Understanding High Performance Liquid Chromatography (HPLC): Hands-on Short Course for Intermediate Users


Understanding High Performance Liquid Chromatography (HPLC): Hands-on Short Course for Intermediate Users is a 2-day, hands-on course designed to explain the fundamentals of HPLC for users with some prior experience. The course goes beyond the basics of “how” to explain the “why” for HPLC practitioners seeking a greater understanding of HPLC and its applications to enhance daily use, troubleshooting, and method development skills.

The course is taught through a mixture of lectures, videos, pen and paper activities, hands-on HPLC analyses, chromatography simulations, and group discussions. The most unique feature of this course is the use of lab activities to show theoretical concepts happening in real time. The lab utilizes 4 comparable HPLCs allowing the participants to divide the experimental variations across the instruments and compile the outcomes to cover more ground in the 2-day period. Each activity concludes with strategic questions to illustrate the learning objectives and stimulate the learner to reflect on the demonstrated principles. The lecture content and lab activities are organized in a complementary manner to introduce the concepts, demonstrate through lab, then follow-up with further explanation and address student questions.

The course has a pharmaceutical undertone including discussion of common system suitability tests, quantitation approaches, and control strategies but can be modified as necessary to meet the learner’s specific interests.


The instructor, Dr. Jack Pender, is a curious and creative analytical chemist experienced as an HPLC user, researcher, and trainer with interests primarily supporting the small molecule pharmaceutical and cannabis industries. Dr. Pender specializes in explaining concepts simply, constantly asking questions in search of better understanding, designing practical activities for illustration, and trouble-solving/problem-shooting.

Location and Class Size

East Carolina University, Science and Technology Building, Third Floor, Lab 347 and Lecture (TBD)

Limited to 8 students (2 per HPLC)

Learning Objectives and Outline

Upon successful completion of the 2-day laboratory course the student will:

HPLC Equipment: Under the Covers With Your HPLC

  1. Be able to diagram/label the flow-path of a typical HPLC from the mobile phase sinker to the waste bottle
  2. Become familiar with the function of each component in the HPLC flow-path
  3. Understand the principles of the UV/VIS detector
  4. Become familiar with HPLC detector options beyond the variable-wavelength UV/VIS detector

HPLC Separations: The Basics and Beyond

  1. Understand the relationship between HPLC, liquid extraction, and the processes leading to analytes moving through an HPLC column
  2. Use common terminology and symbols to describe chromatograms and separation performance
  3. Be able to explain differences, benefits, and limitations between isocratic and gradient separations
  4. Gain appreciation for the sources and impact of band broadening on separation performance
  5. Learn theory, calculations, and practical considerations related to resolution, number of theoretical plates, signal-to-noise ratio, and tailing factor
  6. Gain appreciation for characteristics of the mobile phase, stationary phase, analyte, and instrumentation that impact the HPLC separation (%, chemistry, pH, temp, dimensions etc.)
  7. Be able to predict the direction of peak movement expected with changes in common HPLC method parameters
  8. Demonstrate understanding of separation principles through a friendly “Separation Contest”
  9. Become familiar with choosing separation options beyond reversed-phase chromatography

HPLC Troubleshooting: When Good Chromatography Goes Bad

  1. Gain appreciation and understanding of the loss of column performance expected with use including decreased resolution, increased peak tailing, increased pressure, and decreased sensitivity
  2. Gain appreciation for the variation realized between “equivalent” method conditions such as columns, instruments, instrument settings, solution preparations, analysts etc.
  3. Work with the instructor to solve real lab problems as they happen

Analytical HPLC: Integration, System Suitability, and Quantitation

  1. Learn to properly integrate peaks of various size and resolution
  2. Gain appreciation for the impact of tailing and resolution on integration performance
  3. Increase understanding of the role of specific system suitability tests in data reliability
  4. Expand understanding of common pharmaceutical calculations
  5. Increase awareness, understanding, and appropriate use of common quantitation and control strategies such as a multi-point calibration curve, bracketing standards, all standards and more

“The class greatly exceeded my expectations because I run HPLC everyday, but I would say this class is a huge asset to anyone with limited experience like myself.”     – From a confidential satisfied student, March 7th, 2018

Questions? Contact Dr. Jack Pender. Penderj15@ecu.edu

Register here.