High performance liquid chromatography is an important branch of chromatography. This method has become an important separation and analysis technique in disciplines such as chemistry, medicine, industry, agriculture, commodity inspection, and legal inspection. Taking the pharmaceutical industry as an example: a comparison table of the items and quantities of high-performance liquid chromatography methods included in the Chinese Pharmacopoeia.
Given the increasingly widespread use of HPLC in the entire industry, every laboratory analyst should be proficient in and apply HPLC. They should be adept at analyzing common faults in order to solve problems and improve work efficiency even when running faults. For this purpose, the editor has compiled about 30 common HPLC faults and countermeasures analysis plans, don't miss them.
What are the reasons for the drift or rapid change in retention time?
Regarding the issue of drift:
① Poor temperature control, the solution is to use a constant temperature device to maintain a constant column temperature;
② The solution to prevent changes in the mobile phase is to prevent evaporation, reactions, etc;
③ The column is not balanced properly and needs to be balanced for a longer period of time.
Regarding the issue of rapid change:
① The solution to the change in flow rate is to reset it to maintain stability;
② There are bubbles in the pump, which can be expelled through exhaust and other operations;
③ If the mobile phase is not suitable, the solution is to change the mobile phase or mix it appropriately in the control room.
What are the reasons for trailing or bimodal appearance in 02?
① If the sieve plate is blocked or the column fails, the solution is to backwash the column in reverse, replace the sieve plate or replace the column;
② There are interference peaks, and the solution is to use longer columns; Change the mobile phase or replace the column with good selectivity;
③ Possible column overload, reduce injection volume.
Main reasons and solutions for insufficient sensitivity of HPLC in 03
① Insufficient sample size, the solution is to increase the sample size;
② The sample did not flow out of the column. The mobile phase or column can be changed according to the chemical properties of the sample;
③ The sample does not match the detector. Adjust the wavelength or change the detector based on the chemical properties of the sample;
④ The detector attenuates too much. Adjust the attenuation;
⑤ The detector time constant is too large. The solution is to reduce the time parameter;
⑥ Detector pool window contamination. The solution is to clean the pool window;
⑦ There are bubbles in the detection pool. The solution is exhaust;
⑧ The pressure measurement range of the recorder is inappropriate. Adjust the voltage range;
⑨ The flow rate of the mobile phase is inappropriate. Adjust the flow rate;
⑩ The detector and recorder exceed the calibration curve. The solution is to check the recorder and detector and redo the calibration curve.
What is the reason for unstable column pressure during HPLC analysis? How to solve it?
① There is air inside the pump. The solution is to remove the air inside the pump and degas the solvent;
② Proportional valve failure, replace the proportional valve;
③ The pump gasket is damaged, replace the gasket;
④ The solution to the bubbles in the solvent is to degas the solvent, and if necessary, change the degassing method;
⑤ System leak detection, identify the leak point, and seal it;
⑥ Gradient elution, at which point pressure fluctuations are normal.
05 replaced another brand of ODS column, but the retention time cannot be reproduced. Why?
This is because the analyte may have the ability to form hydrogen bonds. Although the manufacturing technology of fillers has greatly improved in the past few years, the concentration of silanol groups on the surface of ODS fillers varies among different manufacturers. It is precisely these silanol groups that may interact with the sample. Therefore, the relative retention time of each component in the same analyte may vary on ODS columns of different grades. Adding a small amount of competitor, such as triethylamine (TEA), to the mobile phase will saturate the bonding ability of the silanol group, thereby ensuring good reproducibility of the relative retention time on different grades of columns. If the separation situation is feasible, the system is stable and meets the requirements of system applicability, there is no need to retain the reproduction of time.
Why is the column pressure too high during the acceptance test of HPLC column 06?
High column pressure is the most common problem encountered by HPLC column users. There are multiple reasons for this, and it is often not a problem with the pillar itself. You can follow the steps below to check the cause of the problem.
① Remove the protective pre column and check if the column pressure is still high. Otherwise, it is a problem with the protective column. If the column pressure is still high, check again;
② Remove the chromatography column from the instrument and check if the pressure drops. Otherwise, it may be a blocked pipeline and needs to be cleaned. If the pressure drops, check again;
③ Connect the inlet and outlet of the column to the instrument in reverse, and rinse the column with a mobile phase of 10 times the volume of the column (do not connect the detector at this time to prevent solid particles from entering the flow cell). At this point, if the column pressure still does not decrease, check again; Only for used pillars.
④ Replace the column inlet sieve plate. If the column pressure drops, it indicates that your solvent or sample contains particulate impurities, which block the sieve plate and cause pressure rise. If the column pressure is still high, please contact the manufacturer. In general, connecting an online filter between the injector and the protective column can avoid the problem of high column pressure. The Rheodyne 7315 filter provided by SGE is the best choice to solve this problem.
What is the displacement of the mobile phase in the 07 chromatographic column?
Many people who conduct chromatographic separation experiments have encountered situations where the mobile phase is not replenished in a timely manner, and the pump sucks up the mobile phase in the solvent bottle, causing the HPLC system to stop working. Will this situation damage the chromatography column? Has the pump drained all the mobile phase from the chromatographic column? Can the chromatographic column still be used? In fact, if the pump sucks up the mobile phase in the solvent bottle, it will not cause damage to the chromatographic column. Even if the pump is filled with air, it will not discharge air into the chromatographic column. Because pumps can only transport liquids, not air.
In contrast, another more likely scenario is forgetting to cover the sealing caps at both ends of the chromatographic column or the caps being too loose, causing the column to dry out. Similarly, it is not easy for the entire chromatographic column to dry up, and it is likely that only a few millimeters at both ends of the column have dried up, as it takes a considerable amount of time for the column to dry due to the evaporation of all solvents. Even if the chromatography column really dries up, it doesn't necessarily mean it's hopeless. You can try rinsing the chromatography column with a completely degassed, low surface tension solvent (such as methanol degassed with helium gas) to remove the gas. Lower surface tension helps to infiltrate the surface of the filler; The degassed solvent should be able to dissolve and remove the gas trapped in the packing. The chromatographic column requires approximately one hour or more of flushing (at a flow rate of 1mL/min) to thoroughly infiltrate and return to a normal state.
Reasons and solutions for baseline drift in 08
1. Cause analysis
① Column temperature fluctuation. (Even small temperature changes can cause fluctuations in the baseline. They typically affect differential detectors, conductivity detectors, lower sensitivity ultraviolet detectors, or other optoelectronic detectors.)
② The mobile phase is uneven. The baseline drift caused by changes in mobile phase conditions is greater than the drift caused by temperature
③ The circulation pool is contaminated or has gas
④ The detector outlet is blocked. (High pressure causes the flow pool window to rupture, resulting in noise baseline)
⑤ Improper flow matching ratio or changes in flow velocity.
⑥ Slow column balance, especially when there is a change in the mobile phase.
⑦ Mobile phase contamination, deterioration, or preparation from low-quality solvents.
⑧ The strongly retained substance (high K 'value) in the sample was washed out with the Mantou peak sample, thus showing a gradually rising baseline.
⑨ Using recycled solvents is not recommended. The detector has not been adjusted.
⑩ The detector is not set at the maximum absorption wavelength.
2. Solution
① Control the temperature of the column and mobile phase, and use a heat exchanger before the detector
② Use HPLC grade solvents, high-purity salts, and additives. The mobile phase should be degassed before use, and online degassing or helium degassing should be used during use.
③ Rinse the flow tank with methanol or other strongly polar solvents. If necessary, 1N nitric acid can be used. (Do not use hydrochloric acid)
④ Remove the obstruction or replace the pipe. Refer to the detector manual to replace the flow pool window.
⑤ Change the ratio or flow rate. To avoid this problem, it is recommended to regularly check the composition and flow rate of the mobile phase.
⑥ Rinse with a medium strength solvent, and when changing the mobile phase, rinse the column with 10-20 times the volume of new mobile phase before analysis. When using ion pair reagents and buffer salts, more attention should be paid to the equilibrium column.
⑦ Check the composition of the mobile phase. Use high-quality chemical reagents and HPLC grade solvents.
⑧ Change the analysis conditions. Use a protective column and, if necessary, regularly rinse the column with strong solvent between injections or during the analysis process.
⑨ Reset the baseline. Use a new mobile phase.
⑩ Adjust the wavelength to the maximum absorption wavelength. Re select the detection wavelength.
What are the reasons for the baseline noise generated by Rule 09?
1. Cause of occurrence
① There is air (sharp peak) in the mobile phase, detector or pump.
② Leakage of liquid.
③ Incomplete mixing of mobile phase.
④ Temperature impact (column temperature too high, detector not heated).
⑤ There are other electronic devices on the same line (occasional noise).
⑥ Pump vibration.
2. Solution method
① Mobile phase degassing. Flush the system to remove air from the detector or pump.
② Check if the pipeline joints are loose, if the pump is leaking, if there is salt precipitation and abnormal noise. If necessary, replace the pump seal.
③ Shake by hand to mix evenly or use a low viscosity solvent.
④ Reduce differences or add heat exchangers.
⑤ Disconnect the LC, detector, and recorder, check if the interference comes from external sources, and make corrections. Adopting precision grade regulated power supply.
⑥ Add pulse dampers to the system.
What are the reasons for irregular baseline noise?
1. Cause of occurrence
① Leakage of liquid.
② Mobile phase contamination, deterioration, or preparation from low-quality solvents.
③ The solvents in the mobile phase are immiscible.
④ Problems with electronic components of detectors/recorders.
⑤ There are bubbles in the system.
⑥ There are bubbles inside the detector.
⑦ Circulating pool pollution (even minimal pollutants can produce noise)
⑧ The detector light is running low on energy.
⑨ Loss or blockage of chromatographic column packing.
⑩ Uneven mixing of mobile phase or abnormal operation of mixer.
2. Solution method
① Check if the joints are loose, if the pump is leaking, if there is salt precipitation and abnormal noise. If necessary, replace the seal. Check if there is any leakage in the circulation pool.
② Check the composition of the mobile phase.
③ Choose a compatible mobile phase.
④ Disconnect the power supply of the detector and recorder, check and correct.
⑤ Clean the system with a strong polar solution.
⑥ Clean the detector and install a background pressure regulator behind it.
⑦ Clean the circulation tank with 1N nitric acid (not phosphoric acid).
⑧ Replace the lamp.
⑨ Replace the chromatographic column.
⑩ When repairing or replacing the mixer, it is recommended not to use the mixing device of the pump when the mobile phase does not follow the gradient.
Troubleshooting of 11 retention time drift
There are two different situations where retention time does not reproduce: retention time drift and retention time fluctuation. The former refers to the retention time changing only in one direction, while the latter refers to the fluctuation of retention time without a fixed pattern. Distinguishing between these two situations is often helpful in identifying the cause of the problem. For example, the drift in retention time is often caused by column aging; And column aging cannot cause irregular fluctuations in retention time. In fact, most of the reasons for retention time drift are due to different mechanisms of chromatographic column aging, such as stationary phase loss (e.g. through hydrolysis), chromatographic column contamination (caused by samples or mobile phases), etc.
The most common reasons for retaining time drift are as follows:
1. Chromatographic column equilibrium
If we observe retention time drift, we should first consider whether the chromatographic column has been completely equilibrated with the mobile phase. Usually, equilibrium requires 10-20 column volumes of the mobile phase, but if a small amount of additives (such as ion pair reagents) are added to the mobile phase, it takes a considerable amount of time to equilibrate the chromatographic column.
Mobile phase pollution may also be one of the reasons. A small amount of pollutants dissolved in the mobile phase may slowly accumulate on the chromatographic column, causing a drift in retention time. Attention should be paid: Water is a mobile phase component that is easily contaminated.
2. Fixed phase stability
The stability of stationary phases is limited, and even when used within the recommended pH range, the stationary phase will slowly hydrolyze. For example, the hydrolysis stability of silicone matrix is best at pH 4. The hydrolysis rate is related to the type of mobile phase and ligand. The bonding phase of bifunctional and trifunctional ligands is more stable than that of monofunctional ligands; Long chain bonding is more stable than short chain bonding; Alkyl bonding is much more stable than cyano bonding.
Frequent cleaning of the chromatographic column can also accelerate the hydrolysis of the stationary phase of the column. Other silicone matrix bonding phases can also undergo hydrolysis in aqueous solution environments, such as amino bonding.
3. Column contamination
Another common reason for retention time drift is column contamination. HPLC chromatography columns are highly effective adsorbent filters that can filter and adsorb any substance carried by the mobile phase. The sources of pollution can be: the mobile phase itself, flow compatibilizers, connecting tubes, pumps, injectors and instrument seals, as well as samples, etc. Usually, the source of pollution can be determined through experiments.
If there are strong components retained on the chromatographic column in the sample, it may be a potential source of retention time drift. These roots are usually sample matrices. The simplest way to avoid column contamination is to take preventive measures. Strong solvents are typically used under given chromatographic conditions, but not all pollutants can dissolve in the mobile phase. Using protective pillars is a very effective method. Backwash chromatography columns are only used as a last resort.
4. Composition of mobile phase
The slow change in the composition of the mobile phase is also a common cause of retention time drift. The volatilization of volatile components in the mobile phase and the flow of recycling are equal.
5. Hydrophobic collapse
When a reverse phase packed chromatography column with small pore size and well sealed end groups uses nearly 100% water as the mobile phase, there may be a sudden loss of separation and a significant decrease or complete absence of the analyte retention, which is known as hydrophobic collapse. This phenomenon is caused by the mobile phase not infiltrating the surface of the stationary phase. The rescue method is to infiltrate the fixed phase with a mobile phase containing a large amount of organic components, and then balance it with a mobile phase with high water content. This phenomenon can also occur during long-term storage of chromatography columns. The use of reverse phase chromatography columns with embedded polar groups (such as Waters SymmetryShield RP columns) or non end capped chromatography columns (such as Waters Resolve columns) can also avoid collapse.
Why do shoulder peaks or bifurcations appear?
① Excessive sample volume - using flow matching, the total sample volume is less than 15% of the first peak;
② The sample solvent is too strong - use a weaker sample solvent;
③ Column collapse or formation of short circuit channel - replace the chromatographic column and use less corrosive conditions;
④ Failure of sintered stainless steel in the column - replace the sintered stainless steel, add an online filter, and filter the sample;
⑤ Damaged injector - replace the injector rotor.
Why did Ghost Peak appear on the 13th?
① Residual peak of injection valve - clean the valve with strong solvent after each use to improve the cleaning of the valve and sample;
② Unknown substances in the sample - processing the sample;
③ Column imbalance - rebalance the column and use mobile phase as the sample solvent (especially ion pair chromatography);
④ Trifluoroacetic acid
