HPLC and UHPLC for Practicing Scientists

Michael W. Dong

• Publisher: Wiley
• Year: 2019
• ISBN:
  9781119313779
• Edition: 2
• Topic: Naturwissenschaften
• Subtopic: Analytische Chemie

A concise yet comprehensive reference guide on HPLC/UHPLC that focuses on its fundamentals, latest developments, and best practices in the pharmaceutical and biotechnology industries

Written for practitioners by an expert practitioner, this new edition of HPLC and UHPLC for Practicing Scientists adds numerous updates to its coverage of high-performance liquid chromatography, including comprehensive information on UHPLC (ultra-high-pressure liquid chromatography) and the continuing migration of HPLC to UHPLC, the modern standard platform. In addition to introducing readers to HPLC's fundamentals, applications, and developments, the book describes basic theory and terminology for the novice, and reviews relevant concepts, best practices, and modern trends for the experienced practitioner.

HPLC and UHPLC for Practicing Scientists, Second Edition offers three new chapters. One is a standalone chapter on UHPLC, covering concepts, benefits, practices, and potential issues. Another examines liquid chromatography/mass spectrometry (LC/MS). The third reviews at the analysis of recombinant biologics, particularly monoclonal antibodies (mAbs), used as therapeutics. While all chapters are revised in the new edition, five chapters are essentially rewritten (HPLC columns, instrumentation, pharmaceutical analysis, method development, and regulatory aspects). The book also includes problem and answer sections at the end of each chapter.

• Overviews fundamentals of HPLC to UHPLC, including theories, columns, and instruments with an abundance of tables, figures, and key references
• Features brand new chapters on UHPLC, LC/MS, and analysis of recombinant biologics
• Presents updated information on the best practices in method development, validation, operation, troubleshooting, and maintaining regulatory compliance for both HPLC and UHPLC
• Contains major revisions to all chapters of the first edition and substantial rewrites of chapters on HPLC columns, instrumentation, pharmaceutical analysis, method development, and regulatory aspects
• Includes end-of-chapter quizzes as assessment and learning aids
• Offers a reference guide to graduate students and practicing scientists in pharmaceutical, biotechnology, and other industries

Filled with intuitive explanations, case studies, and clear figures, HPLC and UHPLC for Practicing Scientists, Second Edition is an essential resource for practitioners of all levels who need to understand and utilize this versatile analytical technology. It will be a great benefit to every busy laboratory analyst and researcher.

1
Introduction

1.1 INTRODUCTION

1.1.1 Scope

High‐performance liquid chromatography (HPLC) is a versatile analytical (separation) technique widely used for the analysis of pharmaceuticals, biomolecules, polymers, and many organic and ionic compounds. There is no shortage of excellent books on chromatography [1–3] and on HPLC [4–11], though many are outdated and others tend to focus more on academic theories or specialized topics. This book strives to be a concise and an all‐inclusive text that “capsulizes” the essence of HPLC fundamentals, applications, and developments. It describes the fundamental theories and terminologies for the novice and reviews relevant concepts, best practices, and modern trends for the experienced practitioner. While broad in scope, this book focuses on reversed‐phase HPLC (the most common separation mode) and pharmaceutical applications (the most significant user segment). Information is presented straightforwardly and is illustrated with an abundance of figures, chromatograms, tables, and case studies, supported by selected key references or web resources.

Most importantly, this book was written as an updated reference guide for busy laboratory analysts and researchers. Topics covered include HPLC operation, method development, maintenance/troubleshooting, and the regulatory aspects of pharmaceutical analysis. This book can serve as a supplementary text for students pursuing a career in analytical chemistry and pharmaceutical science. A reader with a science degree and a basic understanding of chemistry is assumed. This second edition continues the same theme as the first edition [4] with updates on all chapters plus three new chapters on ultra‐high‐pressure liquid chromatography (UHPLC), liquid chromatography–mass spectrometry (LC/MS), and the analysis of recombinant biologics (biopharmaceuticals). A quiz section at the end of each chapter serves as a teaching/evaluation aid.

This book offers the following benefits:

• A broad‐scope overview of fundamental principles, instrumentation, columns, and applications.
• A concise review of concepts and trends of modern HPLC.
• An update of best practices in HPLC operation, method development, maintenance, troubleshooting, and regulatory aspects in analytical testing.
• New standalone overview chapters on UHPLC, LC/MS, and analysis of recombinant biologics.

1.1.2 What Is HPLC?

Liquid chromatography (LC) is a physical separation technique conducted between two phases – a solid phase and a liquid phase. A sample is separated into its constituent components (or analytes) by distributing (via partitioning, adsorption, or other interactions) between the mobile phase (a flowing liquid) and a solid stationary phase (sorbents packed inside a column). For example, the flowing liquid can be an organic solvent such as hexane and the stationary phase can be the porous silica particles packed into a column. HPLC is a modern form of LC that uses small‐particle columns through which the mobile phase is pumped at high pressure.

Figure 1.1a is a schematic of the chromatographic process, where a mixture of components A and B are separated into two distinct bands as they migrate down the column filled with packing (stationary phase). Figure 1.1b is a microscopic representation of the dynamic partitioning process of the analytes between the flowing liquid and the stationary phase attached to a spherical packing particle. Note that the movement of component B is retarded in the column because each B molecule has a stronger affinity for the stationary phase than the A molecule. An inline detector monitors the concentration of each separated component band in the effluent and generates a signal trace called the “chromatogram,” shown in Figure 1.1c.

Figure 1.1 (a) Schematic of the chromatographic process showing the migration of two bands of components down a column. (b) Microscopic representation of the partitioning process of analyte molecules A and B into the stationary phase bonded to a solid spherical support. (c) A chromatogram plotting the signal from a UV detector displays the elution of components A and B.

1.1.3 A Brief History

The term chromatography meaning “color writing” was first used by Mikhail Tsvet, a Russian botanist who separated plant pigments on chalk (CaCO₃) packed in glass columns in 1903. Since the 1930s, chemists have used gravity‐fed silica columns to purify organic materials and ion‐exchange resin columns to separate ionic compounds and radionuclides. The invention of gas chromatography (GC) by the British biochemists A. J. P. Martin and R. L. M. Synge in 1952 and its successful applications provided the theoretical foundation and the incentive for the development of LC. In the late 1960s, LC turned “highperformance” with the use of small‐particle columns that required high‐pressure pumps. The first generation of HPLCs was developed by researchers in the 1960s, including Joseph Huber in Europe and Csaba Horváth and Jack Kirkland in the United States. Commercial development of ...