Column life

Q: my chromatographic column only has about 100 samples. Then, the peak shape becomes worse and the number of theoretical plates becomes very low. What is the reason for this?
A: 100 times is really very short. Under normal circumstances, we all want the column to last longer. In order to determine what the problem is, we need to first determine whether your method has caused the column life to be shortened.
There are two situations:
Whether the previous column used for the same test can be used for a longer time.
Whether all chromatographic columns used in this method have the same problem when running about the same number of needles.
In the first case, make sure that all conditions of the test are consistent. Has the composition of the sample changed? The strong adsorption of pollutants in the sample destroyed the performance of the chromatographic column. Are the seals in the instrument flow path intact? Whether the detached seal will block the filter and top packing of chromatographic column, thus affecting the distribution of samples.
If it can reasonably ensure that the chromatographic conditions do not change, it can be conservatively considered that the reason is the physical damage of packed bed. This includes rough handling of the column in the laboratory or during transportation (is it dropped?) Or manufacturing defects. Such defects can not be detected by the quality control of standard chromatographic column, and can only be revealed after use. In this case, the column manufacturer will replace the column free of charge.
Q: these manufacturers are good, but that's not my problem. My column is always used for a short time. Sometimes 100 injections, sometimes 200 injections. I can take 200 injections, but only 100 is really too little. It's too expensive. What do I need to do?
A: I totally agree with you. What we have to do together is find out the cause of the problem and see what we can do.
The most likely cause of the problem is that the sample component is adsorbed on the top of the column. They may precipitate due to their low solubility in the mobile phase, or they may be strongly adsorbed. As more and more samples are injected, these pollutants will accumulate on the top of the column, thus preventing the reasonable adsorption and distribution of samples. This leads to a poor peak pattern. Usually, this problem is accompanied by an increase in back pressure.
Q: OK, maybe that's it. So how can I solve this problem?
A: there are several ways to prevent this. One is to use appropriate sample preparation techniques to purify samples. SPE column with similar chemical properties can be used as separation column for solid phase extraction.
A better alternative is to use protective columns. The pre column can be used as the top of the analytical column and replaced when problems arise. In order to obtain the best performance, the protective column should have the same packing material as the analytical column, and should be filled with the same high-performance packing technology as the analytical column. If the precolumn made of different brands of fillers is used, the best performance can not be obtained in terms of separation capacity and protection of analytical column. In addition, do not use larger particle size. Larger particles or poorly packed precolumn will lead to the broadening of spectral band and the decrease of separation effect.
Q: it sounds good to use a guard post. Are there any other solutions?
A: Well, yes. There are several other possibilities, but they all have disadvantages.
I'm not advocating "washing" with solvents that may dissolve contaminants at the top of the column. In many cases, this process simply doesn't work. For example, if the contaminants deposited on the top of the column are proteins, then when you try to rinse them, they may not be dissolved again due to denaturation or even crosslinking. In addition, each rinse will remove the hydrolytic bonded phase, otherwise it will maintain local equilibrium at the site where hydrolysis occurs. Therefore, repeated washing can actually lead to accelerated aging of chromatographic column. Also, after washing, the column must be rebalanced with the mobile phase, and in some cases (e.g. ion pair chromatography) it may take a lot of time.
Another frequently tried method is backwashing. If you use a different solvent than the mobile phase, the same objection applies to column cleaning. If you only use the mobile phase, it will take a long time to get the pollutants out, or it may not work at all. Moreover, any backwashing will weaken the performance of the column. Although the current chromatographic columns are well packed and can withstand backwash, I would like to reiterate that this is not a standard practice.
Q: so, the best suggestion is to use precolumn?
A: Yes. They are inexpensive - between $10 and $50, depending on the brand and type - and the columns they protect usually cost about ten times as much. In addition, it protects the column from other contaminants that are more difficult to trace. The root cause of your column problem may be caused by dust in the mobile phase or debris caused by the pump gasket falling off. It may also be the adsorption of pollutants in the mobile phase. Some of them may be difficult to solve, but pre column can better solve these problems.
Q: are there any other reasons for shortening the column life?
A: Yes, but it's unlikely if you follow the manufacturer's advice.
One possibility is that the pH value of the mobile phase is beyond the recommended range, resulting in column collapse. This may also be caused by the sample dissolving in strong acid or alkaline solution.
In addition, there are some chromatographic columns for specific projects.
For example, amino columns react with aldehydes and ketones. In the buffer free aqueous solution, the amino column will produce strong alkalinity, resulting in partial dissolution of silica gel.
If the wrong solvent is used, the column will collapse. The reason is that the column is fundamentally very loose. They are partially held together by the adhesion of particles to each other. When placed in a mobile phase capable of breaking this adhesion, the possibility of collapse increases. This occasionally occurs in cyano columns in moderately polar solvents or phenyl columns in THF.
Another reason is to stay away from column "cleaning.".
Summary
The reasons for the short service life of chromatographic column, possible sample reasons, physical damage and unreasonable use:
If the reason is the sample, the sample can be pretreated, or the precolumn (first push) can be used. In addition, cleaning and backwashing can also be carried out (not recommended).
The physical damage is irreversible and the column needs to be replaced.
Unreasonable use includes exceeding the pH range, using the wrong solvent, and some chromatographic columns can only be used for specific projects.
For more related technologies, please consult MS technologies technicians