Troubleshooting Guide | ChromatoHub Academy

Systematic troubleshooting starts with reproducing the problem, then isolating the variable. Change one thing at a time — run a blank (no sample) first to distinguish system artefacts from sample-related issues.

Peak tailing

Likely causes

Column packed too loosely or bed damaged
Non-specific ionic or hydrophobic interactions with the medium (for example in SEC)
Target not binding properly under the start conditions (IEX/HIC)
Extra-column volume in tubing and connections

What to try

Measure asymmetry (As = b/a at 10% of peak height) and efficiency, and compare with earlier tests of the same column on the same system
In SEC, adjust buffer composition, for example ionic strength, pH or an additive, within the column limits
Check start-buffer pH and salt, and whether the load is within capacity
Minimise tubing length and connections between injector, column and detector

Poor resolution between adjacent peaks

Likely causes

Gradient too steep for the separation (IEX/HIC)
Flow rate above the range that gives adequate resolution
Loss of column performance (packing, fouling)
Sample volume or load too high
Selectivity mismatch (wrong resin or conditions for the separation)

What to try

Try a shallower gradient over the elution window
Test a lower flow rate within the column specification and compare resolution
Clean the column, then re-test efficiency; efficiency depends on the system, so compare with the column baseline rather than a fixed fraction of the specification
Reduce sample volume or load and compare
Scout different pH, salt or an alternative resin chemistry

Ghost peaks or extra peaks

Likely causes

Buffer impurities concentrated on the column and released during elution
Material retained from a previous run
Sample and running buffer mismatch causing refractive-index disturbances in the UV trace (often negative peaks near the solvent front)
Air bubble passing through the UV flow cell (spikes)

What to try

Run a blank gradient with no sample to see which peaks come from the system or buffers
Clean the column as described in its instructions
Exchange or dilute the sample into start or running buffer
Use degassed buffers and check the pump and inlets for air

High system pressure

Likely causes

Column or column filter fouled or blocked
Particulates in sample or buffer
Viscous sample or buffer, or low temperature
Flow rate too high for the column and conditions
Blockage in system tubing, valves or in-line filters

What to try

Clean the column with the procedure in its instructions; use reverse flow only if the column instructions allow it
Filter or centrifuge samples and filter buffers, choosing filter pore size to suit the medium; check recovery if the target may adsorb to the filter
Dilute viscous samples and reduce flow for viscous buffers or cold operation
Check the column instructions for pressure and flow limits, and set system alarm limits accordingly
Run the system without the column to locate where the pressure arises

Baseline drift

Likely causes

Buffers A and B absorb differently at the monitored wavelength
Temperature changes in the lab, buffers or column
Contamination or deposits in the UV flow cell
Air in the flow cell

What to try

Balance UV absorbance of buffers A and B, or monitor at a different wavelength
Let instrument, column and buffers reach the same temperature before the run
Clean the flow cell following the system manual
Degas buffers and purge air as described in the system manual

Sources and scope

Causes and remedies follow the troubleshooting tables in Cytiva™’s Size Exclusion Chromatography, Ion Exchange Chromatography and Hydrophobic Interaction and Reversed Phase Chromatography handbooks (official, accessed 2026-09-25). They are diagnostic prompts, not fixed recipes: take flow, pressure, cleaning and buffer limits from the instructions for the exact column, resin and system.

Try it on a trace

Open a two-column chromatogram in the workbench, or load the synthetic example. No account needed.

Open the workbench