Daily Maintenance and Common problems in HPLC

HPLC Problems and Solutions | HPLC Troubleshooting Guide | Common error occurs during HPLC Analysis |

Topic covers below points:

  • Pre-Analysis Checks
    • Before “RUN”
  • Pre-Stabilization Checks
    • After “RUN”
  • Post-Analysis Checks
    • After Analysis
    • Mobile Phase – Solvent Grade
    • Mobile Phase – Use of buffer solutions
    • Mobile Phase – Premixing of Solvents
    • Waste Bottle Maintenance
    • Pump
    • Injector
    • Detector
    • Problems in Retention Time
      • Decreasing Retention Time :
      • Increasing Retention Time :
  • Treatment of Column
  • Useful Tips

Pre-Analysis Checks

Before “RUN”

  • Filter the Mobile Phase & Sample
    • Always filter the mobile phase & Sample with 0.45μ & 0.2 μ membrane filter respectively
  • Wait the mobile phase to reach room temperature
    • Because the temperature of recently mixed solution can vary greatly from the ambient temperature, verify that the mobile phase is at room temperature before analysis. Until the mobile phase reaches room temperature, there may be considerable baseline instability, especially drift. Bubble may also form in flow lines
  • Purge the flow lines
    • Ensure the flow lines are free from bubbles.
    • Bubble in flow lines may result in inaccurate concentrations and instability, causing changes in retention time and poor reproducibility
  • Replace mobile phase as necessary
    • Replacing mobile phase with a miscible solvent.
    • Replacing mobile phase with an immiscible solvent.
    • Replacing mobile phase if either old or new solvent is a buffer solution.
  • Replacing mobile phase with a miscible solvent
    • Pour some of the new mobile phase into a clean beaker.
    • Remove the solvent reservoir bottle, insert it into the beaker and rinse the filter well by shaking.
    • Purge the connected flow lines.
    • Insert the solvent reservoir filter into the new mobile phase reservoir bottle and rinse the flow lines.
    • Connect the column and rinse the flow lines.
  • Replacing mobile phase with an immiscible solvent
    • First replace the old mobile phase with an intermediate solution that is miscible to both the old and new mobile phases. Then replace the intermediate solution with the new mobile phase.
    • Follow the procedure for replacing the mobile phase with a miscible solvent for both the intermediate solution and new mobile phase.
  • Replacing mobile phase if either old or new solvent is a buffer solution
    • First replace the old mobile phase with distilled water as an intermediate solution. Then replace the water with the new mobile phase. This prevents the buffer solution salts from precipitating.
    • If this procedure is not followed, organic solvents, such as isopropyl alcohol, may precipitate buffer crystals or other residual material and cause the flow lines to clog.
  • Verify that the seal rinse bottle is prepared for use
    • If the mobile phase contains buffer solution or phosphoric acid, ensure that the rinse bottle has been filled so that the seal will be rinsed. If the mobile phase is distilled Water or contains an immiscible solvent, the rinse bottle should be empty.
    • Use distilled water to fill the rinse bottle.
    • Replace the water every two or three days to prevent bacterial growth
  • Verify that the autosampler rinse bottle is full
    • Use mobile phase as the rinse solution. If the mobile phase contains a salt, use the equivalent solution without the salt.
    • The rinse solution mixes with the mobile phase in the high pressure valve; this will ensure that no crystals form.
  • Purge the auto sampler measuring pump
    • If there is a large change in room temperature between uses, or if the instrument remains idle for several days, bubbles may form in the pump head, resulting in inconsistent injection volume. Purge the measuring pump to eliminate bubbles

Pre-Stabilization Checks

After “RUN”

  • Check for leaks
    • After the instrument starts running an analysis, verify that there are no leaks in the flow lines.
    • If a leak is discovered, either tighten the connection or replace the damaged part.
  • Check the pump pressure
    • The pump pressure varies with the entered pressure parameter and type of mobile phase used
  • Perform a baseline check
    • Verify that the baseline is stable.
  • Rinse the seal
    • If the mobile phase contains buffer solution or phosphoric acid, rinse the seal. Otherwise, salt or phosphoric acid may precipitate out at the rear seal surface where it can scratch the seal and plunger, shortening the life of these parts.
    • Rinse the seal with distilled water.
  • Note: Delivering Rinse Solution
    • If the rinse solution is not delivered when Pump is pressed, insert the injector syringe as shown in the figure, and aspirate the rinse solution into the syringe.

Post-Analysis Checks

After Analysis

  • Rinse the column
    • If the instrument will remain idle for some time, rinse the column with storage solution and detach it from the instrument.
    • The cleaning and storage procedure varies by column, so refer to the documentation provided with the column or contact the manufacturer.
  • Clean the flow line
    • If a buffer solution is used, rinse the contact areas with water.
    • If the instrument is to remain idle for some time, rinse the column, and detach it from the instrument. Connect the inlet and outlet column ports with a coupling, then rinse the flow lines. To prevent the growth of mold or bacteria, rinse the flow lines with an alcohol, such as isopropyl alcohol or methanol, and leave the alcohol in the system.
    • Also disconnect and rinse the solvent reservoir filter and UV-VIS detector outlet tubing. Store them in a beaker of rinse solution to prevent them from drying out.

Mobile Phase – Solvent Grade

  • If the solvent has background absorption,
  • linear range will be smaller, noise will be larger
  • baseline stability especially during gradient runs will be poor.
  • Water grade
    • Pure water
    • Distilled water
    • Deionized water
  • Due to existence of impurity, deionized water shows higher absorption.
  • The use of pure water systems (such as those supplied by Millipore or Barnstead) is ideal as both inorganic and organic contaminants are removed.
  • Modern pure water systems give TOC values as indication of total organic content in water.
  • Ghost peak will appear in gradient elution if water is of bad quality
  • Difference of analytical and HPLC grade solvent

Mobile Phase – Use of buffer solutions

  • Buffer solutions must be filtered before using.
Solvent Filtration Kit
  • Buffer solutions must be filtered before use.
  • Buffer solutions must not be left in system to avoid crystallisation.
    • effect on pump – damage plunger and seal
    • effect on column – creation of column voids
    • effect on flow line – corrosion of stainless steel lines
  • Possible bacterial growth
    • especially phosphate buffers – good medium for bacterial and fungus growth.
  • •Ideally, solutions should be prepared fresh everyday.

Mobile Phase – Premixing of Solvents

  • For isocratic systems, different solvents are premixed.
    • E.g. To prepare 1000ml of 50:50 mixture of water/methanol,
      • Measure out 500ml of water and 500ml of methanol separately, and then mix them together.
      • Measure 500ml of water, and top up to 1000ml mark of measuring cylinder/volumetric flask with methanol.
      • Measure 500ml of methanol and top up to 1000ml mark of measuring cylinder/volumetric flask with water.

All three methods will give different compositions. Whichever method selected must be used consistently for repeatable results.

Waste Bottle Maintenance

  • Remember to empty waste bottles regularly.
  • Do not mix concentrated nitric acid with organic solvents.
  • Do not immerse the drain tubing in the waste liquid as this may create back pressure on the flow cell.



Leaks :
Leakage from the pump head.Replace the pump seal; Check the piston for scratches and if necessary replace.

Decreasing Pressure :
Insufficient flow from pumpLoosen cap on mobile phase reservoir
Leak in lines from pump to columnTighten or replace fittings; tighten rotor in injection valve
Leaking pump check valve or sealReplace or clean check valve; replace pump seals
Suction filter cloggedSonicate or replace

Increasing Pressure :
Blocked flow linesSystematically disconnect components from pump to column end to find blockage; replace or clean blocked component
Particulate buildup at head of columnFilter sample; use 0.45μ filter; disconnect and back flush column; replace line filter

Fluctuating Pressure :
Bubble in pump headSuck up bubbles through the drain tubing with a syringe.
Leaking pump seals, flow lines or faulty check valve.Replace pump seals, tight the flow line connection; Sonicate check valves or replace.
Old Solvents remains in the pump chamberPurge or increase the flow rate.

High Back Pressure :
Column BlockedImprove sample cleanup; use guard column; reverse flush column with strong solvent to dissolve blockage.
Microbial growth on column.Store column in at least 25% organic solvent without buffer.
Mobile phase viscosity too highUse lower viscosity solvents or high temperature
Plugged frit in line filter or guard columnReplace frit or guard column
Plugged inlet fritReplace end fitting or frit assembly
Salt precipitation (especially in reversed phase chromatography with high conc. of organic solvent in mobile phase)Ensure mobile phase compatibility with buffer conc.


Unstable Pressure during Sample injecting :
Valve CloggedClean the valve or replace rotor seal.

Leaks :
Needle seal has deterioratedReplace the needle seal
Valve Rotor or Stator has deterioratedReplace the rotor or stator seal

Area Reproducibility :
Auto sampler was not rinsedCheck the level of rinsing solvent and ensure that it is being rinsed
Column has deterioratedCheck the column performance under known analysis condition or replace the column.
Sample flow line is cloggedVerify the sample is completely dissolve, Clean the clogged flow line or replace.
Valve rotor or stator has deterioratedReplace the rotor or stator seal

Ghost Peak :
Contamination in columnFlush column to remove contamination; use HPLC grade solvent
Elution of analytes retained from previous injectionFlush column with strong solvents at end of run; end gradient at higher solvent concentration
Unknown interferences in sampleUse sample cleanup or prefractionation before injection


Leak :
Blocked column outlet (leak in flowcell)Replace outlet tube or change gasket in flowcell.

Noise :
Bubble flowing or trapped in the flow cellApply pressure to the flow cell outlet, use MeOH to flush flow cell
Cell window is strainedCell rinsing.
D2 Lamp DegradationReplace a new D2 Lamp
Strained lens and gasketReplace with a new one
Mobile phase pulsationEliminate pulsation from pump by dampener

Drift :
Cell Window is strainedCell  Rinsing
Impurities in mobile phaseUse fresh, covered mobile phase
Unit is in the presence of strong air currentChange the location of the instrument or place it in a protective environment.

Problems in Retention Time

Contamination BuildupFlush the column occasionally with suitable solvents
Improper flow or pressureCheck the leaks in flow lines; check the check valves
Equilibration time insufficient for gradient run or changes in isocratic mobile phaseCondition the column
Inconsistent on-line mobile phase mixingEnsure the gradient system is delivering a constant composition
Selective evaporation of mobile phase componentCover solvent reservoirs; Prepare fresh mobile phase
Varying column temperatureThermostat or insulate column; ensure laboratory temperature is constant.

Decreasing Retention Time :
Column overloaded with sampleDecrease sample amount or use large diameter column
Increase flow rateCheck the reset the pump flow rate
Varying column temperatureThermostat or insulate column; ensure laboratory temperature is constant

Increasing Retention Time :
Decreasing flow rateCheck and reset the flow rate. Check for leaking pump seals and other leak in system.
Changing mobile phase compositionCover solvent reservoirs; ensure that gradient system is delivering correct composition
Varying column temperatureThermostat or insulate column; ensure laboratory temperature is constant

Treatment of Column

  • For new columns, it is best to condition under low flow rate (0.2 – 0.3 ml/min) for about 30 mins before operating under analytical flow rate. It applies for columns that have not been used for long time.
  • During shutdown, it is best to gradually reduce the flow rate so as not to subject column to sudden pressure drop.
  • When using buffer solutions, remember to flush with water before changing to organic solvents to prevent crystallization.
  • It is best to filter mobile phase and sample before use to reduce clogging by particulates in column frit.
  • Sample is best dissolved in mobile phase or if not possible then as clse to mobile phase composition as possible. This reduce chances that sample may participate in column bed in mixing with mobile phase.
  • Flush with stronger solvent after daily use. For Eg. ODS columns may flushed with Acetonitrile anger use daily to remove strong absorbed materials from column.
  • Prevention is by filtering both sample and mobile phase through 0.45um. Use 0.2 um membrane filter if it is very dirty.
  • Normal flushing with solvents may dissolve and remove some of these particles but in cases where the particulates are insoluble, flushing the column in reverse may help.
  • Replace filter frit if it cannot be washed. Be careful not to disturb column bed or performance may drop and in some cased, the stainless steel frits are fused to the columns and these cannot be replaced.
  • As HPLC columns are pressure-packed, subjecting them to pressure shock such as dropping the column or opening of drain valve during operation many result in formation of voids.

Useful Tips

  • Good habit
    • Log book – record pressure and mobile phase
    • Read manual / instruction
  • If any problem happens
    • Observe the instrument carefully – look, smell, and feel
    • Check the simple things first
    • Remember the last thing that happen before the problem started
    • Isolate the source of the problem

Reference : Shimadzu Maintenance & Troubleshooting Webpage