1) PRESSURE PROBLEMS IN HPLC?
Low or no pressure:
› The solvent inlet lines are not being immersed in the solvent. (Immerse the inlets into the solvents)
› No solvent in the reservoir. (Fill the reservoir with mobile phase)
› Leaks in the system. (Identify leaks and close the leaks)
› Air lock in the pump line. (Remove all the air and purge the system with mobile phase)
High pressure:
› Blocked tubing around injector or column inlet. (Wash the system with proper solvent)
› High viscous solvents. (Better to use less viscous solvents)
Fluctuating pressure:
› Poorly primed (purged) lines. (Purge sufficient time)
› Badly degassed solvents. (Degas the mobile phase properly)
› Column contamination. (Check the system with known good column)
2) RETENTION PROBLEMS IN HPLC?
Retention time changes from injection to injection:
› Insufficiently equilibrated system. (Equilibrate the system up to steady baseline)
› Ambient Temperature fluctuation. (Maintain the system environment properly)
› Pressure fluctuating. (See above)
› Column contamination. (Check the system with known good column)
Continuously increasing or decreasing Retention time:
› Solvent preparation. (Prepare M.P freshly by mixing correct proportions)
› Flow rate changes. (Check the flow rate by graduated volumetric flask)
› Solvent delivery system blockage. (Wash the system with proper solvent)
› Ambient Temperature variation. (Maintain the system environment properly)
› Insufficient equilibration. (Equilibrate the system up to steady baseline)
› System leak. (Identify leaks and close the leaks)
› Column contamination. (Check the system with known good column)
Increasing or decreasing to a new constant Retention time:
› Mobile phase inconsistencies. (Prepare M.P freshly by mixing correct proportions)
› Gradient delays. (Check the gradient and curves)
› Incorrect column. (Use proper column)
› Changes in the solvent flow rate. (Check the flow rate by graduated volumetric flask)
› Temperature change. (Maintain the system environment properly)
› Column contamination. (Check the system with known good column)
› Leak in the system. (Identify leaks and close the leaks)
3) WHAT ARE THE REASON FOR NEGATIVE PEAKS IN HPLC?
› Absorption of analyte is lower than the absorption of mobile phase. (Use less absorptive mobile phase- use all HPLC grade solvents)
Note: Generally in QC, all validated methods are used so Mobile phase may not be a problem. But we look for any contamination to mobile phase which are highly absorbing than analyte and also quality of solvents used.
› Air bubble observed in system. (Give purge injector & Needle wash)
› Detector signal polarity setting reversed-all peaks negative. (Correct it)
› Cabels reversed-all peaks negative. (Plug the cables correctly)
4) WHAT ARE THE REASONS FOR FLUCTUATION IN PEAK AREA WHEN THE SAME SAMPLE IS INJECTED MULTIPLE TIMES IN HPLC?
› Air bubble in the injector. (Perform needle wash sufficient time)
› Carry over. (Needle wash with proper solvent)
› Temperature changes. (Inject the sample after attaining sample temperature or Injector temperature).
Explanation: If you take the sample out of refrigerator and put it into auto injector it will slowly warm up to the injector temperature and the sample will expand. So that initially you will inject a larger mass of the sample than later. (when the sample has reached the injector temperature of the sample compartment). So peak areas decreases from run to run.
› Random variation of flow rate. (Monitor the back pressure & rectify if any problem).
› Integration problems. (Check and reprocess the data)
Ghost peaks & negative peaks produced by the mobile phase contaminations affect the integration.
› Sample itself can be a source of problem. (Samples like proteins).
a) Some proteins do not elute completely during the initial runs. An additional quantity is eluted in the subsequent runs. (If this occurs blank need to be run between the analysis)
b) Some proteins irreversibly adsorbed (bind to active sites) on some columns. The peak area obtained with brand new columns may increase slowly with respective injections. (Once these active sites are saturated reproducible peak areas obtained. Saturate the active sites with your analyte or other proteins before the actual injection).
c) Some proteins can also bind to the wall of the vial. So that peak area decreases from injection to injection. (Use specially made vials)
5) WHAT ARE THE REASONS FOR GHOST PEAKS?
› Column contamination (Flush the column)
› Carry over (Flush the column with strong solvent)
› Contaminated water (Use HPLC grade water)
› Unknown interferences in sample (Check for interferences)
6) WHAT ARE THE REASONS FOR PEAK DOUBLING?
› Blocked frit (Replace or clean the frit)
› Co-elution of interfering compound (Check for interferences)
› Column over loaded (Flush the column)
› Sample volume too large (Decrease the sample amount)
7) HOW TO TROUBLE SHOOT NOISE,DRIFT & SPIKE?
Noise:
☻Detector lamp problem or Dirty flow cell (Replace UV lamp or clean dirty flow cell).
☻Gradient or Isocratic proportioning (Lack of proper solvent mixing) - (Check the mixing device)
☻Homogeneity of Mobile phase - (Degas and sonicate well the M.P before)
Drift:
☻Positive drift (Absorbance of M.P-B) - (Use non uv absorbing M.P)
☻Positive drift (Contamination) - (Flush the column with strong solvent)
☻Negative drift (Absorbance of M.P-A) - (Use non uv absorbing M.P)
☻Wavy or Undulating drift (Temperature changes) -(Control the environment)
Spike:
☻Column stored without caps (Store column always in solvent by tightly capping)
☻Bubble in the detector (Degas the M.P)
☻External electrical interferences (Use Voltage stabilizer or UPS power supply)
☻Column temperature higher than boiling point of solvent (Use low column temperature than solvent boiling point)
Low or no pressure:
› The solvent inlet lines are not being immersed in the solvent. (Immerse the inlets into the solvents)
› No solvent in the reservoir. (Fill the reservoir with mobile phase)
› Leaks in the system. (Identify leaks and close the leaks)
› Air lock in the pump line. (Remove all the air and purge the system with mobile phase)
High pressure:
› Blocked tubing around injector or column inlet. (Wash the system with proper solvent)
› High viscous solvents. (Better to use less viscous solvents)
Fluctuating pressure:
› Poorly primed (purged) lines. (Purge sufficient time)
› Badly degassed solvents. (Degas the mobile phase properly)
› Column contamination. (Check the system with known good column)
2) RETENTION PROBLEMS IN HPLC?
Retention time changes from injection to injection:
› Insufficiently equilibrated system. (Equilibrate the system up to steady baseline)
› Ambient Temperature fluctuation. (Maintain the system environment properly)
› Pressure fluctuating. (See above)
› Column contamination. (Check the system with known good column)
Continuously increasing or decreasing Retention time:
› Solvent preparation. (Prepare M.P freshly by mixing correct proportions)
› Flow rate changes. (Check the flow rate by graduated volumetric flask)
› Solvent delivery system blockage. (Wash the system with proper solvent)
› Ambient Temperature variation. (Maintain the system environment properly)
› Insufficient equilibration. (Equilibrate the system up to steady baseline)
› System leak. (Identify leaks and close the leaks)
› Column contamination. (Check the system with known good column)
Increasing or decreasing to a new constant Retention time:
› Mobile phase inconsistencies. (Prepare M.P freshly by mixing correct proportions)
› Gradient delays. (Check the gradient and curves)
› Incorrect column. (Use proper column)
› Changes in the solvent flow rate. (Check the flow rate by graduated volumetric flask)
› Temperature change. (Maintain the system environment properly)
› Column contamination. (Check the system with known good column)
› Leak in the system. (Identify leaks and close the leaks)
3) WHAT ARE THE REASON FOR NEGATIVE PEAKS IN HPLC?
› Absorption of analyte is lower than the absorption of mobile phase. (Use less absorptive mobile phase- use all HPLC grade solvents)
Note: Generally in QC, all validated methods are used so Mobile phase may not be a problem. But we look for any contamination to mobile phase which are highly absorbing than analyte and also quality of solvents used.
› Air bubble observed in system. (Give purge injector & Needle wash)
› Detector signal polarity setting reversed-all peaks negative. (Correct it)
› Cabels reversed-all peaks negative. (Plug the cables correctly)
4) WHAT ARE THE REASONS FOR FLUCTUATION IN PEAK AREA WHEN THE SAME SAMPLE IS INJECTED MULTIPLE TIMES IN HPLC?
› Air bubble in the injector. (Perform needle wash sufficient time)
› Carry over. (Needle wash with proper solvent)
› Temperature changes. (Inject the sample after attaining sample temperature or Injector temperature).
Explanation: If you take the sample out of refrigerator and put it into auto injector it will slowly warm up to the injector temperature and the sample will expand. So that initially you will inject a larger mass of the sample than later. (when the sample has reached the injector temperature of the sample compartment). So peak areas decreases from run to run.
› Random variation of flow rate. (Monitor the back pressure & rectify if any problem).
› Integration problems. (Check and reprocess the data)
Ghost peaks & negative peaks produced by the mobile phase contaminations affect the integration.
› Sample itself can be a source of problem. (Samples like proteins).
a) Some proteins do not elute completely during the initial runs. An additional quantity is eluted in the subsequent runs. (If this occurs blank need to be run between the analysis)
b) Some proteins irreversibly adsorbed (bind to active sites) on some columns. The peak area obtained with brand new columns may increase slowly with respective injections. (Once these active sites are saturated reproducible peak areas obtained. Saturate the active sites with your analyte or other proteins before the actual injection).
c) Some proteins can also bind to the wall of the vial. So that peak area decreases from injection to injection. (Use specially made vials)
5) WHAT ARE THE REASONS FOR GHOST PEAKS?
› Column contamination (Flush the column)
› Carry over (Flush the column with strong solvent)
› Contaminated water (Use HPLC grade water)
› Unknown interferences in sample (Check for interferences)
6) WHAT ARE THE REASONS FOR PEAK DOUBLING?
› Blocked frit (Replace or clean the frit)
› Co-elution of interfering compound (Check for interferences)
› Column over loaded (Flush the column)
› Sample volume too large (Decrease the sample amount)
7) HOW TO TROUBLE SHOOT NOISE,DRIFT & SPIKE?
Noise:
☻Detector lamp problem or Dirty flow cell (Replace UV lamp or clean dirty flow cell).
☻Gradient or Isocratic proportioning (Lack of proper solvent mixing) - (Check the mixing device)
☻Homogeneity of Mobile phase - (Degas and sonicate well the M.P before)
Drift:
☻Positive drift (Absorbance of M.P-B) - (Use non uv absorbing M.P)
☻Positive drift (Contamination) - (Flush the column with strong solvent)
☻Negative drift (Absorbance of M.P-A) - (Use non uv absorbing M.P)
☻Wavy or Undulating drift (Temperature changes) -(Control the environment)
Spike:
☻Column stored without caps (Store column always in solvent by tightly capping)
☻Bubble in the detector (Degas the M.P)
☻External electrical interferences (Use Voltage stabilizer or UPS power supply)
☻Column temperature higher than boiling point of solvent (Use low column temperature than solvent boiling point)
