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Troubleshooting the Agilent 1100 HPLC

1. Troubleshooting the Agilent 1100 HPLC Agilent Technologies Innovating the HP Way In This Section You Will Learn e General troubleshooting strategy e Common Problems Pressure Reproducibility e Retention time Rt e Area Linearity Baseline noise Sensitivity Peak shape yl Agilent Technologies T Innovating the HP Way Important HPLC Parameters Pressure Baseline Noise These parameters can tell you if your er MOE PISO B is experiencing problems eArea precision ePeak height precision ePeak shape Sensitivity se Agilent Technologies Innovating the HP Way Why Do You Care if Your System Is Functioning Properly Quantitative Analysis Changes in flow rate caused by pumping problems result in incorrect area counts eNoisy baselines will interfere with integration and decrease signal to noise ratio ePoor autosampler maintenance will result in poor injection volume reproducibility Qualitative Analysis Retention time fluctuations resulting in incorrect identifications Spectral analysis difficulties se Agilent Technologies Innovating the HP Way Troubleshooting Key Concept Divide and Conquer Test Mix 01080 68704 AJL Test column Record length internal diameter stationary phase particle diameter HPLC Test Sample Keep it simple Test sample 2 3 Analytes chemically stable in sufficient concentration Separation method Solvent flow rate tempe
2. n Volume 1pL Injection 10 Aoinwe EX LS COINWU Use short small internal diameter tubing between the injector and the column and between the column and the detector Make certain all tubing connections are made with matched fittings Use a low volume detector cell Inject small sample volumes Agilent Technologies Innovating the HP Way 10 Peak Shape Tailing Symmetry gt 1 2 y Causes Some Peaks Tail Secondary Retention Effects Residual Silanol Interactions Small Peak Eluting on Tail of Larger Peak Normal Tailing All Peaks Tail Extra Column Effects Build up of Contamination on Column Inlet sHeavy Metals m Bad Column Normal Tailing Agilent Technologies Innovating the HP Way Peak Shape Fronting Peaks i all P Time min Normal Fronting Symmetry lt 0 9 Causes gt Column Overload gt Small Band Eluting Before Large Band Agilent Technologies Innovating the HP Way Review of Important Tests Leak test Temperature stability amp accuracy Baseline noise drift Wavelength accurac ee Detector linearity Autosampler carry over a os Gee eer Autosampler linearity Composition ripple o Agilent Technologies Innovating the HP Way Summary Troubleshooting Exclude application errors Take a methodical and systematic approach Localize error in a part of the system Change only one thing at a time Ask for help where appr
3. oducibility Peak retention time precision with oven lt 0 3 without oven lt 0 7 Typically Peak area precision lt 1 5 eArea and Peak Height problems together point to the autosampler system eArea and Retention Time problems together point to the pump 7 8 1 h 10th inj INTE JUUS 5th inj 1st inj a ii 0 4 12 Agilent Technologies a Innovating the HP Way Problems with Reproducibility Peak Areas Peak Areas not Reproducible With peak height Rotor seal cross port leak or injection valve not tight Piston seal of metering unit leaking eNeedle partially blocked With retention time Variable pump flow rate Other Capillary from injector to detector not tight Detector equilibration problems Agilent Technologies Innovating the HP Way Problems with Reproducibiliy Retention Time A Retention Times not Reproducible e Pump Problems Mobile phase composition problems Valves AIV ball valve defective Flow rate problems eColumn Oven Problems Temperature fluctuations Other Column equilibration Column deterioration att Agilent Technologies a Innovating the HP Way Linearity Problems Peak Areas not Linear Autosampler Rotor seal cross port leak or injection valve not tight Piston seal of metering unit defective Needle partially blocked Detector Satuaration Agilent Technologies Innovating the HP Way Baseline Fluctuation
4. opriate Plan course of action Verify if problem has been eliminated test injection Periodic maintenance of the Agilent 1100 prevents downtime o Agilent Technologies Innovating the HP Way 12
5. rature pressure detection parameters Results tp peak width resolution peak area peak height baseline noise signal noise ratio of analytes Agilent Technologies Innovating the HP Way Problems with the System Pressure Pressure problems Pressure too high Pressure too low Pressure Unstable lt Agilent Technologies Innovating the HP Way Pressure Problem Pressuren Measurement Pressure Too High e Column inlet frit contaminated eFrit in purge valve contaminated e Column contaminated e Blockage in a capillary particularly needle seat capillary Ih Use this valve e Rotor in injection valve plugged 7 oo to divide the e Injection needle or needle seat plugged system ae Agilent Technologies o s Innovating the HP Way Pressure Measurement ra Pressure Problem Il Pressure Too Low e Solvent inlet frit plugged eLeak in a capillary connection or other part pump seals Solvent inl Wrong solvent or flow rate frits AIV Active inlet valve defective Multichannel Gradient valve incorrectly proportioning Ball valve defective Column defective stationaryphase Agilent Technologies Innovating the HP Way Pressure Problem Ill Pressure Fluctuation e Solvent inlet frits plugged e Solvent not degassed e Seals of the pump defective e Ball valve defective e AIV defective Solvent inl frits Usually an indication there is air in the pump Innovating the HP Way Repr
6. s Possible Causes Dirty Flow Cell Dirty mobile phase Detector Lamp Failing Pulses from Pump if Periodic Temperature Effects on Detector RI Air Bubbles passing through Detector Gradient elution Immiscible Solvents Agilent Technologies Innovating the HP Way Troubleshooting asell ol no gt Pump or Mobile Phase Detector Other Questions to Ask Have you changed your mobile phase composition gt Have you changed your acquisition wavelength gt What mobile phase was last used in your instrument gt Do you have a miscibility problem gt Are your solvents dirty Agilant Technologies Irina the HP Way Example Ghost Peaks Ghost Peaks Peaks which appear even on blank injections Problem Dirty Mobile Phase 20 100 MeOH Gradient No Sample Injected Agilant Technologies Innovating the HP Way Sensitivity Problems Peak response too low Causes e insufficient injection amount e Detector problems e Lamp aging e Detector cell contamiated e Solvent absorption high Agilent Technologies Innovating the HP Way Peak Problems Split Tailing Fronting Appendix age Agilent Technologies 2 Innovating the HP Way Peak Shape Doublets Void Volume in Column ol Normal Doublet Peaks Void Volume in Column Partially Blocked Frit Only One Peak a Doublet Coeluting Components oS Agilent Technologies T Innovating the HP Way Effect of Extra Colum

Troubleshooting the Agilent 1100 HPLC

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