Common Faults and Troubleshooting Methods in HPLC Column

The hardware of the HPLC column includes the column tube and fittings which contain the chromatographic packing material used for the effect of the separation. The hardware not only needs to be strong enough to stand back pressure in the process of separation, but also needs to be well-controlled, acting leak-free, minimum-volume, and zero-dead-volume. It provides a flowing path for the sample at the inlet and the analyte bands at the outlet。

The hardware of the HPLC needs to be chemically inert relative to the separation system, including sample, mobile, and stationary phases. The columns which are made of stainless steel have the character of the highest pressure resistance. If you need to complete special chemical or biological applications, where inert surfaces are required, the engineered plastic and glass columns with less pressure tolerance, will be the better choice.

The chromatographic resolution shows the degree of how two compounds are separated. The ability of mechanical and chemical separation of the HPLC column are the key factors to determine the separation. The ability of mechanical separation is created by the length, particle size, and packed-bed uniformity of the HPLC column. The ability of chemical separation is created by the physicochemical competition for compounds between the packing material in the column and the mobile phase.

Operating Conditions of HPLC Column

HPLC column is preferably constant temperature and humidity. The temperature is between 15-30 degrees Celsius, relative humidity is less than 80%, the room should be well-ventilated, and air flows generated by air conditioning or other equipment do not be directly below the instrument. What’s more, it is better to avoid direct light and stay away from high electrical interference and high-vibration equipment.

Troubleshooting Methods in HPLC Column

1. High-column pressure

(1) A buffer salt such as (ammonium acetate or the like) is deposited in the column;
(2) Sample contamination deposition. For this case, use 40~50°C pure water first, and rinse the column at a low speed. After the column pressure is gradually decreased, the flow rate is increased accordingly. After the column pressure is greatly reduced, rinse with normal-temperature pure water, then use pure methanol.

Rinse the column for 30 minutes; for the second case, contaminate the C18 column by deposition of the sample, and rinse the column back with pure water, replace it with methanol, then rinse the column with methanol + isopropanol (4+6). Rinse with methanol, rinse with pure water, and finally rinse with methanol for more than 30 minutes.

2. Unstable

Cause There is a foreign matter between the gem ball and the valve seat with air or check valve in the system so the two cannot be sealed. Pay attention to the amount of mobile phase in the treatment work, ensure that the stainless steel filter sinks into the bottom of the reservoir, avoid inhaling air, and the mobile phase should be fully degassed.

If there is a foreign object between the check valve and the valve seat, remove the check valve and put it into the beaker containing acetone for ultrasonic cleaning. Liquid chromatography column troubleshooting method.

Common Faults in HPLC Column

High Quality HPLC Columns
1. Irreproducible Chromatography:

  • Possible Causes:
    • Inconsistent mobile phase preparation.
    • Column contamination.
    • Fluctuations in temperature or pressure.
  • Troubleshooting:
    • Ensure consistent mobile phase preparation.
    • Check for column contamination and clean or replace the column if necessary.
    • Stabilize temperature and pressure conditions.

2. Poor Peak Shape:

  • Possible Causes:
    • Column overloading.
    • Sample solvent mismatch with mobile phase.
    • Mobile phase compatibility issues.
  • Troubleshooting:
    • Reduce sample load or dilute the sample.
    • Optimize sample solvent to match the mobile phase.
    • Check mobile phase composition and compatibility.

3. Baseline Drift:

  • Possible Causes:
    • Contaminated mobile phase or solvent.
    • Detector issues.
    • Sample matrix interference.
  • Troubleshooting:
    • Purify or filter the mobile phase.
    • Check and maintain the detector.
    • Evaluate the sample matrix for interference.

4. Retention Time Variability:

  • Possible Causes:
    • Fluctuations in mobile phase flow rate.
    • Changes in temperature.
    • Column degradation.
  • Troubleshooting:
    • Stabilize the mobile phase flow rate.
    • Maintain a consistent temperature.
    • Check column integrity and replace if needed.

5. Low Sensitivity:

  • Possible Causes:
    • Detector issues.
    • Mobile phase composition not suitable for detection.
    • Column degradation.
  • Troubleshooting:
    • Check and maintain the detector.
    • Optimize mobile phase for detection.
    • Verify column integrity.

6. Loss of Resolution:

  • Possible Causes:
    • Column aging.
    • Mobile phase composition issues.
    • Inadequate column equilibration.
  • Troubleshooting:
    • Replace the column if aging is confirmed.
    • Optimize mobile phase composition.
    • Ensure proper column equilibration.

7. Excessive Pressure:

  • Possible Causes:
    • Column blockage or packing issues.
    • Clogging of frit or guard column.
    • Mobile phase viscosity mismatch.
  • Troubleshooting:
    • Check for column blockage or packing issues.
    • Clean or replace frit or guard column.
    • Adjust mobile phase viscosity or solvent composition.

8. Peak Splitting or Tailing:

  • Possible Causes:
    • Sample matrix interference.
    • Column contamination.
    • Mobile phase issues.
  • Troubleshooting:
    • Evaluate sample matrix for interference.
    • Check and clean or replace the column.
    • Optimize mobile phase composition.

9. Ghost Peaks:

  • Possible Causes:
    • Sample carryover.
    • Mobile phase contamination.
    • Column degradation.
  • Troubleshooting:
    • Use appropriate cleaning procedures for sample carryover.
    • Purify or filter mobile phase.
    • Check and replace the column if necessary.

10. Excessive Baseline Noise:

  • Possible Causes:
    • Detector issues.
    • Mobile phase impurities.
    • Sample matrix interference.
  • Troubleshooting:
    • Check and maintain the detector.
    • Purify or filter the mobile phase.
    • Evaluate sample matrix for interference.

One common fault is that HPLC column pressure is too high. In this situation, the following methods can be adopted:
1. Remove the protective column, and see if the column pressure is still high, otherwise, it is the protection column problem, if the column pressure is still high, re-check;
2. Take the column from the instrument, if the pressure is still not down, it is pipe blocked, to be cleaned, if the pressure drop, the column inlet, and outlet in turn connected to the instrument, with 10 times the volume of the column flow phase washing column, if column pressure still not down, re-check;
3. Replace the column inlet sieve plate, if column pressure drops, indicating that the solvent sample contains solid particles if column pressure is still high, can be connected between the sampler and the protective column online filter.

Another problem is that baseline is instability, fluctuation, or drift, in this situation, the following methods can be adopted:
1. The mobile phase has dissolved gas, ultrasonic degassing can be used for 15-30 minutes;
2. Single-way valve blockage can remove the ultrasonic plug;
3. There are leak points in the system, determine the leak location and repair;
4. Column balance is slow, especially when the mobile phase changes, it can be washed with a medium-strength solvent. When changing the mobile phase, it is washed with 10-20 times the volume of the flow relative column before analysis.

General Tips for Troubleshooting HPLC Columns:

  • Regular Maintenance: Implement routine column checks and maintenance procedures as recommended by the column manufacturer.
  • Use Quality Solvents: Ensure the use of high-quality solvents to minimize impurities and contaminants.
  • Guard Columns: Consider using guard columns to protect the analytical column from contamination.
  • System Checks: Regularly perform system suitability tests and checks to monitor the overall performance of the HPLC system.
  • Record Keeping: Keep detailed records of column usage, maintenance, and troubleshooting steps for future reference.