The mobile phase is not filtered
Because dust or any other impurity particles will wear the plunger, sealing ring, cylinder and one-way valve, any solid particles in the mobile phase should be removed in advance. The mobile phase is best distilled in a glass container, and the common method is filtration, which can be used with filters such as Millipore filter membranes (0.2μm or 0.45μm). The inlet of the pump should be connected to a sand filter rod (or sheet). The filter of the infusion pump should be cleaned or replaced frequently.
The pump is not cleaned in time after use
The mobile phase should not contain any corrosive substances, and the mobile phase containing buffer should not be retained in the pump, especially when the pump is stopped overnight or longer. If the mobile phase containing buffer is left in the pump, due to evaporation or leakage, or even just due to the static state of the solution, fine crystals of salt may precipitate, which will damage the sealing ring and plunger, etc. like the above-mentioned solid particles. Therefore, pure water must be pumped into the pump to fully clean it, and then replaced with a solvent suitable for the preservation of the chromatographic column and conducive to pump maintenance (for reversed-phase bonded silica gel stationary phase, it can be methanol or methanol-water).
The mobile phase is empty
It may be that there is a lot of gas in the pump. At this time, you can open the pressure relief valve and run the pump at a larger flow rate (such as 5ml/min) to exhaust all the bubbles. You can also use a 50ml syringe to help extract the gas at the pump outlet. Another possible reason is that the sealing ring is worn and needs to be replaced.
No mobile phase flows out, and there is no pressure indication?
It may be that there is a lot of gas in the pump. At this time, you can open the pressure relief valve and run the pump at a larger flow rate (such as 5ml/min) to exhaust all the bubbles. You can also use a 50ml syringe to help extract the gas at the pump outlet. Another possible reason is that the sealing ring is worn and needs to be replaced.
The pressure and flow are unstable?
The reason may be bubbles, which need to be eliminated; or there is foreign matter in the one-way valve. The one-way valve can be removed and immersed in acetone for ultrasonic cleaning. Sometimes there may be bubbles in the sand filter stick, or it may be partially blocked by fine salt crystals or growing microorganisms. At this time, you can remove the sand filter stick and immerse it in the mobile phase to remove bubbles by ultrasound, or immerse the sand filter stick in dilute acid (such as 4mol/L nitric acid) to quickly remove microorganisms, or dissolve the salt and then clean it immediately.
Is the pressure too high or too low?
It may be that the pipeline is blocked and needs to be cleared and cleaned. The reason for the pressure drop may be that there is a leak in the pipeline. Check for blockage or leakage section by section.
When gradient elution is performed, due to the mixing of multiple solvents and the constant change in composition, some special problems arise, which must be fully taken into account:
PS: (In liquid chromatography, gradient elution is used for samples with complex components. In the same analysis cycle, the concentration ratio of the mobile phase is continuously changed according to a certain procedure, which is called gradient elution. This allows components with large differences in properties in a complex sample to achieve good separation according to their appropriate capacity factors k. The advantages of gradient elution are: 1. Shorten the analysis cycle; 2. Improve separation ability; 3. Improve the peak shape, with little tailing; 4. Increase sensitivity. But sometimes it causes baseline drift.)
Improper selection of mobile phase for gradient elution
Pay attention to the miscibility of solvents. Immiscible solvents cannot be used as mobile phases for gradient elution. Some solvents are miscible within a certain ratio, but they are not miscible beyond the range, so they should be used with more attention. When organic solvents and buffers are mixed, salt crystals may also precipitate, especially when using phosphates.
Ignore blank gradient elution
The purity of the solvent used for gradient elution is required to be higher to ensure good reproducibility. Before performing sample analysis, a blank gradient elution must be performed to identify the solvent impurity peak, because the impurities in the weak solvent will be eluted by the strong solvent after being enriched at the head of the chromatographic column. The solvent used for gradient elution needs to be thoroughly degassed to prevent bubbles from being generated during mixing.
Ignore the viscosity changes caused by solvent mixing
The viscosity of the mixed solvent often changes with the composition, so pressure changes often occur during gradient elution. For example, methanol and water have low viscosities. When the two are mixed in a similar proportion, the viscosity increases a lot. At this time, the column pressure is about twice that when methanol or water is the mobile phase. Therefore, care should be taken to prevent the pressure from exceeding the maximum pressure that the infusion pump or chromatographic column can withstand during gradient elution.
About the correct use and maintenance of the six-way valve
1. The sample solution must be filtered with a 0.45 micron filter membrane before injection to reduce the wear of the injection valve by particles.
2. The valve core should not be turned too slowly, and it should not stay in the middle position, otherwise the mobile phase will be blocked, causing the pressure in the pump to increase sharply, and even exceed the maximum pressure of the pump; when it is turned to the injection position, the excessive pressure will damage the column head.
3. To prevent buffer salts and samples from remaining in the injection valve, the injection valve should be flushed after each analysis. Usually, it can be flushed with water, or first with a solvent that can dissolve the sample, and then with water.
About the use and maintenance of chromatographic columns
The correct use and maintenance of chromatographic columns are very important. Any carelessness will reduce the column efficiency, shorten the service life, or even damage it. During chromatographic operation, you need to pay attention to the following issues to maintain the chromatographic column.
Adjust the flow rate too fast
Avoid sudden changes in pressure and temperature and any mechanical vibration. Sudden changes in temperature or dropping the chromatographic column from a high place will affect the filling condition in the column; sudden increases or decreases in column pressure will also affect the filling material in the column. Therefore, the flow rate should be adjusted slowly, and the valve should not be rotated too slowly when the valve is injected (as mentioned above).
Backflushing chromatographic columns
Generally speaking, chromatographic columns cannot be backflushed. Only when the manufacturer indicates that the column can be backflushed can the impurities left at the column head be backflushed to remove them. Otherwise, backflushing will quickly reduce the column efficiency.
Pre-column and guard column
Choose and use the appropriate mobile phase (especially pH) to avoid the stationary phase from being destroyed. Sometimes a pre-column can be connected in front of the injector. When the analytical column is bonded silica gel, the pre-column is silica gel, which can make the mobile phase "saturated" with silica gel before entering the analytical column to avoid the silica gel matrix in the analytical column from being dissolved.
Avoid injecting samples with complex matrices, especially biological samples, directly into the column. It is necessary to pretreat the samples or connect a guard column between the injector and the chromatographic column. The guard column is generally a short column filled with a similar stationary phase. The guard column can and should be replaced frequently.
How to troubleshoot small probability failures?
1. Pump pressure jumps, with a range of about 10 bar
Solutions:
(1) The original white head was a bit dirty, and the problem was not solved after replacement;
(2) After removing the valve core from the active valve, similar bubbles were found. It was immersed in methanol-water solution for 5 minutes to try to remove the bubbles. After reinstalling it and observing it again, it was still not solved;
(3) After replacing the new valve core and installing it, there was no more jumping. After observing for a period of time, it was relatively stable and successfully solved.
2. Faults occurred:
(1) Plug homing over pressure;
(2) There was no pressure and no liquid outflow during infusion and flow rate setting;
(3) The detector shutter could not be reset;
(4) After the high-concentration sample was introduced, a peak also appeared at the corresponding position of the blank. Possible reasons:
(1) The online filter is blocked;
(2) The check valve under the pump head is blocked;
(3) The detection cell may be dirty, or the optical path may be a little blocked. If that doesn't work, it may be that the lamp has reached the end of its life;
(4) The small rag is dirty and needs to be replaced.
Solution:
(1) Remove the online filter, put it in a beaker, add purified water and boil it for 5-10 minutes, then use methanol to supersonicate. Generally, it will be fine. If it is still blocked, soak it in dilute nitric acid. If that doesn't work, replace it with a new one. This is a consumable.
(2) Remove the check valve under the pump head. Both may be blocked. When it is just removed, you can't hear any sound when you shake it vertically next to your ear. Simply use an ear cleaning bulb to blow the check valve (pay attention to the direction). If there is no response after blowing, you can use a solvent to ultrasonicate it, but don't boil it in water. Generally, if you can hear a sound when you shake it next to your ear, it will not affect the experiment.
(3) First, rinse the detection pool with a solvent. The solvent to be used depends on the specific sample. If it still doesn't work after flushing, remove the detection pool and check the lamp energy. If the energy is OK (about a few thousand), you may need to clean the light path. For the first cleaning, it is recommended to call an engineer because the part to be cleaned is at the bottom of the detector. There is a black piece with a crack in the middle, and the screws of that piece are all octagonal, which is very troublesome to remove.
(4) Take out all 5 sample trays. You can see a black protrusion inside (use a flashlight to see it clearly). The injection needle usually draws samples from that place. You can remove it by reaching in. There is a small white circle inside (it will turn black naturally after being used for a long time). Just replace it with a new one. Generally, 10 are provided when you buy the instrument.
Four key factors in the use of liquid chromatography
1. Liquid chromatograph itself:
There are generally no problems with the instrument itself, especially for new instruments that have just been purchased. The most important thing for the instrument itself is the calibration of the instrument. Generally, the seller will first do a system confirmation of the instrument (including IQ, PQ, OQ) after the instrument is purchased. After the confirmation is passed, the national or provincial instrument calibration bureau will be asked to calibrate it. After the calibration is passed, it can be used with confidence. In addition, some well-performing companies will conduct internal calibration of the instrument every six months to ensure the normal operation of the instrument.
2. Chromatographic conditions:
Chromatographic conditions generally include mobile phase, chromatographic column, detection wavelength, flow rate and injection volume. The mobile phase involves the water and reagent solution used to prepare the mobile phase. Water is generally required to be ultrapure water or other water with a resistivity of no more than 18.2 megohms. The reagent solution is required to be chromatographic grade. The reason is to prevent impurities in water and reagent solution from interfering with the chromatographic results.
The chromatographic column will have different requirements for different varieties, but each chromatographic column has its cleaning requirements, which can be found in the chromatographic column manual. It must be carried out according to the above requirements, otherwise it will cause the chromatographic column efficiency to decrease and the service life of the chromatographic column to be shortened. The detection wavelength will also have different requirements for different varieties. It should be pointed out here that attention should be paid to observing the changes in the energy of the detector deuterium lamp, such as large baseline fluctuations, sudden decreases in the response value of active ingredients, etc., which may be caused by insufficient energy of the deuterium lamp. The deuterium lamp is generally recommended to be used for 800 hours, but in fact, the use time is more than that, and it can be replaced according to actual conditions.
The flow rate also varies with different varieties, generally 1.0ml/min, and the flow rate cannot be changed at will, especially as a QC personnel, but as a research and development personnel, sometimes in order to explore conditions or verify the durability of the method, the flow rate will be changed to evaluate the applicability of the method. There are also different regulations for the injection volume due to different varieties. The injection volume now uses automatic injection, so the injection precision is very good, and there is no need to emphasize anything.
3. Operators: First of all, as a liquid phase operator, you must carefully study the liquid phase manual, master the operation of liquid phase hardware and software, master daily maintenance, and master the most basic troubleshooting and repair. The general liquid phase operating procedures are also drafted by operators, so as long as the operation is strictly carried out in accordance with the operating requirements, there will generally be no problems. Of course, it should be pointed out here that daily maintenance of liquid phase is very important, such as filtering and degassing of mobile phase, cleaning of pump seals, cleaning of system pipes, cleaning of solvent filter heads, etc. If you do not insist, it is easy to cause unnecessary failures such as chromatographic column blockage, excessive system pressure, abnormal chromatographic peaks, etc., which wastes time. Secondly, when the operator prepares the sample solution for liquid phase analysis, it is best to have the same person complete it alone to avoid unnecessary accidental errors. 4. Liquid phase installation environment: The liquid chromatograph should be installed on a horizontal table in the room away from vibration and electromagnetic interference as much as possible. If conditions permit, the liquid phase data processing system should be separated as much as possible. One is to prevent the chromatographic baseline noise from being too large, the baseline is unstable, etc., and the other is to reduce the harm to the human body.
2026
