Operation Specification of Loading and Elution of Packing Medium of Chromatography Columns
Operation specification of loading and elution of packing medium of chromatography columns can influence efficiency of chromatography separation and purification directly. With many-year practical experience and guides of related experts, Malston writes the “Operation Specification of Loading and Elution of Packing Medium of Chromatography Columns”, which is reference of users and scientific researchers.
Installation and strict test for column head
1. To check whether it is installed according to drawings.
2. To check whether it has filter screen or strainer to protect from leaking adsorbent (such as silica gel).
3. Material of filter screen should be corrosion protection, and small mesh. In general, it should use more than 500 mesh filter screen.
4. Install lower column well and fasten it, add the smallest polarity of solvent (such as hexane, petroleum ether, toluene), let level of solvent is higher 20-750px than lower column. Check whether there is any leak. If there is leakage, fasten well or re-install until no leakage.
5. After check without leakage, let solvent in column empty, then prepare to put adsorbent (such as silica gel)
A same as dry method filling to prepare some silica gel and record quantity.
B Prepare an anti-corrosion plastic bucket that can contain 30-50 L solvent, and pour into low polarity solvent that will be used with eluent, such as hexane, petroleum ether, toluene etc. And pour into silica gel to stir together. Then pour the mixture separately into chromatography column. And grout outlet under the column connected with a buffer tank and vacuum system. Vacuum switch, upper grout will fall down, and add the grout continuously (It is better to let silica gel under the liquid so that there is no air in the column) until fill up. And open vacuum to pump the liquid, and add solvent from the head of column until the silica gel does not fall down.
C Wet method filling can cause loss of solvent and big smell so that environment is not good. But dust is very small so that it can not damage outside wall of column.
D Quantity of silica gel in dry filling may be more than that wet filling.
1. Dry method adding sample
A Sample preparation: Dissolve material into slurry with the solvent that is big polarity and used in eluent, then add diatomite (or silica gel)mixed, airing, and smash into powder and mix evenly.
B Flatten the preparation sample and put it into chromatography column above silica gel, then put filter screen and sieve tray, finally install the upper head according to the drawing.
2. Wet method adding sample
A. Sample preparation: Dissolve the material into slurry with the solvent that polarity is smaller than silica gel (such as dichlorom ethane, acetic acid ethanol, ethane mixture ethane, toluene, etc.), then pour the slurry above the silica gel of chromatography column, and flatten (If the liquor is much more, pump can press it into the column.)
B. Same as above, put filter screen and sieve tray, install upper head.
1. Balance the column
Small polarity solvent in eluent (such as ethane, dichlorom ethane, toluene, etc. ) will be put into the upper side of chromatography column by metering pump or nitrogen. Polarity of the solvent is smaller than silica gel, and it can not clean away the material adsorbed in silica gel, it can clean the impurity away. And the process can let much air in silica gel out of the column. It is balancing column or adsorbent. Silica gel can be infiltrated by solvent, and it can discharge the air and is good for elution.
When the solvent used in balance process flows out of column, the process can stop, then do elution process with eluent. Balancing continues and let more air discharge out, but consumption of solvent will be added.
According to result of test, using some eluent clean all materials adsorbed in silica gel of columns reach separation. Flow rate of solvent in elution is 1-50px/min.
3. Washing the column
When there are some useful material flowing out of the column, wash impurities that have big polarity in silica gel level with big polarity solvent, then discharge it out.
4. Re-balancing column
Using small polarity solvent pushes out the big polarity solvent that left in column, then doing next batch of material adding sample process.
Source from: Malston (Beijing) technology Co., Ltd.