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The choice of gel depends on the purpose of the experimentGel: Set & Match。If the purpose of the experiment is toMacromolecular substancesSephadexG-25 and G-50 can be used to separate from small molecular substances due to their significant difference in partition coefficient. SephadexG-10 can be used for desalination of small peptides and low molecular weight substances (1000-5000),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.Generally, the gel with the exclusion limit slightly greater than the highest molecular weight substance in the sample is selected. During chromatography, these substances can penetrate into the gel to varying degrees. Due to different Kd, they are finally separated.
Chinese name
Exclusion limit
Foreign name
Exclusion limit
The diameter and length of the column According to experience, when separating groups, the chromatographic column with a length of 2-30 cm is mostly used. When separating by grades, the chromatographic column with a length of about 100 cm is generally required, and its diameter is within 1-5 cm, and less than 1 cm produces pipe wallseffectIf 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.Preparation of gel column After the gel model is selected, suspend the dry gel particles in 5-10 times amount of distilled water or eluent for full swelling, and pour out the very fine particles after swelling.Natural swelling takes a long time, and heating can accelerate the swelling, that is, the wet gel slurry is gradually heated to near boiling in a boiling water bath, and the gel can be fully expanded and dissolved in 1-2 hours.Heating method can save time and sterilize.Filling of gel: vertically fix the chromatographic column on the shelf with the groundClipClamp, a large container with a stirring device can be installed on the top of the column, the column is filled with eluent, the gel is mixed into a thinner slurry and put into the container on the top of the column, and then the gel sinks into the column under slight stirring, so that the gel particles rise horizontally until the required height, and the column top is removeddeviceCover the surface of the gel bed gently with the 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.During the balance process, the flow rate gradually increases to the chromatography flow rate, which must not exceed the final flow rate.To balance the gel bed overnight, before use, check whether the chromatography bed is even, whether there are "lines" or bubbles, or add some colored substances to observe the movement of the color band. If the band is narrow, even and flat, it indicates that the performance of the chromatography column is good. If the color band is distorted, scattered, or widened, the column must be reinstalled.After the balance of the sample adding and eluting gel bed, leave a few milliliters of eluent at the top of the bed to saturate the gel bed, and then add the sample with a dropper.Generally, the sample volume is not more than 5% - 10% of the total bed volume of the gel.The sample concentration is independent of the distribution coefficient, so the sample concentration can be increased, but the molecular weight ofmaterialThe viscosity of the solution will increase with the increase of the concentration, which will limit the molecular movement, so the relative viscosity of the sample and the eluent should not exceed 1.5-2. After the sample is added, open the outflow port 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 eluant to wash the pipe wall, so that all of it enters the gel bed,Connect the chromatographic bed with the eluent storage bottle and collector, design the flow rate in advance, then collect the eluent separately, and conduct qualitative and quantitative determination for each fraction.The repeated use of gel column, gel recovery and preservation can be used repeatedly after being packed once, without special treatment, and will not affect the separation effect.In order to prevent the gel from being recovered if it is no longer used, the general method is to wash the gel with water and filter it dry. Then use 70%, 90%, and 95% ethanol to dehydrate and balance it until the ethanol concentration reaches 90% or more, filter it dry, and then use ether to wash off the ethanol, filter it dry, and store it dry.The wet storage method is to add bacteriostatic agent or water in the gel slurry to wash it to neutral, seal it and then autoclave it for storage.
