Liquid Chromatography Detectors

T. M. Vickrey

• Publisher: CRC Press
• Year: 2020
• ISBN:
  9781000146219
• Edition: 1
• Topic: Business
• Subtopic: Pharmaceutical, Biotechnology & Healthcare Industry
• Index: Business

This book documents the principles of operation, the advantages and drawbacks, and the potential future of currently available liquid chromatographic detectors. In offering a snapshot of the current technology, it provides clear explanations and possible new horizons for both beginners and experts.

1
Description of Concentration Profiles Entering an LC Detector

Thomas M. Vickrey^* Texas A&M University, College Station, Texas

^* Present affiliation: Nissei Sangyo America, Hitachi Scientific Instruments Division, Mountain View, California

I. INTRODUCTION

Modern liquid chromatography (LC) dates back to the original liquid-solid chromatography work of Tswett [1–3] and Day [4,5]. The word chromatography is derived from the Greek chromatos, meaning color. The separation of colored substances was the sole use of liquid chromatography until the advent of nonvisual detectors [6], The first paper describing liquid-liquid chromatography, by Martin and Synge [7; see also 8], describes the use of a stationary phase impregnated with acid-base indicator to aid in the visualization of amino acids as they eluted. The problem of determination of the compounds which have been separated is a problem in detection. This book will attempt to survey the detection systems which are currently in widespread use or have the potential of becoming widespread.

This chapter will define the concentration profiles which enter the detectors described in later chapters. Further, the terminology used in comparison of detectors is presented here. This ensures a uniform use of terms.

Below is a brief discussion of the separation processes that lead to differential migration. This is neither detailed nor complete; if the reader is interested in more complete treatment of chromatography, reviews of the theory [9,10] or practice of chromatography [11–15] should be consulted. For specific questions most LC manufacturers have literature services that aid in the choice of column and mobile phase. The applications presented in subsequent chapters are illustrative of the use of detectors that may or may not have been run under optimum chromatographic conditions. With this in mind, let us review the separation process.

II. METHODS OF COMPONENT SEPARATION

A. Packed Column Methods

There are basically four types of packed column liquid chromatography: partition, adsorption, ion exchange, and gel permeation. Complementing these methods are “open column” methods of hydrodynamic chromatography and field-flow fractionation. These techniques differ in principle of separation but are similar in result. That is, the components of a mixture interact differentially with an agent external to the mobile phase. This differential interaction leads to a differential migration rate, and therefore to individual retention times for each component. A brief description of each interaction will be given and the reader directed to more complete reviews for detailed coverage.

1. Partition Chromatography

The mechanism of separation in partition chromatography is based on the competition between the mobile phase and stationary phase for the solute species. The original concept of a series of liquid-liquid separations of the sample in the mobile phase is instructive [7,8,11,16–18]. The basic equation of separation is

$${\text{V}}_{\text{r}}={\text{V}}_{\text{m}}+{\text{K}}_{\text{p}}{\text{V}}_{\text{s}}$$ (1)

where

$${\text{K}}_{\text{p}}=\frac{{\text{C}}_{\text{s}}}{{\text{C}}_{\text{m}}}$$ (2)

V_r is the retention volume, V_m the volume in the column occupied by mobile phase, and V_s the accessible stationary phase volume. K_p is the partition constant which describes the equilibrium distribution of the component between the two phases (see Fig. 1.1). [In practice, nonequilibrium effects must be considered, but Eq. (1) is a useful pedagogical tool.] In this case the retention time is a function of the affinity of the solute for the stationary phase. The higher the partitioning into the stationary phase (and therefore the higher K_p), the larger the retention volume.

Fig. 1.1 Liquid-liquid partitioning is based on differential solubility of S into the mobile phase and stationary phase. Mobile phase and stationary phase compete for solute.

The polarity of the stationary phase can be greater or less than the mobile phase. When the stationary phase is more polar than the mobile phase (as in 1₂0 on silica) the partition method is called normal phase liquid-liquid chromatography. When the reverse is true, a polar mobile phase and nonpolar stationary phase, the method is termed reverse phase (RP) partition chromatography. Although the interactions are quite similar, reverse phase chromatography has become widespread in the past several years due to unique experimental advantages.

The partition constant K_p can be changed during the course of elution. This is accomplished by modifying the mobile phase as a function of time [19,20]. This formation of mobile phase gradients can improve resolution and solve the general elution problem for multicomponent mixtures. Although this is often beneficial chromatographically, the detector may demonstrate an artifactual response.

The artifact may be due to changes in the bulk properties of the mobile phase, or elution of a trace mobile phase contaminant which is held up under initial conditions but which is eluted during the course of gradient formation.

Partition chromatography has been applied to a variety of problems. The partitioning phenomenon in general is best applied to components of similar chemical properties that differ slightly in size or structure. Compounds of a homologous series are generally best separated using the differential solubility in the stationary phase to cause differential migration.

2. Adsorption Chromatography

The mechanism of separation in adsorption chromatography (liquid-solid chromatography) differs from partition chromatography in that the differential migration depends on the mobile phase competing with the solute for the stationary phase [1–5,21–24]. The stationary phase has a fixed number of adsorption sites and the migration of the solute occurs by displacement from these sites by the mobile phase. This is depicted diagrammatically in Fig. 1....

Table of contents

• Cover
• Half Title
• Series Page
• Title Page
• Copyright Page
• Preface
• Contributors
• Contents
• 1. Description of Concentration Profiles Entering an LC Detector
• 2. UV-VIS Absorption Detectors for HPLC
• 3. Fluorescence Detectors in High-Performance Liquid Chromatography
• 4. Electrochemical Detectors
• 5. Refractive Index Detectors
• 6. The Use of Element-Specific Detectors Coupled with High-Performance Liquid Chromatographs
• 7. Mass Spectrometric Detectors
• 8. Less Popular Detectors
• 9. Data Handling for LC Detectors
• Author Index
• Subject Index