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HPLC troubleshooting Guide


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Cleaning Dirty columns
Changing column Frit

Column leaks, Retention time, Back pressure, peaks and baseline problems

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Start up - Preliminary checks

Problem Possible cause Solution
No peaks or very small peaks Detector off Check detector
Broken connections to recorder Check connections
No sample/Wrong sample Check sample. Be sure it is not deteriorated. Check for bubbles in the vials
Wrong settings on recorder or detector Check attenuation. Check gain
No Flow Pump off Start Pump
Flow interrupted Check reservoirs. Check position of the inlet tubing. Check loop for obstruction or air. Check degasing of mobile phase. Check compatibility of the mobile phase components.
Leak Check fittings. Check pump for leaks and precipitates. Check pump seals.
Air trapped in the system Disconnect column and prime pump. Flush system with 100% methanol or isopropanol. Contact servicing if necessary.

Column and Fittings Leaks

Problem Possible cause Solution
Column end leaks Loose fitting
White powder at loose fitting
Tighten or replace fitting
Cut tubing and replace ferrule; disassemble fitting, rinse and reassemble.
Leak at detector Detector-seal failureReplace detector seal or gaskets.
Leak at injection valve Worn or scratched valve rotor Replace valve rotor
Leak at pump Pump seal failure Replace pump seal; check piston for scratches and, if necessary, replace

Change in Retention time

Problem Possible cause Solution
Changing Retention Times Buffer retention times Use buffer with concentration greater than 20 mM.
Contamination buildup Flush column occasionally with strong solvent
Equilibration time insufficient for gradient run or changes in isocratic mobile phase Pass at least 10 column volumes through the column for gradient regeneration or after solvent changes
First few injections - active sites Condition column by injecting concentrated sample
Inconsistent on-line mobile-phase mixing Ensure gradient system is delivering a constant composition; compare with manually prepared mobile phase; partially premix mobile phase
Selective evaporation of mobile-phase component Cover solvent reservoirs; use less-vigorous helium purging; prepare fresh mobile phase
Varying column temperature Thermostat or insulate column; ensure laboratory temperature is constant.
Decreasing Retention Times Active sites on column packing Use mobil-phase modifier, competing base (basic compounds), or increase buffer strength; use higher coverage column packing.
Column overloaded with sample Decrease sample amount or use larger-diameter column.
Increasing flow rate Check and reset pump flow rate.
Loss of bonded stationary phase or base silica Use mobile-phase pH between pH 2 and pH 8
Varying column temperature Thermostat or insulate column; ensure laboratory temperature is constant
Increasing Retention Times Decreasing flow rate Check and reset pump flow rate; check for pump cavitation; check for leaking pump seals and other leaks in system
Changing mobile-phase composition Cover solvent reservoirs; ensure that gradient system is delivering correct composition.
Loss of bonded stationary phase Use mobile-phase pH between pH 2 and pH 8
Slow column equilibration time Reversed phase ion pairing - long chain ion pairing reagents require longer equilibration time Use ion-pairing reagent with shorter alkyl chain length

Baseline

Problem Possible cause Solution
Void Time noise Air bubbles in mobile phase Degas or use back pressure restricor on detector
Positive-negative - difference in refractive index of injection solvent and mobile phase Normal with many samples; use mobile phase as sample solvent
Drifting baseline Negative direction (gradient elution) - absorbance of mobile-phase A Use non-UV absorbing mobile phase solvents; use HPLC grade mobile phase solvents; add UV absorbing compound to mobile phase B.
Positive direction (gradient elution) - absorbance of mobile phase B Use higher UV absorbance detector wavelength; use non-UV absorbing mobile phase solvents; use HPLC grade mobile phase solvents; add UV absorbing compound to modile phase A.
Positive direction - contamination buildup and elution Flush column with strong solvent; clean up sample; use HPLC grade solvents
Wavy or undulating - temperature changes in room Monitor and control changes in room temperature; insulate column or use column oven; cover refractive index detector and keep it out of air currents.
Baseline noise Continous - detector lamp problem or dirty cell Replace UV lamp( each should last 2000 h; clean and flush flow cell.
Gradient or isocratic proportioning - lack of solvent mixing Use proper mixing device; check proportioning precision by spiking one solvent with UV absorbing compound and mointor UV absorbance detector outputl.
Gradient or isocratic proportioning - malfunctioning proportioning valvesl Clean or replace proportioning precision valves; partially remix solventsl.
Occasional sharp spikes - external electrical interference Use voltage stabilizer for LC system; use independent electrical circuit.
Periodic - pump pulses Service or replace pulse damper; purge air from pump; clean or replace check valves.
Random - contamination buildup Flush column with strong solvent; clean up sample; use HPLC grade solvent
Spikes - bubble in detector Degas mobile phase; use back pressure restrictor at detector outlet.
Spikes - column temperature higher than boiling point of solvent Use lower column temperature.

Pressure

Problem Possible cause Solution
Decreasing Pressure Insufficient flow from pump Loosen cap on mobile phase reservior
Leak in hydralic lines from pump to column Tighten or replace fittings; tighten rotor in injection valve
Leaking pump check valve or seals Replace or clean check valves; replace pump seals.
Pump cavitation Degas solvent; check for obstruction in line from solvent reservoir to pump; replace inlet-line frit
Fluctuating pressurre Bubble in pump Degas solvent; purge solvent with helium
Leaking pump check valve or seals Replace or clean check valves; replace pump seals
High Back Pressure Column blocked wth irreversibly adorbed sample Improve sample cleanup; use guard column; reverse-flush column with strong solvent to dissolve blockage
Column particle size too small (for example 3 micrometers) Use larger particle size (for example 5 micrometer)
Microbial growth on column Use at least 10% organic modifier in mobile phase; use fresh buffer daily; add 0.02% sodium azide to aqueous mobile phase; store column in at least 25% organic solvent without buffer
Mobile phase viscosity too high Use lower viscosity solvents or higher temperature
Plugged frit in in-line filter or guard column Replace frit or guard column
Plugged inlet frit Replace endfitting or frit assembly
Polymetric columns - solvent change causes swelling of packing Use correct solvent with column; change to proper solvent compositionl consult manufacturer's solvent-compatibility chartl use a column with a higher percentage of cross-linking
Salt precipitation (especially in reversed-phase chromatography with high concentration of organic solvent in mobile phase) concentration of organic solvent in mobile phase) Ensure mobile phase compatibility with buffer concentration; decrease ionic strength and water-organic solvent ratio; premix mobile phase
When injector disconnected from column - blockage in injector Clean injector or replace rotor
Increasing Pressure Blocked flow lines Systematically disconnect components from detector end to column end to find blockage; replace or clean blocked component
Particulate buildup at head of column Filter sample; use .5 micrometer in-line filter; disconnect and backflush column; replace inlet frit
Water-organic solvent systems - buffer precipitation Ensure mobile phase compatibility with buffer concentration; decrease ionic strength or water organic solvent ratio

Peaks

Problem Possible cause Solution
Broad peaks Analytes eluted early due to sample overload Dilute sample 1:10 and reinject
Detector-cell volume too large Use smallest possible cell volume consistent with sensitivity needs; use detector with no heat exchanger in system
Injection volume too large Decrease solvent strength of injection solvent to focus solute; inject smaller volume
Large extra column volume Use low- or zero-dead-volume endfittings and connectors; use smallest possible diameter of connecting tubing (<0.10 in. i.d.); connect tubing with matched fittings
Mobile-phase solvent viscosity too high Increase column temperature; change to lower viscosity solvent
Peak dispersion in injector valve Decrease injector sample loop size; introduce air bubble in front and back of sample in loop
Poor column efficiency Use smaller-particle-diameter packing, lower-viscosity mobile phase, higher column temperature, or lower flow rate
Retention time too long Use gradient elution or stronger isocratic mobile phase
Sampling rate of data system too low Increase sampling frequency.
Slow detector time constant Adjust time constant to match peak width
Some peaks broad - late elution of analytes retained from previous injection Flush column with strong solvent at end of run; end gradient at higher solvent concentration
Ghost peaks Contamination Flush column to remove contaminatint; use HPLC-grade solven
Elution of analytes retained from previous injection Flush column with strong solvent at end of run; end gradient at higher solvent concentration
Ion-pair chromatography - upset equilibrium Prepare sample in mobile phase; reduce injection volume
Oxidation of trifluoroacetic acid in peptide mapping Prepare trifluoroacetic acid solutions fresh daily; use antioxidant
Reversed-phase chromatography - contaminated water Check suitability of water by running different amounts through column and measure peak height of interferences as function of enrichment time; clean water by running it through old reversed-phase column; use HPLC-grade water.
Unknown interferences in sample Use sample cleanup or prefractionation before injection.
Negative peaks Refractive index detection - refractive index of solute less than that of mobile phase Reverse polarity to make peak positive
UV-absorbance detection - absorbance of solute less than that of mobile phase Use mobile phase with lower UV absorbance; if recycling solvent, stop recycling when recycled solvent affects detection
Peak Doubling Blocked Frit Replace or clean frit; install 0.5-um porosity in-line filter between pump and injector to eliminate mobile-phase contaminants or between injector and column to eliminate sample contaminants
Coelution of interfering compound Use sample cleanup or prefractionation; adjust selectivity by changing mobile or stationary phase
Coelution of interfering compound from previous injection Flush column with strong solvent at end of ran; end gradient at higher solvent concentration
Column overloaded Use higher-capacity stationary phase; increase column diameter; decrease sample amount
Column void or channeling Replace column, or, if possible, open top endfitting and clean and fill void with glass beads or same column packing; repack column
Injection solvent too strong Use weaker injection solvent or stronger mobile phase
Sample volume too large Use injection volume equal to one-sixth of column volume when sample prepared in mobile phase for injection
Unswept injector flow path Replace injector rotor
Peak Fronting Channeling in column Replace or repack column
Column overloaded Use higher-capacity stationary phase; increase column diameter; decrease sample amount
Tailing Peaks Basic solutes - silanol interactions Use competing base such as triethylamine; use a stronger mobile phase; use base-deactivated silica-based reversed-phase column; use polymeric column
Beginning of peak doubling See peak doubling
Chelating solutes - trace metals in base silica Use high purity silica-based column with low trace-metal content; add EDTA or chelating compound to mobile phase; use polymeric column
Silica-based column - degradation at high pH Use polymeric, sterically protected, or high-coverage reversed-phase column; install silica gel saturatorcolumn between pump and injector
Silica-based column - degradation at high temperature Reduce temperature to less than 50 C
Silica-based column - silanol interactions Decrease mobile-phase pH to suppress silanol ionization; increase buffer concentration; derivatize solute to change polar interactions
Unswept dead volume Minimize number of connections; ensure injector rotor seal is tight; ensure all compression fittings arecorrectly seated
Void formation at head of column Replace column, or, if possible, open top endfitting and clean and fill in void with glass beads or samecolumn packing; rotate injection valve quickly; use injection valve with pressure bypass; avoid pressure shock
Spikes Bubbles in mobile phase Degas mobile phase; use back-pressure restrictor at detector outlet; ensure that all fittings are tight
Column stored without caps Store column tightly capped; flush reversed-phase columns with degassed methanol


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Signature: Dhanlal De Lloyd, Chem. Dept, The University of The West Indies, St. Augustine campus
The Republic of Trinidad and Tobago.
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