How to judge the retirement time of a chromatographic column
Recommended steps for opening and installing new chromatographic column1. Use fresh and clean water and acetonitrile. Flush the system to ensure it is clean and free of any buffer salts and contaminants.
2. Avoid bumping and dropping when using chromatographic column.
3. Connect the inlet end of the chromatographic column to the system, and do not connect the outlet end of the column. There is an arrow on the column body to indicate the correct flow direction.
4. Run the column with pure acetonitrile at a flow rate of 0.1 ml / min, and then increase the flow rate to 0.5 ml / min within 2 minutes.
5. When the solvent uniformly flows out from the column outlet, stop the flow rate and connect the column outlet to the system detector (this can prevent bubbles from entering the detection system and quickly achieve baseline equilibrium).
6. Restart the flow rate and gradually increase the flow rate according to step 4 to the flow rate used in routine analysis.
7. Referring to the method in the column efficiency test report, the mobile phase with 5-10 times of column volume is usually required to balance the chromatographic column with the mobile phase condition until the pressure is stable with the baseline.
8. Test column efficiency. If there are no analytes in the column efficiency test report, please contact the manufacturer's department for support and use the appropriate concentration and injection volume. It is normal that the column efficiency is slightly lower than the column efficiency test report. If the result is obviously low and the peak shape is tailed, it indicates that the chromatographic column and / or the liquid phase system used are in an imperfect state, and troubleshooting is needed. When using a 2.1 mm inner diameter column, it is recommended to optimize the system to reduce the band broadening, including using the detector micro flow cell, reducing the injector loop volume, and using the system pipeline with smaller inner diameter (such as 0.005 "or 0.12 mm). And ensure that the pipeline and chromatographic column are connected properly without dead volume. When using small particle packing (e.g. 2.5 μ m) short column for efficient and rapid separation,
The following factors need to be considered:
1. Ensure that the pipeline and column are connected properly without dead volume, and optimize the system as much as possible to reduce the broadening of spectral band;
2. Increase the sampling frequency to more than 10 points / second;
3. The column with smaller particle size produces higher column pressure, while the linear velocity required for smaller particle size to achieve optimal chromatographic efficiency is higher. Please adjust the flow rate according to the actual situation of the LC system used;
4. A higher column temperature can be used to compensate for the pressure rise caused by small particles, such as 40 ℃ C. When using hybrid particle RP column, 45 ℃ C or higher can be used.
Some experiences of using reversed phase C18 column
1. Treatment of new columns: Although the newly purchased columns have been tested and sealed by the manufacturer, some of them have been in our hands for a long time. Therefore, every time we get a new column, we have to deal with the column. Firstly, 65% acetonitrile / water should be prepared, then the chromatographic column should be connected to the chromatograph correctly, and the outlet should be vented (Note: the detector should not be connected at the outlet end to avoid contamination of the detector). The flow rate can be 0.1ml/min ~ 0.5ml/min (for LC / MC column, it should be 0.3ml/min). It can not be carried out at a high flow rate. After seeing the liquid flowing out of the column outlet, 20 minutes should be passed Then connect the detector, adjust the flow rate to 1.0ml/min (0.3ml/min for LC / MC column) and continue to rinse for 2-8 hours.
2. Use of new column: first of all, we confirm whether the pH of the mobile phase of the sample is within the tolerance range of the column pH, so as to avoid damaging the column! If it is confirmed to be within the scope of use of the column, it is necessary to use 5% methanol or 5% acetonitrile for more than 30 minutes before balancing the column. A mobile phase with salt was used to balance the column. ）When the column is balanced enough time and the baseline is very stable, the sample can be injected for analysis. No matter what kind of products are analyzed, there are always impurities in the samples due to various factors. Therefore, it is better to add a protective column at the front of the column. To increase the service life of chromatographic column!
3. Column cleaning: ① if the mobile phases of acetonitrile, methanol and water are used in sample analysis, the chromatographic column can be washed directly with 55% ~ 65% acetonitrile / water or 55% ~ 65% methanol / water for 40 ~ 80 minutes, and then washed with pure methanol or acetonitrile for 30 minutes, then the chromatographic column can be preserved; ② if the mobile phase in the sample contains the mobile phase with buffer solution or acid or ion pair reagent After the sample is finished, the column must be cleaned according to the following steps: first, wash the column with 5% methanol / water or 5% acetonitrile / water at 1ml / min for more than 1.5 hours (if the column is longer, appropriate time should be added); then, wash the column with 55% methanol / ~ water 65% / water for 30-60 minutes; finally, wash the column with methanol or acetonitrile at 1ml / min for 30 minutes.
4. Regeneration of chromatographic column: when the chromatographic column has been used for a long time, the column pressure may be increased, the resolution is not good, the column efficiency is reduced, and the peak shape is not good. At this time, I need to regenerate the chromatographic column to extend the service life of the chromatographic column. The specific methods are as follows: (1) first, use 5% methanol / water or 5% acetonitrile / water, reverse the chromatographic column with 1ml / min for more than 60 minutes; then wash the column with tetrahydrofuran at 1ml / min for more than 30 minutes; and then rinse with 65% methanol / water or 65 acetonitrile / water at a flow rate of 1ml / min for 60 minutes. Finally, rinse with pure methanol or acetonitrile for 30 minutes. Remarks: methanol, acetonitrile and tetrahydrofuran should be pure with good quality and color spectrum. Water use is heavy steam retention water.
When is the time for a new post?
1. When the number of column trays is reduced to the percentage of new columns, what percentage should be replaced?
Chromatographic workers often encounter such problems: when should the chromatographic column be replaced? Can the number of plates of chromatographic column reflect the state of chromatographic column? Determine the standard indicating that a chromatographic column should be "retired". From experience, when the number of plates of a chromatographic column is reduced to what percentage of a new column, it can be considered that the chromatographic column can no longer be used? Take the number of trays as an evaluation How can you judge the quality of a column in the laboratory?
The number of plates is a rough index and can not be used as a standard for evaluating chromatographic columns. The number of trays is usually used to examine the chromatographic system: connect the new column to the instrument, test the column according to the test conditions provided by the column manufacturer, and you should get the same results as in the analysis report. The worst case should also be a plate count 10% lower than the reported value. If the number of plates you measured is much lower than the reported data provided by the manufacturer, it may indicate that there may be some problems with your chromatographic system. Injector, out of column effect, capillary connection and detector are all prone to problems.
2. The mixed index of pressure, resolution and peak shape was used to investigate the state of a chromatographic column
It is believed that every chromatographic worker will be concerned about the resolution, which is an index to evaluate the quality of a chromatographic column for the separation of specific samples. However, another factor we need to consider is that the effect of the number of plates on the resolution is only the square root relationship, which means that when the plate number of a column decreases by 50%, the resolution only decreases by 7%. And when the number of plates on a column drops to half of that of a new column, you must see other more serious symptoms that tell you that the life of the column is not long. In practical work, most chromatographic methods only need half of the number of plates. Is there room for optimization of these chromatographic methods? The answer is yes. It can be seen that the number of plates of chromatographic column is not the most important parameter in separation.
A better method is to use a mixture of pressure, resolution and peak shape to investigate the state of a column.
(1) Pressure measurement: it is the easiest method. Generally, the chromatographic method we determine should meet the following requirements: when using a new column, the column pressure should not be higher than 2500 psi, and in any case, the pressure should not exceed 3000 psi. Although modern liquid chromatography systems can be used at higher pressures, the problems of system loss and leakage due to high pressure can be very serious. When the pressure exceeds 3000 psi, consider replacing the on-line filter with a diameter of 0.5 mm between the injector and the column. If the pressure is still high after replacing the filter, the filter or column of the chromatographic column has been polluted, which indicates that we should replace the column. Statistics show that more than 60% of chromatographic column damage is due to high pressure.
(2) Resolution: it is a very important index for us to investigate the chromatographic column. As long as the two components can be separated by baseline, there will be no problem in the quantification of the analyte. For symmetrical Gaussian peaks, baseline separation can be achieved at a resolution of 1.5, and better if RS is between 1.7 and 2.0. If the tailing is serious, a higher resolution is needed. The advantage of using resolution as the evaluation index of chromatographic column is that we can directly see the separation of adjacent peaks.
(3) Peak shape tailing: another: one of the standards for inspecting the quality of chromatographic column is the tailing of peak shape. The peak shape of the manufacturer's test report is very beautiful, but this does not represent the actual sample analysis of the chromatographic column. The actual samples always contain more or less components that cause the tailing of peaks. The compounds containing nitrogen atoms are the most worthy to be mentioned. These compounds are closely bound with the free silica hydroxyl groups on the silica gel skeleton. With the longer the chromatographic column is used, the bond phase will be lost and the tailing will be more serious. However, the change in tailing is not as immediately visible as a change in resolution. Based on this, it is better to set an upper limit of the tailing factor. For example, the United States Pharmacopoeia stipulates that the tailing factor should not exceed 1.8.
3. Determine the best time for column abandonment
The best time to determine the "discard" standard of chromatographic column is when we develop analytical methods or conduct method certification. Usually, in the process of developing analytical methods, you test several columns to analyze enough actual samples, and you know what happens when the separation becomes poor. I tend to start with a wide range. Our experience is this: if you tighten the system applicability requirements at the beginning, it is very difficult to relax the requirements after the method certification. Before analyzing the actual sample, the applicability of the system should be inspected to ensure that the chromatographic method meets the standards acceptable to the analyst.
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