HPLC TROUBLESHOOTING

There are five major of symptoms to help you quickly identify the source of the problem(s): I. Pressure abnormalitiesII. LeaksIII. Problems with the chromatogramIV. Injector problemsV. 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.

HPLC - Modes

• Normal Phase.- Polar stationary phase and non-polar solvent.

• Reverse Phase. - Non-polar stationary phase and a polar solvent.

WHOLE APPARTUS LOOKS LIKE

I- Abnormal Pressure

Causes SolutionsPower off Turn on powerFuse blown Replace fuseBroken piston Replace pistonAir trapped in pump head Purging & Degas solventsInsufficient mobile phase Replenish reservoirFaulty check valve(s) Replace check valve(s)Major leak Tighten or replace fittings

Problem-1: No pressure reading, no flow

Problem-2: No pressure reading, flow is normal

Causes Solutions

Faulty meter Replace meter

Faulty pressure transducer Replace transducer

Problem-3: Steady, high pressureCauses Solutions

Flow rate set too high Adjust setting

Blocked column frit Replace fritReplace column

Improper mobile phase Use correct mobile phase

Improper column Use proper column

Injector blockage Clear blockage or replace injector

Column temperature too low Raise temperature

Blocked guard column Remove/replace guard column

Blocked in-line filter Remove/replace in-line filter

Problem-4: Steady, low pressure

Causes Solutions

Flow set too low Adjust flow rate

Leak in system Locate and correct

Improper column Use proper column

Column temperature too high

Lower temperature

Controller malfunction Repair or replace controller

Problem-3: Pressure cycling

Causes Solutions

Air in pump Degas solvent

Pump seal failure Replace pump seal

Insufficient degassing Degas solvent

Leak in system Locate and correct

Faulty check valve(s) Replace check valve(s)

II- Leaks

Causes Solutions

Loose fitting Tighten

Over tightened* fitting Replace

Stripped fitting Loosen and retightenReplace

Dirty fitting Disassemble and cleanReplace

Problem-1: Leaky fittings

Problem-2: Leaks at pump

Causes SolutionsLoose fittings Tighten fittings (do not over

tighten)

Loose Purge valve Tighten valveReplace purge valve

Pump seal failure Repair or replace

Mixer seal failure Repair or replace

Pressure transducer failure Repair or replace

Pulse damper failure Repair or replace

Loose check valves Tighten check valve (do not over tighten)Replace check valve

Problem-3: Injector leaks

Causes Solutions

Improper syringe-needle diameter

Use correct syringe

Loose injection-port seal Adjust

Blocked loop Replace loop

Waste-line blockage Replace waste line

Problem-4: Column leaks

Causes Solutions

Loose end-fitting Tighten end-fitting

Column packing in ferrule Disassemble, rinse ferrule,reassemble

Improper frit thickness Use proper frit

Problem-4: Detector leaks

Causes Solutions

Blocked waste line Rebuild or replace

Leaky fittings Tighten or replace

Cell gasket failure Replace gasket

Problem-1: Peak tailing

III- Problems with the Chromatogram

Peak tailing

Causes Solutions

Blocked frit Replace inlet fritReplace column

Column void Fill void

Wrong mobile phase pH Adjust pH

Problem-2: Peak fronting

Causes Solutions

Low temperature Increase column temperature

Sample overload Decrease sample concentration

Wrong sample solvent Use mobile phase forinjection solvent

Problem-3: Split peaks

Causes Solution

Contamination on guard andanalytical column inlet.

Remove guard column and replace if necessary. If analytical column is obstructed, Change frit or replace column.

Problem-3: Baseline Drift

Causes Solutions

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 orchange in flow rate

Correct composition / flow rate

Problem-4: Baseline noise (regular)

Causes Solutions

Air in mobile phase, detector cellor pump

Degas mobile phase. Flush system to remove air from detector cell or pump

Leak Check system for loose fittings. Change pump seals if necessary

Incomplete mobile phase mixing Mix mobile phase by hand or useless viscous solvent.

Problem-5: Baseline Noise (irregular)

Causes Solutions

Leak Check system for loose fittings. Change pump seals if necessary

Air trapped in system. Flush system with strong solvent.

Air bubbles in detector Purge detector

Weak detector lamp. Replace lamp.

Column leaking silica or packingmaterial

Replace column and clean system.

Problem-6: broad peak

Causes Solutions

Mobile phase flow rate too low Adjust flow rate

Mobile phase composition changed. Prepare new mobile phase.

Leak Check 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 out Replace column with new one ofsame type.

Column temperature too low Increase temperature. Do notexceed 75 °C.

Problem-7: loss of Resolution

Causes Solutions

Mobile phase contaminated /deteriorated

Prepare new mobile phase

Obstructed guard or andanalytical column

Remove guard column , Replace if necessary. If analytical column is obstructed, Change frit or replace column.

IV- Injector problems

Causes Solutions

Damaged rotor seal Rebuild or replace valve

Rotor too tight Adjust rotor tension

Problem-1: Manual injector, hard to turn

Causes Solutions

Valve misaligned Adjust alignment

Blocked loop Replace loop

Dirty syringe Clean or replace syringe

Blocked lines Clear or replace lines

Problem-2: Manual injector, hard to load

V- Other problems: Detected by the senses of smell, sight, and sound

Causes Solutions

Leak Check system for loose fittings. Change pump seals if necessary.

Problem-1: smell

Causes SolutionsDetector lamp failing Replace lampPressure abnormality Control Pressure

Problem-2: Abnormal meter readings

Problem-3: Warning lamps

Causes Solutions

Pressure limit exceeded Check for blockage

Other warning lamps See service manual

Causes Solutions

Solvent leak Locate and correct

Other warning buzzers See service manual

Problem-4: Warning buzzers

Problem-5: Squeaks and squeals

Causes Solutions

Poor lubrication Lubricate as necessary

Bearing failure See service manual

Mechanical wear See service manual

THANK YOU

Manish Kumar (M.Pharm. Pharmacology)