During the use of the liquid chromatographic 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. According to the bonded phase used, the liquid chromatographic column can be divided into reversed-phase column and non reversed-phase column. For reversed-phase bonded phases, such as C18, C8, phenyl, etc., due to their wide range of applications and complex mobile phase composition, it is difficult to have fixed ideas and modes for the maintenance of such chromatographic columns, and in most cases, specific problems need to be analyzed; For non reversed phase fillers and bonding phases, such as silica gel, gel, amino, cyano and ion exchange, due to the relatively specific items used or the short span of mobile phase composition, there are also relatively fixed ideas to follow in dealing with problems.
Next, We will sort out some troubleshooting ideas and common maintenance methods for non reversed phase chromatographic columns.
G-10 is a soft gel filler, and its mechanical strength is naturally not as strong as that of silica gel filler. Therefore, it is necessary to avoid organic solvents in the mobile phase or sample during use, otherwise the column bed is easy to collapse. If problems occur 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 instructions. The washing time can be longer, and then the mobile phase can be used to balance overnight at a low flow rate; If there is no obvious improvement, flush according to the regeneration flushing method in the manual. See the following for the regeneration flushing steps.
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 wash again. It is recommended that the customer re purchase a new chromatographic column.
G-10 chromatographic column regeneration washing:
- 0.5mol/l NaOH and 0.5mol/l NaCl, 1:1 mixing (v/v), washing 20-30 times the column volume;
- Wash the chromatographic column with distilled water until it is neutral;
- Suitability test of dextran 2000 control system according to pharmacopoeia method;
SEC filler is hydrophilic globular 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. Wash it with pure water for a long time, and then the mobile phase is balanced; If there is no obvious improvement, wash according to the abnormal washing method in the manual. See the following for the washing method. As for the resolution, if the resolution decreases, the resolution can be increased by adjusting the flow rate.
SEC chromatographic column abnormal flushing steps:
- High concentration neutral salt with low pH (such as 0.5m sodium sulfate solution, pH 3.0 adjusted by sulfuric acid) washes 15 times the column volume, which is helpful to remove basic protein;
- The buffer salt solution containing organic solvent (such as 10%-20% methanol and acetonitrile) (such as 50mm phosphate buffer, pH 7.0) is used to wash 15 times the column volume, which is helpful to wash away hydrophobic proteins;
- The addition of cosolvent is helpful to remove the strongly adsorbed substances on the stationary phase (such as adsorption through hydrogen bonding);
- Inject the reference substance according to the injection conditions of the chromatographic column test report to test the column efficiency;
The use of cosolvent (e.g. 6-8 mol/l urea or 0.2-0.3% sodium dodecyl sulfate) is recommended only when there is no significant improvement in the washing of neutral salt solution and organic solvent.
Silica Gel Column
Common problems of silica gel column (SiO2) in normal phase mode are usually caused by insufficient balance, insufficient transition or water content. Water content in normal phase chromatography is an important parameter affecting retention and selectivity. Most solvents contain a small amount of dissolved water (for example, the water content of n-hexane at 20 ℃ is 0.0111% w/w). The common problems of resolution and retention time drift in normal phase chromatography can be attributed to the change of water in the stationary phase and mobile phase, and the filler may be intact.
The following methods are recommended:
- 100% isopropanol–100% methanol–100% isopropanol, pay attention to the full transition of isopropanol (isopropanol has high viscosity and high pressure, pay attention to adjusting the flow rate within the appropriate range), and then balance the mobile phase overnight;
- Remove the water on the stationary phase, rinse the column with n-hexane containing 2.5% dimethoxypropane and 2.5% glacial acetic acid for 30 column volumes;
- Using a semi-saturated mobile phase, divide the anhydrous non-polar mobile phase into two halves, add a certain amount of water to half of them, and mix and stir for one hour. Some non-polar solvents are mixed together to form a “semi-saturated mobile phase” (choose one of Method 2 and Method 3);
- Inject the sample in the order of empty sample–blank solvent–reference substance–test sample, and see the peak situation;
Amino column is one of the common chromatographic columns that can be used in both normal phase and reverse phase. When the normal phase is used, the treatment and maintenance method of the amino column is the same as that of the silica gel column;
In reverse phase use, especially in sugar projects, peak broadening, tailing and service life are easy to occur. First of all, it should be understood that the amino propyl group bonded to the amino column itself is easier to hydrolyze than the conventional C18, C8 and other reversed-phase groups. Therefore, when using the amino column, it is necessary to be prepared to have a shorter service life than the conventional chromatographic column.
If the above problems occur, it is suggested that:
- 100% acetonitrile –100% methanol –100% isopropanol –100% acetonitrile, all items shall be washed for more than 40min (isopropanol has high viscosity and pressure, and the flow rate shall be adjusted within the appropriate range);
- After washing according to method 1, the mobile phase shall be balanced overnight, and priming and sampling shall be carried out if necessary;
- If there is still no obvious improvement, use pure methanol with a flow rate of 1.0ml/min and positive impulse for more than 3 hours, then prepare all mobile phases, solvents and samples, and balance them with the newly prepared mobile phase for 1 hour, and inject samples in the order of empty sample – Blank solvent – control sample – test sample;
- If the above treatment is not satisfied, it is most likely caused by the breakage of silica gel matrix during use. At this time, if it takes time and solvent to continue washing, and the improvement is of little significance, it is recommended to directly purchase a new chromatographic column.
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