① It is recommended to filter the sample and mobile phase before testing.
② It is recommended to clean the samples in time after finishing every day.
③ Routine detection: After testing, the column was directly reversed connected, rinsed with 90% organic phase for 45 minutes, and finally stored in pure methanol or pure acetonitrile.
④ Conditions for using buffer salt:
A. Isodegree condition: washing at a flow rate of 1ml/min for 45min before and after the application of buffer salt.
b. Gradient condition: The same transition mobile phase as the initial mobile phase was rinsed for 45min at a flow rate of 1ml/min before the buffer salt was used.
Pay attention
(1) refers to the transition flow phase ratio and analysis of the organic phase and water phase flow is the same, but does not contain the buffer salt.
(2) the buffer salt after washing, use 90% organic instead flushing 60 minutes, finally in the pure organic solvent.
(3)The buffer solution should not be left in the column overnight.
When 2 amino column into acid samples, is very harmful to column, if use after a period of time, lower efficiency, peak shape change, how to restore?
A: 5-10 times the column volume contains 0.5-1.0% acetonitrile-water (50:50) solution to wash the column (after washing, of course, with an alkali free mobile phase to wash the excess ammonia), and then when analyzing such acidic analytes, it is recommended to add a small amount of ammonia, such as 0.1%, to the mobile phase.
3. Liquid chromatography 27 trailing or bimodal reason is what?
Answer: （1）The sieve plate is blocked or the column fails. The solution is to reverse wash the column and replace the sieve plate or column.
(2) there are interference peak, the solution is to use a longer pillars, change of mobile phase pillars or replace good selectivity.
(3) post may be overloaded, reduce the sample quantity.
4. The main reasons of the lack of sensitivity HPLC and what is the solution?
Answer: ① The sample size is insufficient, the solution is to increase the sample size.
Do not issue from the pillar (2) the sample, according to the chemical properties of the samples change mobile phase or pillars.
③ The sample does not match the detector, adjust the wavelength or replace the detector according to the chemical properties of the sample.
(4) detector attenuation is overmuch, can adjust the attenuation.
(5) the probe’s time constant is too large, the solution is to reduce time parameters.
⑥ detector pool window pollution, the solution is to clean the pool window.
7 the cell inside the bubble, the solution is to exhaust.
⑧ If the recorder voltage range is not appropriate, the voltage range can be adjusted.
⑨ The flow rate of mobile phase is not suitable, adjust the flow rate.
Attending detector and recorder than calibration curve, the solution is to check the recorder and detector, the calibration curve.
5. When performing HPLC analysis, the column pressure is not stable. What is the reason? How to solve?
Answer: ① There is air in the pump. The solution is to remove the air in the pump and degassing the solvent.
② Proportional valve failure, replace the proportional valve.
③ The sealing pad of the pump is damaged, replace the sealing pad.
(4) in the solvent of bubbles, the solution is for solvent degassing, if necessary, change the degassing method.
(5) system leak detection, find the leak point, seal.
⑥ gradient elution, at this time the pressure fluctuation is normal.
Liquid chromatography operation in these 10 common classic
6. The acceptance test of the HPLC column when the pressure is too high, could you tell me why?
Answer: the pressure is the most commonly encountered in HPLC column users were too high. There are many reasons for this, often not the column itself. You can follow the steps below to check the cause of the problem:
1) remove the protect column, the column pressure is still high, otherwise is to protect column, if the pressure is high, check again.
Remove the chromatographic column (2) from the instrument, to see if pressure drop, otherwise any blockage, need to be cleaned, if the pressure drop, check again.
③ Connect the inlet and outlet of the column to the instrument in reverse, and wash the column with 10 times the column volume of mobile phase (do not connect the detector at this time to prevent solid particles from entering the flow cell). At this point, if the pressure is still not down, please check again. Used only for the pole.
4. Replace the sieve plate column entry. If the column pressure decreases, it indicates that your solvent or sample contains particulate impurities. It is these impurities clogging the sieve plate, causing the pressure rise. If the column pressure is still high, contact the manufacturer. In general, the problem of high column pressure can be avoided by connecting an in-line filter between the injector and the guard column.
7. Why does peak width occur?
Answer: ① The sample volume is too large: the total sample volume is less than 15% of the first peak.
② Cause peak spread in the injection valve: air bubbles are expelled before and after injection to reduce diffusion.
③ The sampling speed of data system is too slow: the set rate should be greater than 10 points per peak.
(4) mobile phase viscosity too high: improve the column temperature, with low viscosity fluid.
⑤ The detection cell volume is too large: with a small volume cell, remove the heat exchanger.
6 retention time is too long: the degree of des such as increasing strong solvent content, and gradient elution can be applied.
All landowners column outside the volume is too large, the connecting pipe diameter and length to a minimum.
Today the sample overload: enter the small concentration of small sample volume.
8. What causes bubbles in the mobile phase?
Answer: (1) with the dissolution of oxygen or air, mobile phase often form bubbles in the solution.
(2) fluid road resistance is larger, there was a vacuum suction fluid bubbles.
(3) when the system began to work, unable to remove the air flow.
④ Mix with air when injecting the sample.
9. What are the commonly used method laboratory degassing?
Answer: (1) heating reflux degassing and degassing effect is best, but you can’t keep.
② Helium degassing, this method degassing effect is good, can remove more than 90% of the air, but the price of helium is too expensive, so it is not used much.
(3) vacuum degassing, the effect after helium gas, but easy to cause the sample solution in the process of degassing volatilization losses.
(4) the ultrasonic degassing, can remove about 30% of the air, but the most commonly used in laboratory. Still use online degassing as far as possible, convenient, and the effect is good.
10. In acidic amino column sample, is very harmful to column, if use after a period of time, lower efficiency, peak shape change, how to restore?
Post time: Jul-25-2024