Quick Troubleshooting Guide For HPLC Column Usage

One rule you need to know what the normal situation is beforehand so you can tell if something is wrong. The following two points should be done in the laboratory, which will be very helpful for troubleshooting:

  • Keep a chromatogram of the test sample for each new column - start by looking at the test chromatogram provided with the column. In most cases, test samples are readily available chemicals, commonly used in laboratories, or available from chemical suppliers. Prepare a test sample (a starting concentration of 0.1 mg/mL for each component is a good choice), analyze it with a new column on your instrument, and then compare. The initial test sample injection will help determine if there is a system problem that prevents you from achieving the best results. Some people prefer to use their own samples or standards because the test mix may not be relevant to their application. It is best to use equal-degree conditions because sometimes gradients can "compress" peaks and mask bad column effects, making them artificially sharp. Comparing your own test chromatogram to this initial chromatogram over time can help you determine if column efficiency has decreased or if there are other changes affecting column efficiency.
  • Keep a system diagram of the optimized instrument -- When you install the instrument and optimize the method, document in detail how your instrument is stacked, the part numbers of all accessories and the lengths of all connecting pipes, and all electrical connections. This diagram serves as a handy reference if there is a problem, to ensure that the configuration has not changed, and therefore the result or instrument performance.
Problem Possible causes Solution
High back pressure Column inlet screen plate blocked Cleaning column in a reversed direction.
Column blockage (chemical contamination) Clean column with solvent, replace column if not improved.
The particle size of the column filler is too small Select an appropriate HPLC column
In-line filter or guard column blocked Check the filter head of the in-line filter and replace it if necessary
Blockage of line Remove lines for validation and replace them if necessary
Polymer HPLC column.

A change in solvent causes swelling

Check solvent compatibility information
The viscosity of mobile phase is too high Use a solvent with lower viscosity, or raise the temperature
Salt/buffer precipitation Ensure the compatibility of the mobile phase with the buffer
Fluctuation of pressure There are air bubbles in the pump Solvent degassing.

Fill the solvent with helium or use an in-line degasser

Check valve or gasket leakage Replace or clean the check valve.

Replace the pump gasket.

Pressure drop or low pressure Insufficient pump flow Empty the mobile phase container and replace the on-line filter head of the sample inlet in the container.

Check if the pipeline is squeezed.

Check traffic Settings.

System-wide leak detection.

Pump check valve or gasket leakage Replace or clean the check valve.

Replace the pump gasket.

Check for residue from salting out

There are bubbles in the pump Solvent degassing.

Check whether the pipeline from the solvent bottle to the pump is blocked.

Replace the in-line filter head of the injector.

Peak shape
There is no peak Instrument problem Ensure that all HPLC components are on and running.

Check the detector outlet line

Mobile phase or fixed phase combination error Whether there is a liquid outflow.

Compounds are not retained in the injection to ensure system suitability.

Increase solvent strength or use gradient elution.

Accidental peak or "ghost" peak Analyte that was not eluted in the previous analysis Using a rapid gradient, such as from 10% to 90% acetonitrile over a period of 10 to 15 minutes, the number of components in the sample and their relative retention is determined. Start with a strong mobile phase, such as 75% methanol and/or a higher flow rate to allow the components to flow out of the column faster.
Mobile phase pollution Use only high-purity chromatographic grade and above (LC/MS or gradient grade) solvents. Use high-purity water from an internal pure water system. TFA was used in the aqueous mobile phase and a lower concentration of TFA was used in the organic mobile phase solvent (i.e. 0.1% TFA in water / 0.086% TFA in acetonitrile). Longer detection wavelengths are used so that TFA absorption is weaker.
Sample preparation/Sample preparation contamination Use the usual sample preparation methods to reduce contamination - filtration, SPE, liquid-liquid extraction, centrifugation, etc.
Contamination of system Sample solvent, make sure there is no problem with the sample solvent.

Blank injection was performed during multiple runs to prove that there were no ghost peaks caused by cross contamination.

Remove the automatic sampler from the flow path and run the blank program to check whether the ghost peak disappeared. If it disappears, clean the autosampler. If not, check other system components in the flow path to find the source.
Chromatographic column contamination (Note: less cause ghost peak) Recoil column (consulting manufacturer).

Cleaning chromatographic column.

Forward extension peak Column groove flow Replace the column with a protective column.
Column overload Use higher capacity columns (increase column length or inner diameter).

Reduce the sample size.

Tailing Peak Silanol group interaction (Silica gel column) Use closed-end or special column.

Increase the buffer concentration.

Reduce the pH of the mobile phase to inhibit silanol interaction.

Use a competing base.

Using a derivative solution of changing polarity interactions.

If that doesn't work, try running the column in reverse.

If the results improve, it indicates that column contamination is most likely the cause.

Clean or replace the column.

Out of column effect Check the system, whether the connecting pipeline of each component is too long, and replace it with a shorter pipeline.

If using a high-efficiency column, replace the line with a thick inner diameter with a thin inner diameter.

Chromatographic column at high-temperature degradation (silica-gel based column) Reduce the temperature below 40 °C, especially when using a high pH mobile phase.
Use high-temperature compatible columns, such as stereo-protected silica gel column, mixed column, polymer column, zirconia column, etc.
The column is decomposed at high pH (Silica gel column) Use a high coverage or double cross-linked stationary phase especially suitable for operation at higher pH, or use polymer, hybrid or oxygen pick reverse column.
HPLC column cavity Run in the opposite direction.

If the peak shape of all the peaks is poor or double peaks appear, it indicates that the column cavity may exist.

Discard the column.

Interference co-elution peaks Adjust the mobile phase to improve selectivity or select a new stationary phase to improve sample purification.
Peak split/Double peak Component of interference Preparation of purified sample by sample.

Change the mobile or stationary phase to adjust selectivity

If the component is suspected to be from a previous injection, flush the column with a strong solvent after analysis.

Increase the concentration of strong solvents in the gradient.

Extended elution time.

The column sieve plate is partially blocked Recoil column (if recoil is possible).

U s e a 0.2µm or 0.5µm (UHPLC) in-line filter between the injector and the column.

Filter samples using protective columns.

Chromatographic column cavity Replace the column.

A guard column is later used to protect the analysis column. Mobile phase conditions with less condensation are used.

Solvent effect of injection Use a mobile phase or a weak injection solvent.
Sample volume


Use a smaller sample injection volume.
The sample solvent is incompatible with the mobile phase Use a mobile phase or a weak solvent that is soluble in the injection solvent.
The sampler rotor is damaged Replace the sampler rotor.
Peak broadening/peak broadening The connector/connection is not suitable Make sure your connector is properly installed.
External pipeline volume in the system Make sure the line is thin and as short as possible to avoid column volume.
The sample volume is too large Reduce the injection volume.
System Settings

(For example, the data collection rate is too low)

Check the data collection rate. Adjust detector Settings and/or time constants to use the fastest acquisition rate without reducing the signal-to-noise ratio.
Sample dilution is too high Reduce dilution strength.
For gradient elution: resident volume Reduce the initial gradient concentration for peak concentration or start the gradient using the sampler procedure prior to sample injection. Note that the peaks are elution in the gradient phase and not in equal degree elution.
Retention time drift The column is getting old Compared with the test chromatogram, the chromatographic column changes were found.

Use protective columns to extend column life.

Column size and flow rate changes Make sure your method parameters are adjusted according to changes in flow rate and column specifications.

This is especially important for gradient elution.

Column/mobile phase mismatch for your analyte (retention too low for bonded phase, pH too close to pKa. The pH range of the column is incompatible with the

mobile phase, etc.)

Your mobile phases may not be consistent. Make sure you use the same mobile phase each time.

The buffer and solvent should be measured.

separately in clean glassware and then mixed. Mobile phase degassing. Replace the aging mobile phase.

For the mobile phase: Column rebalancing time is insufficient Measure the dead volume of your column and the resident volume of your system to optimize the balance time and method.
Shortened retention time The active site of column packing Use mobile phase modifiers, compete for bases (alkaline compounds such as triethylamine) or increase buffer strength, and use high coverage column fillers.
Sample overload Reduce sample size or use a larger bore or longer column.
Bonding phase or silica gel group loss Use a mobile phase with pH between 2 and 8.

Use high or low pH special silica gel column, polymer column or other high/low pH columns.

Aging of the HPLC column Use protective columns, or high-stability bonded polymer, hybrid or high-stability columns (e.g., zirconia, titanium, graphitic carbon).
Extension of retention time Flow rate decrease Check and reset the flow rate. Check whether the pump has bubbles.

Check pump gaskets for leakage, and check valves and other systems for leakage.

The mobile phase composition changes Refill the solvent bottle.

Ensure the scale of the gradient system is correct.

Equigradient elution was performed on the pre-mixed

mobile phase.

Loss of bonding phase For common silica gel columns, keep the mobile phase pH between 2 and 8.

For extremely high pH (> 10) or extremely low pH (< 2) work, use high stability stationary phase, polymer, or high stability stationary phase column.

Drift of baseline For gradient elution: The absorption of mobile phase A or B is different For negative drift: use mobile phase solvent without UV absorption.

Use chromatography-grade mobile phase solvent; The UV absorption additive in mobile phase A is added to mobile phase B to balance/compensate for the drift.

For positive drift: Use a higher UV absorption detector wavelength that the analyte still absorbs; Use mobile phase solvent without UV absorption.

Reduce the amount of UV-absorbing compounds in mobile phase B to balance/compensate for drift.

Fluctuation - changes in room temperature Isolation column or use column temperature chamber.

Mask the differential refraction detector or insulate it from airflow.

Forward - LC/MS, stationary phase loss Use low drain, MS compatible, or high stability stationary phase column.
Positive - MS pollution Clean the mass spectrum interface, avoid using THF and halogenated solvents, and use PEEK line columns.
Column contamination (column loss) Flush the column with a strong solvent. Improve sample purification. Use/replace protective posts. Replacement of analysis column.
For persistent problems: Check the detector light source or flow tank Replace the UV lamp. Clean and flush the circulation tank.
Gradient or equivalence: Lack of solvent mixing Use appropriate mixing devices. Check the precision of the proportional valve by adding a compound containing UV absorption to the solvent and monitoring the output of the UV absorption detector.
Gradient or equal scale – proportional valve failure Clean and replace the proportional valve. Partially premix the solvent. (e.g., 5% B in A, and/or vice versa)
Occasional spikes - interference from external electrical signals LC system uses a voltage regulator/constant voltage supply. Check whether there is a local interference source, such as a circulating furnace, using an independent current.
Periodic - Pump pulse Repair or replace dampers. Remove the air from the pump. Clean or replace the check valve. Mobile phase degassing.
Random - accumulation of pollution Flush the column with strong solvent or recoil column (if recoil is possible). Purified sample. Use chromatography-grade solvents.
Spikes-Bubbles in the detector Mobile phase degassing. Use the reverse pressure limiter at the detector outlet.

Ensure all joints are tight and leak-free.

GALAK ChromatographyAuthor
Tian Jing

Manager & Engineer in GALAK Chromatography. Master of Chemical Engineering.
During my college study, I found liquid chromatography to be a profound subject. I know the painful struggle a novice needs to go through to get started. I share this article to help you solve your problems quickly.

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