ Polymer solution Concentration: usually, the Sephadex G-25 or 50 dry glue is put into the dilute polymer solution. At this time, the water and low molecular weight substances will enter the pores inside the gel particles, while the polymer substances Obstruction removal In addition to the gel particles, the swelled gel is separated by centrifugation or filtration to obtain a concentrated polymer solution

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