Chromatographic column maintenance mode, take it away!

In the use of liquid chromatography column, it is inevitable that there will be pollution, loss and other problems. At this time, it is necessary to wash and maintain the chromatographic column. From the point of view of the bonded phase, the HPLC column can be divided into reversed phase column and non reversed phase column. For the reverse phase bonding phase, such as C18, C8, phenyl and so on, because of its wide application and complex composition of the mobile phase, it is difficult to maintain a fixed thinking and mode for the maintenance of such columns. In most cases, specific problems need to be analyzed. The project used is relatively specific, or its mobile phase composition span is not large, so there are more fixed ideas to follow in dealing with problems.
Below, the small editor for you to sort out some of the non reversed phase chromatographic column troubleshooting ideas and common maintenance methods.
G10
G-10 is a soft gel filler, and its mechanical strength is not as strong as that of silica gel filler. Therefore, in the process of using, we should strongly avoid the organic solvents in the mobile phase or sample, otherwise the column will collapse easily. If there are problems after using for a period of time, such as the peak shape and resolution decrease, the column can be reactivated with distilled water according to the instruction manual. The washing time can be a little longer, and then the low flow rate of mobile phase can be used to balance overnight. If there is no obvious improvement, it can be washed according to the regeneration washing method in the manual. The regeneration washing steps are shown below.
If there is no obvious improvement in the above operations, the most likely case is that the packing is seriously polluted or even the packing collapses. In this case, it is of little significance to carry out washing again. It is recommended that customers purchase new chromatographic columns.
Regeneration and washing of G-10 column:
1. 5 mol / L NaOH and 0. 5 mol / L NaCl, 1:1 mixture (V / V), washing 20-30 times of column volume;
2. Wash the column with distilled water until it is neutral;
3. According to the Pharmacopoeia method, dextran 2000 reference material system suitability test was tested;
SEC
SEC packing is hydrophilic spherical protein silica gel, which is often used for molecular weight exclusion separation of high molecular polymers. The treatment method is similar to G-10. If there is a problem, it is also reactivated. Rinse with pure water for a long time, and then the mobile phase is balanced. If there is no obvious improvement, flush according to the abnormal flushing method in the manual. See the following for the flushing method. As for the resolution, if the resolution decreases, the separation can be increased by adjusting the flow rate.
Abnormal washing steps of SEC column:
1. Low pH and high concentration neutral salt (such as 0.5m sodium sulfate solution, adjusted to pH 3.0 with sulfuric acid) can wash 15 times the column volume, which is helpful to remove the basic protein;
2. Washing 15 times the column volume with buffer salt solution containing organic solvent (such as 10% - 20% methanol and acetonitrile) (such as 50 mM phosphate buffer solution, pH 7.0) is helpful to wash out the hydrophobic protein;
3. The addition of cosolvent helps to remove the strongly adsorbed substances on the stationary phase (such as hydrogen bonding);
4. According to the injection conditions of the column test report, the reference substance was injected to test the column efficiency;
be careful
Cosolvents (e.g., 6-8 mol / L urea or 0.2-0.3% sodium dodecyl sulfate) are recommended only when there is no significant improvement in both neutral salt solution and organic solvent washing.

Silica gel column
The common problems of silica gel column (SiO2) in normal phase mode are usually caused by insufficient balance, insufficient transition or water content. Water content is an important parameter affecting retention and selectivity in normal chromatography. Most solvents contain a small amount of dissolved water (for example, the moisture content of n-hexane at 20 ℃ is 0.0111% w / W). The common problems in normal chromatography, such as resolution and retention time drift, can be attributed to the change of water content in stationary phase and mobile phase, while the packing may be intact.
The following methods are recommended:
1. 100% isopropanol -- 100% methanol -- 100% isopropanol, pay attention to the sufficient transition of isopropanol (isopropanol has high viscosity and high pressure, pay attention to adjust the flow rate within a suitable range), and then the mobile phase is balanced overnight;
2. After removing the water on the stationary phase, 30 columns were washed with n-hexane containing 2.5% dimethoxypropane and 2.5% glacial acetic acid;
3. Using semi saturated mobile phase, the anhydrous non-polar mobile phase is divided into two parts, one half of which is added with a certain amount of water, mixed and stirred for an hour. After standing and layering, the water phase is removed, and then the two parts of non-polar solvent are mixed together to form a "Semi saturated mobile phase" (one of methods 2 and 3 is selected);
4. According to the order of empty sample, blank solvent, reference substance and test sample, the peak situation can be seen;
Amino column
Amino column is one of the common normal and reversed phase chromatographic columns. When the normal phase is used, the treatment and maintenance of amino column are the same as that of silica gel column;
Under the reversed phase use, especially in sugar projects, it is easy to have problems such as peak broadening, tailing and service life. First of all, it needs to be understood that the amino acid column bound aminopropyl group itself is easier to hydrolyze than the conventional C18, C8 and other reverse groups. Therefore, when using the amino column, it is necessary to be prepared for a shorter service life than the conventional chromatographic column
In case of the above problems, it is suggested that:
1. 100% acetonitrile -- 100% methanol -- 100% isopropanol -- 100% acetonitrile, each flushing for more than 40min (isopropanol has high viscosity and high pressure, pay attention to adjust the flow rate within an appropriate range);
2. After washing according to method 1, the mobile phase was balanced overnight, and the sample should be injected when necessary;
3. If there is no obvious improvement, use pure methanol, flow rate of 1.0 ml / min, forward flushing for more than 3 hours, and then the mobile phase, solvent and sample are all newly prepared, and then balance with the newly prepared mobile phase for 1 hour, and inject the sample according to the sequence of empty sample, blank solvent, reference substance and test sample;
4. If the above treatment is not satisfied, it is most likely caused by the breakage of silica gel matrix in the process of use. At this time, if the solvent is washed continuously and the improvement is not significant, it is recommended to purchase a new chromatographic column directly