HPLC Troubleshooting
Contents
1. Problem System Related Low unsteady system pressure High system pressure Noisy fluctuating drifting baseline Regular pulsing of the baseline The Chromatogram Tailing peaks Fronting tailing peaks Reason Leak Air in pump head Dirt in check valve check whether valve cannot close Blockage contamination Blockage precipitated buffer salts can happen when the system or user suddenly changes mobile phase composition from high organic to aqueous buffer or vice versa High viscosity mobile phase Small stationary phase particles Crushed particles sudden pressure spikes can cause porous silica to fracture and generate fines System contamination Age of the UV lamp Temperature fluctuations Higher UV absorption of either mobile phase A or B causes drift in gradient elution Air in pump head also causes pulsing of the back pressure Dirt in check valve also causes pulsing of the back pressure Bubble trapped in the flow cell the detector response changes dramatically when the detector outlet is temporarily blocked with a finger Wrong pH some peaks are tailing while others are symmetrical Void volumes all peaks are tailing Non specific interactions some all sample components can interact with active sites in the flowpath silanol groups metal surfaces of tubes and frits Channeling Viscous fingering happens when there is a large di2. Second disassemble check valve and sonicate Degas mobile phase and purge system The pH of the mobile phase should be 1 5 units or more above or below the pKa value of the analyte to have all molecules either in the charged or in the neutral state Check connections replace guard column replace column Replace column with an inert column replace metal tubing see inert PEEKsil tubing pages 238 239 add additives e g EDTA into mobile phase lower pH to lt 2 5 in order to protonate silanol groups Channeling indicates a serious problem with he column and the column needs replacing For the interim one can try to reverse the column flow direction Ey Try to match the viscosity of the sample wi he mobile phase Ideally always use mobi phase as diluent D Loss of ligands when the column is exposed to extreme pH or when the column is very old can ead to peak fronting Replace the column Reduce the amount of sample injected or use a column with a larger ID sce Analytical Science Method Development and Troubleshooting 253 Method Development and Troubleshooting 254 Problem Broad but symmetrical peaks Ghost peaks Shifting retention times Low sensitivity SGE Analytical Science Reason Column over loading sample volume too large Poor column efficiency Late eluting sample components from the previous injection Carry over from contaminated injector Co
3. fference between the viscosity of the sample and the viscosity of the mobile phase Stationary phase degradation Column over loading HPLC Troubleshooting Resolution Check all connections and tighten connections replace seals Degas mobile phase and purge system Firstly try purging system at high flow rate to dislodge contamination Secondly disassemble check valve and sonicate Open connections sequentially from the detector back to the pump to locate blockage Flush capillaries replace in line filters or guard columns clean injector valve reverse column low without detector in line depending on where the blockage was located Disconnect column and flush with pure water at low flow rate to dissolve buffer salts again ncrease temperature change mobile phase or decrease flow rate ncrease temperature reduce flow rate use shorter column Replace the column see ProteCol HPLC columns pages 208 210 Disconnect column and rinse system with a combination of acid 10 nitric acid or 15 phosphoric acid for a short period of time followed by water and a organic wash of 75 acetonitrile 25 IPA over night Do NOT run the acid through the column Replace the UV lamp Use column oven Use HPLC grade solvents check UV cut off values for mobile phase components change to higher wavelength Degas mobile phase and purge system First try purging system at high flow rate to dislodge contamination
4. mn with smaller particles reduce extra column volumes Use stronger eluent use gradient elution Use inert HPLC system use inert HPLC columns use mobile phase additives to reduce non specific binding
5. ntaminated mobile phase A in a gradient elution Air bubbles Electronic interference Change in temperature Mobile phase not mixed properly Solvent evaporation Column contamination Wrong wavelength weak chromophore Broad peaks Sample loss due to non specific binding HPLC Troubleshooting Resolution Reduce the amount of sample injected or use a column with a larger ID Optimize running conditions flow rate temperature use column with smaller particles reduce extra column volumes Use double injections late eluters only appear in the second run Extend run time use strong eluting wash step use gradient Clean system injector until obtaining a clean blank Make fresh mobile phase Use only HPLC grade solvents Air bubble cause very sharp spikes Degas solvents Check for source of interference Use independent power source Use column oven or operate in a temperature controlled laboratory Make sure the mobile phase is well mixed isocratic or the solvent mixer proportioning valve or pump heads A and B is working correctly gradient Make sure solvent bottles are capped Build up of non eluting sample components change the selectivity of the column Introduce washing procedure at regular intervals Use photodiode array detector change detection mode for example to to fluorescence RI or electrochemical etc Optimize running conditions flow rate temperature use colu
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