Solutions for High Pressure and Baseline Drift in HPLC Columns

This is the most common problem in the use of high-performance liquid phase column (such as C8 Low PH HPLC Columns, Amino Acid HPLC Column), which refers to the sudden rise of pressure, generally due to the blockage in the flow path. At this time, we should check by section.

High pressure and baseline drift are common issues encountered in High-Performance Liquid Chromatography (HPLC) systems. These problems can adversely affect the accuracy and reproducibility of chromatographic results. Here are some potential solutions for addressing high pressure and baseline drift in HPLC columns:

Solutions for high pressure in HPLC columns

  1. Check Column Integrity:

    • Examine the HPLC column for damage, blockages, or leaks. Ensure that the column is properly installed and that there are no issues with the frit or end fittings.
  2. Column Equilibration:

    • Allow the column to equilibrate at the initial mobile phase conditions for an adequate amount of time before starting the analysis. This ensures that the column is in a stable state.
  3. Optimize Flow Rate:

    • Adjust the flow rate within the recommended range for the column. High flow rates can lead to increased backpressure.
  4. Mobile Phase Compatibility:

    • Ensure that the mobile phase is compatible with the column material. Incompatible solvents or additives may lead to increased pressure.
  5. Particle Size and Pore Size:

    • Choose the appropriate particle size and pore size for the column based on the requirements of the separation. Smaller particles may result in higher pressure but better resolution.
  6. Column Temperature:

    • Check and optimize the column temperature. Elevated temperatures can reduce viscosity and pressure.
  7. Use Guard Columns:

    • Employ guard columns to protect the analytical column from particulate matter and contaminants, which can lead to pressure buildup.
  8. Sample Filtration:

    • Filter samples to remove particulate matter that could cause blockages in the column.
  9. Check Check Valves:

    • Inspect and clean or replace check valves in the pump if they are not functioning properly. Malfunctioning check valves can lead to pressure fluctuations.

Solutions for baseline drift in HPLC columns

Generally speaking, when the machine is just started, the baseline is easy to drift, and the equilibration time is about half an hour. If you use a buffer solution or buffer salt, the equilibration time is relatively long at low wavelengths (220nm).

However, if you find baseline drift during your experiment, you need to consider the following reasons:
① The energy of the UV lamp is insufficient. Solution: Replace with a new UV lamp;
② Column temperature fluctuates. Solution: Control the temperature of the column and mobile phase, and check whether there are open windows or air conditioners facing the column oven.
③ The flow cell has gas or is polluted. Solution: Rinse the flow cell with methanol or other strongly polar solvents (preferably disconnect the column).
④ The mobile phase is contaminated, deteriorated or formulated with low-quality solvents. Solution: Check the composition of the mobile phase, using high-quality chemicals and HPLC-grade solvents.
⑤ The strongly retained substance (high K ‘value) in the sample was eluted as a hoe-like peak, thus showing a gradually rising baseline. Workaround: Use guard columns, if necessary, between injections. During the analysis, the column was periodically flushed with a strong solvent.
⑥ The detector is not set at the maximum absorption wavelength. Solution: Adjust the wavelength to the maximum absorption wavelength;
The PH of the mobile phase is not well adjusted. Solution: Add an appropriate amount of acid or alkali to adjust the optimal pH value.

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Disconnect the inlet of the vacuum pump

At this time, the peek tube is filled with liquid, so that the peek tube is lower than the solvent bottle, to see whether the liquid drops freely. If the liquid does not drop or drops slowly, the solvent filter head is blocked. Treatment method: Soak in 30% nitric acid for half an hour, then rinse with ultra-pure water. If the liquid drips freely and the solvent filter head is normal, check again;

Open the purge valve

If the pressure does not drop obviously, it will be blocked by a filter white head. Treatment method: Take out the filtered white head and use 10% isopropanol for ultrasonic for half an hour. If the pressure drops below 100psi (6.7bar), the filter white head is normal, and recheck;

Remove the column outlet end

If the pressure does not drop, the HPLC column is blocked. Treatment: If the buffer salt is blocked, flush 95% water to normal pressure. If some strongly retained substances cause blockage, the mobile phase stronger than the current one should be used to rush to normal pressure. If the washing pressure does not drop for a long time according to the above method, the inlet and outlet of the HPLC columns can be connected to the instrument in turn, and the column can be washed with a mobile phase. At this time, if the column pressure still does not drop, only change the column inlet sieve plate, but once the operation is not very good, it is easy to cause column efficiency to drop, so try to be useless.