HPLC troubleshooting

Abnormal pressure

No pressure reading, no flow

Possible cause	Solution
Power off	Turn on power
Fuse blown	Replace fuse
Controller setting or failure	Verify proper settings, repaire or replace controller
Broken piston	Replace piston
Air trapped in pump head	Degas mobile phase, bleed air from pump and prime pump
Insufficient mobile phase	Replenish reservoir, replace inlet frit if it is blocked
Faulty check valves	Replace check valves
Major leak	Tighten or replace fittings

Now pressure reading, flow is normal

Possible cause	Solution
Faulty meter	Replace meter
Faulty pressure transducer	Replace transducer

High back pressure

Possible cause	Solution
Flow rate set too high	Adjust settings
Blocked column frit	Backflush column if it is permitted, replace frit according to the manufacturer's instructions and warratny conditions or replace column
Improper mobile phase, precipitated buffer	Use correct mobile phase, wash column
Improper column	Use proper column
Injector blockage	Clear blockage or replace injector
Column temperature too low	Raise temperature
Controller malfunction	Repair or replace controller
Blocked guard column	Remove/replace guard column
Blocked in-line filter	Remove/replace in-line filter

You should find out, what caused high back pressure - column or system? We recommend following procedure:

Remove column from the system and turn on pump. If high back pressure still appears, then the blockage is in the system:

• blocked or crimped tubing
• dirty pump frit
• or clogged injection valve

If the pressure is normal, there is a problem with the column:

• clogged or damaged pre-column filter, guard column or frit
• precipitation of sample or buffer in column

Low back pressure

Possible cause	Solution
Flow set too low	Adjust flow rate
Leak in the system	Locate and correct
Improper column	Use proper column
Column temperature too high	Lower temperature
Controller malfunction	Repair or replace controller

Fluctuating pressure

See section High back pressure.

Pressure dropping to zero

See sections No pressure reading, no flow and No pressure reading, flow is normal

Pressure dropping, but not to zero

See section Low pressure

Pressure cycling

Possible case	Solution
Air in pump	Degas solvent and/or bleed air from the pump
Faulty check valve(s)	Replace check valve(s)
Pump seal failure	Replace pump seal
Insufficient degassing	Degas solvent and/or change degassing methods (e.g. use vacuum degasser)
Leak in system	Locate leak and correct it
Using gradient elution	Pressure cycling is normal due to viscosity change

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Leaks

Leaks are usually stopped by tightening or replacing a fitting. Be aware, however, that overtightened metal compression fittings can leak and plastic fingertight fittings can wear out. If a fitting leak does not stop when the fitting is tightened a little, take the fitting apart and inspect for damage (e.g. distored ferrule, or particles on the sealing surface). If the fitting or ferrule is damaged, replace it with new one.

Leaky fittings

Possible cause	Solution
Loose fitting	Tighten the fitting
Stripped fitting	Replace the fitting
Overtightened fitting	Loosen and retighten the fitting. If the fitting is damaged, replace it.
Dirty fitting	Disassemble fitting and clean it. If the fitting is damaged, replace it.
Mismatched parts	Use all parts from the same brand/type.

Leaks at the pump

Possible cause	Solution
Loose check valves	Tighten check valve (do not overtighten) or replace check valve
Loose fittings	Tighten fittings (do not overtighten)
Mixer seal failure	Repair or replace
Pump seal failure	Repair or replace the seal
Pulse damper failure	Replace pulse damper
Proportionin valve failure	Check diaphragms, replace if leaky and/or check for fitting damage, replace
Purge valve	Tighten valve or replace it if it is faulty

Injector leaks

Possible cause	Solution
Rotor seal failure	Rebuild or replace rotor
Blocked loop	Clean or replace loop
Loose injector port seal	Adjust
Improper syringe needle diameter	Use correct syringe
Waste line siphoning	Keep waste line above surface vaste, with proper slope
Waste line blockage	Replace waste line

Column leaks

Possible cause	Solution
Loose endfitting	Tighten endfitting
Column packing in ferrule	Disassemble, rinse ferrule, reassemble
Improper frit thickness	Use proper frit(*)

*Note: When the particle size of stationary phase is 3 to 4 µm, use frit 0.5 µm. When particle size of stationary phase is 5 to 20 µm, use frit 2 µm.

Picture: Correct fitting/capillary connection (dead volume in red)

Detector leaks

Possible cause	Solution
Cell gasket failure	Prevent excessive backpressure or replace gasket
Cracked cell window(s)	Replace cell window(s)
Leaky fittings	Tighten or replace fittings
Blocked waste line	Replace waste line
Blocke flow cell	Rebuild or replace flow cell

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Problems with the chromatogram

Many issues in the LC system appear as changes in the chromatogram. Some of these can be solved by changes in the instrument, however, other problems require modification of the assay procedure. Setting the proper column type, pre-column or guard column, tubings, detector cell and mobile phase are keys to good chromatography.

Peak tailing

Possible cause	Solution
Blocked frit	Reverse flush column (if it is allowed) or replace frit (if it is allowed) or replace column
Column void	Fill void
Interfering peak	Use longer column or change mobile phase and/or column selectivity
Wrong mobile phase pH	Adjust pH. For basic compounds, lower pH usually provides more symmetric peaks
Sample reacting with active sites	Add ion pair reagent or volatile basic modifier or change column

Peak fronting

Possible cause	Solution
Low temperature	Increase column temperature
Wrong sample solvent	Use mobile phase for injection solvent
Sample overload	Decrease sample concentration
Bad column	Reverse flush column (if it is allowed) or replace inlet frit (if it is allowed) or replace the column.

Split peaks

Possible cause	Solution
Contamination on guard or analytical column inlet	Remove guard column and attempt analysis. replace guard column if necessary. If analytical column is obstructed, reverse and flush (if it is allowed). If problem persists, column may be fouled with strongly retained contaminants. Use appropriate restoration procedure (see column care information). If problem persists, inlet is probably plugged. Change frit or replace column.
Sample solvent incompatible with mobile phase	Change solvent. whenever possible, inject samples in mobile phase.

Distortion of larger peaks

The peak distortion can be caused by sample overload. Reduce the sample size.

Distortion of early peaks

The distortion of early eluting peaks can be caused by wrong injection solvent. Reduce the injection volume, or use weaker injection solvent.

Tailing, early peaks mor than later ones

Possible cause	Solution
Extra-column effects	Replumb the system (shorter, narrower tubing), use smaller volume detector cell

Increased tailing as k' increases

Possible cause	Solution
Secondary retention effects, reversed-phase mode	Add triethylamine (basic sampes) or add acetate (acidic samples) or add salt or buffer (ionic samples) or try a different column.
Secondary retention effects, normal-phase mode	Add triethylamine (basic compounds) or add acetic acid (acidic compounds) or add water (poly-functional compounds). Only for normal-phase methods which use water-miscible solvents. You can also try a different LC method.
Secondary retention effects, ion-pair chromatography	Add triethylamine (basic samples)

Acidic or basic peaks tail

Possible cause	Solution
Inadequate buffering	Use 50 to 100 mM buffer concentration, use buffer with pKa equal to pH of mobile phase

Extra peaks

Possible cause	Solution
Ghost peaks	Impurities in the sample, reagents or material used. Change clean-up procedure and/or check possible source of contamination (glassware, vials, used reagents, solvents, etc...)
Late eluting peak from previous injection	Increase run time or gradient slope and/or increase flow rate

Retention time drifts

Possible cause	Solution
Poor temperature control	Use column termostat. If you use it, check its proper functionality.
Mobile phase changing	Prevent change (evaporation, reaction, etc...)
Poor column equilibration	Allow more time for column equilibration between runs

Abrupt retention time changes

Possible cause	Solution
Flow rate change	Reset flowrate
Air bubble in pump	Bleed air from pump
Improper mobile phase	Replace mobile phase with proper one and/or set proper mobile phase mixture on controller

Baseline drift

Possible cause	Solution
Column temperature fluctuation. Even small changes cause cyclic baseline rise and fall. Most often affects refractive index, UV and conductivity detectors at high sensitivity	Control column and mobile phase temperature, use heat exchanger before detector
Nonhomogenous mobile phase. Baseline drift is usually to higher absorbance, rather than cyclic pattern from temperature fluctuation.	Use HPLC grade solvents, high purity salts, and additives. Degas mobile phase before use, sparge with helium during use.
Contaminant or air buildup in detector cell	Flush cell with methanol or other strong solvent. If necessary, clean cell with 1N HNO3 (never use HCl).
Plugged outlet line after detector. High pressure cracks cell windoe, producing noisy baseline.	Unplug or replace line. Refere to detector manual to replace window.
Mobile phase mixing problem or change in flow rate	Correct composition or flow rate. To avoid this issue, routinely monitor composition and flow rate.
Slow column equilibration, especially when changing mobile phase	Flush with intermediate strength solvent, run 10 to 20 column volumes of new mobile phase before analysis
Mobile phase contaminated, deteriorated or prepared from low quality materials	Chack mobile phase preparation. Use highest grade chemicals and HPLC solvents
Strongly retained materials in sample (high k') can elute as very broad peaks and appear to be a rising baseline (gradient analyses canaggravate problem).	Use guard column. If necessary, flush column with strong solvent between injections or periodically during analysis.
Mobile phase recycled but detector not adjusted	Reset baseline. Use new mobile phase when dynamic range of detector is exceeded.
Detector (UV) not set at absorbance maximum but at slope of curve	Change wavelength to UV absorbance maximum

Baseline noise (regular)

Possible cause	Solution
Air in mobile phase, detector cell or pump	Degas mobile phase. Flush system to remove air from detector cell or pump.
Leak	See section Leaks. Check system for loose fittings. Check pump for leaks, salt build-up, unusual noise. Change pump seals if necessary.
Incomplete mobile phase mixing	Mix mobile phase by hand or use less visous sample.
Temperature effect (column at high temperature or detector unheated)	Reduce differential or add heat exchanger
Other electronic equipment on the same line	isolate LC, detector or recorder/PC to determine if source of issue is external. Correct as necessary.
Pump pulsation	Incorporate pulse dampener into system.

Baseline noise (irregular)

Possible cause	Solution
Leak	See section Leaks. Check system for loose fittings. Check pump for leaks, salt build-up, unusual noise. Change pump seals if necessary.
Mobile phase contaminated, deteriorated or prepared from low quality materials	Check mobile phase preparation
Mobile phase solvents immiscible	Select and use only miscible solvents
Air trapped in system	Flush system with strong solvent
Air bubbles in detector	Purge detector. Install back-pressure device after detector
Detector/recorder electronics	Isolate detector and recorder/PC electronically. Refer to isntruction manual to correct problem.
Detector cell contaminated (even small amounts of contaminants can cause noise)	Clean cell by flushing 1N HNO3 (never use HCl)
Weak detector lamp	Replace lamp
Column leaking silica or packing material	Replace column
Mobile phase mixer inadequate or malfunctioning	Repair or replace the mixer or mix off-line if isocratic.

Broad peaks

Possible cause	Solution
Mobile phase composition changed	Prepare new mobile phase
Mobile phase flow rate too low	Adjust flow rate
Leaks (especially between column and detector)	See section Leaks. Check system for loose fittings. Check pump for leaks, salt build-up, unusual noise. Change pump seals if necessary.
Detector settings are incorrect	Adjust settings
Buffer concentration too low	Increase buffer concentration
Guard column contaminated/worn out	Replace guard column
Column contaminated/worn out. Low plate number.	Replace column with new one of same type.
Void at column inlet	Open inlet end and fill void or replace column
Peak represents two or more poorly resolved compounds	Change column type to improve separation
Column temperature too low	Increase temperature. Do not exceed 60°C unless higher temperatures are acceptable to column manufacturer.
Detector time constant too large	Use smaller time constant

Broad peaks can be caused by extra-column effects:

• Column overloaded - inject smaller volume (e.g. 10µl instead of 100µl) or dilute sample
• Detector response time or cell volume too large - reduce response time or use smaller cell
• Tubing between column and detector too long ir ID too large - use as short a piece of 0.007" to 0.010" ID tubing as practical (UHPLC techniques require use of 0.005" ID tubing)
• Recorder response time too high - Reduce response time

Loss of resolution

Possible cause	Solution
Mobile phase contaminated(deteriorated (causing retention time to change)	Prepare new mobile phase
Obstructed guard or analytical column	Remove guard column and attepmt analysis. Replace guard if necessary. If analytical column is obstructed, reverse and flush. If problem persists, column may be fouled with strongly retained contaminants. Use appropriate restoration procedure according to the manufacturer's manual. If problem persists, inlet is probably plugged. Change frit or replace column.

All peaks too small

Possible cause	Solution
Detector attenutation too high	Reduce attenuation
Detector time constant too large	Use smaller time constant
Injection size too small	Increase sample concentration or increase injection volume, if column size allows it
Improper recorder connection	Use correct connection

All peaks too large

Possible cause	Solution
Detector attenuation too low	Use larger attenuation
Injection size too large	Reduce sample concentration or decrease injection volume, use smaller sample loop or use partial loop filling
Improper recorder connection	Use correct connection

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Problems with the injector

Manual injector, hard to turn

Possible cause	Solution
Damaged rotor seal	Rebuilt or replace seal/valve
Rotor too tight	Adjust rotor tension

Manual injector, hard to load

Possible cause	Solution
Valve misaligned	Adjust alignment
Blocked loop	Replace loop
Dirty syringe	Clean or replace syringe
Blocked lines	Clear or replace lines

Autosampler injector - won't turn

Possible cause	Solution
No air pressure or power	Supply proper pressure/power
Rotor too tight	Adjust
Valve misaligned	Adjust alignment

Autosampler - other problems

Possible cause	Solution
Blockage	Clear or replace blocked portion
Jammed mechanism	See service manual
Faulty controller	Repair or replace controller

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Problems detected by smell, sight or sound

Solvent smell

Possible cause	Solution
Leak	See section Leaks
Spill	Check for overflowing waste container or locate spill and clean up

"Hot" smell

Possible cause	Solution
Overheating module	Check for proper ventilation, adjust it or check temperature setting, adjust or shut module off, see service manual

Abnormal meter readings

Possible cause	Solution
Pressure abnormality	See section Abnormal pressure
Column oven problem	Check settings and adjust, see service manual
Detector lamp failing	Replace lamp

Warning lamps

Possible cause	solution
Pressure limit exceeded	Check for blockage or check limit settings and adjust them
Other warning lamps	See operation/service manual

Warning buzzers

Possible cause	Solution
Solvent leak/spill	Locate and correct
Other warning buzzers	See operation/service manual

Squeaks and squeals

Possible cause	Solution
Bearing failure	See service manual
Poor lubrication	Lubricate as necessary
Mechanical wear	See service manual

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Key problem areas and preventive maintenance

Reservoir

Problem	Solution/Preventive maintenace
Blocked inlet frit	Replace every 3 to 6 months and filter mobile phase through 0.5µm filter
Gas bubbles	Degas mobile phase

Pump

Problem	Solution/Preventive maintenance
Air bubbles	Degas mobile phase, check for leaks
Pump seal failure	Replace pump seal every 3 months
Check valve failure	Filter mobile phase, use inlet-line frit. Keep spare one.

Injector

Problem	Solution/Preventive maintenance
Rotor seal wear	Do not overtighten and filter samples

Column

Problem	Solution/Preventive maintenance
Blocked frit	Filter mobile phase, filter samples, use in-line filter and/or guard column
Void at head of column	Avoid mobile phase pH>8 (most silica-based columns), use guard column, use precolumn (saturator column)

Detector

Problem	Solution/Preventive maintenance
Lamp failure	Replace the lamp
Decreased detector response	Replace the lamp on regular basis according to the operation manual (keep spare lamp)
Increased detector noise	Replace the lamp on regular basis according to the operation manual (keep spare lamp)
Bubbles in cell	Keep cell clean or use restrictor after cell or degas mobile phase. Keep attention on maximum allowable pressure in the cell.

General

Problem	Solution/Preventive maintenance
Corrosive/abrasive damage	Flush buffer from LC and clean when not in use