High Performance Liquid Chromatography

Theory, Instrumentation and Application in Drug Quality Control

Omar Al Sayed Omar, Moustafa A. Khalifa

• Publisher: De Gruyter
• Year: 2022
• ISBN:
  9783110528954
• Edition: 1
• Topic: Physical Sciences
• Subtopic: Analytic Chemistry

During the past decade, modern high-performance liquid chromatography (HPLC) utilization has expanded greatly, especially in the quality control of pharmaceutical products in drug quality control laboratories. This book provides an extensive collection of technical information about HPLC-Columns (physicochemical properties and chromatographic characteristics), from various manufacturers, and helps analysts to decide on the ideal approach for their analysis according to the requirements of drug manufacturers specifi cations and the desired Pharmacopeia. In addition, the authors give practical advice on how to prepare mobile phases, choose a suitable detector, and set up an HPLC analysis. This book is comprehensive for the average professional or technician who plans to work with modern HPLC. This book is useful for most Drug Quality Control Laboratories where modern HPLC is utilized. Following a hands-on approach, the book gives key insights into the pharmaceutical applications of HPLC and the latest requirements of the major regulatory agencies such as ICH, FDA, or USP.

1 High-performance liquid chromatography (HPLC): theoretical principles

1.1 Introduction

High-performance liquid chromatography (HPLC, formerly referred to high-pressure liquid chromatography) is, for many scientists, an essential piece of apparatus for the separation, identification, purification and quantification of various compounds, especially very polar (organic) in their complex mixtures. Users of HPLC work in a variety of fields, including analysis of pharmaceuticals, foods, waters, cosmetics, biochemicals and environmental samples [1, 2, 3, 4, 5, 6]. For HPLC analysis, mixtures from interested compounds are first dissolved in a liquid solvent and then forced to flow with mobile phase through a stationary phase in the form of column (usually stainless steel column) under high pressure. On this stationary phase (column), the mixture of chemical compounds is resolved into its components. The amount of resolution is important and is dependent upon the extent of interaction between the solute components and the stationary phase. The stationary phase is defined as the immobile packing material of the column. The moving part of the system is the mobile phase, which is a liquid. The interaction of the solute with mobile and stationary phases can be manipulated through different choices of solvents and columns. As a result, HPLC acquires a high degree of versatility not found in other chromatographic systems. Thus, HPLC has the ability to easily separate a wide variety of chemical mixtures, especially which contains thermally labile or easily oxidized compounds.

In the opposite, gas chromatography (GC) is limited in its applications, and the analysis of compounds which are thermally labile or easily oxidized is not possible. So, HPLC can fill this gap and has thus become an essential addition to GC in every analytical laboratory. Advances in HPLC technology have been assisted by the continual development of new stationary phases, improvements in instrumentation and the facility the method offers for the application of computer technology and developments in automation techniques.

It is, therefore, not surprising that the number of publications which are now dealing with HPLC exceeded than GC [7].

In this chapter we will discuss the following topics:

• What is HPLC in relation to other chromatographic techniques?
• Components of an HPLC system
• History of HPLC
• Types of separation in HPLC
• Mechanism of separation in HPLC
• Where HPLC can be used as an analytical tool?
• Advantages of modern HPLC

In this part, we will discuss the previously mentioned topics in detail.

1.2 What is HPLC in relation to other chromatographic techniques?

In chromatographic technique, the components of a chemical mixture are separated based upon the rates at which they are carried through a stationary phase (solid or liquid on solid support) by gaseous or liquid phases (mobile phase). Chromatographic method is categorized into three types based on the nature of the mobile phase [7]. The three types of phases include liquid, gases, and supercritical fluids as shown in Fig.1.1. Liquid chromatography (LC) can be performed in columns and on planar surfaces, but GC and supercritical fluid chromatography are restricted to column procedures. Column chromatography is often described as elution chromatography in which solutes are washed through stationary phase by the movement of the mobile phase (eluent).

The chromatographic separation of a mixture of various compounds depends primarily on the fact that each of them is physicochemically different from two nonmiscible phases (the stationary phase and the mobile phase). Thus, thermodynamic effects such as partition and absorption are mostly involved. Depending on the physical state of the mobile phase, a distinction is made between GC and LC (as shown in Fig.1.1). A further distinction depends on the form of stationary phase, layer or column, which leads to other classifications, thin-layer chromatography and column chromatography. The stationary phase may be solid, porous chemically modified or liquid film on solid support.

Fig.1.1: Categories of chromatography and their relationship to each other.

1.3 Components of an HPLC system

Schematic representation of the essential components of an HPLC system is found ...