Instruction: Fix peak tailing/broadening/splitting.

Input: Matrix: river water; Analytes: azo dyes; Column: C18, 2.1×100 mm, 1.7 µm; Oven 25°C; A: 5 mM ammonium acetate in water (pH ~5.0); B: MeOH; Gradient 10→95% B over 6 min; Flow 0.30 mL/min. Observation: tailing factor ~1.8; occasional split peaks on main analyte.

Output:
Likely causes and actions:
1) Match sample solvent to initial mobile phase; dilute strong solvents.
2) Lower pH to suppress ionization for bases (pH 2.8–3.2) or raise for acids (pH 6.5–7.5).
3) Consider polar-embedded/C18-AQ or PFP to reduce secondary interactions.
4) Replace guard; check for column void (reverse flush briefly).
Acceptance: backpressure within ±10% of baseline; plate count within spec.


Instruction: Reduce carryover observed in blanks after high standards.

Input: Matrix: QC standard in water; Analytes: azo dyes; Column: C18, 2.1×100 mm, 1.7 µm; Oven 45°C; A: 10 mM ammonium bicarbonate (pH 9.0); B: ACN; Isocratic 50% B; Flow 0.50 mL/min. Observation: blank after high standard shows 0.5–2% residual peak.

Output:
Likely causes and actions:
1) Strengthen needle wash (≥80% organic + 0.1% acid); extend wash cycles.
2) Include a high-organic purge step between runs; evaluate backflush if available.
3) Use low-adsorption vials/caps; check sample loop/syringe cleanliness.
4) Set acceptance carryover <0.1% of high standard peak area.
Acceptance: backpressure within ±10% of baseline; plate count within spec.


Instruction: Diagnose column frit clogging or void formation.

Input: Matrix: serum; Analytes: sugars; Column: C8, 4.6×100 mm, 3.5 µm; Oven 35°C; A: 10 mM ammonium formate + 0.1% FA in water (pH ~3.2); B: ACN; Gradient 10→80% B over 6 min; Flow 0.50 mL/min. Observation: plate count dropped 40%; asymmetry 1.7; possible void.

Output:
Likely causes and actions:
1) Check asymmetry and plates with a system suitability compound.
2) Reverse-flush 5–10 min; replace guard; inspect for voids (shoulder/doublet).
3) Reduce max pressure; verify fittings/ferrules to prevent channeling.
4) Log column volumes; retire if performance out of spec.
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Diagnose bubble/degassing issues causing ripple.

Input: Matrix: wastewater effluent; Analytes: acidic herbicides (pKa 2–4); Column: PFP, 2.1×100 mm, 1.9 µm; Oven 30°C; A: 10 mM ammonium formate + 0.1% FA in water (pH ~3.2); B: ACN; Isocratic 50% B; Flow 0.50 mL/min. Observation: periodic bubble bursts; UV shows spikes.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: recovery 80–120%, precision (%RSD) ≤5% for n=6.


Instruction: Diagnose bubble/degassing issues causing ripple.

Input: Matrix: wine; Analytes: quinolones; Column: Phenyl-hexyl, 3.0×100 mm, 3 µm; Oven 35°C; A: 50 mM ammonium formate (pH 3.2); B: MeOH; Gradient 10→60% B over 15 min; Flow 0.30 mL/min. Observation: periodic bubble bursts; UV shows spikes.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Fix peak tailing/broadening/splitting.

Input: Matrix: urine; Analytes: azo dyes; Column: C18-AQ, 2.1×150 mm, 3 µm; Oven 25°C; A: 20 mM ammonium acetate (pH 6.8); B: ACN; Isocratic 35% B; Flow 0.50 mL/min. Observation: tailing factor ~1.8; occasional split peaks on main analyte.

Output:
Likely causes and actions:
1) Match sample solvent to initial mobile phase; dilute strong solvents.
2) Lower pH to suppress ionization for bases (pH 2.8–3.2) or raise for acids (pH 6.5–7.5).
3) Consider polar-embedded/C18-AQ or PFP to reduce secondary interactions.
4) Replace guard; check for column void (reverse flush briefly).
Acceptance: recovery 80–120%, precision (%RSD) ≤5% for n=6.


Instruction: Fix peak tailing/broadening/splitting.

Input: Matrix: tablet dissolution (pH 6.8); Analytes: phenols; Column: HILIC amide, 2.1×100 mm, 1.7 µm; Oven 30°C; A: 5 mM ammonium acetate in water (pH ~5.0); B: MeOH; Isocratic 50% B; Flow 0.50 mL/min. Observation: tailing factor ~1.8; occasional split peaks on main analyte.

Output:
Likely causes and actions:
1) Match sample solvent to initial mobile phase; dilute strong solvents.
2) Lower pH to suppress ionization for bases (pH 2.8–3.2) or raise for acids (pH 6.5–7.5).
3) Consider polar-embedded/C18-AQ or PFP to reduce secondary interactions.
4) Replace guard; check for column void (reverse flush briefly).
Acceptance: backpressure within ±10% of baseline; plate count within spec.


Instruction: Resolve pump pulsation/check valve problems.

Input: Matrix: QC standard in water; Analytes: steroids; Column: Polar-embedded C18, 2.1×150 mm, 3 µm; Oven 30°C; A: 5 mM ammonium acetate in water (pH ~5.0); B: MeOH; Isocratic 50% B; Flow 1.0 mL/min. Observation: pulsation visible on pressure trace; peak area RSD ~6%.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: carryover <0.1% of high standard; blank shows no analyte peaks.


Instruction: Resolve pump pulsation/check valve problems.

Input: Matrix: cosmetics extract (IPA); Analytes: alkylbenzene sulfonates; Column: C18, 2.1×100 mm, 1.7 µm; Oven 35°C; A: 10 mM ammonium formate + 0.1% FA in water (pH ~3.2); B: ACN; Gradient 2→95% B over 6 min; Flow 1.0 mL/min. Observation: pulsation visible on pressure trace; peak area RSD ~6%.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: Rs>1.5 for critical pair, asymmetry 0.9–1.3, %RSD RT <1.0%.


Instruction: Diagnose column frit clogging or void formation.

Input: Matrix: milk; Analytes: beta-lactams; Column: C18-AQ, 2.1×150 mm, 3 µm; Oven 45°C; A: water + 0.05% TFA; B: ACN; Gradient 10→80% B over 12 min; Flow 0.30 mL/min. Observation: plate count dropped 40%; asymmetry 1.7; possible void.

Output:
Likely causes and actions:
1) Check asymmetry and plates with a system suitability compound.
2) Reverse-flush 5–10 min; replace guard; inspect for voids (shoulder/doublet).
3) Reduce max pressure; verify fittings/ferrules to prevent channeling.
4) Log column volumes; retire if performance out of spec.
Acceptance: carryover <0.1% of high standard; blank shows no analyte peaks.


Instruction: Reduce carryover observed in blanks after high standards.

Input: Matrix: milk; Analytes: acidic herbicides (pKa 2–4); Column: C18-AQ, 2.1×150 mm, 3 µm; Oven 40°C; A: 10 mM ammonium formate + 0.1% FA in water (pH ~3.2); B: ACN; Gradient 10→60% B over 8 min; Flow 0.30 mL/min. Observation: blank after high standard shows 0.5–2% residual peak.

Output:
Likely causes and actions:
1) Strengthen needle wash (≥80% organic + 0.1% acid); extend wash cycles.
2) Include a high-organic purge step between runs; evaluate backflush if available.
3) Use low-adsorption vials/caps; check sample loop/syringe cleanliness.
4) Set acceptance carryover <0.1% of high standard peak area.
Acceptance: k′ between 2–10; tailing factor <1.3; RT window ±2%.


Instruction: Diagnose retention time drift >5%.

Input: Matrix: oil matrix (hexane); Analytes: polyphenols; Column: HILIC amide, 2.1×100 mm, 1.7 µm; Oven 35°C; A: 10 mM ammonium bicarbonate (pH 9.0); B: ACN; Gradient 10→95% B over 6 min; Flow 1.0 mL/min. Observation: RT drifts +6–10% over a batch of 30 samples.

Output:
Likely causes and actions:
1) Verify oven setpoint holds within ±0.1°C; allow ≥15 min stabilization.
2) Increase post-gradient equilibration to ≥10–15 column volumes.
3) Prime A/B 5 min to remove composition drift; verify mixer volume.
4) Run bracketing standards every 10 injections; apply RT window ±2%.
Acceptance: carryover <0.1% of high standard; blank shows no analyte peaks.


Instruction: Reduce carryover observed in blanks after high standards.

Input: Matrix: drinking water; Analytes: acidic herbicides (pKa 2–4); Column: C18-AQ, 2.1×150 mm, 3 µm; Oven 25°C; A: 50 mM ammonium formate (pH 3.2); B: MeOH; Gradient 2→40% B over 10 min; Flow 0.40 mL/min. Observation: blank after high standard shows 0.5–2% residual peak.

Output:
Likely causes and actions:
1) Strengthen needle wash (≥80% organic + 0.1% acid); extend wash cycles.
2) Include a high-organic purge step between runs; evaluate backflush if available.
3) Use low-adsorption vials/caps; check sample loop/syringe cleanliness.
4) Set acceptance carryover <0.1% of high standard peak area.
Acceptance: recovery 80–120%, precision (%RSD) ≤5% for n=6.


Instruction: Stabilize noisy or drifting baseline.

Input: Matrix: tablet dissolution (pH 6.8); Analytes: azo dyes; Column: C18-AQ, 2.1×150 mm, 3 µm; Oven 40°C; A: 20 mM ammonium acetate (pH 6.8); B: ACN; Isocratic 50% B; Flow 1.0 mL/min. Observation: baseline ripple and drift during gradient; S/N reduced.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Troubleshoot high backpressure increase vs baseline.

Input: Matrix: pharma API in diluent; Analytes: sugars; Column: C18, 2.1×100 mm, 1.7 µm; Oven 35°C; A: 50 mM ammonium formate (pH 3.2); B: MeOH; Isocratic 60% B; Flow 0.30 mL/min. Observation: pressure increased 30–50% vs yesterday; pulses under gradient.

Output:
Likely causes and actions:
1) Bypass column to assess system backpressure; replace inline filter and frits (0.2 µm).
2) Flush column: 50:50 water:organic (10 min) → 100% organic (10 min) → 100% water (10 min).
3) Check solvent miscibility; remove salts before 100% organic to avoid precipitation.
4) Inspect pump seals/check valves; verify degasser performance (vacuum level OK).
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Stabilize noisy or drifting baseline.

Input: Matrix: soil extract (MeOH); Analytes: acidic herbicides (pKa 2–4); Column: PFP, 2.1×100 mm, 1.9 µm; Oven 45°C; A: 10 mM ammonium bicarbonate (pH 9.0); B: ACN; Gradient 2→40% B over 6 min; Flow 0.30 mL/min. Observation: baseline ripple and drift during gradient; S/N reduced.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Troubleshoot high backpressure increase vs baseline.

Input: Matrix: tablet dissolution (pH 6.8); Analytes: beta-lactams; Column: C18-AQ, 2.1×150 mm, 3 µm; Oven 25°C; A: 10 mM ammonium bicarbonate (pH 9.0); B: ACN; Isocratic 50% B; Flow 1.0 mL/min. Observation: pressure increased 30–50% vs yesterday; pulses under gradient.

Output:
Likely causes and actions:
1) Bypass column to assess system backpressure; replace inline filter and frits (0.2 µm).
2) Flush column: 50:50 water:organic (10 min) → 100% organic (10 min) → 100% water (10 min).
3) Check solvent miscibility; remove salts before 100% organic to avoid precipitation.
4) Inspect pump seals/check valves; verify degasser performance (vacuum level OK).
Acceptance: backpressure within ±10% of baseline; plate count within spec.


Instruction: Ensure sufficient column equilibration between runs.

Input: Matrix: milk; Analytes: alkylbenzene sulfonates; Column: C18-AQ, 2.1×150 mm, 3 µm; Oven 25°C; A: 10 mM ammonium formate + 0.1% FA in water (pH ~3.2); B: ACN; Gradient 10→60% B over 6 min; Flow 1.0 mL/min. Observation: first run after gradient shift differs from subsequent runs.

Output:
Likely causes and actions:
1) Verify oven setpoint holds within ±0.1°C; allow ≥15 min stabilization.
2) Increase post-gradient equilibration to ≥10–15 column volumes.
3) Prime A/B 5 min to remove composition drift; verify mixer volume.
4) Run bracketing standards every 10 injections; apply RT window ±2%.
Acceptance: backpressure within ±10% of baseline; plate count within spec.


Instruction: Diagnose column frit clogging or void formation.

Input: Matrix: wastewater effluent; Analytes: steroids; Column: C18, 4.6×150 mm, 5 µm; Oven 30°C; A: water + 0.05% TFA; B: ACN; Isocratic 60% B; Flow 0.30 mL/min. Observation: plate count dropped 40%; asymmetry 1.7; possible void.

Output:
Likely causes and actions:
1) Check asymmetry and plates with a system suitability compound.
2) Reverse-flush 5–10 min; replace guard; inspect for voids (shoulder/doublet).
3) Reduce max pressure; verify fittings/ferrules to prevent channeling.
4) Log column volumes; retire if performance out of spec.
Acceptance: carryover <0.1% of high standard; blank shows no analyte peaks.


Instruction: Diagnose retention time drift >5%.

Input: Matrix: serum; Analytes: sugars; Column: C8, 4.6×100 mm, 3.5 µm; Oven 35°C; A: 5 mM ammonium acetate in water (pH ~5.0); B: MeOH; Gradient 5→40% B over 6 min; Flow 1.0 mL/min. Observation: RT drifts +6–10% over a batch of 30 samples.

Output:
Likely causes and actions:
1) Verify oven setpoint holds within ±0.1°C; allow ≥15 min stabilization.
2) Increase post-gradient equilibration to ≥10–15 column volumes.
3) Prime A/B 5 min to remove composition drift; verify mixer volume.
4) Run bracketing standards every 10 injections; apply RT window ±2%.
Acceptance: k′ between 2–10; tailing factor <1.3; RT window ±2%.


Instruction: Address temperature-related retention and viscosity issues.

Input: Matrix: serum; Analytes: acidic herbicides (pKa 2–4); Column: PFP, 2.1×100 mm, 1.9 µm; Oven 35°C; A: 5 mM ammonium acetate in water (pH ~5.0); B: MeOH; Gradient 2→95% B over 15 min; Flow 0.50 mL/min. Observation: RT shortens 3–5% when lab ambient increases; viscosity changes.

Output:
Likely causes and actions:
1) Verify oven setpoint holds within ±0.1°C; allow ≥15 min stabilization.
2) Increase post-gradient equilibration to ≥10–15 column volumes.
3) Prime A/B 5 min to remove composition drift; verify mixer volume.
4) Run bracketing standards every 10 injections; apply RT window ±2%.
Acceptance: k′ between 2–10; tailing factor <1.3; RT window ±2%.


Instruction: Fix peak tailing/broadening/splitting.

Input: Matrix: milk; Analytes: steroids; Column: C18, 4.6×150 mm, 5 µm; Oven 45°C; A: 10 mM ammonium formate + 0.1% FA in water (pH ~3.2); B: ACN; Isocratic 35% B; Flow 0.40 mL/min. Observation: tailing factor ~1.8; occasional split peaks on main analyte.

Output:
Likely causes and actions:
1) Match sample solvent to initial mobile phase; dilute strong solvents.
2) Lower pH to suppress ionization for bases (pH 2.8–3.2) or raise for acids (pH 6.5–7.5).
3) Consider polar-embedded/C18-AQ or PFP to reduce secondary interactions.
4) Replace guard; check for column void (reverse flush briefly).
Acceptance: recovery 80–120%, precision (%RSD) ≤5% for n=6.


Instruction: Troubleshoot high backpressure increase vs baseline.

Input: Matrix: urine; Analytes: acidic herbicides (pKa 2–4); Column: Polar-embedded C18, 2.1×150 mm, 3 µm; Oven 40°C; A: 50 mM ammonium formate (pH 3.2); B: MeOH; Isocratic 35% B; Flow 0.50 mL/min. Observation: pressure increased 30–50% vs yesterday; pulses under gradient.

Output:
Likely causes and actions:
1) Bypass column to assess system backpressure; replace inline filter and frits (0.2 µm).
2) Flush column: 50:50 water:organic (10 min) → 100% organic (10 min) → 100% water (10 min).
3) Check solvent miscibility; remove salts before 100% organic to avoid precipitation.
4) Inspect pump seals/check valves; verify degasser performance (vacuum level OK).
Acceptance: recovery 80–120%, precision (%RSD) ≤5% for n=6.


Instruction: Reduce carryover observed in blanks after high standards.

Input: Matrix: pharma API in diluent; Analytes: acidic herbicides (pKa 2–4); Column: HILIC amide, 2.1×100 mm, 1.7 µm; Oven 30°C; A: 50 mM ammonium formate (pH 3.2); B: MeOH; Gradient 5→60% B over 12 min; Flow 0.30 mL/min. Observation: blank after high standard shows 0.5–2% residual peak.

Output:
Likely causes and actions:
1) Strengthen needle wash (≥80% organic + 0.1% acid); extend wash cycles.
2) Include a high-organic purge step between runs; evaluate backflush if available.
3) Use low-adsorption vials/caps; check sample loop/syringe cleanliness.
4) Set acceptance carryover <0.1% of high standard peak area.
Acceptance: carryover <0.1% of high standard; blank shows no analyte peaks.


Instruction: Diagnose column frit clogging or void formation.

Input: Matrix: wastewater influent; Analytes: steroids; Column: C8, 4.6×100 mm, 3.5 µm; Oven 45°C; A: water + 0.1% FA; B: ACN; Gradient 10→60% B over 8 min; Flow 1.0 mL/min. Observation: plate count dropped 40%; asymmetry 1.7; possible void.

Output:
Likely causes and actions:
1) Check asymmetry and plates with a system suitability compound.
2) Reverse-flush 5–10 min; replace guard; inspect for voids (shoulder/doublet).
3) Reduce max pressure; verify fittings/ferrules to prevent channeling.
4) Log column volumes; retire if performance out of spec.
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Ensure sufficient column equilibration between runs.

Input: Matrix: drinking water; Analytes: azo dyes; Column: Phenyl-hexyl, 3.0×100 mm, 3 µm; Oven 30°C; A: water + 0.1% FA; B: ACN; Gradient 5→40% B over 12 min; Flow 0.30 mL/min. Observation: first run after gradient shift differs from subsequent runs.

Output:
Likely causes and actions:
1) Verify oven setpoint holds within ±0.1°C; allow ≥15 min stabilization.
2) Increase post-gradient equilibration to ≥10–15 column volumes.
3) Prime A/B 5 min to remove composition drift; verify mixer volume.
4) Run bracketing standards every 10 injections; apply RT window ±2%.
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Address temperature-related retention and viscosity issues.

Input: Matrix: wine; Analytes: basic amines (pKa 8–10); Column: PFP, 2.1×100 mm, 1.9 µm; Oven 30°C; A: 50 mM ammonium formate (pH 3.2); B: MeOH; Isocratic 40% B; Flow 1.0 mL/min. Observation: RT shortens 3–5% when lab ambient increases; viscosity changes.

Output:
Likely causes and actions:
1) Verify oven setpoint holds within ±0.1°C; allow ≥15 min stabilization.
2) Increase post-gradient equilibration to ≥10–15 column volumes.
3) Prime A/B 5 min to remove composition drift; verify mixer volume.
4) Run bracketing standards every 10 injections; apply RT window ±2%.
Acceptance: k′ between 2–10; tailing factor <1.3; RT window ±2%.


Instruction: Resolve buffer pH/precipitation/mixing problems.

Input: Matrix: leaf extract; Analytes: phenols; Column: C8, 4.6×100 mm, 3.5 µm; Oven 45°C; A: 5 mM ammonium acetate in water (pH ~5.0); B: MeOH; Isocratic 50% B; Flow 0.50 mL/min. Observation: salt precipitation suspected at high % organic.

Output:
Likely causes and actions:
1) Use volatile buffers (ammonium formate/acetate) at ≤10–20 mM for MS; check pH at run temp.
2) Avoid high-salt transitions to 100% organic; flush with salt-free aqueous before.
3) Filter and degas buffers; replace after 48 h; verify miscibility A/B.
4) At low pH for bases, add 0.1% FA; for strong tailing, consider 2–5 mM TFA (UV penalty).
Acceptance: carryover <0.1% of high standard; blank shows no analyte peaks.


Instruction: Address temperature-related retention and viscosity issues.

Input: Matrix: food homogenate; Analytes: phenols; Column: C18, 4.6×150 mm, 5 µm; Oven 40°C; A: 10 mM ammonium formate + 0.1% FA in water (pH ~3.2); B: ACN; Gradient 10→60% B over 10 min; Flow 0.30 mL/min. Observation: RT shortens 3–5% when lab ambient increases; viscosity changes.

Output:
Likely causes and actions:
1) Verify oven setpoint holds within ±0.1°C; allow ≥15 min stabilization.
2) Increase post-gradient equilibration to ≥10–15 column volumes.
3) Prime A/B 5 min to remove composition drift; verify mixer volume.
4) Run bracketing standards every 10 injections; apply RT window ±2%.
Acceptance: carryover <0.1% of high standard; blank shows no analyte peaks.


Instruction: Stabilize noisy or drifting baseline.

Input: Matrix: oil matrix (hexane); Analytes: vitamins (B-group); Column: C8, 4.6×100 mm, 3.5 µm; Oven 40°C; A: water + 0.1% FA; B: ACN; Gradient 5→80% B over 15 min; Flow 0.50 mL/min. Observation: baseline ripple and drift during gradient; S/N reduced.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: recovery 80–120%, precision (%RSD) ≤5% for n=6.


Instruction: Address temperature-related retention and viscosity issues.

Input: Matrix: leaf extract; Analytes: basic amines (pKa 8–10); Column: Phenyl-hexyl, 3.0×100 mm, 3 µm; Oven 35°C; A: water + 0.1% FA; B: ACN; Isocratic 50% B; Flow 0.40 mL/min. Observation: RT shortens 3–5% when lab ambient increases; viscosity changes.

Output:
Likely causes and actions:
1) Verify oven setpoint holds within ±0.1°C; allow ≥15 min stabilization.
2) Increase post-gradient equilibration to ≥10–15 column volumes.
3) Prime A/B 5 min to remove composition drift; verify mixer volume.
4) Run bracketing standards every 10 injections; apply RT window ±2%.
Acceptance: backpressure within ±10% of baseline; plate count within spec.


Instruction: Decide on ion-pairing conditions and caveats.

Input: Matrix: QC standard in water; Analytes: polyphenols; Column: Polar-embedded C18, 2.1×150 mm, 3 µm; Oven 45°C; A: water + 0.05% TFA; B: ACN; Gradient 2→60% B over 20 min; Flow 0.50 mL/min. Observation: basic amines tail at pH>6; poor retention at low organic.

Output:
Likely causes and actions:
1) Try without ion-pair first using acidic pH and endcapped C18; assess tailing.
2) If needed, test HFBA/TFA (UV penalty) or volatile IP reagents for MS with caution.
3) Document equilibration burden and memory effects; dedicate system if adopting IP.
4) Consider polar-embedded or mixed-mode columns as alternatives.
Acceptance: backpressure within ±10% of baseline; plate count within spec.


Instruction: Stabilize noisy or drifting baseline.

Input: Matrix: groundwater; Analytes: caffeine analogs; Column: C18, 2.1×100 mm, 1.7 µm; Oven 25°C; A: water + 0.05% TFA; B: ACN; Isocratic 50% B; Flow 1.0 mL/min. Observation: baseline ripple and drift during gradient; S/N reduced.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: backpressure within ±10% of baseline; plate count within spec.


Instruction: Decide on ion-pairing conditions and caveats.

Input: Matrix: groundwater; Analytes: azo dyes; Column: C18, 2.1×100 mm, 1.7 µm; Oven 45°C; A: 5 mM ammonium acetate in water (pH ~5.0); B: MeOH; Gradient 2→60% B over 6 min; Flow 1.0 mL/min. Observation: basic amines tail at pH>6; poor retention at low organic.

Output:
Likely causes and actions:
1) Try without ion-pair first using acidic pH and endcapped C18; assess tailing.
2) If needed, test HFBA/TFA (UV penalty) or volatile IP reagents for MS with caution.
3) Document equilibration burden and memory effects; dedicate system if adopting IP.
4) Consider polar-embedded or mixed-mode columns as alternatives.
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Address temperature-related retention and viscosity issues.

Input: Matrix: QC standard in water; Analytes: glycosides; Column: C8, 4.6×100 mm, 3.5 µm; Oven 40°C; A: 5 mM ammonium acetate in water (pH ~5.0); B: MeOH; Isocratic 40% B; Flow 1.0 mL/min. Observation: RT shortens 3–5% when lab ambient increases; viscosity changes.

Output:
Likely causes and actions:
1) Verify oven setpoint holds within ±0.1°C; allow ≥15 min stabilization.
2) Increase post-gradient equilibration to ≥10–15 column volumes.
3) Prime A/B 5 min to remove composition drift; verify mixer volume.
4) Run bracketing standards every 10 injections; apply RT window ±2%.
Acceptance: backpressure within ±10% of baseline; plate count within spec.


Instruction: Stabilize noisy or drifting baseline.

Input: Matrix: urine; Analytes: azo dyes; Column: Polar-embedded C18, 2.1×150 mm, 3 µm; Oven 35°C; A: water + 0.1% FA; B: ACN; Gradient 10→40% B over 8 min; Flow 0.50 mL/min. Observation: baseline ripple and drift during gradient; S/N reduced.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: recovery 80–120%, precision (%RSD) ≤5% for n=6.


Instruction: Diagnose column frit clogging or void formation.

Input: Matrix: groundwater; Analytes: bile acids; Column: C18-AQ, 2.1×150 mm, 3 µm; Oven 45°C; A: water + 0.1% FA; B: ACN; Isocratic 50% B; Flow 1.0 mL/min. Observation: plate count dropped 40%; asymmetry 1.7; possible void.

Output:
Likely causes and actions:
1) Check asymmetry and plates with a system suitability compound.
2) Reverse-flush 5–10 min; replace guard; inspect for voids (shoulder/doublet).
3) Reduce max pressure; verify fittings/ferrules to prevent channeling.
4) Log column volumes; retire if performance out of spec.
Acceptance: k′ between 2–10; tailing factor <1.3; RT window ±2%.


Instruction: Stabilize noisy or drifting baseline.

Input: Matrix: drinking water; Analytes: amino acids; Column: HILIC amide, 2.1×100 mm, 1.7 µm; Oven 40°C; A: water + 0.1% FA; B: ACN; Isocratic 35% B; Flow 1.0 mL/min. Observation: baseline ripple and drift during gradient; S/N reduced.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Diagnose retention time drift >5%.

Input: Matrix: drinking water; Analytes: beta-lactams; Column: HILIC amide, 2.1×100 mm, 1.7 µm; Oven 40°C; A: 20 mM ammonium acetate (pH 6.8); B: ACN; Gradient 10→40% B over 12 min; Flow 0.40 mL/min. Observation: RT drifts +6–10% over a batch of 30 samples.

Output:
Likely causes and actions:
1) Verify oven setpoint holds within ±0.1°C; allow ≥15 min stabilization.
2) Increase post-gradient equilibration to ≥10–15 column volumes.
3) Prime A/B 5 min to remove composition drift; verify mixer volume.
4) Run bracketing standards every 10 injections; apply RT window ±2%.
Acceptance: recovery 80–120%, precision (%RSD) ≤5% for n=6.


Instruction: Reduce carryover observed in blanks after high standards.

Input: Matrix: food homogenate; Analytes: neutral pesticides; Column: Phenyl-hexyl, 3.0×100 mm, 3 µm; Oven 40°C; A: water + 0.1% FA; B: ACN; Isocratic 40% B; Flow 0.50 mL/min. Observation: blank after high standard shows 0.5–2% residual peak.

Output:
Likely causes and actions:
1) Strengthen needle wash (≥80% organic + 0.1% acid); extend wash cycles.
2) Include a high-organic purge step between runs; evaluate backflush if available.
3) Use low-adsorption vials/caps; check sample loop/syringe cleanliness.
4) Set acceptance carryover <0.1% of high standard peak area.
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Ensure sufficient column equilibration between runs.

Input: Matrix: urine; Analytes: neutral pesticides; Column: C18, 4.6×150 mm, 5 µm; Oven 30°C; A: water + 0.1% FA; B: ACN; Gradient 10→40% B over 6 min; Flow 0.30 mL/min. Observation: first run after gradient shift differs from subsequent runs.

Output:
Likely causes and actions:
1) Verify oven setpoint holds within ±0.1°C; allow ≥15 min stabilization.
2) Increase post-gradient equilibration to ≥10–15 column volumes.
3) Prime A/B 5 min to remove composition drift; verify mixer volume.
4) Run bracketing standards every 10 injections; apply RT window ±2%.
Acceptance: Rs>1.5 for critical pair, asymmetry 0.9–1.3, %RSD RT <1.0%.


Instruction: Decide on ion-pairing conditions and caveats.

Input: Matrix: oil matrix (hexane); Analytes: caffeine analogs; Column: Phenyl-hexyl, 3.0×100 mm, 3 µm; Oven 25°C; A: 50 mM ammonium formate (pH 3.2); B: MeOH; Gradient 5→80% B over 10 min; Flow 0.40 mL/min. Observation: basic amines tail at pH>6; poor retention at low organic.

Output:
Likely causes and actions:
1) Try without ion-pair first using acidic pH and endcapped C18; assess tailing.
2) If needed, test HFBA/TFA (UV penalty) or volatile IP reagents for MS with caution.
3) Document equilibration burden and memory effects; dedicate system if adopting IP.
4) Consider polar-embedded or mixed-mode columns as alternatives.
Acceptance: backpressure within ±10% of baseline; plate count within spec.


Instruction: Decide on ion-pairing conditions and caveats.

Input: Matrix: milk; Analytes: sugars; Column: C18, 4.6×150 mm, 5 µm; Oven 30°C; A: 10 mM ammonium bicarbonate (pH 9.0); B: ACN; Gradient 10→60% B over 6 min; Flow 0.30 mL/min. Observation: basic amines tail at pH>6; poor retention at low organic.

Output:
Likely causes and actions:
1) Try without ion-pair first using acidic pH and endcapped C18; assess tailing.
2) If needed, test HFBA/TFA (UV penalty) or volatile IP reagents for MS with caution.
3) Document equilibration burden and memory effects; dedicate system if adopting IP.
4) Consider polar-embedded or mixed-mode columns as alternatives.
Acceptance: k′ between 2–10; tailing factor <1.3; RT window ±2%.


Instruction: Address temperature-related retention and viscosity issues.

Input: Matrix: wastewater influent; Analytes: acidic herbicides (pKa 2–4); Column: C8, 4.6×100 mm, 3.5 µm; Oven 35°C; A: water + 0.05% TFA; B: ACN; Isocratic 50% B; Flow 0.30 mL/min. Observation: RT shortens 3–5% when lab ambient increases; viscosity changes.

Output:
Likely causes and actions:
1) Verify oven setpoint holds within ±0.1°C; allow ≥15 min stabilization.
2) Increase post-gradient equilibration to ≥10–15 column volumes.
3) Prime A/B 5 min to remove composition drift; verify mixer volume.
4) Run bracketing standards every 10 injections; apply RT window ±2%.
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Fix peak tailing/broadening/splitting.

Input: Matrix: groundwater; Analytes: caffeine analogs; Column: HILIC amide, 2.1×100 mm, 1.7 µm; Oven 40°C; A: 10 mM ammonium bicarbonate (pH 9.0); B: ACN; Isocratic 40% B; Flow 0.50 mL/min. Observation: tailing factor ~1.8; occasional split peaks on main analyte.

Output:
Likely causes and actions:
1) Match sample solvent to initial mobile phase; dilute strong solvents.
2) Lower pH to suppress ionization for bases (pH 2.8–3.2) or raise for acids (pH 6.5–7.5).
3) Consider polar-embedded/C18-AQ or PFP to reduce secondary interactions.
4) Replace guard; check for column void (reverse flush briefly).
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Reduce carryover observed in blanks after high standards.

Input: Matrix: polymer extract; Analytes: caffeine analogs; Column: HILIC amide, 2.1×100 mm, 1.7 µm; Oven 35°C; A: 20 mM ammonium acetate (pH 6.8); B: ACN; Isocratic 40% B; Flow 0.40 mL/min. Observation: blank after high standard shows 0.5–2% residual peak.

Output:
Likely causes and actions:
1) Strengthen needle wash (≥80% organic + 0.1% acid); extend wash cycles.
2) Include a high-organic purge step between runs; evaluate backflush if available.
3) Use low-adsorption vials/caps; check sample loop/syringe cleanliness.
4) Set acceptance carryover <0.1% of high standard peak area.
Acceptance: k′ between 2–10; tailing factor <1.3; RT window ±2%.


Instruction: Diagnose bubble/degassing issues causing ripple.

Input: Matrix: urine; Analytes: azo dyes; Column: C18, 2.1×100 mm, 1.7 µm; Oven 40°C; A: 50 mM ammonium formate (pH 3.2); B: MeOH; Isocratic 50% B; Flow 1.0 mL/min. Observation: periodic bubble bursts; UV shows spikes.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: Rs>1.5 for critical pair, asymmetry 0.9–1.3, %RSD RT <1.0%.


Instruction: Eliminate ghost peaks seen in solvent blanks.

Input: Matrix: wastewater effluent; Analytes: azo dyes; Column: PFP, 2.1×100 mm, 1.9 µm; Oven 45°C; A: water + 0.1% FA; B: ACN; Isocratic 60% B; Flow 0.50 mL/min. Observation: ghost peaks in solvent blanks at random RTs.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: recovery 80–120%, precision (%RSD) ≤5% for n=6.


Instruction: Reduce carryover observed in blanks after high standards.

Input: Matrix: pharma API in diluent; Analytes: azo dyes; Column: PFP, 2.1×100 mm, 1.9 µm; Oven 30°C; A: 10 mM ammonium formate + 0.1% FA in water (pH ~3.2); B: ACN; Isocratic 35% B; Flow 0.50 mL/min. Observation: blank after high standard shows 0.5–2% residual peak.

Output:
Likely causes and actions:
1) Strengthen needle wash (≥80% organic + 0.1% acid); extend wash cycles.
2) Include a high-organic purge step between runs; evaluate backflush if available.
3) Use low-adsorption vials/caps; check sample loop/syringe cleanliness.
4) Set acceptance carryover <0.1% of high standard peak area.
Acceptance: recovery 80–120%, precision (%RSD) ≤5% for n=6.


Instruction: Reduce carryover observed in blanks after high standards.

Input: Matrix: polymer extract; Analytes: polyphenols; Column: PFP, 2.1×100 mm, 1.9 µm; Oven 35°C; A: 5 mM ammonium acetate in water (pH ~5.0); B: MeOH; Gradient 5→40% B over 20 min; Flow 0.40 mL/min. Observation: blank after high standard shows 0.5–2% residual peak.

Output:
Likely causes and actions:
1) Strengthen needle wash (≥80% organic + 0.1% acid); extend wash cycles.
2) Include a high-organic purge step between runs; evaluate backflush if available.
3) Use low-adsorption vials/caps; check sample loop/syringe cleanliness.
4) Set acceptance carryover <0.1% of high standard peak area.
Acceptance: Rs>1.5 for critical pair, asymmetry 0.9–1.3, %RSD RT <1.0%.


Instruction: Propose starting conditions for HPLC method development.

Input: Matrix: wastewater influent; Analytes: steroids; Column: PFP, 2.1×100 mm, 1.9 µm; Oven 25°C; A: 20 mM ammonium acetate (pH 6.8); B: ACN; Gradient 5→95% B over 12 min; Flow 0.50 mL/min. Goal: fast baseline method to separate target class within 8–12 min.

Output:
Likely causes and actions:
1) Start: C18 or PFP; 35–40°C; 1.0 mL/min (4.6 mm) or 0.3–0.5 mL/min (2.1 mm).
2) Acidic mobile for bases: A 10 mM ammonium formate + 0.1% FA; B ACN.
3) Gradient 5→40% B in 10–12 min; adjust to hit k′ 2–10 and Rs>1.5 for critical pairs.
4) If poor retention at low %B, try C18-AQ or phenyl-hexyl; consider HILIC for very polar.
Acceptance: carryover <0.1% of high standard; blank shows no analyte peaks.


Instruction: Fix peak tailing/broadening/splitting.

Input: Matrix: tablet dissolution (pH 6.8); Analytes: caffeine analogs; Column: C18, 4.6×150 mm, 5 µm; Oven 45°C; A: 10 mM ammonium formate + 0.1% FA in water (pH ~3.2); B: ACN; Gradient 2→60% B over 15 min; Flow 0.30 mL/min. Observation: tailing factor ~1.8; occasional split peaks on main analyte.

Output:
Likely causes and actions:
1) Match sample solvent to initial mobile phase; dilute strong solvents.
2) Lower pH to suppress ionization for bases (pH 2.8–3.2) or raise for acids (pH 6.5–7.5).
3) Consider polar-embedded/C18-AQ or PFP to reduce secondary interactions.
4) Replace guard; check for column void (reverse flush briefly).
Acceptance: Rs>1.5 for critical pair, asymmetry 0.9–1.3, %RSD RT <1.0%.


Instruction: Address temperature-related retention and viscosity issues.

Input: Matrix: cosmetics extract (IPA); Analytes: glycosides; Column: HILIC amide, 2.1×100 mm, 1.7 µm; Oven 35°C; A: 10 mM ammonium bicarbonate (pH 9.0); B: ACN; Gradient 5→40% B over 15 min; Flow 1.0 mL/min. Observation: RT shortens 3–5% when lab ambient increases; viscosity changes.

Output:
Likely causes and actions:
1) Verify oven setpoint holds within ±0.1°C; allow ≥15 min stabilization.
2) Increase post-gradient equilibration to ≥10–15 column volumes.
3) Prime A/B 5 min to remove composition drift; verify mixer volume.
4) Run bracketing standards every 10 injections; apply RT window ±2%.
Acceptance: backpressure within ±10% of baseline; plate count within spec.


Instruction: Reduce carryover observed in blanks after high standards.

Input: Matrix: urine; Analytes: beta-lactams; Column: C18, 4.6×150 mm, 5 µm; Oven 25°C; A: water + 0.05% TFA; B: ACN; Isocratic 60% B; Flow 0.40 mL/min. Observation: blank after high standard shows 0.5–2% residual peak.

Output:
Likely causes and actions:
1) Strengthen needle wash (≥80% organic + 0.1% acid); extend wash cycles.
2) Include a high-organic purge step between runs; evaluate backflush if available.
3) Use low-adsorption vials/caps; check sample loop/syringe cleanliness.
4) Set acceptance carryover <0.1% of high standard peak area.
Acceptance: Rs>1.5 for critical pair, asymmetry 0.9–1.3, %RSD RT <1.0%.


Instruction: Address temperature-related retention and viscosity issues.

Input: Matrix: plasma; Analytes: acidic herbicides (pKa 2–4); Column: PFP, 2.1×100 mm, 1.9 µm; Oven 30°C; A: 50 mM ammonium formate (pH 3.2); B: MeOH; Gradient 10→60% B over 12 min; Flow 0.50 mL/min. Observation: RT shortens 3–5% when lab ambient increases; viscosity changes.

Output:
Likely causes and actions:
1) Verify oven setpoint holds within ±0.1°C; allow ≥15 min stabilization.
2) Increase post-gradient equilibration to ≥10–15 column volumes.
3) Prime A/B 5 min to remove composition drift; verify mixer volume.
4) Run bracketing standards every 10 injections; apply RT window ±2%.
Acceptance: backpressure within ±10% of baseline; plate count within spec.


Instruction: Reduce carryover observed in blanks after high standards.

Input: Matrix: urine; Analytes: beta-lactams; Column: Phenyl-hexyl, 3.0×100 mm, 3 µm; Oven 35°C; A: 20 mM ammonium acetate (pH 6.8); B: ACN; Isocratic 40% B; Flow 0.50 mL/min. Observation: blank after high standard shows 0.5–2% residual peak.

Output:
Likely causes and actions:
1) Strengthen needle wash (≥80% organic + 0.1% acid); extend wash cycles.
2) Include a high-organic purge step between runs; evaluate backflush if available.
3) Use low-adsorption vials/caps; check sample loop/syringe cleanliness.
4) Set acceptance carryover <0.1% of high standard peak area.
Acceptance: k′ between 2–10; tailing factor <1.3; RT window ±2%.


Instruction: Decide on ion-pairing conditions and caveats.

Input: Matrix: cosmetics extract (IPA); Analytes: neutral pesticides; Column: Polar-embedded C18, 2.1×150 mm, 3 µm; Oven 35°C; A: 10 mM ammonium bicarbonate (pH 9.0); B: ACN; Gradient 2→40% B over 8 min; Flow 0.50 mL/min. Observation: basic amines tail at pH>6; poor retention at low organic.

Output:
Likely causes and actions:
1) Try without ion-pair first using acidic pH and endcapped C18; assess tailing.
2) If needed, test HFBA/TFA (UV penalty) or volatile IP reagents for MS with caution.
3) Document equilibration burden and memory effects; dedicate system if adopting IP.
4) Consider polar-embedded or mixed-mode columns as alternatives.
Acceptance: recovery 80–120%, precision (%RSD) ≤5% for n=6.


Instruction: Diagnose bubble/degassing issues causing ripple.

Input: Matrix: urine; Analytes: vitamins (B-group); Column: C18, 2.1×100 mm, 1.7 µm; Oven 40°C; A: 50 mM ammonium formate (pH 3.2); B: MeOH; Gradient 2→95% B over 20 min; Flow 0.50 mL/min. Observation: periodic bubble bursts; UV shows spikes.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: carryover <0.1% of high standard; blank shows no analyte peaks.


Instruction: Diagnose retention time drift >5%.

Input: Matrix: food homogenate; Analytes: beta-lactams; Column: C8, 4.6×100 mm, 3.5 µm; Oven 40°C; A: water + 0.1% FA; B: ACN; Isocratic 30% B; Flow 1.0 mL/min. Observation: RT drifts +6–10% over a batch of 30 samples.

Output:
Likely causes and actions:
1) Verify oven setpoint holds within ±0.1°C; allow ≥15 min stabilization.
2) Increase post-gradient equilibration to ≥10–15 column volumes.
3) Prime A/B 5 min to remove composition drift; verify mixer volume.
4) Run bracketing standards every 10 injections; apply RT window ±2%.
Acceptance: carryover <0.1% of high standard; blank shows no analyte peaks.


Instruction: Fix autosampler needle seat leak/cross-contamination.

Input: Matrix: urine; Analytes: vitamins (B-group); Column: C8, 4.6×100 mm, 3.5 µm; Oven 25°C; A: 20 mM ammonium acetate (pH 6.8); B: ACN; Isocratic 60% B; Flow 1.0 mL/min. Observation: variable injection volumes; leaks near needle seat.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Resolve buffer pH/precipitation/mixing problems.

Input: Matrix: pharma API in diluent; Analytes: PFAS (PFOS/PFOA); Column: C18, 2.1×100 mm, 1.7 µm; Oven 40°C; A: water + 0.1% FA; B: ACN; Gradient 2→40% B over 20 min; Flow 1.0 mL/min. Observation: salt precipitation suspected at high % organic.

Output:
Likely causes and actions:
1) Use volatile buffers (ammonium formate/acetate) at ≤10–20 mM for MS; check pH at run temp.
2) Avoid high-salt transitions to 100% organic; flush with salt-free aqueous before.
3) Filter and degas buffers; replace after 48 h; verify miscibility A/B.
4) At low pH for bases, add 0.1% FA; for strong tailing, consider 2–5 mM TFA (UV penalty).
Acceptance: backpressure within ±10% of baseline; plate count within spec.


Instruction: Stabilize noisy or drifting baseline.

Input: Matrix: milk; Analytes: azo dyes; Column: C18, 2.1×100 mm, 1.7 µm; Oven 25°C; A: 20 mM ammonium acetate (pH 6.8); B: ACN; Isocratic 40% B; Flow 1.0 mL/min. Observation: baseline ripple and drift during gradient; S/N reduced.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Ensure sufficient column equilibration between runs.

Input: Matrix: tablet dissolution (pH 6.8); Analytes: sugars; Column: C18-AQ, 2.1×150 mm, 3 µm; Oven 45°C; A: 5 mM ammonium acetate in water (pH ~5.0); B: MeOH; Isocratic 40% B; Flow 0.40 mL/min. Observation: first run after gradient shift differs from subsequent runs.

Output:
Likely causes and actions:
1) Verify oven setpoint holds within ±0.1°C; allow ≥15 min stabilization.
2) Increase post-gradient equilibration to ≥10–15 column volumes.
3) Prime A/B 5 min to remove composition drift; verify mixer volume.
4) Run bracketing standards every 10 injections; apply RT window ±2%.
Acceptance: Rs>1.5 for critical pair, asymmetry 0.9–1.3, %RSD RT <1.0%.


Instruction: Resolve buffer pH/precipitation/mixing problems.

Input: Matrix: milk; Analytes: glycosides; Column: PFP, 2.1×100 mm, 1.9 µm; Oven 40°C; A: water + 0.05% TFA; B: ACN; Gradient 5→95% B over 12 min; Flow 0.50 mL/min. Observation: salt precipitation suspected at high % organic.

Output:
Likely causes and actions:
1) Use volatile buffers (ammonium formate/acetate) at ≤10–20 mM for MS; check pH at run temp.
2) Avoid high-salt transitions to 100% organic; flush with salt-free aqueous before.
3) Filter and degas buffers; replace after 48 h; verify miscibility A/B.
4) At low pH for bases, add 0.1% FA; for strong tailing, consider 2–5 mM TFA (UV penalty).
Acceptance: backpressure within ±10% of baseline; plate count within spec.


Instruction: Resolve pump pulsation/check valve problems.

Input: Matrix: polymer extract; Analytes: neutral pesticides; Column: HILIC amide, 2.1×100 mm, 1.7 µm; Oven 40°C; A: water + 0.1% FA; B: ACN; Gradient 5→60% B over 12 min; Flow 1.0 mL/min. Observation: pulsation visible on pressure trace; peak area RSD ~6%.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: k′ between 2–10; tailing factor <1.3; RT window ±2%.


Instruction: Eliminate ghost peaks seen in solvent blanks.

Input: Matrix: beer; Analytes: vitamins (B-group); Column: C18, 4.6×150 mm, 5 µm; Oven 25°C; A: 10 mM ammonium bicarbonate (pH 9.0); B: ACN; Isocratic 50% B; Flow 0.40 mL/min. Observation: ghost peaks in solvent blanks at random RTs.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: k′ between 2–10; tailing factor <1.3; RT window ±2%.


Instruction: Resolve buffer pH/precipitation/mixing problems.

Input: Matrix: leaf extract; Analytes: alkylbenzene sulfonates; Column: C18, 4.6×150 mm, 5 µm; Oven 40°C; A: 50 mM ammonium formate (pH 3.2); B: MeOH; Gradient 10→40% B over 8 min; Flow 0.30 mL/min. Observation: salt precipitation suspected at high % organic.

Output:
Likely causes and actions:
1) Use volatile buffers (ammonium formate/acetate) at ≤10–20 mM for MS; check pH at run temp.
2) Avoid high-salt transitions to 100% organic; flush with salt-free aqueous before.
3) Filter and degas buffers; replace after 48 h; verify miscibility A/B.
4) At low pH for bases, add 0.1% FA; for strong tailing, consider 2–5 mM TFA (UV penalty).
Acceptance: Rs>1.5 for critical pair, asymmetry 0.9–1.3, %RSD RT <1.0%.


Instruction: Eliminate ghost peaks seen in solvent blanks.

Input: Matrix: food homogenate; Analytes: beta-lactams; Column: C18, 4.6×150 mm, 5 µm; Oven 35°C; A: 50 mM ammonium formate (pH 3.2); B: MeOH; Isocratic 40% B; Flow 0.30 mL/min. Observation: ghost peaks in solvent blanks at random RTs.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Fix peak tailing/broadening/splitting.

Input: Matrix: groundwater; Analytes: beta-lactams; Column: C18-AQ, 2.1×150 mm, 3 µm; Oven 25°C; A: 5 mM ammonium acetate in water (pH ~5.0); B: MeOH; Gradient 10→60% B over 12 min; Flow 1.0 mL/min. Observation: tailing factor ~1.8; occasional split peaks on main analyte.

Output:
Likely causes and actions:
1) Match sample solvent to initial mobile phase; dilute strong solvents.
2) Lower pH to suppress ionization for bases (pH 2.8–3.2) or raise for acids (pH 6.5–7.5).
3) Consider polar-embedded/C18-AQ or PFP to reduce secondary interactions.
4) Replace guard; check for column void (reverse flush briefly).
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Address temperature-related retention and viscosity issues.

Input: Matrix: urine; Analytes: glycosides; Column: Polar-embedded C18, 2.1×150 mm, 3 µm; Oven 45°C; A: water + 0.1% FA; B: ACN; Isocratic 60% B; Flow 0.40 mL/min. Observation: RT shortens 3–5% when lab ambient increases; viscosity changes.

Output:
Likely causes and actions:
1) Verify oven setpoint holds within ±0.1°C; allow ≥15 min stabilization.
2) Increase post-gradient equilibration to ≥10–15 column volumes.
3) Prime A/B 5 min to remove composition drift; verify mixer volume.
4) Run bracketing standards every 10 injections; apply RT window ±2%.
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Resolve buffer pH/precipitation/mixing problems.

Input: Matrix: urine; Analytes: sugars; Column: Polar-embedded C18, 2.1×150 mm, 3 µm; Oven 40°C; A: 20 mM ammonium acetate (pH 6.8); B: ACN; Isocratic 40% B; Flow 0.30 mL/min. Observation: salt precipitation suspected at high % organic.

Output:
Likely causes and actions:
1) Use volatile buffers (ammonium formate/acetate) at ≤10–20 mM for MS; check pH at run temp.
2) Avoid high-salt transitions to 100% organic; flush with salt-free aqueous before.
3) Filter and degas buffers; replace after 48 h; verify miscibility A/B.
4) At low pH for bases, add 0.1% FA; for strong tailing, consider 2–5 mM TFA (UV penalty).
Acceptance: carryover <0.1% of high standard; blank shows no analyte peaks.


Instruction: Address temperature-related retention and viscosity issues.

Input: Matrix: drinking water; Analytes: neutral pesticides; Column: HILIC amide, 2.1×100 mm, 1.7 µm; Oven 40°C; A: water + 0.05% TFA; B: ACN; Isocratic 35% B; Flow 0.50 mL/min. Observation: RT shortens 3–5% when lab ambient increases; viscosity changes.

Output:
Likely causes and actions:
1) Verify oven setpoint holds within ±0.1°C; allow ≥15 min stabilization.
2) Increase post-gradient equilibration to ≥10–15 column volumes.
3) Prime A/B 5 min to remove composition drift; verify mixer volume.
4) Run bracketing standards every 10 injections; apply RT window ±2%.
Acceptance: recovery 80–120%, precision (%RSD) ≤5% for n=6.


Instruction: Resolve buffer pH/precipitation/mixing problems.

Input: Matrix: groundwater; Analytes: glycosides; Column: C18, 4.6×150 mm, 5 µm; Oven 45°C; A: 10 mM ammonium formate + 0.1% FA in water (pH ~3.2); B: ACN; Isocratic 40% B; Flow 0.50 mL/min. Observation: salt precipitation suspected at high % organic.

Output:
Likely causes and actions:
1) Use volatile buffers (ammonium formate/acetate) at ≤10–20 mM for MS; check pH at run temp.
2) Avoid high-salt transitions to 100% organic; flush with salt-free aqueous before.
3) Filter and degas buffers; replace after 48 h; verify miscibility A/B.
4) At low pH for bases, add 0.1% FA; for strong tailing, consider 2–5 mM TFA (UV penalty).
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Diagnose bubble/degassing issues causing ripple.

Input: Matrix: soil extract (MeOH); Analytes: vitamins (B-group); Column: C18-AQ, 2.1×150 mm, 3 µm; Oven 35°C; A: water + 0.05% TFA; B: ACN; Gradient 5→95% B over 10 min; Flow 0.40 mL/min. Observation: periodic bubble bursts; UV shows spikes.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: Rs>1.5 for critical pair, asymmetry 0.9–1.3, %RSD RT <1.0%.


Instruction: Fix autosampler needle seat leak/cross-contamination.

Input: Matrix: groundwater; Analytes: acidic herbicides (pKa 2–4); Column: C18-AQ, 2.1×150 mm, 3 µm; Oven 40°C; A: 20 mM ammonium acetate (pH 6.8); B: ACN; Isocratic 40% B; Flow 0.30 mL/min. Observation: variable injection volumes; leaks near needle seat.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Diagnose column frit clogging or void formation.

Input: Matrix: wine; Analytes: basic amines (pKa 8–10); Column: C18, 4.6×150 mm, 5 µm; Oven 45°C; A: water + 0.05% TFA; B: ACN; Isocratic 30% B; Flow 0.50 mL/min. Observation: plate count dropped 40%; asymmetry 1.7; possible void.

Output:
Likely causes and actions:
1) Check asymmetry and plates with a system suitability compound.
2) Reverse-flush 5–10 min; replace guard; inspect for voids (shoulder/doublet).
3) Reduce max pressure; verify fittings/ferrules to prevent channeling.
4) Log column volumes; retire if performance out of spec.
Acceptance: recovery 80–120%, precision (%RSD) ≤5% for n=6.


Instruction: Stabilize noisy or drifting baseline.

Input: Matrix: milk; Analytes: neutral pesticides; Column: HILIC amide, 2.1×100 mm, 1.7 µm; Oven 35°C; A: 10 mM ammonium formate + 0.1% FA in water (pH ~3.2); B: ACN; Gradient 2→40% B over 8 min; Flow 1.0 mL/min. Observation: baseline ripple and drift during gradient; S/N reduced.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Diagnose bubble/degassing issues causing ripple.

Input: Matrix: wine; Analytes: steroids; Column: HILIC amide, 2.1×100 mm, 1.7 µm; Oven 40°C; A: 20 mM ammonium acetate (pH 6.8); B: ACN; Gradient 5→40% B over 20 min; Flow 1.0 mL/min. Observation: periodic bubble bursts; UV shows spikes.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Address temperature-related retention and viscosity issues.

Input: Matrix: plasma; Analytes: amino acids; Column: C8, 4.6×100 mm, 3.5 µm; Oven 40°C; A: 10 mM ammonium formate + 0.1% FA in water (pH ~3.2); B: ACN; Gradient 2→40% B over 12 min; Flow 0.50 mL/min. Observation: RT shortens 3–5% when lab ambient increases; viscosity changes.

Output:
Likely causes and actions:
1) Verify oven setpoint holds within ±0.1°C; allow ≥15 min stabilization.
2) Increase post-gradient equilibration to ≥10–15 column volumes.
3) Prime A/B 5 min to remove composition drift; verify mixer volume.
4) Run bracketing standards every 10 injections; apply RT window ±2%.
Acceptance: recovery 80–120%, precision (%RSD) ≤5% for n=6.


Instruction: Eliminate ghost peaks seen in solvent blanks.

Input: Matrix: oil matrix (hexane); Analytes: azo dyes; Column: C18, 2.1×100 mm, 1.7 µm; Oven 45°C; A: 5 mM ammonium acetate in water (pH ~5.0); B: MeOH; Gradient 2→60% B over 15 min; Flow 0.40 mL/min. Observation: ghost peaks in solvent blanks at random RTs.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: backpressure within ±10% of baseline; plate count within spec.


Instruction: Decide on ion-pairing conditions and caveats.

Input: Matrix: oil matrix (hexane); Analytes: PFAS (PFOS/PFOA); Column: C18, 2.1×100 mm, 1.7 µm; Oven 35°C; A: water + 0.05% TFA; B: ACN; Isocratic 30% B; Flow 0.40 mL/min. Observation: basic amines tail at pH>6; poor retention at low organic.

Output:
Likely causes and actions:
1) Try without ion-pair first using acidic pH and endcapped C18; assess tailing.
2) If needed, test HFBA/TFA (UV penalty) or volatile IP reagents for MS with caution.
3) Document equilibration burden and memory effects; dedicate system if adopting IP.
4) Consider polar-embedded or mixed-mode columns as alternatives.
Acceptance: backpressure within ±10% of baseline; plate count within spec.


Instruction: Diagnose bubble/degassing issues causing ripple.

Input: Matrix: polymer extract; Analytes: phenols; Column: C8, 4.6×100 mm, 3.5 µm; Oven 30°C; A: 10 mM ammonium formate + 0.1% FA in water (pH ~3.2); B: ACN; Isocratic 30% B; Flow 1.0 mL/min. Observation: periodic bubble bursts; UV shows spikes.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: carryover <0.1% of high standard; blank shows no analyte peaks.


Instruction: Stabilize noisy or drifting baseline.

Input: Matrix: QC standard in water; Analytes: neutral pesticides; Column: C18, 4.6×150 mm, 5 µm; Oven 45°C; A: 50 mM ammonium formate (pH 3.2); B: MeOH; Isocratic 50% B; Flow 0.50 mL/min. Observation: baseline ripple and drift during gradient; S/N reduced.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: recovery 80–120%, precision (%RSD) ≤5% for n=6.


Instruction: Stabilize noisy or drifting baseline.

Input: Matrix: river water; Analytes: acidic herbicides (pKa 2–4); Column: Polar-embedded C18, 2.1×150 mm, 3 µm; Oven 45°C; A: water + 0.1% FA; B: ACN; Isocratic 60% B; Flow 0.40 mL/min. Observation: baseline ripple and drift during gradient; S/N reduced.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: k′ between 2–10; tailing factor <1.3; RT window ±2%.


Instruction: Ensure sufficient column equilibration between runs.

Input: Matrix: QC standard in water; Analytes: steroids; Column: PFP, 2.1×100 mm, 1.9 µm; Oven 25°C; A: 20 mM ammonium acetate (pH 6.8); B: ACN; Isocratic 50% B; Flow 0.40 mL/min. Observation: first run after gradient shift differs from subsequent runs.

Output:
Likely causes and actions:
1) Verify oven setpoint holds within ±0.1°C; allow ≥15 min stabilization.
2) Increase post-gradient equilibration to ≥10–15 column volumes.
3) Prime A/B 5 min to remove composition drift; verify mixer volume.
4) Run bracketing standards every 10 injections; apply RT window ±2%.
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Diagnose retention time drift >5%.

Input: Matrix: leaf extract; Analytes: bile acids; Column: HILIC amide, 2.1×100 mm, 1.7 µm; Oven 35°C; A: water + 0.1% FA; B: ACN; Gradient 2→95% B over 6 min; Flow 0.50 mL/min. Observation: RT drifts +6–10% over a batch of 30 samples.

Output:
Likely causes and actions:
1) Verify oven setpoint holds within ±0.1°C; allow ≥15 min stabilization.
2) Increase post-gradient equilibration to ≥10–15 column volumes.
3) Prime A/B 5 min to remove composition drift; verify mixer volume.
4) Run bracketing standards every 10 injections; apply RT window ±2%.
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Fix autosampler needle seat leak/cross-contamination.

Input: Matrix: river water; Analytes: alkylbenzene sulfonates; Column: C8, 4.6×100 mm, 3.5 µm; Oven 25°C; A: 50 mM ammonium formate (pH 3.2); B: MeOH; Isocratic 30% B; Flow 1.0 mL/min. Observation: variable injection volumes; leaks near needle seat.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: carryover <0.1% of high standard; blank shows no analyte peaks.


Instruction: Decide on ion-pairing conditions and caveats.

Input: Matrix: wine; Analytes: caffeine analogs; Column: C18, 4.6×150 mm, 5 µm; Oven 25°C; A: 5 mM ammonium acetate in water (pH ~5.0); B: MeOH; Gradient 10→80% B over 6 min; Flow 0.50 mL/min. Observation: basic amines tail at pH>6; poor retention at low organic.

Output:
Likely causes and actions:
1) Try without ion-pair first using acidic pH and endcapped C18; assess tailing.
2) If needed, test HFBA/TFA (UV penalty) or volatile IP reagents for MS with caution.
3) Document equilibration burden and memory effects; dedicate system if adopting IP.
4) Consider polar-embedded or mixed-mode columns as alternatives.
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Diagnose retention time drift >5%.

Input: Matrix: cosmetics extract (IPA); Analytes: beta-lactams; Column: C18, 2.1×100 mm, 1.7 µm; Oven 35°C; A: water + 0.1% FA; B: ACN; Isocratic 35% B; Flow 0.50 mL/min. Observation: RT drifts +6–10% over a batch of 30 samples.

Output:
Likely causes and actions:
1) Verify oven setpoint holds within ±0.1°C; allow ≥15 min stabilization.
2) Increase post-gradient equilibration to ≥10–15 column volumes.
3) Prime A/B 5 min to remove composition drift; verify mixer volume.
4) Run bracketing standards every 10 injections; apply RT window ±2%.
Acceptance: backpressure within ±10% of baseline; plate count within spec.


Instruction: Diagnose column frit clogging or void formation.

Input: Matrix: drinking water; Analytes: bile acids; Column: C18, 4.6×150 mm, 5 µm; Oven 35°C; A: 10 mM ammonium bicarbonate (pH 9.0); B: ACN; Gradient 5→80% B over 20 min; Flow 0.40 mL/min. Observation: plate count dropped 40%; asymmetry 1.7; possible void.

Output:
Likely causes and actions:
1) Check asymmetry and plates with a system suitability compound.
2) Reverse-flush 5–10 min; replace guard; inspect for voids (shoulder/doublet).
3) Reduce max pressure; verify fittings/ferrules to prevent channeling.
4) Log column volumes; retire if performance out of spec.
Acceptance: Rs>1.5 for critical pair, asymmetry 0.9–1.3, %RSD RT <1.0%.


Instruction: Decide on ion-pairing conditions and caveats.

Input: Matrix: groundwater; Analytes: phenols; Column: Phenyl-hexyl, 3.0×100 mm, 3 µm; Oven 40°C; A: 10 mM ammonium bicarbonate (pH 9.0); B: ACN; Gradient 10→95% B over 12 min; Flow 1.0 mL/min. Observation: basic amines tail at pH>6; poor retention at low organic.

Output:
Likely causes and actions:
1) Try without ion-pair first using acidic pH and endcapped C18; assess tailing.
2) If needed, test HFBA/TFA (UV penalty) or volatile IP reagents for MS with caution.
3) Document equilibration burden and memory effects; dedicate system if adopting IP.
4) Consider polar-embedded or mixed-mode columns as alternatives.
Acceptance: k′ between 2–10; tailing factor <1.3; RT window ±2%.


Instruction: Fix autosampler needle seat leak/cross-contamination.

Input: Matrix: river water; Analytes: polyphenols; Column: C8, 4.6×100 mm, 3.5 µm; Oven 45°C; A: 50 mM ammonium formate (pH 3.2); B: MeOH; Isocratic 35% B; Flow 1.0 mL/min. Observation: variable injection volumes; leaks near needle seat.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: Rs>1.5 for critical pair, asymmetry 0.9–1.3, %RSD RT <1.0%.


Instruction: Troubleshoot high backpressure increase vs baseline.

Input: Matrix: drinking water; Analytes: PFAS (PFOS/PFOA); Column: C18-AQ, 2.1×150 mm, 3 µm; Oven 35°C; A: 20 mM ammonium acetate (pH 6.8); B: ACN; Isocratic 50% B; Flow 0.50 mL/min. Observation: pressure increased 30–50% vs yesterday; pulses under gradient.

Output:
Likely causes and actions:
1) Bypass column to assess system backpressure; replace inline filter and frits (0.2 µm).
2) Flush column: 50:50 water:organic (10 min) → 100% organic (10 min) → 100% water (10 min).
3) Check solvent miscibility; remove salts before 100% organic to avoid precipitation.
4) Inspect pump seals/check valves; verify degasser performance (vacuum level OK).
Acceptance: backpressure within ±10% of baseline; plate count within spec.


Instruction: Fix autosampler needle seat leak/cross-contamination.

Input: Matrix: leaf extract; Analytes: sugars; Column: C18-AQ, 2.1×150 mm, 3 µm; Oven 25°C; A: 20 mM ammonium acetate (pH 6.8); B: ACN; Isocratic 40% B; Flow 1.0 mL/min. Observation: variable injection volumes; leaks near needle seat.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Fix peak tailing/broadening/splitting.

Input: Matrix: food homogenate; Analytes: basic amines (pKa 8–10); Column: PFP, 2.1×100 mm, 1.9 µm; Oven 30°C; A: water + 0.1% FA; B: ACN; Gradient 10→80% B over 6 min; Flow 1.0 mL/min. Observation: tailing factor ~1.8; occasional split peaks on main analyte.

Output:
Likely causes and actions:
1) Match sample solvent to initial mobile phase; dilute strong solvents.
2) Lower pH to suppress ionization for bases (pH 2.8–3.2) or raise for acids (pH 6.5–7.5).
3) Consider polar-embedded/C18-AQ or PFP to reduce secondary interactions.
4) Replace guard; check for column void (reverse flush briefly).
Acceptance: S/N > 10 at LOQ; baseline drift <5% over run.


Instruction: Resolve pump pulsation/check valve problems.

Input: Matrix: polymer extract; Analytes: polyphenols; Column: HILIC amide, 2.1×100 mm, 1.7 µm; Oven 35°C; A: 50 mM ammonium formate (pH 3.2); B: MeOH; Gradient 5→60% B over 6 min; Flow 0.40 mL/min. Observation: pulsation visible on pressure trace; peak area RSD ~6%.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: k′ between 2–10; tailing factor <1.3; RT window ±2%.


Instruction: Troubleshoot high backpressure increase vs baseline.

Input: Matrix: serum; Analytes: azo dyes; Column: C8, 4.6×100 mm, 3.5 µm; Oven 30°C; A: 10 mM ammonium formate + 0.1% FA in water (pH ~3.2); B: ACN; Gradient 2→60% B over 15 min; Flow 0.50 mL/min. Observation: pressure increased 30–50% vs yesterday; pulses under gradient.

Output:
Likely causes and actions:
1) Bypass column to assess system backpressure; replace inline filter and frits (0.2 µm).
2) Flush column: 50:50 water:organic (10 min) → 100% organic (10 min) → 100% water (10 min).
3) Check solvent miscibility; remove salts before 100% organic to avoid precipitation.
4) Inspect pump seals/check valves; verify degasser performance (vacuum level OK).
Acceptance: recovery 80–120%, precision (%RSD) ≤5% for n=6.


Instruction: Fix autosampler needle seat leak/cross-contamination.

Input: Matrix: tablet dissolution (pH 6.8); Analytes: alkylbenzene sulfonates; Column: Polar-embedded C18, 2.1×150 mm, 3 µm; Oven 40°C; A: 5 mM ammonium acetate in water (pH ~5.0); B: MeOH; Isocratic 40% B; Flow 0.30 mL/min. Observation: variable injection volumes; leaks near needle seat.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: Rs>1.5 for critical pair, asymmetry 0.9–1.3, %RSD RT <1.0%.


Instruction: Diagnose bubble/degassing issues causing ripple.

Input: Matrix: wine; Analytes: glycosides; Column: C18, 4.6×150 mm, 5 µm; Oven 25°C; A: 10 mM ammonium formate + 0.1% FA in water (pH ~3.2); B: ACN; Isocratic 30% B; Flow 0.50 mL/min. Observation: periodic bubble bursts; UV shows spikes.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: k′ between 2–10; tailing factor <1.3; RT window ±2%.


Instruction: Resolve pump pulsation/check valve problems.

Input: Matrix: cosmetics extract (IPA); Analytes: alkylbenzene sulfonates; Column: C18, 2.1×100 mm, 1.7 µm; Oven 40°C; A: water + 0.05% TFA; B: ACN; Gradient 2→95% B over 6 min; Flow 0.40 mL/min. Observation: pulsation visible on pressure trace; peak area RSD ~6%.

Output:
Likely causes and actions:
1) Run solvent and system blanks; isolate source by bypassing modules.
2) Replace suspect solvents; ensure fresh buffer (≤48 h) and proper filtration (0.2 µm).
3) Inspect degasser (vacuum level, tubing kinks) and pump check valves/seals.
4) Perform needle-seat test, adjust wash, and verify no leaks (draw-air test).
Acceptance: recovery 80–120%, precision (%RSD) ≤5% for n=6.