Friends who use liquid chromatographs will definitely encounter air leakage and liquid leakage. The mobile phase is the main source of various problems in liquid chromatography. One of the most common failures of liquid chromatography is blockage and the other is leakage. Today we will discuss the two parts separately.
 
Why is it blocked? The manifestation of "blocking" is the abnormal increase in column pressure, and the direct cause is that the flow path is not smooth. The main location of blockage is at the front end of the chromatographic column. The main reason is that there are impurities in the mobile phase. The main source of impurities is bacteria.
 
1. Bacterial contamination in pure water
 
We must realize that the general domestic products do not need additional filtration treatment, and there is no problem in using them directly. Even some solid particulate impurities can be removed from the filter head at the front of the liquid flow path system. What really easily causes problems is the bacteria in the water. Freshly prepared pure water will grow bacteria when placed indoors for a few days. Although these bacteria are invisible to the naked eye, they are enough to block the gaps of the column packing particles, causing the column to be quickly scrapped. This is the cause of bacterial contamination during the preparation of the mobile phase. The solution to it is very simple, which is to ensure the reliability of the water.
 
Liquid chromatography solution:
 
(1) The ideal way is of course to purchase a laboratory-specific water purifier, which is convenient and reliable, and the quality is assured. The disadvantage is that it is expensive.
 
(2) Buy commercially available branded purified water in boxes, such as 500ml of Cestbon or Wahaha. The quality of the water is sufficient to meet the requirements of liquid chromatography. First, randomly select a bottle to do a bacterial plate experiment, and use it only after the number of colonies is qualified. In this way, you only need to open one bottle at a time, which is also very convenient. The cost is about 2 yuan each time. Here is a particular detail: in most books, when it comes to preparing the mobile phase, the last filtration step will be mentioned. But from the actual effect of our long-term use, as long as the quality of the water can be guaranteed, this step can and should be eliminated.
 
Water is guaranteed, can it be filtered?
 
(1) Mobile phase filtration is beneficial in theory, but in actual operation, it is impossible to specialize in special bottles, but it is easy to cause cross-contamination, which has a greater impact on mobile phases with complex ratios.
 
(2) Mobile phase filtration is not economical in terms of cost. It costs more than 6000 yuan to buy a set of filtering device, and the filter is recognized as a relatively easy to damage equipment. The most important thing is that the cost of the filter is too high, one piece costs tens of yuan. Calculated according to the normal service life of a general liquid phase column, the cost of the filter will be much higher than the cost increase of the chromatographic column.
 
2. Bacterial contamination of mobile phase
 
The mobile phase does not grow bacteria at first, but it produces bacterial contamination during use. This is mainly caused by a bad habits when using a multi-component liquid chromatograph. Take the simplest example: 50% water mobile phase, there are two ways to use it. One way is to mix them together before getting on the machine, and the other way is to put pure water in flow path A and flow path B with pure water. In terms of pure experimental results, the latter has obvious advantages: firstly, it is simple and does not require the experimenter to calculate the ratio and mixing, and secondly, the ratio is accurate, and the effect of retention time repeatability can be obtained.
 
However, liquid chromatography has a fatal flaw, that is, the pure water in the mobile phase bottle will grow bacteria within a few days (in many cases, not only pure water is used as the mobile phase, but a buffer salt solution is used, which is itself a high-quality fertilizer. , Bacteria grow more quickly), once there is a bacterial column, it will deteriorate quickly. Therefore, this method requires the operator to use freshly prepared pure water for each experiment, and it also requires that the water phase be replaced after each experiment and replaced with flushing. This is not a point that many people are aware of in actual work. Aware of it, but there will always be one or two misses in repeated use, but often one or two times are enough to have a fatal effect. Because the clogging of the liquid chromatography column is irreversible.
 
Therefore, it is better to sacrifice the reproducibility of the small retention time than to use a pure aqueous solution as the mobile phase. In terms of actual experimental results, I suggest using 10% water instead of aqueous solution (I have done experiments on the total number of bacteria in different proportions of water before, and it is basically antibacterial at 5%, and it can be completely sterilized at 10% and above), This can effectively eliminate the hidden danger of growing bacteria, and it can be used as a mobile phase or as a column flush. Even if it is more troublesome to calculate when preparing the mobile phase, it is troublesome once and you will benefit for life.
 
3, improper operation of liquid chromatography
 
(1) The wrong model was selected when replacing the parts, the interface was not very matched, and it was deformed when tightening and the pipeline was blocked.
 
(2) The sample treatment solution is not cleanly purified, and it will be blocked between the six-way valve and the column for a long time.
 
(3) When using a manual six-way valve, some people may not turn in place due to small hand strength, so that the flow path is blocked and the pressure quickly rises above the warning value.