LC Troubleshooting Essentials: A Guide to Common Problems and Solutions for Peak Tailing, Ghost Peaks, and Pressure Spikes

From common problems encountered by liquid chromatography (LC) users to practical solutions, this LC resource helps you optimize your analysis and solve problems with confidence, with advice from two true masters in the practical science of LC problem-solving: Dwight Stoll and John Dolan.

1. Why are my peaks tailing or fronting?

Tailing and fronting are asymmetrical peak shapes that signal something is off in your chromatographic system.

What causes tailing and fronting:

• Tailing often arises from secondary interactions between analyte molecules and active sites (for example, residual silanol groups) on the stationary phase.
• Also, column overload (too much analyte mass) can lead to tailing due to slower‐equilibrating retention sites.
• Fronting is typically caused by column overload (too large injection volume or too high concentration) or by a physical change in the column (collapse) that disturbs the packing.
• Injection solvent mismatch (sample in strong solvent relative to mobile phase) or too large an injection volume can distort peaks (fronting or splitting), particularly early eluting peaks.
• Physical problems like voids at the column inlet/frit blockage affect all peaks (so if all peaks are tailing, suspect a physical cause).

What to do:

• Check sample load: reduce injection volume or dilute sample to see if tailing/fronting improves.
• Ensure sample solvent strength is compatible with the initial mobile phase.
• Use a column with less active residual sites (end-capped silica, or a more inert stationary phase) for analytes prone to interaction.
• If physical: examine inlet frit, guard cartridge, in-line filter; consider reversing/flushing the column if allowed.
• Monitor system suitability (tailing factor, asymmetry factor) over time.

2. What causes ghost peaks or unexpected signals?

Common causes:

• Carryover from prior injections (insufficient cleaning of autosampler or injection needle).
• Contaminants in the mobile phase, solvent bottle, or sample vial (leachables, plasticizers).
• Column bleed or decomposition of the stationary phase, especially with high temperature or extreme pH.
• Sample matrix components that weren’t removed during preparation, generating late‐eluting or low‐level peaks.
• System hardware contamination (pump seals, injector rotor, tubing), depositing compounds that show up later.

What to do:

• Run blank injections (solvent only) and compare chromatograms to spot ghost peaks.
• Clean autosampler, change or clean injection needle/loop, purge injection path.
• Use fresh mobile phase, check solvent bottles for contamination or precipitates; filter solvents.
• Replace or clean column if suspect bleed or degradation (especially if ghost peaks increase with column usage).
• Use a guard column or in-line filter to protect the column and capture contaminants early.

3. Why has my retention time shifted?

Possible causes:

• Change in mobile phase composition, pH, or buffer strength (especially for ionizable analytes).
• Change in flow rate or pump performance (if flow is higher, retention decreases; if lower, retention increases).
• Column temperature change (higher temp. reduces retention, lower temp. increases retention).
• Column aging or stationary phase degradation (ligand loss, silica dissolution) altering retention.
• Change of column lot (lot-to-lot variability) causing slightly different chemistry/selectivity.
• Pump mixing problems (in gradient systems) or solvent delivery variances.

What to do:

• Verify mobile-phase preparation: composition, pH, buffer lot, freshness.
• Check flow rate: collect mobile phase for a minute and measure volume to verify.
• Check column oven/thermostat: ensure set-point is stable and matches method.
• Compare current retention times with historical controls; if shift is uniform for all peaks, likely flow or mobile phase; if selective to some peaks, likely chemical/column issue.
• If the column was recently changed, consider lot variability; try the old column if available.
• Check for system leaks or degasser/pump issues influencing solvent composition/delivery.

4. What should I do if pressure suddenly spikes or drops?

Sudden pressure spike (too high):

• Likely blockage somewhere: inlet frit clogged, guard column blocked, particulate buildup in tubing or column.
• Plugged filter or debris in the mobile phase line.
• Use of too viscous a mobile phase or incorrect solvent causing higher backpressure.
• Column collapse or packing disruption causing abnormal pressure behaviour.

Sudden pressure drop (too low):

• Leak in tubing/fittings, broken pump seal, air entering pump head.
• Pump malfunction or solvent starvation (inlet filter blocked, insufficient feed to pump).
• Column lost packing or void formed inside column (reducing pressure).

What to do:

• Record “normal” system pressure under standard conditions to use as a reference.
• When pressures spike start at downstream end. Disconnect column and measure pressure without it; if lower, column likely culprit. Reverse‐flush column if permitted.
• When pressures drop: check pump flow rate, collect output, check for leaks or air bubbles, check solvent levels and filters.
• Ensure mobile‐phase viscosity is expected, solvent composition correct, tubing condition normal.
• Maintain in-line filters and guard columns to prevent frit/column damage.

5. How can I differentiate between column, injector, or detector problems?

Differentiating the source of a problem is key. Here’s a structured approach:

• Column issues often affect all peaks, especially if efficiency falls or tailing increases across the board, or resolution drops for many analytes. Physical changes in the packing (voids, bed collapse) will show broad effects.
• Injector issues tend to show problems in the early part of the chromatogram (peak distortion, split peaks, inconsistent injection volume) and may vary sample to sample. If the injection loop or rotor seal is faulty, you might see inconsistent area/height, carryover.
• Detector issues often manifest as baseline noise, drift, sudden loss of sensitivity, or a subset of peaks inexplicably altered (detector saturates). Detector hardware problems might not shift retention but will affect response.
• Use the “does the problem affect all peaks or only one/a few?” rule: If only one or two specific peaks are distorted → likely chemical or column-analyte interaction; if all peaks are similarly affected → more likely physical (column/injector) or system-wide.

Practical tests:

• Replace/inject a standard sample under known good conditions: If performance returns, suspect sample/column; if not, suspect injector, detector, or system.
• Disconnect column (or replace with a short “dummy” column) and check injection and detector behavior: if problem disappears, likely column; if persists, injector or detector.
• Check detector baseline and signal with a known solution; ensure detector is working properly.
• Check injection precision (multiple injections of the same standard) to assess injector reproducibility.
• Check column efficiency metrics (plates, tailing factor) to evaluate column health.
• Check injector carryover by running a blank immediately after a high concentration sample.

6. What steps should I follow for a systematic troubleshooting approach?

A structured, step-by-step process helps minimize wasted time and guesswork.

Step-by-step:

1. Recognize and quantify the deviation: Note what’s changed (retention time, peak shape, resolution, pressure). Refer to previous “good” runs.
2. Check the simplest cause first: Mobile phase preparation (composition, pH), solvent bottles, sample preparation, injection volume.
3. Check system conditions: Flow rate, column temperature, detector settings, baseline stability.
4. Isolate where the problem originates:

• Remove/replace column (or bypass) to test column health.
• Run system without a sample (blank) to test for ghost/contaminant peaks.
• Check injection reproducibility to test the injector.
• Check the detector with a known solution to test the detector/response.
• Monitor pressure behaviour (high/low/fluctuating) to identify blockage/leak.

1. Check for hardware maintenance issues: Filters/frits, guard column, tubing, pump seals, check-valves.
2. If column suspected: Check age, history (number of injections), cleaning/back-flushing history, manufacturer instructions (pH/temperature limits).
3. If method suspected: Consider mobile phase changes, gradient shift, sample matrix effects, column lot change.
4. Make one change at a time, then test. Avoid changing multiple variables simultaneously so you can identify the cause.
5. Document results: Record what you changed and what the effect was; build a log for recurring issues.
6. Preventive actions: Once resolved, implement steps to avoid recurrence (for example, use inline filters, improved sample prep, periodic column performance checks, system suitability tracking).

Dwight Stoll is the editor of “LC Troubleshooting,” which provides practical, nuts-and-bolts style advice to analysts working in liquid chromatography.

John Dolan contributed "LC Troubleshooting" to LCGC for over 25 years. One of the industry's most respected professionals, John is currently a principal instructor for LC Resources, Walnut Creek, California.

A more extensive discussion on the points above can be found by clicking the following links:

Troubleshooting Basics, Part 4: Peak Shape Problems

Essentials of LC Troubleshooting, Part III: Those Peaks Don’t Look Right

Essentials of LC Troubleshooting, Part II: Misbehaving Retention Times

Essentials of LC Troubleshooting, Part I: Pressure Problems