16 common problems in the use of liquid chromatography colum

The following is the text:
1. For a commonly used C18 column, how to activate and maintain a new column? Why do you do this?
A: in fact, the activation of a new column is a process of equilibrium. In addition to the mobile phase equilibrium, sometimes it is necessary to balance the new column with the measured sample, especially for the peptide with high molecular weight. Because of the high molecular weight material molecules, the diffusion speed is slow, and the equilibrium time is also longer. It is simple to determine the sample size and injection time until the sample is stable.
If you want to speed up the equilibrium time, increase the concentration of the sample used to balance in front of you, or do not wait until the elution is completed, and then inject multiple needles continuously. The purpose of equilibrating the new column with the substance to be tested is to saturate the adsorption capacity of the non-specific adsorption sites on the surface of the silica matrix filler.
2. What kind of column is usually used for peptide determination? The mobile phase consisted of acetonitrile and water, and a small amount of TFA. Especially for short peptides like tripeptide, how to choose the column?
A: peptides with low molecular weight can be determined by conventional C18 column, and also by ion exchange column, water-based C18 column and HILIC hydrophilic interaction column.
3. When the amino acid column enters the acid sample, it will hurt the column. If it is used for a period of time, the column efficiency will decrease and the peak shape will change. How to recover it?
A: the HILIC model of amino column should be used for acid sample determination. The presence of acids may protonate the slightly negatively charged amino functional groups, resulting in changes in retention properties of some analytes or a decrease in column efficiency after a period of use. Suggestion: 5-10 times of column volume of acetonitrile water (50:50) solution containing 0.5-1.0% NH3 should be used to wash the column (after washing, of course, the excess ammonia should be washed out with the mobile phase without alkali). Then, when analyzing such acid analytes, it is recommended to add a little ammonia such as 0.1% to the mobile phase.
4. What are the techniques of chromatographic column? Such as sealing, where are these technologies applied?
A: chromatographic column technology includes packing technology and column loading technology. It goes without saying that the quality of packing has a decisive impact on the separation performance and selectivity of chromatographic column. It is different in diameter and technology, but different in length and stability in the same column.
In my opinion, the gap between domestic and foreign chromatographic columns is that domestic companies would not develop their own fillers before. Generally, they buy foreign ready-made packing columns, and the quality control right of the fillers purchased is not in their own hands. In addition, due to the short history of column installation and less experience accumulation, the column installation technology has not fully reached the foreign level. In addition, the bare silica gel, which is a key basic material for the performance of chromatographic column, is not up to standard. Compared with foreign products in purity, particle size and pore size uniformity, domestic silica gel has a big gap.
5. What is the gap of chromatographic column technology?
A: in fact, there are certain skills and procedures for filling the HPLC column, and there may be some luck. Generally, high pressure homogenization method is used for loading. In other words, the filler can be evenly suspended in the solvent. And then it's compacted with instantaneous high pressure, which actually uses different proportions of homogenate liquid and the right pressure. The pressure is too high, the particles are broken, the pressure is too small, and the number of trays is small. At the same time, the pressure needs to be stable, otherwise the distribution is uneven and the tailing is serious. At the same time, the head is flat. Put it on and you can use it.
6. Under what circumstances can the column clean the sieve plate? This problem was also discussed. I took it down to clean it, but I saw that the pollution in the front section of the column was more serious, so I scraped it with a blade, and then installed the cleaned sieve plate. The problem is solved, but will the service life be reduced?
Answer: if the stigma is polluted, take out the contaminated one and then install some fillers. Because you scrape some packing, the number of micro trays is less. If you don't shave much, just the surface, it may be some dirt, so the problem is solved. But there will be the same problem in the future. If you hang it again, it will affect the column efficiency. It is suggested to install a pre column.
7. If the column is removed and placed for a period of time, what protection should be done?
A: for the general reverse phase column, that is, after washing, put it into pure methanol (acetonitrile) or about 80% methanol (acetonitrile) water, and then plug the two ends of the column with plugs to avoid the volatilization of the preservation solvent. There should be no special protection.
8. What is the mechanism of peak shape improvement by adding appropriate amount of tetrahydrofuran into the mobile phase?
A: according to the article compiled by Yu Shilin, methods and applications of high performance liquid chromatography (HPLC): methanol is the proton donor, acetonitrile is the proton acceptor, and tetrahydrofuran is the dipolar solvent. In addition to the polarity effect, there are other influencing factors. As for the separation mechanism, it is relatively complex, and can not be regarded as a universal method.
9. About the packing of chromatographic column. In my opinion, there are generally three situations in the packing of chromatographic columns
(1) Foreign production of fillers and filling finished products sold to the domestic;
(2) Foreign production of fillers, domestic filling sales;
(3) Domestic production filler, domestic filling sales.
In general, the first case is the most expensive and the best quality! But if the production of fillers is very complicated, can't the domestic packing keep up with it? Why is there more or less small problems in domestic filling?
A: there will be small quality problems in domestic filling, which is related to the fact that domestic work is not as rigorous as abroad. If there are no technical problems and a set of strict production quality management measures can be formulated and implemented, there is no difference between domestic filling and foreign filling.
10. How about the market share of domestic chromatographic column?
No one has made scientific statistics on the domestic chromatographic column market, and even the total sales volume of chromatographic column in one year is still controversial, not to mention the share of chromatographic column produced in China. I think it's about 30%.
11. Pre column or protection column with or without the problem! Originally, when analyzing the varieties of traditional Chinese medicine, I always used the protective column. However, when I came to the new company, I found that none of us had used it. In other words, we had never used a protective column in several laboratories. Later, I asked an old employee that it might affect drug analysis. Would you like to ask: will the sample analysis be affected after installing the protective column? Most of what we do is cephalosporins.
A: it should be said that adding a protective column will certainly help to protect the chromatographic column from being blocked by some particulate matter, which is certainly harmful to the resolution and column efficiency, because there is a dead volume in the middle of the protective column. It should be replaced with the protection column as often as possible. Cephalosporin antibiotics also depends on whether it is a drug substance or a preparation. For some raw materials, you can choose to add a pre column (a sieve plate in the middle) according to the loss of chromatographic column. If there is serious interference from excipients or the salt content of mobile phase is relatively large, it is better to prepare a protective column.
12. The use of a four-dimensional gradient pump a50% methanol B50% water often stop or enter the bubble, what is the reason?
A: when the water / methanol ratio is 55:45, the viscosity and column pressure have a maximum value. 50:50 is close to this extreme value. The column pressure is relatively high, but the most important factors affecting the column pressure are the particle size of the packing and the inner diameter of the chromatographic column. What kind of chromatographic column do you not know? If the system pressure is high, bubbles may enter the system due to the liquid feeding speed of the filter head in the solvent pump can not keep up with it. The shutdown should also be due to the drop of bubble entry pressure. It is suggested to replace the filter head with higher liquid flux.
13. The data showed that under normal conditions, the dry filling method was more suitable when the filler particle size was more than 20 μ m, and the wet filling method was more ideal when the particle size was less than 20 μ M. What is the difference between the filling methods?
A: there are generally four filling methods
① High pressure homogenization method is mostly used to fill analysis column and small-scale preparation column;
② Radial pressure method, waters patent;
③ The axial compression method is mainly used for loading large diameter columns;
④ Dry method. The technique of column filling is very strong, and most laboratories use filled commercial columns. Why take 20um as the dividing point? Are the first three filling methods wet? Can you give a brief description of the four filling methods?
14. Would you like to specify the method of recoil column without detector?
A: recoil is the reverse connection of columns to the system. Because there are pollutants backwashing out, of course, without the detector, the liquid outlet can be directly connected to the waste liquid bottle.
15. There has been a debate on whether columns can be recoiled on the Internet. What kind of columns can be recoiled and what can't be. After recoil, is it used by the positive or the reverse. For each type of column, not only ODS column, but also other columns such as positive column, amino column, ion exchange column and so on, are best explained.
A: the general positive and negative phase columns should be able to recoil. Only columns with asymmetric sieve plate aperture at both ends can not recoil, but at present, such columns are relatively rare. The purpose of backwashing is to wash off the pollutants in the column head. It is better to use it in front after backwashing, so as to avoid contamination at both ends of the column. What we have been advocating is: forward use, reverse flushing.
16. In the process of method development, acetonitrile and water were used as mobile phases. When adjusting the gradient, it was found that when 60% acetonitrile was used at the beginning, RT was 2.5 minutes. When it was adjusted to 40% acetonitrile, RT did not change and 30% did not change. When it was adjusted to 20%, RT suddenly changed to about 13 minutes. Please ask why? I use an ion exchange column.
A: the retention time of the ion exchange column is mainly determined by the ionic strength and pH of the eluent. What you are talking about is relatively simple. You need to make a detailed analysis of your method. For example, what is the situation of the analyte, and what are the properties of the polar ionizing group and the non-polar group? Is the ion exchange column polymer matrix or silica gel matrix? What buffer salt is the aqueous phase?
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