Molecular sieve definition
Molecular sieve chromatography, also known as gel filtration chromatography or size exclusion chromatography. Molecular sieve chromatography is a chromatographic technique in which a porous gel having a certain pore size range is used as a stationary phase to separate the components in the mixture by molecular size. There are many substances having molecular sieve action, such as pumice, agar, agarose, polyvinyl alcohol, polyacrylamide, dextran gel and the like.
Avoid the cause one: the process is difficult to enlarge
Molecular sieve chromatography cannot follow the linear amplification principle, and even if the process flow rate is adjusted according to the principle that the height of the bed is constant, it is also a problem to be faced. In order to ensure good separation, molecular sieve chromatography must require a relatively high filling height. However, the filling height is high and the back pressure is bound to be high. Due to the limited pressure resistance of the chromatography packing, the process flow rate is significantly limited under the premise of ensuring no overpressure, which makes the entire purification process take a long time.
Avoid the second reason: the column is difficult to fill
When performing column packing, the concentration of the homogenate must be within a certain range (50%-70%) to ensure uniform sedimentation and good column efficiency, which has special requirements for the specification of the column. For a column with a height of 100 cm, at a homogenization concentration of 50%-70%, assuming a compression factor of 1.15, the actual use height is 43.5-60.9 cm, which often fails to meet the requirements of some users. If the column packing device is selected and the space occupied by the column head is taken into consideration, this requires that the experimental space must be highly adequate. If two columns are considered to be used in series, the column efficiency of the two columns is required to ensure a good separation effect, which also has high requirements for the filling technology.
Avoid the cause three: the difficulty of measuring the column
The conventional efficiency test method requires an equilibrium of 1.5-2 CV. After injection, it elutes to the peak (reference value: 2 CV) at a flow rate of 30 cm/h. If the filling height is assumed to be 60 cm, the equilibrium and elution volume totals 3.5 CV. After calculation, it takes 7 hours to measure the efficiency of the column. It is conceivable that the process is extremely cumbersome and time consuming when it is found that the column efficiency is not ideal and needs to be refilled and the efficiency of the column is determined.
Avoid the cause four: maintenance and operation difficulties
When the column is used up and stored in 20% ethanol, it is also extremely time consuming. Moreover, when used again, 20% ethanol is replaced by water, generally requiring more than 3 CV, and since the back pressure is large in the process, it is usually necessary to reduce the flow rate.
In short, the use of molecular sieve chromatography, each link is extremely cumbersome and time consuming, therefore, in the design of the chromatographic process, try to avoid molecular sieve chromatography.
Application field
Molecular sieve chromatography is not without its merits. Its application fields mainly include the following aspects:
1) Desalting:
Low molecular weight impurities in a solution of a polymer (such as a protein, a nucleic acid, a polysaccharide, etc.) can be removed by molecular sieve chromatography, and this operation is called desalting. The desalination operation of the method is simple and rapid, and the protein and the enzyme are not easily deactivated in the desalting process, and the sample volume can reach 25%-30% of the volume of the column bed. In order to prevent the solubility of the protein after desalting, a precipitate is adsorbed on the column, The column is usually equilibrated with a volatile salt buffer such as ammonium acetate, then the sample is added, and the same buffer is used for elution. The collected eluate is subjected to freeze drying to remove volatile salts.
2) Separation and purification:
Molecular sieve chromatography has been widely used in the separation and purification of enzymes, proteins, amino acids, polysaccharides, hormones, alkaloids and other substances.
3) Determination of the molecular weight of the polymer material:
A series of known molecular weight standards were placed in the same gel column, and chromatographed under the same conditions, and the elution volume of each minute component was recorded, and the elution volume was plotted against the logarithm of the molecular weight at a certain molecular weight. A straight line is available within the range, ie a standard curve of molecular weight. When the molecular weight of the unknown substance is determined, the sample can be added to the gel column in which the standard curve is determined, and the molecular weight of the substance can be detected on the standard curve based on the elution volume of the substance.
4) Concentration of polymer solution:
Usually, Sephadex G-25 or 50 dry glue is put into a dilute polymer solution, when moisture and low molecular weight substances enter the pores inside the gel particles, and the high molecular substances are excluded from the gel particles. Further, the swollen gel is separated by centrifugation or filtration to obtain a concentrated polymer solution.
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