Slide 1: Tips and Tricks of HPLC System Troubleshooting
Tips and Tricks of Faster LC Analysis Without Capital Investment It may be easier than you think! Agilent Technologies Page 1 Tips and Tricks of HPLC System Troubleshooting Agilent Technologies Page 2 1
Slide 2: Troubleshooting Steps and HPLC System Components
Trouble Shooting Steps You Have Recognized There is a Problem! How Do You Fix It? •1st Did System Suitability or Calibration Sample Fail? – Make Blank Runs •2nd Review Method for Compliance – Is The Procedure Being Followed Properly? – Are Instrument Settings Correct? •3rd Ask More Questions! – When
Slide 3: Column Problems From LC GC User Survey
Column Problems From LC GC User Survey Column Problem Percentage of Respondents Back Pressure, Plugged Frits 24% Poor Reproducibility 16% Sample Recovery 14% Loss of Resolution 13% Instability 11% Voids 8.1% Leaks, Fittings 4.8% pH Range 2.0% Low Plate Count 2.0% Column Overload 2.0% Cost 1.7% Misce
Slide 4: Pressure Issues and Correcting High Back Pressure
Pressure Issues Column Observations Potential Problems High pressure - Plugged frit - Column contamination - Plugged packing Low Pressure - Leak - Flow Incorrect Page 7 Determining the Cause and Correcting High Back Pressure • Check pressure with/without column - many pressure problems are due to bl
Slide 5: Correcting Overpressure
Correcting Overpressure Determining the Cause and Correcting High Back Pressure Many pressure problems relate to blockages in the system. Check system pressure with / without column If column pressure is high: • Back flush column (care regarding future performance) • Clear blocked frit (reverse flus
Slide 6: Buffer solubility
Buffer solubility Table I presents an estimate of the soluble concentration of the least soluble buffer (potassium phosphate at pH 7.0) in three organic cosolvents Page 11 Solubility of five buffers in mixtures with (a) methanol, (b) acetonitrile, and (c) tetrahydrofuran methanol acetonitrile THF So
Slide 7: Changing a Frit May Not Be a Good Idea
Changing a Frit May Not Be a Good Idea May not be possible with new generation columns May damage high performance columns Compression Column Ferrule Inlet Frit Wear gloves Do not allow bed to dry Do not touch the column - body heat will extrude packing Column Body Do not overtighten Female End Fitt
Slide 8: Inexpensive Filters Prevent Column Frit Plugging
Inexpensive Filters Prevent Column Frit Plugging Regenerated Cellulose (RC) Recommended •Universal hydrophilic membrane, compatible with most solvents - aqueous and organic •High purity, extremely low extractables •High recovery •More Uniform Surface •Different than Other Cellulose Filters!! In-line
Slide 9: Column Cleaning – Normal Phase
Column Cleaning – Normal Phase Use at least 50mL or 20−30 column volume changes for analytical columns • Typical normal phase solvent choices in order of increasing strength: Solvent Composition Methanol:Chloroform 50:50% Ethyl Acetate 100% Page 17 What Are Common Peak Shape Issues? 1. Split peaks 2
Slide 10: Peak Splitting Caused By Disrupted Sample Path
Peak Splitting Caused By Disrupted Sample Path •Flow Path Disrupted by Void •Sample Allowed to Follow Different Paths Through Column •Poorly Packed Bed Settles in Use •High pH Dissolves Silica Normal Double Peaks Split or Double Peaks Tip: Similar Effect Can be Caused by Partially Plugged Frit Page
Slide 11: Split Peaks from Injection Solvent Effects
Split Peaks from Injection Solvent Effects Column: StableBond SB-C8, 4.6 x 150 mm, 5 µm Mobile Phase: 82% H2O : 18% ACN Injection Volume: 30 µL Sample: 1. Caffeine 2. Salicylamide 1 A. Injection Solvent B. Injection Solvent 100% Acetonitrile Mobile Phase 2 2 1 0 10 0 10 Time (min) Time (min) Tip: In
Slide 12: Peak Shape: Tailing Peaks
Peak Shape: Tailing Peaks Causes Symmetry > 1.2 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. Heavy Metals. Bad Column. Normal T
Slide 13: Low pH Minimizes Secondary Interactions for Amines
Peak Tailing Low pH Minimizes “Secondary Interactions” for Amines Column: Alkyl-C8, 4.6 x 150 mm, 5µm Mobile Phase: 85% 25 mM Na2HPO4 : 15% ACN Flow Rate: 1.0 mL/min Temperature: 35°C Sample: 1. Phenylpropa nolamine 2. Ephedrine 3. Amphetamine 4. Methamphetamine 5. Phenteramine 1 1 2 3 pH 3.0 pH 7.0
Slide 14: Peak Tailing - Column Contamination
Peak Tailing - Column Contamination Tip: Quick Test to Determine if Column is Dirty or Damaged Trick: Reverse Column and Run Sample –If Improved, Possible Cleaning Will Help -No improvement-Column Damaged and Needs to be Replaced QC test after cleaning QC test forward QC test reverse direction 100%
Slide 15: Peak Tailing/Broadening Sample Load Effects
Peak Tailing/Broadening Sample Load Effects Columns: 4.6 x 150 mm, 5µm Mobile Phase: 40% 25 mM Na2HPO4 pH 7.0 : 60% ACN Flow Rate: 1.5 mL/min Temperature: 40°C Sample: 1. Desipramine 2. Nortriptyline 3. Doxepin 4. Imipramine 5. Amitriptyline 6. Trimipramine A. High Load Broadening Tailing C. x10 Com
Slide 16: Unknown Phantom Peaks
Unknown “Phantom” Peaks Column: Extend-C18, 4.6 x 150 mm, 5 µm Mobile Phase: 40% 10 mM TEA, pH 11 : 60% MeOH Flow Rate: 1.0 mL/min Temperature: R.T. Detection: UV 254 Sample: 1. Maleate 2. Pseudoephedrine 3. Chlorpheniramine 1 1 Sample 1: Chlorpheniramine maleate Sample 2 : Chlorpheniramine Peak 1:
Slide 17: Poorly Made HPLC System Connections Can Cause Peak Broadening
Peak Broadening Extra-Column Volume Column: StableBond SB-C18, 4.6 x 30 mm, 3.5 µm Mobile Phase: 85% H2O with 0.1% TFA : 15% ACN Flow Rate: 1.0 mL/min Temperature: 35°C Sample: 1. Phenylalan ine 2. 5-benzyl-3,6-dioxo-2-piperazine acetic acid 3. Asp-phe 4. Aspartame 1 1 10 mL extra-column 50 mL extra
Slide 18: Column Connectors Used in HPLC
Column Connectors Used in HPLC Troubleshooting LC Fittings, Part II. J. W. Dolan and P. Upchurch. LC/GC Magazine 6:788 (1988) Swagelok Waters 0.090 0.130 in. in. Parker Rheodyne 0.170 0.090 in. in. Valco Uptight 0.080 0.090 in. in. Page 35 What Happens If the Connections Poorly Made ? Wrong … too lo
Slide 19: Stainless Steel and Polymer Fittings
Stainless Steel and Polymer Fittings Which type is used and when? Stainless Steel (SS) fittings are the best choice for reliable high pressure sealing • Agilent uses Swagelok type fittings with front and back ferrules – which give best sealing performance – throughout all our LC systems PEEK (<400b
Slide 20: Metal Sensitive Compounds Can Chelate
Column Aging/Equilibration Causes Retention/Selectivity Changes Column 1 - After Cleaning Column 1 - Initial Column 1 - Next Day with 1% H3PO4 /Equilibration 1 1 2 2 0 3 5 9 12 15 0 3 5 9 12 15 Time (min) Time (min) • The primary analyte was sensitive to mobile phase aging/ conditioning of the colum
Slide 21: Acid Wash Can Improve Peak Shape
Acid Wash Can Improve Peak Shape Before Acid Wash After Acid Wash 50 – 100 mLs 1% H3PO4 HO OH OH HO OH OH 1. 2. OH 1. 2. OH Columns: ZORBAX SB-Phenyl 1 4.6 x 150 mm 2 1 Mobile Phase: 75% 25 mM ammonium phosphate buffer 25% ACN Flow Rate: 1.0 mL/min. 2 Temperature: RT Sample Size: 5 mL Tf: 3.7 Tf: 1.
Slide 22: Maintain Resolution by Minimizing Extra-Column Volume
Maintain Resolution for Low Volume Peaks by Minimizing Extra-Column Volume ECV = sample volume + connecting tube volume + fitting volume + detector cell volume Page 43 Tip: Dwell Volume Differences Between Instruments Can Cause Changes in Retention and Resolution VD = 0.43 mL Column: ZORBAX Rapid Re
Slide 23: Measure and Correct for Dwell Volume
Trick: Measure and Correct for Dwell Volume (VD) If VD1 > VD2 Compensate for longer VD1 by adding an isocratic hold to VD2, such that Hold + VD2 = VD1 If VD1 < VD2 Delay injection, such that VD2 - delay = VD1 Page 45 Mobile Phase pH and pH Buffers Why Are These So Important in HPLC? •pH Effects Ioni
Slide 24: Lot-to-Lot Selectivity Change Related to pH Choice
Minimize Change in Retention/Selectivity Lot-to-Lot Evaluate: • All causes of column-to-column change* • Method ruggedness (buffers/ionic strength) • pH sensitivity (sample/column interactions) *All causes of column-to-column change should be considered first, especially when only one column from a
Slide 25: Why Worry About pH? Weak Acids
Why Worry About pH? pH, pKa and Weak Acids [RCOO-][H+] RCOOH RCOO- _ + H+ Ka = [RCOOH] COOH COO + H+ Ka = 6.4 x 10-5 pKa = 4.2 At pH 4.2 – the sample exists as benzoic acid and the benzoate ion in a ratio of 1:1. Peak shape can be poor At pH 5.2 – 91% of the sample exists as the benzoate ion. RP ret
Slide 26: Why Worry About pH? Weak Bases
Why Worry About pH? pH, pKa and Weak Bases R3NH+ R3N + H+ [R3N][H+] Ka = [R3NH+] + Ka = 1 x 10-9 ∅ CH ∅ CH H 3 3 + H+ pKa = 9 CHOCH CH N 2 2 CHOCH CH N 2 2 ∅ CH ∅ CH 3 3 At pH 9 – the sample exists as protonated and unprotonated diphenhydramine in a ratio of 1:1. Peak shape can be poor. At pH 10 – 9
Slide 27: Importance of pH and Buffers: A Practical Example
Importance of pH and Buffers A Practical Example •Why the Sample Dictates Use •What Happens When Buffer Used Effectively •What Happens When Buffer Ignored or Used Improperly Page 53 Importance of pH and Buffers - A Practical Example Optimized Isocratic Conditions for Cardiac Drugs 1 Column: StableBo
Slide 28: I Don't Have Time to Make Buffers or Adjust pH
I Don’t Have Time to Make Buffers or Adjust pH … Column: StableBond SB-C18 4.6 x 150 mm, 5 mm Mobile Phase: A: 20% H2O B: 80% MeOH Flow Rate: 1.0 mL/min. Temperature: 35°C UV Detection: 254 nm Sample: Cardiac Drugs Even at very high % MeOH Most Components Strongly Retained with Poor peak Shape Due t
Slide 29: Commonly Used Buffers for Reversed Phase HPLC
Commonly Used Buffers for Reversed Phase HPLC Buffer pKa Buffer Range UV Cutoff (nm) Phosphate 2.1 1.1-3.1 200 7.2 6.2-8.2 12.3 11.3-13.3 Formic acid* 3.8 2.8-4.8 210 Acetic acid* 4.8 3.8-5.8 210 Citrate 3.1 2.1-4.1 230 4.7 3.7-5.7 5.4 4.4-6.4 Tris 8.3 7.3-9.3 205 Triethylamine* 11.0 10.0-12.0 200 P
Slide 30: Detection Issues
Don’t Forget - Match Column to pH of Mobile Phase for Maximum Column Lifetime High pH and Room Temperature (pH 11 RT) Mobile Phase: 50%ACN: 50% Water : 0.2% TEA Initial (~ pH 11) After 30 injections Tip: Use Columns Designed for chosen pH Page 59 Detection Issues Recognize Where the Problem Originat
Slide 31: Peak Shape: Negative Peaks and Ghost Peaks
Peak Shape: Negative Peaks Normal Negative Causes: Absorbance of sample is less than the mobile phase. Equilibrium disturbance when sample solvent passes through the column. Normal with Refractive Index Detectors. Page 61 Ghost Peaks 60 Ghost Peaks - Peaks which appear even when no sample is injecte
Slide 32: Noisy Baselines and Drifting Baselines
Noisy Baselines Time (min.) Possible Causes: Dirty Flow Cell Detector Lamp Failing Pulses from Pump if Periodic Temperature Effects on Detector Air Bubbles passing through Detector Page 63 Drifting Baselines Gradient Elution Temperature Unstable (Refractive Index Detector) Contamination in Mobile Ph
Slide 33: Chromatographic Results with Wrong Lamp at 214 nm
Chromatographic Results with “Wrong” Lamp at 214 nm Wavelength OEM Lamp Lamp from Generic Source Tip: Could also be a symptom of aging lamp Page 65 Expanded View of Chromatographic Results Generic Source Lamp at 214 nm Wavelength Peak 1 S/N = 150 OEM Lamp Peak 2 S/N = 400 Peak 3 S/N = 300 Peak 1 S/N
Slide 34: Effect of Detector Response Time and Conclusions
Effect of Detector Response Time The System is operating well-the settings were poorly made! Slow Data Rates Can Hinder Impurity Detection and Reduce Sensitivity Response Time 0.1 sec Agilent 1100 DAD 1st peak = 1.2 sec At 20 pts/sec = 24 pts/sec 0.2 sec Agilent 1100 WPS with ADVR Column: Poroshell