E24: HPLC and UHPLC for Practicing Scientists 1 and 2: Fundamentals, Method Development, and Troubleshooting

Two-Day Course
Dates to be Determined; 8:30am – 5:00pm

Dr. Michael W. Dong, MWD Consulting, Norwalk, CT

This intermediate course will provide the analytical scientist with a clearer understanding and a solid working knowledge of the concepts, instrumentation, columns, applications, and best practices in method development and troubleshooting of HPLC and UHPLC (ultra-high-pressure liquid chromatography).  The focus is on pharmaceutical analysis of small molecule drugs.

Analysts, scientists, researchers, and managers in pharmaceutical and other industries who want to get an updated overview of fundamentals and best practice of modern HPLC/UHPLC in pharmaceutical analysis and other applications including HPLC method development, operation and troubleshooting.  A basic understanding of chemistry and HPLC with some hands-on experience is assumed.

Day 1: HPLC 1: Fundamentals:
  1. Concepts
    a. Introduction: The chromatographic process, advantages and limitations, and chromatographic modes (NPC, RPC, IEC, SEC)
    b. Concepts: Retention factor (k), separation factor (a), column efficiency (N), resolution (Rs), tailing factor (Tf), the resolution equation, the linear solvent strength theory, and column void volume (VM)
    c. Mobile phase factors (organic modifiers, pH, buffers), mass spec compatibility, isocratic and gradient separations, operating parameters (Flow, gradient time (tG), column temperature (T)), peak capacity (Pc)
  2. HPLC Columns, Trends and Selection Guides
    a. Column fundamentals: Glossary, Characteristics, and types, packing characteristics (support type, particle size, pore size), bonded phases, end-fittings, plate height (H), the van Deemter equation
    b. Trends: Trends of shorter and smaller columns and the use of high-purity silica, traditional and novel bonding chemistries, concept of “orthogonality,”  listing of common RPC columns, column selection guide
    c. Newer column types: Hybrids, charged surface hybrids, HILIC, sub-3 and sub-2 mm, and superficially porous particles (SPP)
  3. HPLC Instrumentation and Operating Principles
    a. System, Pump, and Autosampler. Integrated vs. modular system, solvent delivery system, low-pressure vs. high-pressure mixing, dwell volume, pump trends, autosampler types and trends
    b. Detectors: Operating principles of UV/Vis detection and characteristics, photodiode array detector (DAD), newer detectors for non-chromophoric analytes (ELSD, CAD, and CLND)
    c. MS, CDS, and Instrument Bandwidth. The mass spectrometer (MS), types and ESI interface, Data Chromatography System (CDS), peak integration strategy, instrumental bandwidth
    Day 2: HPLC 2:  Applications, UHPLC, Method Development, and Troubleshooting
  4. HPLC Applications and an Overview of UHPLC
    a. HPLC applications including pharmaceutical, biopharmaceuticals, food, environmental, chemical, and bioseparations
    b. UHPLC overview, perspectives, and benefits: UHPLC concepts, instrumentation, and benefits (fast separations, high-resolution analysis, and rapid method development)
    c. Potential issues and how to mitigate (viscous heating, operating nuances, compatibility to existing methods, injection precision, detector sensitivity vs. mixing volumes), method translation from HPLC to UHPLC, transition from HPLC to UHPLC
  1. HPLC Method Development and Validation
    a. Tradition strategy for HPLC method development and a survey of automated tools and software (DryLab, ACD AutoChrom, and Fusion QbD)
    b. 3-pronged template approach for rapid method development
    c. Use of a universal generic gradient method for the assay of multiple NCEs and stability-indicating assays with simple adjustments
    d. A brief overview of method validation and method transfer with case studies
  2. HPLC Operation and Troubleshooting
    a. Mobile phase and sample preparation and best practice in HPLC operation
    b. Maintenance and troubleshooting guide with case studies
    c. Diagnosing and solving problems (pressure, baseline, peak, data performance)

Dr. Michael W. Dong is a principal of MWD Consulting focusing on training and consulting on HPLC/UHPLC, method development, and pharmaceutical analysis.  He was formerly Senior Scientist at Genentech, Research Fellow at Purdue Pharma, and Senior Staff Scientist at Applied Biosystems/Perkin-Elmer.  He holds a Ph.D. in Analytical Chemistry from the City University of New York, and a certificate in Biotechnology from University California of Santa Cruz. He has 120+ publications including a bestselling book on chromatography (HPLC and UHPLC for Practicing Scientists, Wiley). He is an editorial advisory board member of LCGC North America, American Pharmaceutical Review, and Chinese American Chromatography Association.