HPLC Basics, Equipment, and Troubleshooting
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1 HPLC Basics, Equipment, and Troubleshooting HPLC Basics, Equipment, and Troubleshooting explains chromatography in practical terms from the ground up COURSE HANDOUT: [SECTION 14] Troubleshooting & Diagnostics Preventive Maintenance, Documentation, and Troubleshooting A Troubleshooting Checklist
2 HPLC Basics, Equipment, and Troubleshooting A course in the essentials of liquid chromatography Thomas H. Jupille John W. Dolan Lloyd R. Snyder 14-1 HPLC Basics, Equipment, & Troubleshooting Section 1. Isocratic HPLC Basics Section 2. Gradient HPLC Basics Section 3. HPLC Columns Section 4. Reversed-phase HPLC Section 5. ph & Ionization Effects Section 6. Normal-phase, Ion-exchange, & Size-exclusion Section 7. HPLC Equipment: Reservoirs & Pumps Section 8. HPLC Equipment: Tubing & Injectors Section 9. HPLC Equipment: Columns Section 10. HPLC Equipment: Detectors Section 11. Quantitation Section 12. UHPLC Section 13. Performance Qualification Section 14. Troubleshooting & Diagnostics Preventive Maintenance, Documentation, and Troubleshooting A Troubleshooting Checklist 14-2 HPLC Basics, Equipment, & Troubleshooting Section 1. Isocratic HPLC Basics Section 2. Gradient HPLC Basics Section 3. HPLC Columns Section 4. Reversed-phase HPLC Section 5. ph & Ionization Effects Section 6. Normal-phase, Ion-exchange, & Size-exclusion Section 7. HPLC Equipment: Reservoirs & Pumps Section 8. HPLC Equipment: Tubing & Injectors Section 9. HPLC Equipment: Columns Section 10. HPLC Equipment: Detectors Section 11. Quantitation Section 12. UHPLC Section 13. Performance Qualification Section 14. Troubleshooting & Diagnostics Preventive Maintenance, Documentation, and Troubleshooting A Troubleshooting Checklist 14-3
3 1st Commandment Know Thy System 14-4 The Troubleshooting Process Look for Changes pressure leaks chromatogram Confirm & Isolate Use Module Substitution 14-5 Module Substitution 14-6
4 Rules of Thumb Rule of 1 Rule of 2 Put it Back Crystal Ball Write it Down 14-7 Service Records (W 4 ) Who What When Why 14-8 Recordkeeping System Logbook configuration changes repairs routine maintenance system performance Sample Records LIMS 14-9
5 System Configuration brand model serial number lab i.d. number verify periodically Equipment or Method?? System Reference Test new column test periodic performance test record all settings Maintenance Records instrument: B76-04a system ID when who what why date: 4 April 2007 operator: J Dolan activity: new pump seals comments: routine 6-mo
6 System Suitability Test system suitability ( real( real ) ) sample normal operating conditions system suitability parameters plate number resolution retention pressure keep chromatogram Minimizing Problems It s cheaper to replace it beforeit breaks. target = 75-80% of lifetime /20 Rule We spend 80% of our time on 20% of the problems. 80/20 Corollary A 20% effort at preventive maintenance will prevent 80% of the problems LC Resources, Inc. All rights reserved
7 2nd Commandment Don t Make It a Hassle! HPLC Basics, Equipment, & Troubleshooting Section 1. Isocratic HPLC Basics Section 2. Gradient HPLC Basics Section 3. HPLC Columns Section 4. Reversed-phase HPLC Section 5. ph & Ionization Effects Section 6. Normal-phase, Ion-exchange, & Size-exclusion Section 7. HPLC Equipment: Reservoirs & Pumps Section 8. HPLC Equipment: Tubing & Injectors Section 9. HPLC Equipment: Columns Section 10. HPLC Equipment: Detectors Section 11. Quantitation Section 12. UHPLC Section 13. Performance Qualification Section 14. Troubleshooting & Diagnostics Preventive Maintenance, Documentation, and Troubleshooting A Troubleshooting Checklist What s Wrong With My Method/Results? (assuming an intially validated/working method) 1. No peaks 2. Missing peaks 3. Extra peaks 4. Wrong retention time 5. Poor efficiency or peak shape 6. Poor resolution 7. Poor precision 8. Baseline noise or drift 9. Pressure problems 10. Leaks 11. Column lifetime 14-18
8 Troubleshooting CheckList (1/7) 1. No peaks (at all!) No flow Mobile phase (%B, ph) Detector (functioning? wavelength?) Injector Sample stability Column chemistry (sorption?) 2. Missing (some) peaks Mobile phase (%B, ph) Sample stability Column chemistry (sorption?) Troubleshooting CheckList (2/7) 3. Extra peaks Carryover Late eluters Contamination Contamination Gradient? Autosampler? Sample prep Sample stability They are real? Troubleshooting CheckList (3/7) 4. Wrong retention Flow Mobile phase Column chemistry Temperature 14-21
9 Troubleshooting CheckList (4/7) 5. Poor efficiency or peak shape Injection volume / Strong diluent Overload Column chemistry Partial co-eluting peak Inlet flow profile anomaly Mobile phase (ph? Buffer?) Temperature Sample stability On-column degradation Extra-column volume Troubleshooting CheckList (5/7) 6. Poor resolution Retention Efficiency Selectivity 7. Poor precision Injector Sample prep Integration (signal/noise) Sample degradation Erratic flow Temperature Detector (wavelength?) Troubleshooting CheckList (6/7) 8. Baseline noise Electronic Flow Bubbles Temperature Contamination (gradient Drift Gradient Equilibration Temperature Electronic 14-24
10 Troubleshooting CheckList (7/7) 9. Pressure Leaks Flow Blockage 10. Leaks Overpressure Fitting (assembly? wear?) 11. Column lifetime Sample prep ph / temperature Column chemistry Method robustness The HPLC Troubleshooting Wizard Getting More Help instrument manuals reference books Dolan & Snyder, Troubleshooting LC Systems,, Humana mfg hot-line service calls john.dolan@lcresources.com tjupille@lcresources.com 14-27
11 HPLC Basics, Equipment, & Troubleshooting Section 1. Isocratic HPLC Basics Section 2. Gradient HPLC Basics Section 3. HPLC Columns Section 4. Reversed-phase HPLC Section 5. ph & Ionization Effects Section 6. Normal-phase, Ion-exchange, & Size-exclusion Section 7. HPLC Equipment: Reservoirs & Pumps Section 8. HPLC Equipment: Tubing & Injectors Section 9. HPLC Equipment: Columns Section 10. HPLC Equipment: Detectors Section 11. Quantitation Section 12. UHPLC Section 13. Performance Qualification Section 14. Troubleshooting & Diagnostics Preventive Maintenance, Documentation, and Troubleshooing A Troubleshooting Checklist LC Resources, Inc. All rights reserved
12 For more information and to discuss your training requirements, contact: FOR NORTH AMERICA & EUROPE Dean Graimes T: M: FOR ASIA PACIFIC Jeroen Reiniers T: Frederick House, Princes Court Nantwich, CW5 6PQ, UK 20 Maxwell Road, Maxwell House Singapore
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