Gel filtration chromatography (GFC) is a technology for elution and separation based on the molecular sieve of porous gel with a porous network structure, which is based on the difference of molecular weight of each component in the separated sample.

Today, we mainly introduce the main factors affecting the separation effect:
  1. Flow Rate

The flow rate has a great influence on the separation effect, so it is very important to maintain an appropriate and constant flow rate when eluting and separating protein samples. The elution flow rate in gel chromatography mainly depends on the diameter and height of gel column, gel type, particle size and separation type. Generally, before starting the formal elution, a preliminary test must be carried out to determine the appropriate flow rate. The slower flow rate can fully balance the protein sample with the gel matrix and achieve the desired separation effect. However, the low flow rate will cause the lateral diffusion of the sample to increase in the gel bed, which will widen the peak width and reduce the resolution. In addition, the elution time is prolonged and the work efficiency is reduced. High flow rate is easy to cause the overlap of elution peaks, so that the components that can be separated overlap, and will increase the column pressure and affect the separation effect. The linear velocity of general gel is controlled at 2-10cm/h. When the commercial gel is released, some flow parameters provided by the merchant can be used for reference by users. In general experiments, the peristaltic pump is often used to adjust the flow rate. If there is no condition, the flow rate can be controlled by controlling a certain height differential pressure. The volumetric flow rate (ml / min) is usually used in the experiment. Sometimes, for the needs of the experiment, the volumetric flow rate needs to be converted into linear flow rate (cm / h). The conversion formula is as follows:

2. Sample volume

The sample volume (VS) can also have a great impact on the separation effect of protein samples. The volume of adjacent peaks will overlap or affect the elution effect; Too few samples are added, the collection amount of target protein components is small, the dilution ratio is increased, and the concentration is low, which reduces the experimental efficiency.

3. sample concentration

The sample should be highly concentrated before injection, but the concentration should not be too large, otherwise the separation effect will be affected. The protein concentration should be less than 70mg / ml, generally 10-20mg / ml. If necessary, a series of sample concentration tests shall be carried out to determine the best value. Too low sample concentration can cause excessive dilution of a protein component and affect the collection; When the concentration is too high, the mobile phase will be unstable, resulting in the deformation or overlap of elution peaks. In addition, the sample concentration is also related to the type of separation and purification. During protein group separation or desalination and impurity removal, due to the large molecular weight difference between the target protein and impurities, the sample concentration can be appropriately increased, while the sample concentration should be lower as far as possible during graded separation or analytical experiment with small molecular weight difference.

4. Ionic strength

The sample eluent contains a certain ionic strength salt solution, which can prevent the interaction between protein and protein or protein and gel medium, and exclude the interference between protein and mobile phase or interaction with gel medium. Some neutral proteins without charge can be effectively separated in pure water. When separating some charged protein samples, we should pay special attention to this effect. We must adjust the ionic strength of the eluent to eliminate the interference of ion adsorption. For example, Tandex is weakly acidic because of its carboxyl group. Therefore, when using this kind of gel to operate chromatography, a certain ionic strength salt solution (usually higher than 0.05) is used as eluent, so that Tandex and basic protein can be adsorbed. When separating and purifying proteins, 20-100mmol / L NaCl is generally used as salt solution. But it should be noted that if the salt concentration is too high, the volume of the gel column will change.

5. pH

Because the equilibrium fluid and eluent used in gel filtration chromatography are consistent, pH will not change during the whole operation. The buffer solution with certain buffering capacity can be used to control pH. To maintain a certain pH of the eluent, on the one hand, consider the stability and solubility of the protein sample, and try to avoid the pH range close to the isoelectric point of the protein sample to prevent protein precipitation. On the other hand, the change of pH in equilibrium and elution will lead to changes in the volume of gel bed, which will affect the efficiency and effect of separation and purification. Therefore, when determining pH, we should consider the two factors of the properties of protein samples and the pH range that the gel can tolerate. The pH is generally controlled at 6-8, and phosphate and Tris HCl buffer are widely used. The range of pH suitable for use in some gels provided by manufacturers is for reference.

6. Molecular shape

The size and molecular weight of solutes depend mainly on the shape of the molecules. The proteins dissolved in the eluent have different molecular shapes (such as globulin, micro asymmetric globulin, fibrous rod protein and denatured bent structure). In the ideal gel filtration chromatography, the protein is spherical, and the retention time in the column is only related to the difference between the molecular size and the gel pore size. Therefore, different shapes of protein molecules also have different degrees of influence on the separation. In the experimental process of determining the molecular weight of some non spherical proteins, a high concentration of denaturant (such as 8mol / L urea) is usually added to the sample to make the protein components curl, and the molecular shape tends to be consistent.

7. Other factors

In addition to the above factors, the operating temperature, the type and diameter of gel particles, the length of the column and the inner diameter all affect the separation effect of the protein sample. Keeping proper temperature during operation is very important for separation. The operation temperature should be controlled in the most suitable range of gel. Otherwise, the classification range, the accuracy of exclusion limit and the volume of column bed will be affected.
Welch Materials gel filtration filler:

  • ¬†Tandex G series and LH20 multi-mode gel filtration series
  • SuperTandex Prep Grade Series
  • Tanrose Fast Flow Series
  • Tanrose/Tanrose CL Series

If you have any problem or require further information, please contact info@welchmat

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