1. HPLC TROUBLESHOOTING

2. There are five major of symptoms to help you quickly identify the source of the problem(s): I.Pressure abnormalities II.Leaks III.Problems with the chromatogram IV.Injector problems V.Other problems detected by the senses of smell, sight, and sound When you have corrected the problem, record the incident in the system record book to help with future problems.

3. HPLC - Modes Normal Phase. - Polar stationary phase and non-polar solvent. Reverse Phase. - Non-polar stationary phase and a polar solvent.

5. I- Abnormal Pressure Causes Solutions Power offTurn on power Fuse blownReplace fuse Broken pistonReplace piston Air trapped in pump headPurging & Degas solvents Insufficient mobile phaseReplenish reservoir Faulty check valve(s)Replace check valve(s) Major leakTighten or replace fittings Problem-1: No pressure reading, no flow

6. Problem-2 : No pressure reading, flow is normal Causes Solutions Faulty meterReplace meter Faulty pressure transducerReplace transducer

7. Problem-3 : Steady, high pressure CausesSolutions Flow rate set too highAdjust setting Blocked column fritReplace frit Replace column Improper mobile phaseUse correct mobile phase Improper columnUse proper column Injector blockageClear blockage or replace injector Column temperature too lowRaise temperature Blocked guard columnRemove/replace guard column Blocked in-line filterRemove/replace in-line filter

8. Problem-4: Steady, low pressure CausesSolutions Flow set too lowAdjust flow rate Leak in systemLocate and correct Improper columnUse proper column Column temperature too high Lower temperature Controller malfunctionRepair or replace controller

9. Problem-3: Pressure cycling CausesSolutions Air in pumpDegas solvent Pump seal failureReplace pump seal Insufficient degassingDegas solvent Leak in systemLocate and correct Faulty check valve(s)Replace check valve(s)

10. II- Leaks CausesSolutions Loose fittingTighten Over tightened* fittingReplace Stripped fittingLoosen and retighten Replace Dirty fittingDisassemble and clean Replace Problem-1: Leaky fittings

11. Problem-2: Leaks at pump CausesSolutions Loose fittingsTighten fittings (do not over tighten) Loose Purge valveTighten valve Replace purge valve Pump seal failureRepair or replace Mixer seal failureRepair or replace Pressure transducer failureRepair or replace Pulse damper failureRepair or replace Loose check valvesTighten check valve (do not over tighten) Replace check valve

12. Problem-3: Injector leaks Causes Solutions Improper syringe-needle diameter Use correct syringe Loose injection-port sealAdjust Blocked loopReplace loop Waste-line blockageReplace waste line

13. Problem-4: Column leaks CausesSolutions Loose end-fittingTighten end-fitting Column packing in ferruleDisassemble, rinse ferrule, reassemble Improper frit thicknessUse proper frit

14. Problem-4: Detector leaks CausesSolutions Blocked waste lineRebuild or replace Leaky fittingsTighten or replace Cell gasket failureReplace gasket

15. Problem-1: Peak tailing III- Problems with the Chromatogram

16. Peak tailing CausesSolutions Blocked fritReplace inlet frit Replace column Column voidFill void Wrong mobile phase pHAdjust pH

17. Problem-2: Peak fronting CausesSolutions Low temperatureIncrease column temperature Sample overloadDecrease sample concentration Wrong sample solventUse mobile phase for injection solvent

18. Problem-3: Split peaks CausesSolution Contamination on guard and analytical column inlet. Remove guard column and replace if necessary. If analytical column is obstructed, Change frit or replace column.

19. Problem-3: Baseline Drift CausesSolutions Column temperature fluctuation.Control column and mobile phase temperature Non-homogenous mobile phase.Use HPLC grade solvents, high purity salts, and additives. Degas mobile phase before use. Contaminant or air build-up in the detector Flush detector with methanol or other strong solvent. If necessary, clean cell with 1N HNO3 (never with HCI.) Mobile phase mixing problem or change in flow rate Correct composition / flow rate

20. Problem-4: Baseline noise (regular) CausesSolutions Air in mobile phase, detector cell or pump Degas mobile phase. Flush system to remove air from detector cell or pump LeakCheck system for loose fittings. Change pump seals if necessary Incomplete mobile phase mixingMix mobile phase by hand or use less viscous solvent.

21. Problem-5: Baseline Noise (irregular) CausesSolutions LeakCheck system for loose fittings. Change pump seals if necessary Air trapped in system.Flush system with strong solvent. Air bubbles in detectorPurge detector Weak detector lamp.Replace lamp. Column leaking silica or packing material Replace column and clean system.

22. Problem-6: broad peak CausesSolutions Mobile phase flow rate too lowAdjust flow rate Mobile phase composition changed.Prepare new mobile phase. LeakCheck system for loose fittings. Change pump seals if necessary. Detector settings incorrect.Adjust settings. Guard column contaminated /worn out.Replace guard column Column contaminated/worn outReplace column with new one of same type. Column temperature too lowIncrease temperature. Do not exceed 75 °C.

23. Problem-7: loss of Resolution CausesSolutions Mobile phase contaminated / deteriorated Prepare new mobile phase Obstructed guard or and analytical column Remove guard column, Replace if necessary. If analytical column is obstructed, Change frit or replace column.

24. IV- Injector problems CausesSolutions Damaged rotor sealRebuild or replace valve Rotor too tightAdjust rotor tension Problem-1: Manual injector, hard to turn CausesSolutions Valve misalignedAdjust alignment Blocked loopReplace loop Dirty syringeClean or replace syringe Blocked linesClear or replace lines Problem-2: Manual injector, hard to load

25. V- Other problems: Detected by the senses of smell, sight, and sound Problem-1: smell

26. CausesSolutions Detector lamp failingReplace lamp Pressure abnormalityControl Pressure Problem-2: Abnormal meter readings Problem-3: Warning lamps CausesSolutions Pressure limit exceededCheck for blockage Other warning lampsSee service manual

27. CausesSolutions Solvent leakLocate and correct Other warning buzzersSee service manual Problem-4: Warning buzzers Problem-5: Squeaks and squeals CausesSolutions Poor lubricationLubricate as necessary Bearing failureSee service manual Mechanical wearSee service manual

28. Manish Kumar (M.Pharm. Pharmacology)