Related links:

• Cleaning Dirty columns
  
• Changing column Frit

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

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 failure	Replace 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

Email About Home Copyright Disclaimer
Signature : Delloyd's Lab Tech resources, reagents and Instrumentation
Go Home