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Causes and Remedy for HPLC Troubleshooting

No Peaks/Very Small Peaks


CAUSE

REMEDY

Detector lamp off.

Turn lamp on.

Loose/broken wire between detector and integrator or recorder.

Check electrical connections and cables.

No mobile phase flow.

See “No Flow”.

No sample/deteriorated sample/ wrong sample.

Be sure automatic sampler vials have sufficient liquid and no air bubbles in the sample. Evaluate system performance with fresh standard to confirm sample as source of problem.

Settings too high on detector or recorder.

Check attenuation or gain settings.

Check lamp status. Auto-zero if necessary.



No Flow


CAUSE

REMEDY

Pump off.

Start pump.

Flow interrupted/obstructed.

Check mobile phase level in reservoir(s). Check flow throughout system. Examine sample loop for obstruction or air lock. Make sure mobile phase components are miscible and mobile phase is properly degassed.

Leak.

Check system for loose fittings. Check pump for leaks, salt buildup, unusual noises. Change pump seals if necessary.

Air trapped in pump head. (Revealed by pressure fluctuations.)

Disconnect tubing at guard column (if present) or analytical column inlet. Check for flow. Purge pump at high flow rate (e.g., 5-10 mL/min.), prime system if necessary. (Prime each pump head separately.) If system has check valve, loosen valve to allow air to escape. If problem persists, flush system with 100% methanol or isopropanol. If problem still persists, contact system manufacturer.


No Pressure/Pressure Lower Than Usual

CAUSE

REMEDY

Leak.

Check system for loose fittings. Check pump for leaks, salt buildup, unusual noises. Change pump seals if necessary.

Mobile phase flow interrupted/ obstructed.

Check mobile phase level in reservoir(s). Check flow throughout system. Examine sample loop for

obstruction or air lock. Make sure mobile phase components are miscible and mobile phase is properly degassed.

Air trapped in pump head. (Revealed by pressure fluctuations.)

Disconnect tubing at guard column (if present) or analytical column inlet. Check for flow. Purge pump at high flow rate (e.g., 10 mL/min.), prime system if necessary. (Prime each pump head separately.) If system has check valve, loosen valve to allow air to escape.

Leak at column inlet end fitting.

Reconnect column and pump solvent at double the flow rate. If pressure is still low, check for leaks at inlet fitting or column end fitting.

Air trapped elsewhere in system.

Disconnect guard and analytical column and purge system. Reconnect column(s). If problem persists, flush system with 100% methanol or isopropanol.

Worn pump seal causing leaks around pump head.

Replace seal. If problem persists, replace piston and seal.

Faulty check valve.

Rebuild or replace valve.

Faulty pump seals.

Replace seals.



Pressure Higher Than Usual

CAUSE

REMEDY

Problem in pump, injector, in-line filter, or tubing.

Remove guard column and analytical column from system. Replace with unions and 0.010'' I.D. or larger tubing to reconnect injector to detector. Run pump at 2-5 mL/min. If pressure is minimal, see No Flow. If not, isolate cause by systematically eliminating system components, starting with detector, then in-line filter, and working back to pump. Replace filter in pump if present.

Obstructed guard column or analytical column.

Remove guard column (if present) and check pressure. Replace guard column if necessary. If analytical column is obstructed, reverse and flush the column, while disconnected from the detector. If problem persists, column may be clogged with strongly retained contaminants. Use appropriate restoration procedure. If problem still persists, change inlet frit or replace column.



Variable Retention Times

CAUSE

REMEDY

Leak.

Check system for loose fittings. Check pump for leaks, salt buildup, unusual noises. Change pump seals if necessary.

Change in mobile phase composition. (Small changes can lead to large changes in retention times.)

Check make-up of mobile phase. If mobile phase is machine mixed using proportioning values, hand mix and supply from one reservoir.

Air trapped in pump. (Retention times increase and decrease at random times.)

Purge air from pump head or check valves. Change pump seals if necessary. Be sure mobile phase is degassed.

Column temperature fluctuations (Especially evident in ion exchange systems).

Use reliable column oven. (Note: higher column temperatures increase column efficiency. For optimum results, heat eluant before introducing it onto column.)

Column overloading. (Retention times usually decrease as mass of solute injected on column exceeds column capacity.)

Inject smaller volume (e.g., 10 μL vs. 100 μL) or inject the same volume after 1:10 or 1:100 dilutions of sample.

Sample solvent incompatible with mobile phase.

Adjust solvent. Whenever possible, inject samples in mobile phase.

Column problem. (Not a common cause of erratic retention. As a column ages, retention times gradually decrease.)

Substitute new column of same type to confirm column as cause. Discard old column if restoration procedures fail


Loss of Resolution

CAUSE

REMEDY

Mobile phase contaminated/deteriorated

(Causing retention times and/or selectivity to change).

Prepare fresh mobile phase

Obstructed guard or analytical column.

Remove guard column (if present) and attempt analysis. Replace guard column if necessary. If analytical column is obstructed, reverse and flush. If problem persists, column may be clogged with strongly retained contaminants. Use appropriate restoration procedure. If problem still persists, change inlet frit or replace column.



Split Peaks

CAUSE

REMEDY

Contamination on guard or analytical column inlet.

Remove guard column (if present) and attempt analysis. Replace guard column if necessary. If analytical column is obstructed, reverse and flush. If problem persists, column may be clogged with strongly retained contaminants. Use appropriate restoration procedure.

If problem still persists, inlet frit is probably (partially) plugged. Change frit or replace column.

Partially blocked frit.

Replace frit.

Small (uneven) void at column inlet.

Repack top of column with pellicular particles of same bonded phase functionality. Continue using the column in reverse flow direction.

Sample solvent incompatible with mobile phase.

Adjust solvent. Whenever possible, inject samples in mobile phase.


Peaks Tail on Initial and Later Injections

CAUSE

REMEDY

Sample reacting with active sites.

First check column performance with standard column test mixture. If results for test mix are good, add ion pair reagent or competing base or acid modifier.

Wrong mobile phase pH.

Adjust pH. For basic compounds, lower pH usually provides more symmetric peaks.

Wrong column type.

Try another column type (e.g., deactivated column for basic compounds).

Small (uneven) void at column inlet.

See Split Peaks

Wrong injection solvent.

Peaks can tail when sample is injected in stronger solvent than mobile phase. Dissolve sample in mobile phase.



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Tailing Peaks

CAUSE

REMEDY

Guard or analytical column contaminated/worn out.

Remove guard column (if present) and attempt analysis. Replace guard column if necessary. If analytical column is source of problem, use appropriate restoration procedure. If problem persists, replace column.

Mobile phase contaminated/ deteriorated.

Check make-up of mobile phase

Interfering components in sample.

Check column performance with standards.




Fronting Peaks

CAUSE

REMEDY

Column overloaded.

Inject smaller volume (e.g., 10 μL vs. 100 μL). Dilute the sample 1:10 or 1:100 fold in case of mass overload.

Sample solvent incompatible with mobile phase.

Adjust solvent. Whenever possible, inject samples in mobile phase. Flush polar bonded phase column with 50 column volumes HPLC grade ethyl acetate at 2-3 times the standard flow rate, then with intermediate polarity solvent prior to analysis.

Shoulder or gradual baseline rise before a main peak may be another sample component.

Increase efficiency or change selectivity of system to improve resolution. Try another column type if necessary (e.g., switch from nonpolar C18 to polar cyano phase).



Rounded Peaks

CAUSE

REMEDY

Detector operating outside linear dynamic range.

Reduce sample volume and/or concentration.

Recorder gain set too low.

Adjust gain.

Column overloaded.

Inject smaller volume (e.g., 10 μL vs. 100 μL) or 1:10 or 1:100 dilution of sample.

Sample-column interaction.

Change buffer strength, pH, or mobile phase composition. If necessary, raise column temperature or change column type. (Analysis of solute structure may help predict interaction.)

Detector and/or recorder time constants are set too high.

Reduce settings to lowest values or values at which no further improvements are seen.



Baseline Drift

CAUSE

REMEDY

Column temperature fluctuation. (Even small changes cause cyclic baseline rise and fall. Most often affects refractive index and conductivity detectors, UV detectors at high sensitivity or in indirect photometric mode.)

Control column and mobile phase temperature, use heat exchanger before detector.

Nonhomogeneous mobile phase. (Drift 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 with HCl and never use nitric acid with

PEEK tubing or fittings.)

Plugged outlet line after detector. (High pressure cracks cell window, producing noisy baseline.)

Unplug or replace line. Refer to detector manual to replace window.

Mobile phase mixing problem or change in flow rate.

Correct composition/flow rate. To avoid problem, routinely monitor composition and flow rate.

Slow column equilibration, especially when changing mobile phase.

Flush column with intermediate strength solvent, run 10-20 column volumes of new mobile phase through column before analysis.

Mobile phase contaminated, deteriorated,

or not prepared from high quality chemicals.

Check make-up of mobile phase

Strongly retained materials in sample (high k’) can elute as very broad peaks and appear to be a rising baseline. (Gradient analyses can aggravate problem.)

Use guard column. If necessary, flush column with strong solvent between injections or periodically during analysis.

Detector (UV) not set at absorbance maximum but at slope of curve.

Change wavelength to UV absorbance maximum.



Baseline Noise (Regular)

CAUSE

REMEDY

Air in mobile phase, detector cell, or pump.

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

Pump pulsations.

Incorporate pulse damper into system.

Incomplete mobile phase mixing.

Mix mobile phase by hand or use less viscous solvent.

Temperature effect (column at high temperature, detector unheated).

Reduce differential or add heat exchanger.

Other electronic equipment on same line.

Isolate LC, detector, recorder to determine if source of problem is external. Correct as necessary.

Leak.

Check system for loose fittings. Check pump for leaks, salt buildup, unusual noises. Change pump seals if necessary.



Baseline Noise (Irregular)

CAUSE

REMEDY

Leak.

Check system for loose fittings. Check pump for leaks, salt buildup, unusual noises. Change pump seals if necessary.

Mobile phase contaminated, deteriorated, or prepared from low quality materials.

Check make-up of mobile phase.

Detector/recorder electronics.

Isolate detector and recorder electronically. Refer to instruction manual to correct problem.

Air trapped in system.

Flush system with strong solvent.

Air bubbles in detector.

Purge detector. Install backpressure regulator after detector. Check the instrument manual, particularly for RI detectors (excessive backpressure can cause the flow cell to crack).

Detector cell contaminated. (Even small amounts of contaminants can cause noise.)

Clean cell.

Weak detector lamp.

Replace lamp.

Column leaking silica or packing material.

Replace column and clean system.



Broad Peaks

CAUSE

REMEDY

Mobile phase composition changed.

Prepare new mobile phase.

Mobile phase flow rate too low.

Adjust flow rate.

Leak (especially between column and detector).

Check system for loose fittings. Check pump for leaks, salt buildup, and unusual noises. Change pump seals if necessary.

Detector settings incorrect.

Adjust settings.

Extra-column effects:

 

Column overloaded

Inject smaller volume (e.g., 10 μL vs. 100 μL) or 1:10 and 1:100 dilutions of sample.

Detector response time or cell volume too large.

Reduce response time or use smaller cell.

Tubing between column and detector too long or I.D. too large.

Use as short a piece of 0.007-0.010" I.D. tubing as practical.

Recorder response time too high.

Reduce response time.

Buffer concentration too low.

Increase concentration.

Guard column contaminated/worn out.

Replace guard column.

Column contaminated/worn out.

Replace column with new one of same type. If new column does not provide narrow peaks, flush old column, then retest.

Void at column inlet.

Replace column or open inlet end and fill void.

Peak represents two or more poorly resolved compounds.

Change column type to improve separation.

Column temperature too low.

Increase temperature. Do not exceed 75°C unless higher temperatures are acceptable to column manufacturer.



Change in Peak Height (one or more peaks)

CAUSE

REMEDY

One or more sample components deteriorated or column activity changed.

Use fresh sample or standard to confirm sample as source of problem. If some or all peaks are still smaller than expected, replace column. If new column improves analysis, try to

restore the old column, following appropriate procedure. If performance does not improve, discard old column.

Leak, especially between injection port and column inlet. (Retention also would

change.)

Check system for loose fittings. Check pump for leaks, salt buildup, unusual noises. Change pump seals if necessary.

Inconsistent sample volume.

Be sure samples are consistent. For fixed volume sample loop, use 2-3 times loop volume to ensure loop is completely filled. Be sure automatic sampler vials contain sufficient sample and no air bubbles. Check syringe-type injectors for air. In systems with wash or flushing step, be sure wash solution does not precipitate sample components.

Detector or recorder setting changed.

Check settings.

Weak detector lamp.

Replace lamp.

Contamination in detector cell.

Clean cell.



Change in Selectivity

CAUSE

REMEDY

Increase or decrease solvent ionic strength, pH, or additive concentration (especially affects ionic solutes).

Check make-up of mobile phase

Column changed; new column has different selectivity from that of old column.

Confirm identity of column packing. For reproducible analyses, use same column type. Establish whether change took place gradually.

If so, bonded phase may have stripped. Column activity may have changed, or column may be contaminated.

Sample injected in incorrect solvent or excessive amount (100-200 μL) of strong solvent.

Adjust solvent. Whenever possible, inject sample in mobile phase.

Column temperature change.

Adjust temperature. If needed, use column oven to maintain constant temperature.



Negative Peak(s)

CAUSE

REMEDY

Recorder leads reversed.

Check polarity.

Refractive index of solute less than that of mobile phase (RI detector).

Use mobile phase with lower refractive index, or reverse recorder leads.

Sample solvent and mobile phase differ greatly in composition (vacancy peaks).

Adjust or change sample solvent. Dilute sample in mobile phase whenever possible.

Mobile phase more absorptive than sample components to UV wavelength.

Change polarity when using indirect UV detection, or

Change UV wavelength or use mobile phase that does not adsorb chosen wavelength.



Ghost Peak

CAUSE

REMEDY

Contamination in injector or column.

Flush injector between analyses (a good routine practice). If necessary, run strong solvent through column to remove late eluters. Include final wash step in gradient analyses, to remove strongly retained compounds.

Late eluting peak (usually broad) present in sample.

Check sample preparation.

Include (step) gradient to quickly elute component.


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