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2010-11. UHPLC Tips and Techniques

November 1, 2010

Here are some practical pointers to get the most out of your UHPLC system.

LCGC28(11) 944-950 (2010)

John W. Dolan

Tags: chromatographic hygiene, cleanliness, column temperature, frictional heating, pressure selectivity, temperature selectivity, UHPLC, viscous heating

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← 2010-10. Are the Method Requirements Reasonable?
2010-12. Selectivity in Reversed-Phase LC Separations, Part I: Solvent-Type Selectivity →

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  • 2017-11s. The Best of LC Troubleshooting: A Farewell to John Dolan
  • 2017-10a. 34 Years of LC Troubleshooting: An Interview with John Dolan
  • 2017-10. All’s Well That Ends Well
  • 2017-9. How to Take Maximum Advantage of Column-Type Selectivity
  • 2017-8. Method Adjustment for Gradient Elution
  • 2017-7. Column Care for the Long Haul — Considerations for Column Storage
  • 2017-6. Method Adjustment the USP Way
  • 2017-5. Count the Cost, Part III: Increasing Resolution by Changing Selectivity
  • 2017-4. Count the Cost, Part II: Increasing Resolution by Increasing Retention
  • 2017-3. Count the Cost, Part I: Increasing Resolution by Increasing Column Efficiency
  • 2017-2. Filters and Filtration in Liquid Chromatography — What to Do
  • 2017-1. Back to Basics: The Role of pH in Retention and Selectivity
  • 2016-12. Failed System Suitability Test: A Case Study
  • 2016-11. Reader’s Question: Early Eluted Peak
  • 2016-10. Reader’s Questions: Gradient Ghost Peaks
  • 2016-9. How Does It Work? Part V: Fluorescence Detectors
  • 2016-8. How Does It Work? Part IV: Ultraviolet Detectors
  • 2016-7. How Does It Work? Part III: Autosamplers
  • 2016-6. How Does It Work? Part II: Mixing and Degassing
  • 2016-5. How Does It Work? Part I: Pumps
  • 2016-4. Retention Time Drift — A Case Study
  • 2016-3. Column Plate Number and System Suitability
  • 2016-2. Detective Work, Part IV: Chemical Problems with the Column — Chemical Attack
  • 2016-1. Detective Work, Part III: Strong Retention and Chemical Problems with the Column
  • 2015-12. Detective Work, Part II: Physical Problems with the Column
  • 2015-11. Detective Work, Part I: Simplify the Choices
  • 2015-10. How to Reduce Mobile-Phase Consumption
  • 2015-9. LC Column Problems Everywhere
  • 2015-8. Overload in Liquid Chromatography
  • 2015-7. Distorted Peaks — A Case Study
  • 2015-6. Internal Standard Calibration Problems
  • 2015-5. Calibration Problems — A Case Study
  • 2015-4. Be Careful of the Flow Rate
  • 2015-3. Listen to the Data
  • 2015-2. Slow Column Equilibration
  • 2015-1. Seven Things to Avoid in the Liquid Chromatography Laboratory
  • 2014-12. Column Protection: Three Easy Steps
  • 2014-11. How Much Can I Inject? Part II: Injecting in Solvents Other than Mobile Phase
  • 2014-10. How Much Can I Inject? Part I: Injecting in Mobile Phase
  • 2014-9. Estimating Resolution for Marginally Separated Peaks
  • 2014-8. How Much Retention Time Variation is Normal?
  • 2014-7. Mobile-Phase Degassing: What, Why, and How
  • 2014-6. UV Detector Problems
  • 2014-5. Readers’ Questions: Calibration
  • 2014-4. Problems with Large-Molecule Separations
  • 2014-3. LC Method Scaling, Part II: Gradient Separations
  • 2014-2. LC Method Scaling, Part I: Isocratic Separations
  • 2014-1. Column Dead Time as a Diagnostic Tool
  • 2013-12. Peak Fronting . . . Some of the Time
  • 2013-11. What’s the Problem with the LLOQ – A Case Study
  • 2013-10. Past, Present, and Future – 30 Years of LC Troubleshooting
  • 2013-9. Variable Retention Times – A Case Study
  • 2013-8. Gradient Elution, Part VI: Ghost Peaks
  • 2013-7. Gradient Elution, Part V: Baseline Drift Problems
  • 2013-6. Gradient Elution, Part IV: Dwell-Volume Problems
  • 2013-5. Gradient Elution, Part III: Surprises
  • 2013-4. Gradient Elution, Part II: Equivalent Separations
  • 2013-3. Gradient Elution, Part I: Intuition
  • 2013-2. The Importance of the Sample Matrix
  • 2013-1. Making the Most of a Gradient Scouting Run
  • 2012-12. Avoiding Refractive Index Detector Problems
  • 2012-11. Evaluation of an LC Method
  • 2012-10. The Role of the Injection Solvent
  • 2012-9. Reader’s Questions
  • 2012-8. Questions from Afar
  • 2012-7. Troubleshooting Basics, Part IV: Peak Shape Problems
  • 2012-6. When Should an Internal Standard Be Used?
  • 2012-5. Extra Chromatographic Peaks – A Case Study
  • 2012-4. Retention Time Changes
  • 2012-3. Why Does an Improvement Make Things Worse?
  • 2012-2. A Case of Sporadic LC Assay Results
  • 2012-1. LC Trouble Everywhere
  • 2011-12. Troubleshooting Basics, Part III: Retention Problems
  • 2011-11. Locating Precision Problems
  • 2011-10. Column Triage
  • 2011-9. Troubleshooting Basics, Part II: Pressure Problems
  • 2011-8. How Fast Can a Gradient Be Run?
  • 2011-7. Troubleshooting Basics, Part I: Where to Start?
  • 2011-6. The Case of the Too Big Little Peak
  • 2011-5. Ghost Peak Investigation in a Reversed-Phase Gradient LC System
  • 2011-4. Selectivity in Reversed-Phase LC Separations, Part IV: Pressure Selectivity
  • 2011-3. Selectivity in Reversed-Phase LC Separations, Part III: Column-Type Selectivity
  • 2011-2. A Picture is Worth a Thousand Words
  • 2011-1. Selectivity in Reversed-Phase LC Separations, Part II: Solvent-Strength Selectivity
  • 2010-12. Selectivity in Reversed-Phase LC Separations, Part I: Solvent-Type Selectivity
  • 2010-11. UHPLC Tips and Techniques
  • 2010-10. Are the Method Requirements Reasonable?
  • 2010-9. Speeding Up Gradients
  • 2010-8. System Contamination
  • 2010-7. Where Did That Peak Come From?
  • 2010-6. 300 and Counting
  • 2010-5. Too Much Scrutiny
  • 2010-4. Getting the Most Out of Calibration Standards
  • 2010-3. Enhancing Signal-to-Noise
  • 2010-2. Column Diameter, Linear Velocity, and Column Efficiency
  • 2010-1. Too Little or Too Much
  • 2009-12. LC Troubleshooting Strategies
  • 2009-11. Kinetic Plots Made Easy
  • 2009-10. Integration Problems
  • 2009-9. Method or System?
  • 2009-8. Too Many Changes
  • 2009-7. Calibration Curves, Part V: Curve Weighting
  • 2009-6. Calibration Curves, Part IV: Choosing the Appropriate Model
  • 2009-5. Calibration Curves, Part III: A Different View
  • 2009-4. Calibration Curves, Part II: What are the Limits?
  • 2009-3. Calibration Curves, Part I: To b or Not to b?
  • 2009-2. Testing Method Performance
  • 2009-1. Problems from East and West
  • 2008-12. LC Pumps
  • 2008-11. Extracolumn Effects
  • 2008-10. Drip, Drip, Drip…
  • 2008-9. Odds and Ends from the Inbox
  • 2008-8. Column Care
  • 2008-7. Peak Shape Problems
  • 2008-6. Check Valves and Acetonitrile
  • 2008-5. How Do I …?
  • 2008-4. Only Three Things
  • 2008-3. Method Transfer Problems
  • 2008-2. Ion Pairing — Blessing or Curse
  • 2008-1. Flow-Rate Adjustment and System Suitability
  • 2007-12. The Perfect Method, Part VII: The Gradient Shortcut
  • 2007-11. The Perfect Method, Part VI: Make It Faster
  • 2007-10. The Perfect Method, Part V: Changing Column Selectivity
  • 2007-9. The Perfect Method, Part IV: Controlling Peak Spacing
  • 2007-8. The Perfect Method, Part III: Adjusting Retention
  • 2007-7. The Perfect Method, Part II: Where to Start?
  • 2007-6. The Perfect Method, Part I: What is Your Goal?
  • 2007-5. How Does Temperature Affect Selectivity?
  • 2007-4. Variability — How to Control It
  • 2007-3. Solvent Conservation
  • 2007-2. How Little Can I See?
  • 2007-1. Questions From India
  • 2006-12. Method Reproducibility — A Case Study
  • 2006-11. On-Column Sample Degradation
  • 2006-10. Making Connections
  • 2006-9. What’s Happening to My Column?
  • 2006-8. Autosampler Carryover
  • 2006-7. Gradient Performance Problems — A Case Study
  • 2006-6. The Power of Mobile Phase Strength
  • 2006-5. Dwell Volume Revisited
  • 2006-4. It’s Gonna Break
  • 2006-3. Chromatography Forum — Use the Braintrust
  • 2006-2. Check Valve Problems
  • 2006-1. Obtaining More Consistent Results
  • 2005-12. The Role of Signal-to-Noise Ratio in Precision and Accuracy
  • 2005-11. Extending Column Life
  • 2005-10. LC Troubleshooting: The Basics
  • 2005-9. A Baker’s Dozen
  • 2005-8. Broad Peaks
  • 2005-7. Reader Questions: Mobile Phase
  • 2005-6. Peak Fronting, Column Life, and Column Conditioning
  • 2005-5. Overload or Minor Peak?
  • 2005-4. The Bermuda Triangle
  • 2005-3. Split Peaks — A Case Study
  • 2005-2. Extracolumn Effects
  • 2005-1. Stay Away from the Cliffs!
  • 2004-12. Retention Time Changes
  • 2004-11. My Favorite Shortcuts
  • 2004-10. Gradient Performance Checks
  • 2004-9. Grandma’s Ham
  • 2004-7. Divide and Conquer
  • 2004-6. But I Can’t Change the Method
  • 2004-5. System Suitability
  • 2004-4. Parallel Chromatography — Double Your Money
  • 2004-3. Success with Evaporative Light-Scattering Detection, Part II: Tips and Techniques
  • 2004-2. Selecting the Best Curve Fit
  • 2004-1. Broad Peaks
  • 2003-12. Understanding Split Peaks
  • 2003-11. Extracolumn Effects
  • 2003-10. How Much Is Enough?
  • 2003-9. Honoring Readers
  • 2003-7. Why Do Peaks Tail?
  • 2003-6. Now What’s Going On?
  • 2003-5. Not Another Leak!
  • 2003-4. Flow Rate and Peak Spacing
  • 2003-3. LC Columns — The Top-10 List
  • 2003-2. Success with Evaporative Light-Scattering Detection
  • 2003-1. Mobile-Phase Buffers, Part III — Preparation of Buffers
  • 2002-12. Mobile-Phase Buffers, Part II — Buffer Selection and Capacity
  • 2002-11. Mobile-Phase Buffers, Part I — The Interpretation of pH in Partially Aqueous Mobile Phases
  • 2002-10. The Hazards of Adjusting Gradients
  • 2002-9. Performance Qualification of LC Systems
  • 2002-7. Resolving Minor Peaks
  • 2002-6. The Importance of Temperature
  • 2002-5. Peak Tailing and Resolution
  • 2002-4. Stability-Indicating Assays
  • 2002-3. The Future of LC Troubleshooting — 10 Years Later
  • 2002-2. Quiet, Please
  • 2002-1. Ion Suppression in LC-MS/MS — A Case Study
  • 2001-12. From the e-Mail Bag
  • 2001-11. Retaining Polar Compounds
  • 2001-10. Attacking Carryover Problems
  • 2001-9. When Ideal Isn’t Practical
  • 2001-7. Communicating with the Baseline
  • 2001-6. Buffers and Baselines
  • 2001-5. Autosamplers, Part II — Problems and Solutions
  • 2001-4. Autosamplers, Part I — Design Features
  • 2001-3. Reproducibility and Carryover — A Case Study
  • 2001-2. Autosampler Carryover
  • 2001-1. Unexpected Peaks
  • 2000-12. Reducing Column Diameter in Gradient Elution — A Case Study
  • 2000-11. The Problem of Late Elution — A Case Study
  • 2000-10. Scaling Gradient LC Methods to LC-MS
  • 2000-9. Reversed-Phase in 100% Water
  • 2000-7. Stainless Steel Surfaces in LC Systems, Part II — Passivation and Practical Recommendations
  • 2000-6. Stainless Steel Surfaces in LC Systems, Part I — Corrosion and Erosion
  • 2000-5. Starting Out Right, Part VI — The Scouting Gradient Alternative
  • 2000-4. Starting Out Right, Part V — Changing Column Conditions
  • 2000-3. Starting Out right, Part IV — Additional Variables to Control Selectivity
  • 2000-2. Starting Out Right, Part III — The Role of the Solvent in Controlling Selectivity
  • 2000-1. Starting Out Right, Part II — Measuring Satisfaction
  • 1999-12. Starting Out Right, Part I — Selecting the Tools
  • 1999-11. Reader’s Questions: Carryover, Mobile-Phase Temperature, and Column Care
  • 1999-10. Mobile-Phase Degassing — Why, When, and How
  • 1999-9. Poor Peak Shape: A Case Study
  • 1999-7. Priming Injections
  • 1999-6. How Much Is Too Much?
  • 1999-5. LC Troubleshooting On-Line with Chromatography Forum
  • 1999-4. Lessons in Column Washing
  • 1999-3. Late Elution and Carryover Peaks — A Case Study
  • 1999-2. Basic Compounds — Starting on the Right Foot
  • 1999-1.UV Detector Noise
  • 1998-12. Column Temperature Effects in Gradient Elution
  • 1998-11. Gradient Background Peaks — A Case Study
  • 1998-10. Autosampler Precision
  • 1998-9. From the Mail Bag — Dirty Loop, Precolumn Use, In-Line Degassing, and Injector Plumbing
  • 1998-7. Useful or Useless? — Part II
  • 1998-6. Useful or Useless? — Part I
  • 1998-5. Sample Overload — Friend or Foe?
  • 1998-4. Column Packing — What’s at the Bottom of It?
  • 1998-3. Extracolumn Band Broadening from Injection and Peak Transfer
  • 1998-2. Ghost Peaks and Aerated Sample Solvent
  • 1998-1. Scaling Gradients
  • 1997-12. Reproducibility Problems
  • 1997-11. A Valuable Source of Help
  • 1997-10. Baseline Problems — A Case Study
  • 1997-9. Retention Time Problems
  • 1997-7. Method Development, Peak Distortion, and Interfering Peaks
  • 1997-6. Maintaining Autosampler Performance
  • 1997-5. Anticipating Problems with a New Method
  • 1997-4. Air: Poison for Liquid Chromatographs
  • 1997-3. The System Logbook
  • 1997-2. Pump Preventive Maintenance
  • 1997-1. Method Quality, Standardization, and Peak Shape
  • 1996-12. When Good Isn’t Good Enough
  • 1996-11. What is the True Temperature?
  • 1996-10. Solvent Changeover and Column Equilibration
  • 1996-9. Improving an Ion-Pairing Method
  • 1996-7. Injection Loop Adsorption
  • 1996-6. Readers’ Questions about Mobile-Phase Additives
  • 1996-5. Noise Problems: A Case Study
  • 1996-4. Problems with Gradient Methods
  • 1996-3. Water Problems: A Case Study
  • 1996-2. Using a Gradient Scouting Run to Get Started
  • 1996-1. Injection Problems
  • 1995-12. A Second Shot at Some Troubleshooting Problems
  • 1995-11. Solvent Selection, Part III: Solvent Life and Degradation
  • 1995-10. Testing Autosampler Performance
  • 1995-9. Internal Standard Problems, Buffer Precipitation, and Column Storage
  • 1995-7. Pump Seal Problems
  • 1995-6. Mobile-Phase Proportioning Problems and Standard Operating Procedures
  • 1995-5. International Liquid Chromatography Problems
  • 1995-4. Odds and Ends from the Mailbox
  • 1995-3. Solvent Selection, Part II: Physical Properties
  • 1995-2. Problems with Water
  • 1995-1. Reader Questions
  • 1994-12. Case Study — Irreproducible Retention Times
  • 1994-11. Questions about Ion Pairing
  • 1994-10. Solvent Selection, Part I: UV Absorption Characteristics
  • 1994-9. Obtaining Separations, Part IV: Putting It All Together
  • 1994-7. Obtaining Separations, Part III: Adjusting Column Conditions
  • 1994-6. Obtaining Separations, Part II: Adjusting Selectivity
  • 1994-5. Obtaining Separations, Part I: A Look at Retention
  • 1994-4. Fixing Problems on the Cheap
  • 1994-3. The Dilemma
  • 1994-2. Choosing the Right Instrument
  • 1994-1. Extra Peaks
  • 1993-12. Worth Repeating
  • 1993-11. To Change or Not to Change?
  • 1993-10. What’s Wrong with Ethylparaben?
  • 1993-9. Readers Respond
  • 1993-7. Gradient Problems at Low Detection Wavelengths
  • 1993-6. Avoiding the Pitfalls of Published Methods
  • 1993-5. Aggressive Mobile Phases — A Case Study
  • 1993-4. Air Problems
  • 1993-3. Recycling Revisited
  • 1993-2. Amine Adsorption — A Case Study
  • 1993-1. Column Care and Feeding
  • 1992-12. Problem Isolation: Three More Things
  • 1992-11. Preventive Maintenance: Just Three Things
  • 1992-10. Ion-Pairing Problems
  • 1992-9. Buffer Precipitation Problems
  • 1992-7. LC Problem Guide
  • 1992-6. Solvent Recycling
  • 1992-5. Column Heating and Resolution — A Case Study
  • 1992-4. The Case of the Mystery Gradient Peak
  • 1992-3. LC Problems – Past, Present, and Future
  • 1992-2. Pump Shutdown, Sparging Problems, Double Peaks: Answers to Readers’ Questions
  • 1992-1. Extracolumn Effects
  • 1991-12. Sample Preparation, Guard Columns: Answers to Readers’ Questions
  • 1991-11. Retention-Time Problems: Answers to Readers’ Questions
  • 1991-10. NISA A Service Alternative
  • 1991-9. Electrochemical Detector and System Pressure Problems
  • 1991-7. Using Dead Time, Retention, and the Capacity Factor as Diagnostic Tools
  • 1991-6. Changing System Conditions
  • 1991-5. Liquid Chromatography Pump Refinements
  • 1991-4. Pumps: Operation and Maintenance
  • 1991-3. Retention-Time Drift A Case Study
  • 1991-2. Method Reproducibility, Leaks, and Check-Valve Problems
  • 1991-1. Sample Adsorption in Liquid Chromatography Injection Valves
  • 1990-12. Pump Problems
  • 1990-11. Retention-Time Variation: A Case Study
  • 1990-10. Column and Mobile Phase Problems
  • 1990-9. Baseline Noise: A Case Study
  • 1990-7. Ghost Peaks and Column Problems
  • 1990-6. Peak Tracking
  • 1990-5. Column and Detector Problems
  • 1990-4. Problems in Size-Exclusion Chromatography
  • 1990-3. Mobile Phase Buffer Problems
  • 1990-2. Problems with Small-Volume Columns
  • 1990-1. System-to-System Variation
  • 1989-12. Isolating and Correcting LC Problems
  • 1989-11. Preventive Maintenance of Sample-Injection Valves
  • 1989-10. Readers’ Questions
  • 1989-9. Passivation of Liquid Chromatography Components
  • 1989-7. Column Flushing Demystified
  • 1989-6. Peak Tailing
  • 1989-5. Readers’ Questions
  • 1989-4. Peak Problems
  • 1989-3. Troubleshooting Pump Problems
  • 1989-2. Readers’ Questions
  • 1989-1. Mobile Phase Proportioning Problems: A Case Study
  • 1988-12. Method Optimization and Maintenance Using a Resolution Map
  • 1988-11. Recognizing and Eliminating Noise Problems in Liquid Chromatography
  • 1988-10. Troubleshooting LC Fittings, Part II
  • 1988-9. Troubleshooting LC Fittings, Part I
  • 1988-7. Mobile Phase Proportioning Problems
  • 1988-6. Blocked Columns and Tubing
  • 1988-5. Late Peaks and Gradient Reproducibility
  • 1988-4. Rules of Thumb
  • 1988-3. Readers’ Questions
  • 1988-2. Ghost and Vacancy Peaks
  • 1988-1. More on Solvents
  • 1987-12. Shortcuts for LC Measurements
  • 1987-11. Column Stability: A Case Study
  • 1987-10. Mobile Phase Problems
  • 1987-9. Capacity Factor, Plate Number, Extracolumn Effects, and More
  • 1987-7. Readers’ Questions
  • 1987-6. Gradient Elution Separation Problems, Part II
  • 1987-5. Gradient Elution Separation Problems, Part I
  • 1987-4. Potpourri
  • 1987-3. Troubleshooting Autosamplers, Part II
  • 1987-2. Troubleshooting Autosamplers, Part I
  • 1987-1. Maintaining an LC System with Ease
  • 1986-12. Problems Resulting from Normal System Variability
  • 1986-11. Extracolumn Effects: Two Case Studies
  • 1986-10. Readers’ Questions
  • 1986-9. Readers’ Questions
  • 1986-7. LC User Survey: Data-System Problems
  • 1986-6. LC User Survey: Detector Problems
  • 1986-5. LC User Survey: Autosampler Problems
  • 1986-4. Readers’ Questions
  • 1986-3. Separation Artifacts III: Secondary-Retention Effects in Reversed-Phase Chromatography
  • 1986-2. Separation Artifacts II: Extracolumn Effects, Tailing, and Strong Retention Sites
  • 1986-1. Separation Artifacts I: Sample Overload and Injection-Solvent Problems
  • 1985-12. Sample Injection Valves
  • 1985.11 Equipment Usage
  • 1985-10. Troubleshooting
  • 1985-9. Troubleshooting
  • 1985-7. Troubleshooting
  • 1985-6. Troubleshooting
  • 1985-5. Common Column Problems
  • 1985-4. Troubleshooting
  • 1985-3. Troubleshooting
  • 1985-2. Connecting Tubing
  • 1985-1. Peak Shape
  • 1984-11. Autosamplers
  • 1984-10. Chromatographic Theory as a Problem-Isolation Aid: Part II
  • 1984-9. Chromatographic Theory as a Problem-Isolation Aid: Part I
  • 1984-8. Mobile Phase Preparation
  • 1984-7. Service Contracts
  • 1984-6. Optical Detectors Part III: Variable-Wavelength UV Detectors
  • 1984-5. Optical Detectors Part II: Fixed-Wavelength UV Detectors
  • 1984-4. Optical Detectors, Part I: General Principles
  • 1984-3. Piston-Diaphragm Pumps
  • 1984.2 Reciprocating Piston Pumps
  • 1984-1. Chromatographic Fittings: Proper Use to Minimize Dead Volume
  • 1983-8. Problem Isolation: Shortening Troubleshooting Time
  • 1984-7. Recordkeeping: Plan Ahead to Minimize Downtime
  • 1983-3. Troubleshooting: HPLC Sample Preparation and Introduction
  • 1983-2. Troubleshooting the Mobile Phase of Your HPLC System
  • 1983-1. An HPLC Troubleshooting Guide