Freeze-drying is the process of making protein sample solution in a state of pre-freezing, using vacuum method, without sublimation, to directly sublimate, remove the water content, and make the sample protein into a dry preparation. In the immunoenzyme technology, the used serum, enzymes, etc. are easily denatured and spoiled in the solution. After being prepared as a dry preparation, it can be stored at room temperature or in a refrigerator (4 ° C) for a long time without inactivation, and is easy to carry.
The reason for freezing is that the moisture content or solvent of the dried sample increases the evaporation rate due to the reduction of the surrounding air pressure, and the higher the degree of vacuum, the lower the boiling point of the solution, the faster the evaporation.
Compared with vacuum drying and spray drying, freeze vacuum drying is more commonly used in experiments.
1. Procedure for freeze-drying serum
(1) First pour the appropriate amount of serum into a petri dish of appropriate size (preferably the thickness of the sample solution is below 1cm), and freeze to a solid state at a low temperature-below 20 ℃.
(2) In the freeze-drying device, place solid sodium hydroxide and phosphorus pentoxide separately, and the suction pipe at the upper end of the vacuum dryer is connected to the vacuum pump through the phosphorus pentoxide drying pipe.
(3) Put the frozen serum block together with the petri dish into the vacuum desiccator, close the desiccator immediately (coat petroleum jelly on the cover edge in advance), and start the oil pump, usually after 5-10h, the freeze-dried preparation can be obtained.
(4) Freeze drying is completed. First, gradually open the glass valve on the top cover of the dryer, and slowly deflate. After the pressure in the entire system rises to atmospheric pressure, remove the sample drying container, and finally turn off the vacuum oil pump.
2. Discussion
(1) The effect of freeze-drying depends on the vacuum degree of the vacuum pump and the pipeline with the appropriate diameter. The vacuum oil pump is best to use a large evacuation capacity, and all the connecting pipes should be thick and short.
(2) If the sample is a solution, it is best to be an aqueous solution to avoid the organic solvent lowering the freezing point and entering the pump. The concentration should be moderate, not less than 1.5%.
(3) If the sample uses a buffer as a solvent, it is best to use a volatile, such as carbonate or acetate buffer; if it is non-volatile, the influence of pH and salt concentration on the sample must be considered. For example, with pH 7.0 phosphate buffer, the Na2 salt crystallizes earlier than the Na salt during freeze-drying, and the pH drops to about 3.5, which is harmful to the sample. Adding a small amount of gelatin, lactose or glucose to the sample solution is conducive to preserving activity.
(4) When pre-freezing the sample, it is best to put it in an ordinary refrigerator to cool to 0 ℃, and then quickly move it into a refrigerator below 20 ℃ to freeze it.
(5) When the sample is melted during vacuuming, it must be re-frozen and the cause must be checked.
(6) Vacuum for about 30 minutes. If the container containing the pre-frozen sample is frosted, it indicates that it is working well. Due to the sublimation of the moisture in the sample, the temperature is reduced, the sample is naturally in a frozen state, and there is no need to take cooling measures outside the dryer.
(7) The dry preparation is packed in a vial, sealed with wax, and stored in an ordinary refrigerator.
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