- What are the possible causes of significantly high pump pressure in an HPLC?
- Excessively high flow rate setting;
- Mechanical impurities in the mobile phase or sample injection, causing blockage of the guard column, pre-column frit, or in-line filter;
- Excessively high viscosity of the mobile phase;
- Too low column temperature;
- Crystallization of buffer salts;
- Malfunction of the pressure sensor.
- What causes unstable baseline (fluctuations or drift), and how to resolve it?
- Dissolved gases in the mobile phase; Degas by ultrasonic treatment for 15-30 minutes or by helium sparging.
- Blockage of the check valve; Remove the check valve and sonicate it in pure water for approximately 20 minutes to clear the blockage.
- Damaged pump seal leading to pressure fluctuations; Replace the pump seal.
- Presence of liquid leakage points in the system; Identify the leakage location and repair it.
- Bubble formation after the column; Install a backpressure regulator at the outlet of the flow cell.
- Detector not set to the absorption wavelength; Adjust the wavelength to the absorption wavelength.
- Slow column equilibration, especially when the mobile phase is changed; Flush the column with a solvent of moderate strength. When changing the mobile phase, flush the column with 10-20 column volumes of the new mobile phase before analysis.
- Why does leakage often occur at the fittings, and how to address it?
- Fittings not tightened properly; Loosen the fitting first, then retighten it. For hand-tight fittings, tighten only by hand (do not use tools). For stainless steel fittings, first tighten by hand, then use a dedicated wrench to tighten an additional 1/4 to 1/2 turn. Ensure the tubing inside the fitting reaches the bottom; otherwise, dead volume will be left.
- Contaminated or worn fittings; Replace the fittings.
- Mismatched fittings; Use accessories from the same brand.
- What causes leakage from the injection valve?
- Damaged rotor seal; Replace the rotor seal.
- Blockage of the sample loop; Clean or replace the sample loop.
- Loose injection port seal; Adjust the tightness.
- Inappropriate size of the injection needle (usually too short); Use a suitable injection needle (pay attention to the needle shape).
- Siphoning in the waste line; Empty the waste line.