Chromatografie / Rady a tipy / HPLC závady a ich odstranenie

HPLC závady a jejich odstranění

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
helpNow 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


Leaks are usually stopped by tightening or replacing a fitting. Be aware, however, that overtightened metal compression fittings can leak and plasrtic 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.

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

Problems with the chromatogram

Many issues in the LC system appear as chenges 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

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

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 aalowed) or replace the column.
Split peaks

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

chromBroad 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

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

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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