High Performance Liquid Chromatography (HPLC): Operation, Calibration, Troubleshooting and Maintenance

Introduction

This course offers no-nonsense, results-oriented training that will help you improve your bottom-line performance. Come prepared to discuss your analytical problems and challenges with expert chromatographers. By enrolling in this course, you will learn to master state-of-the-art techniques and equipment in HPLC with practical, hands-on experience. In the course's hands-on laboratory sessions, you will work on a variety of experiments, and in the process, become familiar with HPLC hardware; column dimensions; mobile phase strength, flow rate, and temperature; and internal vs. external HPLC is a technique for separation, identification and quantification of components in a mixture. It is especially suitable for compounds that are not easily volatilized, thermally unstable, and have high molecular weights. The liquid phase is pumped at a constant rate to the column packed with the stationary phase. Before entering the column, the analysis sample is injected into the carrier stream. On reaching the column the sample components are selectively retained on the basis of physic-chemical interactions between the analytic molecules and the stationary phase. The mobile phase moving at a steady rate elutes the components based on the operating conditions. Detection techniques are employed for the detection and quantification of the eluted components. High-performance liquid chromatography is basically a highly improved form of column chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much faster. It also allows you to use a very much smaller particle size for the column packing material which gives a much greater surface area for interactions between the stationary phase and the molecules flowing past it. This allows a much better separation of the components of the mixture. The other major improvement over column chromatography concerns the detection methods which can be used. These methods are highly automated and extremely sensitive. Standard. By the end of the course, you will know how to develop a method for a complete unknown mixture.

Objectives

By the end of the training, participants will be able to deal with:

• The differences between High-Performance Liquid Chromatography and Gas Chromatography
• The components of the high-performance liquid chromatography (HPLC)
• The separation processes
• The chromatogram
• The most common modes of HPLC

Training Methodology

This is an interactive course. There will be open question and answer sessions, regular group exercises and activities, videos, case studies, and presentations on best practice. Participants will have the opportunity to share with the facilitator and other participants on what works well and not so well for them, as well as work on issues from their own organizations. The online course is conducted online using MS-Teams/ClickMeeting.

Who Should Attend?

This course is intended for Laboratory Personnel and Technical Staff such as Chemists, Analysts, Chemical Engineers, and anybody interested in chemical analysis, research and development, environmental studies, quality control, refineries, petrochemical plants, water, and wastewater plants, hospitals, and medical centers.

Key Features of This Dynamic Course

• Basic instrumentation for HPLC
• Detectors: UV/VIS, PDA, MS, Fluorescence
• Column selection and optimization
• Qualitative and quantitative analysis
• Troubleshooting HPLC systems

Course Outline

Day 1: HPLC Overview

• Introduction to HPLC
• Historical Perspective
• Overview of Liquid Chromatography

Practical and Theoretical Principles

• Resolution
• Capacity Factor
• Efficiency

HPLC Instrumentation

• Columns and fittings
• Solvent Delivery System
• Sample Introductions
• Detector Details
• Laboratory: HPLC Hardware

Performing an Analysis

Day 2: Solvent Preparation, Isocratic, and Gradient-Elution

• Sample Preparation
• Columns
• Pump Maintenance

High-Performance Liquid Chromatography Overview

• Packing Materials
• Normal Phase
• Adsorption and Bonded phase
• Principles and Rules of Retention
• Choosing a Reversed-Phase Column
• Secondary Ion Effects
• Paired Ion Chromatography
• Ion Exchange Chromatography
• Size Exclusion Liquid Chromatograph
• Chiral Chromatography
• Affinity Chromatography
• Preparative Chromatography
• HPLC Instrumentation Overview
• Detector Details
• Laboratory: HPLC Hardware

Day 3: Separation Fundamentals and Mobile Phase

• Practical HPLC Theory
• Important HPLC Parameters
• The Mobile Phase
• Laboratory: Mobile Phase Strength, Flow Rate and Temperature

Separation Modes and Columns

• Reversed-Phase
• Normal Phase
• Ion Exchange
• Size Exclusion
• Laboratory: pH Effects in Reversed-Phase
• Laboratory: Column Dimensions

Day 4: Gradients, Method Development, and Gradient Elution

• Purpose of Gradient Elution
• Scouting Runs and Gradient Profiles
• Fundamentals of Method Development
• Laboratory: Develop a method for a complete unknown mixture
• Separation Modes and Columns
• Normal Phase
• Ion Exchange
• Size Exclusions
• Laboratory: pH Effects in Reversed-Phase
• Laboratory: Column Dimensions

Day 5: Quantitative Analysis and Troubleshooting

• Qualitative and Quantitative Analysis
• LC/MS
• Hardware Troubleshooting
• Separation Troubleshooting
• Laboratory: Internal vs. External Standard
• Hardware Troubleshooting