Trouble shooting in HPLC

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TROUBLESHOOTING IN HPLC : 

TROUBLESHOOTING IN HPLC BY SHEKHAR SHINDE

WHAT IS TROUBLE SHOOTING ? : 

WHAT IS TROUBLE SHOOTING ? Troubleshooting  is a form of problem solving most often applied to repair of failed products or processes. It is a logical, systematic search for the source of a problem so that it can be solved, and so the product or process can be made operational again. Troubleshooting is needed to develop and maintain complex systems where the symptoms of a problem can have many possible causes.

WHAT IS TROUBLE SHOOTING ? : 

WHAT IS TROUBLE SHOOTING ? Troubleshooting requires identification of the malfunction(s) or symptoms within a system. Then, experience is commonly used to generate possible causes of the symptoms. Determining which cause is most likely is often a process of elimination - eliminating potential causes of a problem. Finally, troubleshooting requires confirmation that the solution restores the product or process to its working state.

WHAT IS TROUBLE SHOOTING ? : 

WHAT IS TROUBLE SHOOTING ? In general, troubleshooting is the identification of, or diagnosis of "trouble" in a [system] caused by a failure of some kind. The problem is initially described as symptoms of malfunction, and troubleshooting is the process of determining the causes of these symptoms. This is the short but brief idea about the Trouble shooting.

TROUBLE SHOOTING ON HIGH PERFORMANCE LIQUID CHROMATOGRAPHY : 

TROUBLE SHOOTING ON HIGH PERFORMANCE LIQUID CHROMATOGRAPHY There are many causes in which problem are occured in HPLC. By Instrumental. By Procedure. By Human Errors. Because of this causes user have to face various problem such as Back pressure, Leakage, Change in Retention Time, peak shape and base line problem.

CONTENTS : 

CONTENTS START UP-PRELIMIARY CHECKS COLUMNS AND FITTINGS LEAKS CHANGE IN RETENTION TIME BASELINE PRESSURE PEAKS EQUILIBRATION SENSITIVITY

START UP PRELIMINARY CHECKS : 

START UP PRELIMINARY CHECKS PROBLEM:- No Peaks or Very Small Peaks. POSSIBLE CAUSE:- Detector off. Broken Connections to the Recorder. No sample/Wrong sample. Wrong settings on recorder or detector.

START UP PRELIMINARY CHECKS : 

START UP PRELIMINARY CHECKS SOLUTION:- Check detectors. Check Connections. Check Sample. Be Sure It Is Not Deteriorated. Checks For Bubbles In The Vials. Check Attenuation, check Gains.

START UP PRELIMINARY CHECKS : 

START UP PRELIMINARY CHECKS PROBLEM:- No Flow. POSSIBLE CAUSE:- Pump Off. Flow Interrupted. Leak. Air Trapped in the System.

START UP PRELIMINARY CHECKS : 

START UP PRELIMINARY CHECKS SOLUTION:- Start Pump. Check reservoirs. Check position of the inlet tubing. Check loop for obstruction or air. Check degassing of mobile phase. Check compatibility of the mobile phase components. Check fittings. Check pump for leaks and precipitates. Check pump seals. Disconnect column and prime pump. Flush system with 100% methanol. Contact servicing if necessary.

COLUMN AND FITTINGS LEAKS : 

COLUMN AND FITTINGS LEAKS PROBLEM:- Column End Leaks. POSSIBLE CAUSE:- Loose Fitting. SOLUTION:- Tighten or Replace Fitting Cut Tubing and Replace Ferrule; Disassemble Fitting, Rinse and Reassemble.

COLUMN AND FITTINGS LEAKS : 

COLUMN AND FITTINGS LEAKS PROBLEM:- Leak at Detector. POSSIBLE CAUSE:- Detector-seal Failure. SOLUTION:- Replace Detector Seal or Gaskets.

COLUMN AND FITTINGS LEAKS : 

COLUMN AND FITTINGS LEAKS PROBLEM:- Leak at Injection Valve. POSSIBLE CAUSE: Worn or Scratched Valve Rotor. SOLUTION:- Replace Valve Rotor.

COLUMN AND FITTINGS LEAKS : 

COLUMN AND FITTINGS LEAKS PROBLEM:- Leak at Pump. POSSIBLE CAUSE:- Pump Seal Failure. SOLUTION:- Replace Pump Seal; Check Piston for Scratches And, If Necessary, Replace.

CHANGE IN RETENTION TIME : 

CHANGE IN RETENTION TIME PROBLEM:- Changing Retention Times. POSSIBLE CAUSE:- Contamination Buildup. Equilibration time insufficient for gradient run or changes in isocratic mobile phase.

Slide 16: 

First few injections - active sites. Inconsistent on-line mobile-phase mixing. Selective evaporation of mobile-phase component. Varying column temperature.

CHANGE IN RETENTION TIME : 

CHANGE IN RETENTION TIME SOLUTION:- Use buffer with concentration greater than 20 mM. Flush column occasionally with strong solvent. Pass at least 10 column volumes through the column for gradient regeneration or after solvent changes. Condition column by injecting concentrated sample.

CHANGE IN RETENTION TIME : 

CHANGE IN RETENTION TIME Ensure gradient system is delivering a constant composition; compare with manually prepared mobile phase; partially premix mobile phase. Cover solvent reservoirs; prepare fresh mobile phase. Thermostat or insulate column; ensure laboratory temperature is constant.

CHANGE IN RETENTION TIME : 

CHANGE IN RETENTION TIME PROBLEM:- Decreasing Retention Times. POSSIBLE CAUSE:- Column overloaded with sample. Increasing flow rate. Loss of bonded stationary phase or base silica. Varying column temperature.

CHANGE IN RETENTION TIME : 

CHANGE IN RETENTION TIME SOLUTION:- Decrease sample amount or use larger-diameter column. Check and reset pump flow rate. Use mobile-phase pH between pH 2 and pH 8. Thermostat or insulate column; ensure laboratory temperature is constant.

CHANGE IN RETENTION TIME : 

CHANGE IN RETENTION TIME PROBLEM:- Increasing Retention Times. POSSIBLE CAUSE:- Decreasing flow rate. Changing mobile-phase composition. Loss of bonded stationary phase.

CHANGE IN RETENTION TIME : 

CHANGE IN RETENTION TIME SOLUTION:- Check and reset pump flow rate; check for pump cavitation; check for leaking pump seals and other leaks in system. Cover solvent reservoirs; ensure that gradient system is delivering correct composition. Use mobile-phase pH between pH 2 and pH 8.

BASELINE : 

BASELINE PROBLEM:- Void Time noise. POSSIBLE CAUSE:- Air bubbles in mobile phase. Positive-negative - difference in refractive index of injection solvent and mobile phase. SOLUTION:- Degas or use back pressure restrictor on detector. Normal with many samples; use mobile phase as sample solvent.

BASELINE : 

BASELINE PROBLEM:- Drifting baseline. POSSIBLE CAUSE:- Negative direction (gradient elution) - absorbance of mobile-phase A. Positive direction (gradient elution) - absorbance of mobile phase B. Positive direction - contamination buildup and elution. Wavy or undulating - temperature changes in room.

BASELINE : 

BASELINE SOLUTION:- Use non-UV absorbing mobile phase solvents; use HPLC grade mobile phase solvents; add UV absorbing compound to 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 mobile phase A.

BASELINE : 

BASELINE Flush column with strong solvent; clean up sample; use HPLC grade solvents. 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 : 

BASELINE PROBLEM:- Baseline noise. POSSIBLE CAUSE:- Continuous - detector lamp problem or dirty cell. Gradient or isocratic proportioning - lack of solvent mixing. Gradient or isocratic proportioning - malfunctioning proportioning valves.

BASELINE : 

BASELINE Occasional sharp spikes - external electrical interference. Periodic - pump pulses. Random - contamination buildup. Spikes - bubble in detector. Spikes - column temperature higher than boiling point of solvent

BASELINE : 

BASELINE SOLUTION:- Replace UV lamp( each should last 2000 h; clean and flush flow cell. Use proper mixing device; check proportioning precision by spiking one solvent with UV absorbing compound and monitor UV absorbance detector output. Clean or replace proportioning precision valves; partially remix solvents.

BASELINE : 

BASELINE Use voltage stabilizer for LC system; use independent electrical circuit. Service or replace pulse damper; purge air from pump; clean or replace check valves. Flush column with strong solvent; clean up sample; use HPLC grade solvent. Degas mobile phase; use back pressure restrictor at detector outlet. Use lower column temperature.

PRESSURE : 

PRESSURE PROBLEM:- Decreasing Pressure. POSSIBLE CAUSE:- Insufficient flow from pump. Leak in hydralic lines from pump to column. Leaking pump check valve or seals. Pump cavitation.

PRESSURE : 

PRESSURE SOLUTION:- Loosen cap on mobile phase reservoir. Tighten or replace fittings; tighten rotor in injection valve. Replace or clean check valves; replace pump seals. Degas solvent; check for obstruction in line from solvent reservoir to pump; replace inlet-line frit.

PRESSURE : 

PRESSURE PROBLEM:- Fluctuating pressure. POSSIBLE CAUSE:- Bubble in pump. Leaking pump check valve or seals. SOLUTION:- Degas solvent. Replace or clean check valves; replace pump seals.

PRESSURE : 

PRESSURE PROBLEM:- High Back Pressure. POSSIBLE CAUSE:- Column blocked with irreversibly adsorbed sample. Column particle size too small (for example 3 micrometers). Microbial growth on column. Mobile phase viscosity too high. Plugged frit in in-line filter or guard column.

PRESSURE : 

PRESSURE Plugged inlet frit. Polymetric columns - solvent change causes swelling of packing. Salt precipitation (especially in reversed-phase chromatography with high concentration of organic solvent in mobile phase) concentration of organic solvent in mobile phase). When injector disconnected from column - blockage in injector.

PRESSURE : 

PRESSURE SOLUTION:- Improve sample cleanup; use guard column; reverse-flush column with strong solvent to dissolve blockage. Use larger particle size (for example 5 micrometer). 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. Use lower viscosity solvents or higher temperature. Replace frit or guard column.

PRESSURE : 

PRESSURE Replace end fitting or frit assembly. Use correct solvent with column; change to proper solvent composition consult manufacturer's solvent-compatibility chart use a column with a higher percentage of cross-linking. Ensure mobile phase compatibility with buffer concentration; decrease ionic strength and water-organic solvent ratio; premix mobile phase. Clean injector or replace rotor.

PRESSURE : 

PRESSURE PROBLEM:- Increasing Pressure. POSSIBLE CAUSE:- Blocked flow lines. Particulate buildup at head of column. Water-organic solvent systems - buffer precipitation.

PRESSURE : 

PRESSURE SOLUTION:- Systematically disconnect components from detector end to column end to find blockage; replace or clean blocked component. Filter sample; use 0.5 micrometer in-line filter; disconnect and back flush column; replace inlet frit. Ensure mobile phase compatibility with buffer concentration; decrease ionic strength or water organic solvent ratio.

PEAKS : 

PEAKS PROBLEM:- Broad peaks.

PEAKS : 

PEAKS PROBLEM:- Broad peaks. POSSIBLE CAUSE:- Analytes eluted early due to sample overload. Detector-cell volume too large. Injection volume too large. Mobile-phase solvent viscosity too high.

PEAKS : 

PEAKS Peak dispersion in injector valve. Poor column efficiency. Retention time too long. Sampling rate of data system too low. Slow detector time constant. Some peaks broad - late elution of analytes retained from previous injection.

PEAKS : 

PEAKS SOLUTION:- Dilute sample 1:10 and reinject. Use smallest possible cell volume consistent with sensitivity needs; use detector with no heat exchanger in system. Decrease solvent strength of injection solvent to focus solute; inject smaller volume.

PEAKS : 

PEAKS Use low- or zero-dead-volume end fittings and connectors; use smallest possible diameter of connecting tubing (<0.10 in. i.d.); connect tubing with matched fittings. Increase column temperature; change to lower viscosity solvent. Decrease injector sample loop size; introduce air bubble in front and back of sample in loop. Use smaller-particle-diameter packing, lower-viscosity mobile phase, higher column temperature, or lower flow rate.

PEAKS : 

PEAKS Use gradient elution or stronger isocratic mobile phase. Increase sampling frequency. Adjust time constant to match peak width. Flush column with strong solvent at end of run; end gradient at higher solvent concentration.

PEAKS : 

PEAKS PROBLEM:- Ghost peaks.

PEAKS : 

PEAKS PROBLEM:- Ghost peaks. POSSIBLE CAUSE:- Contamination. Elution of analytes retained from previous injection. Ion-pair chromatography - upset equilibrium. Oxidation of trifluoroacetic acid in peptide mapping. Reversed-phase chromatography - contaminated water. Unknown interferences in sample.

PEAKS : 

PEAKS SOLUTION:- Flush column to remove contamination; use HPLC-grade solvent. Flush column with strong solvent at end of run; end gradient at higher solvent concentration. Prepare sample in mobile phase; reduce injection volume. Prepare trifluoroacetic acid solutions fresh daily; use antioxidant.

PEAKS : 

PEAKS 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.

PEAKS : 

PEAKS PROBLEM:- Negative peaks. POSSIBLE CAUSE:- Refractive index detection - refractive index of solute less than that of mobile phase. UV-absorbance detection - absorbance of solute less than that of mobile phase. SOLUTION:- Reverse polarity to make peak positive. Use mobile phase with lower UV absorbance; if recycling solvent, stop recycling when recycled solvent affects detection.

Slide 51: 

PROBLEM:- Peak Doubling.

PEAKS : 

PEAKS PROBLEM:- Peak Doubling. POSSIBLE CAUSE:- Blocked Frit. Co elution of interfering compound. Co elution of interfering compound from previous injection. Column overloaded. Column void or channeling. Injection solvent too strong. Sample volume too large.

PEAKS : 

PEAKS SOLUTION:- 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. Use sample cleanup or prefractionation; adjust selectivity by changing mobile or stationary phase. Flush column with strong solvent at end of run; end gradient at higher solvent concentration.

PEAKS : 

PEAKS Use higher-capacity stationary phase; increase column diameter; decrease sample amount. Replace column, or, if possible, open top end fitting and clean and fill void with glass beads or same column packing; repack column. Use weaker injection solvent or stronger mobile phase. Use injection volume equal to one-sixth of column volume when sample prepared in mobile phase for injection. Replace injector rotor.

Slide 55: 

PROBLEM:- Peak Fronting.

PEAKS : 

PEAKS PROBLEM:- Peak Fronting. POSSIBLE CAUSE:- Channeling in column. Column overloaded. SOLUTION:- Replace or repack column. Use higher-capacity stationary phase; increase column diameter; decrease sample amount.

Slide 57: 

PROBLEM:- Tailing Peaks.

PEAKS : 

PEAKS PROBLEM:- Tailing Peaks. POSSIBLE CAUSE:- Basic solutes - silanol interactions. Beginning of peak doubling. Chelating solutes - trace metals in base silica. Silica-based column - degradation at high pH. Silica-based column - degradation at high temperature. Silica-based column - silanol interactions.

PEAKS : 

PEAKS SOLUTION:- Use competing base such as triethylamine; use a stronger mobile phase; use base-deactivated silica-based reversed-phase column; use polymeric column. See peak doubling. Use high purity silica-based column with low trace-metal content; add EDTA or chelating compound to mobile phase; use polymeric column.

PEAKS : 

PEAKS Use polymeric, sterically protected, or high-coverage reversed-phase column; install silica gel saturator column between pump and injector. Reduce temperature to less than 50 C. Decrease mobile-phase pH to suppress silanol ionization; increase buffer concentration; derivatize solute to change polar interactions. Minimize number of connections; ensure injector rotor seal is tight; ensure all compression fittings are correctly seated. Replace column, or, if possible, open top end fitting and clean and fill in void with glass beads or same column packing; rotate injection valve quickly; use injection valve with pressure bypass; avoid pressure shock.

PEAKS : 

PEAKS PROBLEM:- Spikes. POSSIBLE CAUSE:- Bubbles in mobile phase. Column stored without caps. SOLUTION:- Degas mobile phase; use back-pressure restrictor at detector outlet; ensure that all fittings are tight. Store column tightly capped; flush reversed-phase columns with degassed methanol.

EQUILIBRATION : 

EQUILIBRATION PROBLEM:- Slow Column Equilibration Times. POSSIBLE CAUSE:- Equilibration time slow for long-chain ion pairing reagents. SOLUTION:- Use shorter alkyl chain reagent.

SENSITIVITY : 

SENSITIVITY PROBLEM:- Sensitivity. POSSIBLE CAUSE:- Peaks are outside Linear range of detector. Absorption of sample in loop or column. Autosampler flow lines blocked. Injector sample loop underfilled. Sample-related losses during preparation, optimize sample prep method.

SENSITIVITY : 

SENSITIVITY SOLUTION:- Dilute or concentrate to bring into linear range. Condition loop/column with concentrated sample. Check flow and make sure no blockages. Make sure that loop is overfilled with sample. Use internal standard during sample preparation.

Slide 65: 

ANY QUESTIONS ?

Slide 66: 

THANK YOU