Disconnect each part systematically from the HPLC system to check and see if you get a pressure drop and determine if the pressure built up is before or after the column
Pressure built up can be from several reasons:
Flow rate is set too high – Lower the HPLC pump flow rate
Dirty inlet frits/Inline filters-Replace frits/filters.
This problem can be identified by releasing the pressure valve on the inlet frits and see if you still observe high pressure. If all the pressure drops when the valve is open, frits are good. Move on to next step.
Next common place on the HPLC where the pressure can be built up is the needle/needle seat. Depending on your HPLC this will be different, Change your setting to bypass the needle and see if the high pressure drops, if yes, then the needle needs to be cleaned or changed.
Column temperature low or heater not turned on – Increase column heater temperature
It is possible to damage some columns by having high flow going through while at room temperture. Always make sure that the column temperature has reached operating temperature and stablized for ~30 minutes before raising the flow rate to operating flow rates.
Blocked guard column – Remove/replace guard column
Fluctuating Back Pressure
Bubble in Pump system or column- Thoroughly degas the solvent; Always only used 0.2U filtereted degassed solvents. Whenever possible use online degasser.
Leak in the Pump System- Most common places for the leaks are check valves, purge valves and any other coonections/seals. Check and make sure there are no leaks, Fix any leaks by tightening or replacing the appropriate part
Faulty check valve(s) – Replace check valve(s)
Using gradient elution – Pressure cycling is normal due to viscosity changes from the gradient.
Faulty Pump/injector system seal(s)- Identify the location and replace the seal(s)
In this case, you should note what normal fluctuation is for each gradient, to help idetify abnormal behaviou
Decreasing Back pressure
Insufficient flow from pump- Make sure the cap on the mobile phase reservoir is not too tight, that a vaccum is being created in the bottle thus making it difficult to pump mobile phase out of the bottle
Leak- Thouroughly check for leaks in pump systerm/ injector system and the column. You might see stable pressure at lower flow rate but low/decreasing pressure as you increase the flow rate. Some seals/connections might not be properly placed and leaks can form as system pressure increases.
Column temperature increasing- Column core temperature might not have stablized even if the controller shows a steady final temperature. Give enough time for column temperature to stablize. In rare instances, a faulty controller might be causing the column to overheat.
Increasing Back pressure
Blocked Flow lines- Systematically disconnect components from detector end to column end to find blockage; replace or clean blocked component
Particulate build up in column or pump- Always filter samples and mobile phase with 0.2U filter
Incompatible buffer/solvent system causing salt formation or precipitation- Ensure the solvent systems used are compatible
Chemistry Interview 