Principles and Applications of Clinical Mass Spectrometry

Small Molecules, Peptides, and Pathogens

Nader Rifai, A. Rita Horvath, Carl T. Wittwer, Andy Hoofnagle

• Publisher: Elsevier
• Year: 2018
• Print ISBN: 9780128160633
• eBook ISBN: 9780128160640
• Topic: Physical Sciences
• Subtopic: Analytic Chemistry

Principles and Applications of Clinical Mass Spectrometry: Small Molecules, Peptides, and Pathogens is a concise resource for quick implementation of mass spectrometry methods in clinical laboratory work. Focusing on the practical use of these techniques, the first half of the book covers principles of chromatographic separations, principles and types of mass spectrometers, and sample preparation for analysis; the second half outlines the main applications of this technology within clinical laboratory settings, including determination of small molecules and peptides, as well as pathogen identification. A thorough yet succinct guide to using mass spectrometry technology in the clinical laboratory, Principles and Applications of Clinical Mass Spectrometry: Small Molecules, Peptides, and Pathogens is an essential resource for chemists, pharmaceutical and biotech researchers, certain government agencies, and standardization groups.- Provides concrete examples of the main applications of mass spectrometry technology- Describes current capabilities of the LC- and MS-based analytical methods- Details methods for successful analytical work in the field

1

Chromatography

David S. Hage^∗

Abstract

Background

Clinical tests often involve the use of one or more separation steps to isolate, enrich, or separate a target compound from other chemicals in the sample. Chromatography is one of the most common methods for achieving this type of separation. In this method, the components of a mixture are separated based on their differential interactions with two chemical or physical phases: a mobile phase and a stationary phase that is held in place by a supporting material. There are many forms of chromatography based on the different mobile phases, stationary phases, and supports that can be used in this method, which has led to a wide range of applications for this technique.

Content

This chapter describes the basic principles of chromatography and discusses various forms of this method that are used for chemical analysis or to prepare specimens for analysis by other techniques. The methods of gas chromatography and liquid chromatography are discussed, as well as the techniques of planar chromatography, supercritical fluid chromatography, and multidimensional separations. The mobile phases, stationary phases, and supports that are used in each of these methods are described. The instrumentation and detection schemes that are employed in these methods are also discussed.

Biological fluids are complex mixtures of chemicals. This means that clinical tests for specific components in these fluids often involve the use of one or more separation steps to isolate, enrich, or separate the target compound of interest from other chemicals in the sample. Chromatography is one of the most common methods for achieving this type of separation. This chapter describes the basic principles of chromatography and discusses various forms of this method that are used for chemical analysis or to prepare specimens for analysis by other techniques.

Basic Principles of Chromatography

General Terms and Components of Chromatography

Chromatography is a method in which the components of a mixture are separated based on their differential interactions with two chemical or physical phases: a mobile phase and a stationary phase.^1-4 The basic components and operation of a typical chromatographic system are illustrated in Fig. 1.1. The mobile phase travels through the system and carries sample components with it once the sample has been applied or injected. The stationary phase is held within the system by a support and does not move. As a sample's components pass through this system, the components that have the strongest interactions with the stationary phase will be more highly retained by this phase and move through the system more slowly than components that have weaker interactions with the stationary phase and spend more time in the mobile phase. This leads to a difference in the rate of travel for these components and their separation as they move through the chromatographic system.

The type of chromatographic system that is shown in Fig. 1.1 uses a column (or a tube) to contain the stationary phase and support, while also allowing the mobile phase and sample to pass through the system. This approach was first described in 1903 by Mikhail Tswett, who used this method to separate plant pigments into colored bands by using a column that contained calcium carbonate as both the support and stationary phase.⁵ Tswett gave the name chromatography to this method. This name is derived from Greek words chroma and graphein, which mean “color” and “to write.” This term is still used to describe this technique, even though most modern chromatographic separations do not involve colored sample components.

The type of chromatography that was used by Tswett, in which the stationary phase and support are held within a column, is known as “column chromatography.” In chromatography, the stationary phase may be the surface of the support, a coating on this support, or a chemical layer that is cross-linked or bonded to the support.^2,6,7 In column chromatography, the support may be the interior wall of the column or it may be a material that is placed or packed into the column. A column is the most common format for chromatography. However, it is also possible to use a support and stationary phase that are present on a plane or open surface. This second format is known as “planar chromatography,” as will be discussed in more detail later in this chapter.^2,7

One way of classifying chromatographic methods is based on the type of support that they employ; two examples are the techniques of column chromatography and planar chromatography. Chromatographic methods also can be classified based on the mobile phase that is present. For instance, a chromatographic method that uses a mobile phase that is a gas is called gas chromatography (GC),⁸ and a chromatographic method that uses a liquid mobile phase is known as liquid chromatography (LC).⁹ It is also possible to divide chromatographic methods according to the type of stationary phase that is present or the way in which this stationary phase is interacting with sample components. Examples of these classifications include the GC methods of gas-solid chromatography (GSC) or gas-liquid chromatography (GLC) and the LC methods of adsorption chromatography, partition chromatography, or ion-exchange chromatography (IEC). Each of these categories, as well as others, will also be discussed later in this chapter.

The instrument that is used to perform a separation in chromatography is known as a chromatograph.^7,10 For instance, in GC the instrument is a gas chromatograph, and in LC the instrument used to carry out this method is a liquid chromatograph. These instruments can provide a response that is related to the amount of a compound that is exiting (or eluting) from the column as a function of the elution time or the volume of mobile phase that has passed through the system. The resulting plot of the response versus time or volume is known as a chromatogram,^7,10 as is illustrated in Figs. 1.1 and 1.2.

The average time or volume that is required for a particular chemical to pass through the column is known as that chemical's retention time (t_R) or retention volume (V_R). These values both increase with the strength and degree to which the chemical is interacting with the stationary phase. The elution time or volume for a compound that is nonretained or that does not interact with the stationary phase is known as the void time (t_M) or void volume (V_M). If the retention time or retention volume is corrected for the void time or void volume, the resulting measure of retention is known as the adjusted retention time (t_R′, where t_R′ = t_R − t_M) or the adjusted retention volume (V_R′, where V_R′ = V_R − V_M). For two chemicals to be separated by chromatography, it is necessary for these chemicals to have different values for t_R and V_R (or t_R′ and V_R′).^2,7,10

Most separations that are used for chemical analysis in column chromatography are carried out by injecting a relatively small volume or amount of sample onto the chromatographic system. This situation results in a chromatogram that consists of a series of peaks that represent the different compounds in the sample as they each elute from the column. The retention time or retention volume of each peak can be used to help identify the eluting compound, whereas the area or height of the peak can be used to measure the amount of the compound that is present.

The width of each peak is also of interest in a chromatogram. The peak width reflects the separating performance or efficiency of the chromatographic system. The width of a peak in a chromatogram is often represented by its baseline width (W_b) or its half-height width (W_h) (Fig. 1.3).^2,7,10 As the widths for the peaks in a chromatogram become sharper, it becomes easier for the chromatographic system to separate two peaks with similar interactions with the system and to separate more peaks in a given amount ...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• Contributors
• Preface
• 1. Chromatography
• 2. Mass Spectrometry
• 3. Sample Preparation for Mass Spectrometry Applications
• 4. Mass Spectrometry Applications in Infectious Disease and Pathogens Identification
• 5. Development and Validation of Small Molecule Analytes by Liquid Chromatography-Tandem Mass Spectrometry
• 6. Proteomics
• Index