troubleshoot-separation
About
This Claude Skill systematically diagnoses and resolves GC and HPLC chromatographic separation issues. It helps document symptoms, identify root causes for peak shape and retention anomalies, and evaluate matrix effects. Developers should use it to implement targeted fixes via a structured, one-variable-at-a-time approach.
Quick Install
Claude Code
Recommendednpx skills add pjt222/agent-almanac -a claude-code/plugin add https://github.com/pjt222/agent-almanacgit clone https://github.com/pjt222/agent-almanac.git ~/.claude/skills/troubleshoot-separationCopy and paste this command in Claude Code to install this skill
Documentation
Troubleshoot Chromatographic Separation
GC/HPLC sep diagnosis → symptom doc → peak shape diag → retention anomaly → matrix effects → verified fix via one-var-at-time.
Use When
- Peaks tail|front|split|broaden
- Retention shifted|irreproducible
- Resolution between critical pairs degraded
- Baseline drift|ghost peaks|negative peaks
- Sensitivity drop|S/N worse
- Working method now fails sys suitability
In
Required
- Problem chromatogram: Current data → issue
- Reference chromatogram: Recent good, same method → compare
- Method conditions: Column, mobile phase|carrier gas, temp|gradient, detector, flow
- System log: Recent maint, col changes, mobile phase prep, instrument events
Optional
- Blank chromatogram: Most recent blank|solvent inj
- Sys suitability trends: Historical tailing, resolution, plates, RT
- Col history: # injections, sample types, age
- Instrument maint log: Pump seal, lamp hrs, detector service
Do
Step 1: Doc Problem
- Symptom precise: which peaks, how differ from ref
- When started: gradual|sudden
- All peaks or specific?
- Standards|samples|both?
- Current sys suitability vs historical
- Photo|export problem chromatogram side-by-side w/ ref
Got: Problem statement w/ timeline, scope (all|specific peaks, std|sample), ref comparison.
If err: No ref → inject fresh standard prep under documented method → establish current baseline before troubleshoot.
Step 2: Diag Peak Shape
| Symptom | Possible Causes | Solutions |
|---|---|---|
| Tailing (T > 1.5) | Secondary interactions (silanol activity), dead volume in fittings, contaminated column frit, overloaded active sites | Add amine modifier (HPLC), deactivate liner (GC), replace frit, trim column inlet, reduce injection mass |
| Fronting (T < 0.8) | Column overload (mass or volume), mismatch between sample solvent and mobile phase strength | Reduce injection volume or concentration, dilute in weaker solvent, use larger-bore column |
| Split / double peaks | Partially blocked frit, void at column head, two polymorphic forms, isomeric interconversion | Replace frit, repack column head, verify sample stability, adjust pH to lock one form |
| Broad peaks (all) | Extra-column band broadening, wrong tubing ID, large detector cell, old column, low plate count | Minimize post-column tubing length and ID, check connections, replace column |
| Broad peaks (early eluters) | Poor focusing at column head, injection solvent too strong (HPLC), cold on-column (GC) | Use weaker injection solvent, reduce injection volume, increase initial oven temp |
| Broad peaks (late eluters) | On-column diffusion, temperature too low (GC), insufficient gradient steepness (HPLC) | Increase final oven temperature, steepen gradient, add organic wash |
| Negative peaks | Sample solvent refractive index/absorbance differs from mobile phase, vacancy peaks (IEX) | Match sample solvent to mobile phase, use different detection wavelength |
| Ghost peaks | Carryover from previous injection, contaminated mobile phase, column bleed, septum bleed (GC) | Run blank to confirm, clean or replace injection system, filter/degas mobile phase, replace septum |
| Baseline drift (upward) | Column bleed (GC at high temp), gradient elution baseline shift (HPLC), lamp instability (UV) | Reduce max temp, use low-bleed column (GC), run blank gradient to characterize (HPLC), replace lamp |
| Baseline noise (high-frequency) | Electrical interference, pump pulsation, air bubbles in detector, contaminated detector | Ground instrument, replace pump seals, degas mobile phase, clean detector cell |
- Match symptom(s) → table
- Narrow causes: all peaks|specific, sudden|gradual
- Prioritize → sys history (recent changes, col age, maint)
Got: 1-2 most-likely causes from sym-cause map, prioritized by history.
If err: No table match or multi-symptom → compound problem (col degradation + leak). Address obvious first → re-eval.
Step 3: Diag Retention
| Symptom | Possible Causes | Solutions |
|---|---|---|
| All peaks shifted earlier | Increased flow rate, higher column temperature, stronger mobile phase, column void | Check flow rate setting and actual delivery, verify temperature, remake mobile phase, inspect column |
| All peaks shifted later | Decreased flow rate, lower column temperature, weaker mobile phase, partially blocked tubing | Check for leaks (pressure drop), verify temperature, remake mobile phase, check inline filter |
| Retention time drift (gradual) | Column degradation, mobile phase evaporation (open reservoir), temperature fluctuation | Replace column, seal reservoir, stabilize oven, use column thermostat |
| Retention time irreproducible | Leak at fitting, check valve malfunction, autosampler timing error, inadequate re-equilibration | Pressure-test fittings, replace check valves, verify autosampler, increase equilibration volume |
| Lost retention (k' near 0) | Phase collapse (RP at high aqueous), column dewetting, wrong mobile phase, reversed connections | Use polar-embedded or AQ-type column, re-wet column with organic, verify mobile phase, check plumbing |
| Co-elution (previously resolved) | Column selectivity lost (bonded phase stripped), mobile phase composition changed, temperature changed | Replace column, verify mobile phase preparation, check temperature setpoint vs. actual |
- Shifts uniform (all) or selective (specific)?
- Uniform → systematic (flow, temp, mobile phase)
- Selective → col chemistry|specific analyte
- Check pressure trace: sudden change → leaks|blockage
- Re-inject ref std → confirm sys vs sample
Got: Retention root cause categorized: systematic (instrument/MP) or col-related.
If err: Re-inject std on new col resolves → orig col = problem. Persists on new col → upstream (MP, instrument, method).
Step 4: Matrix Effects
- Std vs sample chromatogram:
- Extra peaks in sample absent from std?
- Baseline elevated|noisy in retention windows?
- Analyte peaks differ in sample vs std (broader, more tailing)?
- LC-MS → ion suppression/enhancement:
- Post-col infusion: infuse analyte continuous while inject blank matrix → dips = suppression regions
- Analyte RT coincides w/ suppression → shift elution
- Col contamination:
- Solvent blanks after sample seq → persistent peaks = col contamination
- Flush col w/ strong solvent (100% organic for RP, or per mfr)
- Sample prep:
- Dirty injector (autosampler needle, GC liner) → replace|clean
- Insufficient cleanup → add filter, SPE, protein precipitation
- GC: non-volatile residue in inlet liner → causes tailing + ghost over time
Got: Matrix effects characterized (interferents, ion suppression zones for LC-MS, col contamination) w/ actions.
If err: Can't characterize → matrix-matched cal curve vs solvent cal curve. Slope diff > 15% → significant matrix effects → method mod.
Step 5: Fix + Verify
- One var at a time. Doc what + why.
- Per change → re-inject sys suitability std vs ref
- Sequence (least → most disruptive):
- Fresh mobile phase | carrier gas tank
- Replace consumables (septum, liner, frit, inline filter, lamp)
- Tighten|replace fittings + tubing
- Flush|regen col
- Adjust method (temp, flow, gradient, pH)
- Replace col
- Service instrument (pump seals, check valves, detector)
- Fix found → full sys suitability test (n >= 5 injections)
- Compare all params (RT, area, resolution, tailing, plates) vs spec
- Doc root cause, action, verify in instrument|col logbook
- Recurs → preventive maint schedule
Got: Problem resolved, sys suitability params restored to spec. Root cause + action + verify documented.
If err: All single-var changes fail → multi simultaneous failures. Replace all consumables + col together → verify w/ fresh std → rebuild from new baseline. Persists after total replacement → escalate to instrument service.
Check
- Problem documented w/ symptom, timeline, scope
- Root cause via sym-cause maps
- One var at a time
- Fix verified by sys suitability (n >= 5 replicate inj)
- All sys suitability params restored to spec
- Root cause + action in logbook
- Preventive measure ID'd
Traps
- Multi vars at once: Can't ID root cause. One change → test → decide.
- Col first: Expensive, masks real problem (leak, wrong MP, contaminated inlet). Exhaust simpler first.
- Ignore logbook: Many problems → recent maint, MP batch, col swap. Check what changed.
- Blame sample w/o evidence: Run ref std first. Std also shows problem → sys, not sample.
- Incompatible solvents: Never flush RP w/ pure water (phase collapse) or silica HILIC w/ pure aqueous (irreversible). Per mfr protocol.
- No documentation: Failed attempts valuable. Record every change + outcome → avoid repeat + build knowledge.
→
interpret-chromatogram— understanding chromatographic data revealing sep problemsdevelop-gc-method— GC method dev, relevant when troubleshooting requires redesigndevelop-hplc-method— HPLC method dev, relevant when troubleshooting requires redesignvalidate-analytical-method— re-validation may need after significant method changes
GitHub Repository
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