Pitfalls and Errors of HPLC in Pictures

PREFACE

INTRODUCTION

PART I: Fundamentals
Chromatography
Chromatographic Figures of Merit
The Resolution of Two Peaks
Reduced Parameters
The Van Deemter Curve
Peak Capacity and Number of Possible Peaks
Statistical Resolution Probability: Simulation
Statistical Resolution Probability: Example
Precision and Accuracy of an Analytical Result
Standard Deviation
Variability of the Standard Deviation
Uncertainty Propagation
Reproducibility in Trace Analysis
Ruggedness
Calibration Curves
The HPLC Instrument
The Detector Response Curve
Noise
The Playground Presented as an Ishikawa Diagram
The Possible and the Impossible

PART II: Pitfalls and Sources of Error
Mixing of the Mobile Phase
Mobile Phase pH
Adjustment of Mobile Phase pH
Influence of the Acid Type and Concentration in the Eluent
Water as an Unintentional Additive in the Mobile Phase
Inadequate Purity of Mobile Phase Water
Inadequate Purity of a Mobile Phase Solvent
Inadequate Purity of a Mobile Phase Reagent
Incomplete Degassing
System Peaks and Quantitative Analysis
Sample Preparation with Solid Phase Extraction
Inadequate Stabilization of the Extraction Solvent
Poor Choice of Sample Solvent: Peak Distortion
Poor Choice of Sample Solvent: Tailing
Sample Solvent and Calibration Curve
Impurities in the Sample
Formation of a By-Product in the Sample Solution
Decomposition by the Sample Vial
Artifact Peaks from the Vial Septum
Formation of an Associate in the Sample Solution
Precision and Accuracy with Loop Injection
Injection Technique
Injection of Air
Sample Adsorption in the Loop
Extra-Column Volumes
Dwell Volume
Elution at t0
Classification of C18 Reversed Phases
Different Selectivity of C18 Reversed Phases
Different Batches of Stationary Phase
Chemical Reaction within the Column
Tailing of Phosphate Compounds in the Presence of Steel
Recovery and Peak Shape Problems with Proteins
Double Peaks from Stable Conformers
Influence of Temperature on the Separation
Thermal Non-Equilibrium within the Column
Influence of the Flow Rate on the Separation
Influence of Run Time and Flow Rate on Gradient Separations
UV Spectra and Quantitative Analysis
UV Detection Wavelength
Different Detection Properties of Diastereomers
Fluorescence Quenching by Air
Detector Overload in UV
Detector Overload in ELSD
Influence of the Retention Factor on Peak Height
Influence of the Flow Rate on Peak Area
Leaks in the HPLC Instrument
Impairment of Precision as a Result of Noise
Determination of Peak Area and Height at High Noise
Peak Height Ratios
Incompletely Resolved Peaks
Area Rules for Incompletely Resolved Peaks
Areas of a 1 : 10 Peak Pair
Heights of a 1 : 10 Peak Pair
Quantitative Analysis of a Small Peak
Incompletely Resolved Peaks with Tailing
Integration Threshold and Number of Detected Peaks
Detector Time Constant and Peak Shape
Quantitative Analysis in the 99% Range
Correlation Coefficient of Calibration Curves

PART III: Useful Strategies
Column Tests
Apparatus Tests
Wavelength Accuracy of the UV Detector
Internal Standards
A Linearity Test
Rules for Accurate Quantitative Peak Size Determination
High-Low Chromatography
Control Charts
Verification of the Analytical Result by Use of a Second Method
Description of Ruggedness
Rules for Passing On an HPLC Method
Quality Assurance in the Laboratory
Standard Operating Procedures
Method Validation
Some Elements of Validation
A Validation Example
System Suitability Test
From Repeatability to Reproducibility
Measurement Uncertainty
Formal Quality Assurance Systems

INDEX