The clogging phenomenon is an abnormal increase in column pressure and the direct cause is a poor flow path. The main place of clogging is at the front end of the chromatography column. The main reason is that there are impurities in the mobile phase and the main source of the impurities is bacteria.
One of the reasons for blocking: bacterial contamination when preparing dhe mobile phase
First of all, we have to note that the general household methanol does not require additional filter treatment, and it poses no problem for direct use. Also, some solid particle impurities can be removed at the filter head at the top of the liquid-phase flow system. The real problem is the bacteria in the water.
Bacteria grow in newly prepared pure water when Although these bacteria are invisible to the naked eye, they are enough to fill the gaps in the particles of the column packingclogging, causing the column to be scrapped quickly. This is the cause of bacterial contamination in the preparation of the mobile phase. The solution to this is very simple, that is, to ensure the reliability of the water. Here are two recommended options:
(1) Of course, the ideal way is to purchase a laboratory-specific pure water machine, which is convenient, reliable and of good quality. The only downside is that it's expensive.
(2) Buy commercially available branded purified water in boxes, such as a 500ml bottle of Yibao or Wahaha, the quality of the water is enough to meet the requirements of liquid chromatographyllen. First, randomly select one to do a bacterial plate test, then use it after determining the number of colonies. This way, only one bottle is opened each time. This is enough and very practical. The cost is about 2 yuan each time.
※Here's a special note on a detail: In most books, when it comes to preparing mobile phases, there will still be a filtering step at the end, but from our long-term practice this step can and should be eliminated, as long as the quality of the water can be guaranteed.
There are three reasons:
(1) Mobile phase filtration is theoretically beneficial, but in practice it is impossible. If if it is intended for a specific bottle, this easily leads to cross-contamination, which affects the mobile phase more. Phases with complex ratios.
(2) Mobile phase filtration is not economical in terms of economic cost. It costs more than 6,000 yuan to buy a filter device, and the filter is recognized. It is relatively easy to damage devices. Most importantly, the cost of the filter is too high, and one piece will cost several tens of yuan in terms of service life the cost of the filter will be much higher than the cost of the chromatography column.
(3) Mobile phase filtration is for work efficiency not cost effective. Using a solvent filter involves a process of pre-cleaning, equipment, use, post-use cleaning, and drying that takes at least an hour. Again, these costs are not negligible.
(4) In practice it has not been found that mobile phase has no impact on column lifetime. We haven't done mobile phase filtration for at least 6 years, but compared to domestic counterparts, our column lifetime is relatively long with the same usage intensity.
The second reason for blocking: bacterial contamination when using the mobile phase.
refers to:
In the mobile phase, bacteria did not grow at the beginning, but bacterial contamination occurred during use. This is mainly caused by a bad usage habit when using multiple liquid chromatography causes.
The simplest example:
50% methanol-water mobile phase, there are two ways to use it. One way is to prepare and mix it before going to the machine and the other way is to put pure methanol and pure water in channel B.
From the point of view of simple experimental results, the latter has obvious advantages: firstly, it is simple and does not require the experimenter to calculate the mixing ratio separately, secondly, the ratio is accurate and the retention time can be repeated results.
Therefore, sacrificing the repeatability of small retention times is better than using a pure aqueous solution as the mobile phase set. Regarding the actual experimental results, I suggest using 10% methanol water instead of pure aqueous solution (I previously conducted experiments on the total number of bacteria in different proportions of methanol water, 5% can basically inhibit bacteria, and 10% and more can completely sterilize ), which can effectively eliminate the hidden danger of bacterial growth, and can be used as mobile phase and column rinsing. Even if it's a little tedious to calculate when making the mobile phase, but once trouble, lifetime benefit.
Third reason for Blocking: Incorrect operation.
Frequently Asked QuestionsThe types are:
(1) The model selected when replacing parts is incorrect, the interface does not fit very well, and deformation occurs when tightening, causing the pipeline to be blocked.
(2) If the sample treatment solution is not cleaned cleanly, it will cause clogging between the six-way valve and the column for a long time.
(3) When using the manual six-way valve, some people may not turn it due to weak hand strength, causing the flow path to dead-block and the pressure to rise above the warning value quickly.
(4) When using metal tubing to make waste liquid tube, it should be noted that it is best to add some water into the waste liquid bottle firstand hold the outlet end of the waste liquid tubing below the liquid surface. If it is in the liquid phase and the experiment uses a higher concentration of buffered saline, it can crystallize and block the outlet port during shutdown. This is not common, but it does happen.
The reasons behind the blocking have been discussed a lot, and now we will introduce the method to check the blocking.
After the locking phenomenon occurs, it is necessary to find out the reason, mainly which position is locked. Note that in most cases only one location in the entire system will be lockedbecomes.
To check the clogging it is necessary to disassemble it in reverse from the stern to the front and carefully observe the pressure value if a certain value The pressure of any component (except for the column) is very different when installing and removing and can be determined by the Evolution and change are assessed the pressure of the column with the same specification is constant.
