When we plan the analysis on HPLC, our target used to complete the analysis successful and without error. But some time we faced pressure drop issue during chromatographic analysis on HPLC. Here we are going to discuss the reason for pressure drop and how we identify the symptoms during system setup, also we will discuss on control on pressure drop.
During system setup:- At the time of system setup if pressure fluctuations occurred more than 50 psi or above 4 to 5 bar it will impact on analysis. The retention time variation will occur in analysis. Below are the possibility for these fluctuations:-
1- Make sure the mobile phase is properly mixed and degassed. Visually inspect the mobile phase and if any immiscible layer observed, mixed vigorously by making cyclonic nature. Degassed the mobile phase by sonication and vacum pump.
2- Make sure selected mobile phase ports are properly purged and traces of previous mobile phase is completely removed from respective ports.
3- Make sure correct mobile phase are being used. Also insure mobile phase ports or properly dipped in mobile phase.
4- Ensure the generation of air bubbles in mobile phase port. It could be occurred due to improper degassing of mobile phase or blockage of suction filters. Sonicated the suction filters in IPA, Methanol and purified water for 20 minutes in each. If glass filters are used then sonicate the same in diluted nitric acid (10 % is ideal) or as per recommended in manuals.
5- Make sure as flow path is not blocked. Check column inlet or column outlet PEAK tubing. Some time it partially blocked and due to this blockage, flow restrictions occurred. Cut the respective PEAK tubing from each end and properly installed the same or change with new one as required.
6- Ensure active inlet valve or Outlet ball valves are functioning properly. Also ensure both cartridge are working properly. Sonicate the cartridge in IPA and water for 5 to 10 minutes or change the same if required.
7- Ensure frits are not blocked. If found buffer deposition then sonicate the same in IPA and water for 10 minutes in each. In some Instrument, the frits are use and throw then replace the frits with new one for Instrument specific.
During running analysis:- Some time we faced pressure fluctuations in running analysis however, during system setup no any symptoms occurred for pressure fluctuations.
Keep in mind, suddenly no any part will create problems. There is cumulative of cause and we have to identify the problem.
1- May be air bubbles trapped in flow path. The possibile reason for generations of air bubbles is dissolved gases in mobile phase hence properly degassed the mobile phase.
2- May be buffer not filtered hence suction filters getting block. Sonicate the suction filters and purge with mobile phase to remove air bubbles.
3- Blockage of column or column frits may generate pressure variation hence backwash the column to remove column blockage or clean the column frits to remove buffer deposition.
Filtration of buffer is most important hence without filtered buffer should not be used otherwise blockage issues will occurred.
4- Blockage of AIV/ OBV cartridge. Blockage can be removed by sonication of cartridge in IPA and water for 10 minutes in each.
5- Malfunctioning of pump drive will also generate pressure drop hence pump drive need to repair or replace. Mostly when pump drive malfunctioning occurred, more resistant generated and error message occurred.
6- Ensure mobile phase port is properly dipped in mobile phase. Some time in running sequence, mobile phase port got hanged and due to this pressure fluctuations occurred.
By considering above mentioned precautions and troubleshooting, we can save our chromatography analysis and will complete the analysis successfully.
Abhishek Singh is the author of Pharma technical support. He is pharmaceutical professionals, having experience of more than 12 years in Quality.