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I. Introduction
According to the different principles of filling substation and sample distribution and exchange, ion exchange layering, gel
filtration
cilation and affinity layering are three classical layering techniques that separate
proteins
., Tips and Methods
1.Choice of column layering operation method
At present, the operation mode of column
chromatography
separation, including normal pressure separation, decompression separation and pressurized separation 3 modes. Normal pressure separation is the simplest separation mode convenient and simple, but the time of escape is long.Decompression separation, although it saves
fills
, is difficult to obtain due to the large amount of air that evaporates the solvent through the filler, and sometimes water vapor condensation outside the post, as well as some easily decomposable
compounds
, which must also be pumped using a water pump or
vacuum pump
.pressure separation can speed up the flow of gonorrhea, shorten the sample wasostation time, is a better method, similar to the normal pressure column, but with pressure to make gonorrhea faster wash. Pressure can be provided by compressed air, double balls or small air pumps.. 2. The choice of column specifications
column analysis separation columns with various specifications on the market. The column grows, the corresponding number of towers is high, separation is good. At present, the column on the market, its diameter ratio is generally in the 1:5 to 10 range, in actual use, the filler volume is generally 30 to 40 times the sample amount, the specific selection to be based on the nature and content of the sample for specific analysis.If the required parts and impurities are more separated, the amount of filler can be reduced, using a column with a relatively small internal diameter (e.g. 2 cm×20 cm of the column);. 3. Load column
column layering
string column
filling mainly has wet method and dry method two, wet method to save trouble, generally dissolve samples with gonorrhea, can also use dichloromethane, ethyl acetate, etc., but the less solvent the better, otherwise the solvent will become a detergent. Pistons at the bottom of the column must not be coated with
lubricity
, otherwise they will be sprayed into the lotion, you can use teteflon-based valves.there is no substantial difference between dry and wet columns, as long as the columns can be installed. The loaded columns should be moderately tight (too dense for the gonorrhea to flow too slowly) and must be uniform, otherwise the sample will flow diagonally from one side.not have air bubbles in the column at the same time, in most cases some small bubbles do not have much effect, because as long as the pressurized bubbles can disappear. But the column is more taboo is cracking, cracking will affect the separation effect, or even scrap.. 4. The choice of
the selection of a suitable solvent system is the key to column analysis separation. There are three factors to consider when selecting column layering scaffolding agents: solubility, affinity, and separation (Resolution).solvents should be inexpensive, safe and environmentally friendly, oil ether, ethyl acetate, dichloromethane, ether, methanol and positive hexane and so on. However, the price of positive hexane is high, ether is very volatile, dichloromethane and methanol and silicone adsorption is a heating process, easy to make the column bubble. Other solvents are used relatively little and need to be selected according to different needs.Also worth mentioning, since we are carrying out trace analysis, the purity of the gonorrhea must be concerned, generally using pesticide residue grade or HPLC grade, if the analysis is pure must be refined. At the same time solvent in the post after the best recycling, on the one hand environmental protection, on the other hand can also save some money.. 5. Sample
Dissolve the sample with a small amount of solvent, after the addition of the bottom end of the piston open, until the solvent layer is lowered to the quartz sand surface, and then add a small amount of low-polar solvent, and then open the piston, so two or three times, the general quartz sand is basically white.add gonorrhea, do not pressurized at first, such as dissolved sample solvent and sample layer has a distance (2 to 4 cm), and then pressurized, so as to avoid solvent (such as dichloromethane, etc.) with the sample quickly down.many samples in the upper column before the viscosity is greater, after the sample will be presection on the column, which is generally a relatively large number of samples will appear, because the filler on the sample adsorption saturation caused. Some samples are soluble that solvents (e.g. DMF, DMSO, etc.) that can be dissolved cannot be posted, so they must be dried.. 6. The principle of collecting and concentrating
washing liquid and using silicone as a fixed phase over column is a balance between adsorption and desorption. If the adsorption of the sample and silicone is relatively strong, it is not easy to flow out, then alumina can be used as a fixed phase.after column analysis of the gonorrhea, due to the use of more solvents, must be concentrated, if the object to be tested has a certain degree of volatility, it is best to use normal pressure volatile solvent, otherwise it is easy to lead to low detection results.
.