1. TROBLESHOOTING HIGH PERFORMANCE LIQIUD CHROMATOGRAPHY HPLC Reza Daj 6/21/2014

2. Today’s Overview Introduction 1 Troubleshooting Approach 2 Pressure Problems 3 Leaks 4 Component Malfunctions 5 Baseline Problems 6 Peak Problems 7 Data Problems 8

3. Introduction

4. T ROUBLESHOOTING A PPROACH Do It Yourself Reference Points Common Problems Safety Getting the Facts Isolating the Source repair Strategy Preventiv e Maintena nce

5. Pressure Problems High PressureLow Pressure Locating a Blockage Erratic or Cycling Pressure

6. Column Pressure 1.Column Length 2.Inner diameter 3.Packing size High Pressure

7. L OW P RESSURE Low flow resistanceLow flow resistance 1 Low flow rateLow flow rate 2 Low eluent viscosityLow eluent viscosity 3

8. Locating a Blockage

9. Erratic or Cycling Pressure Bubbles 1 Gradient Elution 2 Check valves 3

10. Leaks Leaks Location the source Pump Leaks Tube fitting Leaks Detector Leaks

11. Tube fitting Leaks

12. D ETECTOR L EAKS

13. P UMP L EAKS Piston Seal Tube Fitting Check Valve

14. Location the source

15. Component Malfunctions Component Malfunctions Component Malfunction s Electronic Problems Injector Problems Column Problems Pump and Gradient Detector Problems

16. With today's complex digital systems. Repairing electronic malfunctions usually requires an expert. But we provides suggestions for things that can be done before calling in an expert. Electronic Problem Fans Fuses Cables Boards

17. Detector Problems Bubbles 1 Setting 2 Lamps 3 Leaks/Blockage 4

18. Injector Problems Carryover-Manual Injector 1 Adsorption 2 Internal Leak/Bubbles 3 Siphoning 4 Blockage/External Leaks 5

19. Adsorption

20. Column Problems Repairs Storage Cleaning Connation

21. Pump Gradient Problems Priming/BubblesPriming/Bubbles 1 Grandaunt TestGrandaunt Test 2 Proportional ValvesProportional Valves 3 MixerMixer 4 Integral FiltersIntegral Filters 5

22. B ASELINE P ROBLEMS Noisy Baselines 1.PumpPump 2.GradientGradient 3.DetectorDetector 4.RecorderRecorder 5.Data SystemData System Drifting or Cycling Baseline 1.Equilibration 2.Late Peaks 3.Mixer 4.Temperature 5.Electrical 6.Gradient 7.Cell Problem

23. P UMP

24. G RADIENT

25. R ECORDER

26. D ATA S YSTEM

27. D ETECTOR

28. P EAK P ROBLEMS No Peak or Small PeaksNo Peak or Small PeaksNo Peak or Small PeaksNo Peak or Small Peaks1 Bad Peak ShapeBad Peak ShapeBad Peak ShapeBad Peak Shape2 Extra PeakExtra PeakExtra PeakExtra Peak3 Negative PeakNegative PeakNegative PeakNegative Peak4 Incorrect Retention TimeIncorrect Retention TimeIncorrect Retention TimeIncorrect Retention Time5 Poor ResolutionPoor ResolutionPoor ResolutionPoor Resolution6

29. N O P EAK OR S MALL P EAKS

30. B AD P EAK S HAPE Overload Sample Solvent Bad Columns Dead Volume

31. E XTRA P EAKS Late Peaks Carryover Peaks Ghost Peaks

32. N EGATIVE P EAKS

33. U NCORRECTED RETENTION TIME The Column The Eluent Temperature Equilibration

34. P OOR R ESOLUTION Theory Efficiency N Selectivity Retention K

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