Chapter 2

The Role of Sample Preparation

Abstract

Virtually every analytical method based on a core chromatographic procedure includes a sample preparation step. This chapter contains a discussion regarding the role of sample preparation as the first step in a chromatographic method of analysis. It briefly reviews the simple routine manipulations of a sample such as weighing, volume measuring, homogenization, adjustment of water content, dissolution, resampling, and size reduction. Then it examines the role of cleanup and fractionation of the sample, concentration, and chemical modifications. In this chapter are discussed parameters such as analyte recovery and enrichment factor that must be considered when the sample composition is modified during sample preparation. Also, a list of criteria for the selection of a good sample preparation is provided. At the end, some comments on automation in sample preparation techniques are included.

Keywords

Analyte recovery; Enrichment factor; Routine sample manipulations; Sample cleanup and fractionation; Sample homogenization

Outline

2.1 Routine Manipulations of the Sample 34

General Comments 34

Weighing 34

Volume Measuring 34

Mixing and Dilution 34

Heating and Cooling 34

Other Routine Sample Manipulation 34

Preservation of Sample Integrity during Processing 35

2.2 Preliminary Processing of the Sample 35

General Comments 35

Sample Homogenization 35

Adjustment of Water Content in Samples (Drying) 36

Sample Dissolution 36

Resampling for Further Reduction of Sample Size 37

2.3 Sample Cleanup and Fractionation 37

General Comments 37

Cleanup and Fractionation Procedures 38

Analyte Recovery during Sample Cleanup 39

2.4 Concentration of the Sample 40

General Comments 40

Enrichment Factor 40

2.5 Chemical Modifications of the Sample 42

General Comments 42

Chemical Changes for Sample Dissolution 43

Chemical Changes for Cleanup and Fractionation Purposes 43

Chemical Modification for the Enhancement of Detection 43

Chemical Modification for Enhancing the Separation in Core Chromatography 44

2.6 Selection of a Sample Preparation Procedure 44

General Comments 44

Development of a Sample Preparation Scheme based on Preliminary Information 45

2.7 Automation in Sample Preparation 48

General Comments 48

References 49

2.1. Routine Manipulations of the Sample

General Comments

Sample preparation covers a number of operations that are necessary to modify the sample to make it amenable for chromatographic analysis or to improve the analysis results. The operations used in sample preparation may have various purposes, such as sample fractionation, cleanup, concentration of the analyte content. Among these operations, are some routine sample manipulations such as weighing, mixing (e.g., vortexing), measuring of the volume (e.g., pipetting), diluting by the addition of a solvent, heating, cooling. Weighing and measuring of the volumes may not appear as sample preparation, but only as performing a measurement on the sample before the core analysis. These operations are very common in the laboratory and can be repeated at different points during the analysis.

Weighing

Weighing is a very common operation in chemical analysis. One first use of this operation is that weighing must assure that the appropriate amount of sample is taken for analysis. Weighing is almost always used on raw samples or subsamples before the core analysis. Special attention must be given to the initial measurements on the sample weight because it affects the analysis result as much as the final output of the chromatographic measurement.

Volume Measuring

Similar to weighing, volume measuring is very common in sample preparation as well as in chemical analysis in general. While weighing is applied to solid samples but also to liquid samples, volume measuring is applied to liquid samples and gaseous samples. Sampling of gases involves more than volume measurement since factors such as temperature and pressure of the gas must be taken into consideration (see e.g., Ref. 1).

Mixing and Dilution

Different mixing operations including dilution are common in sample preparation (and in chemical analysis in general). Dilution is the operation of reducing the concentration of a sample (or chemical compound). The most common type of dilution is that of a concentrated solution by the addition of solvent. However, gases can be diluted by mixing with another gas (e.g., an inert gas), and even solids can be diluted by mixing with another solid. The mixing of an initial material with a diluent must assure that the resulting diluted material is as homogeneous as possible. Simple rules are applied to calculate the concentration of the diluted sample based on the concentration and the amount of initial sample and the amount of diluent.

Heating and Cooling

Heating and cooling represent the changes in the temperature of a material, and it is common in sample preparation. For example, heating may be necessary during sample dissolution, and cooling may be necessary for the preservation of the sample. The processes of heating and cooling must be performed such that the nature of the sample is not affected. For example, during heating some more volatile components may be lost. Cooling, in general, does not affect the composition of a sample, but in the case of special biological samples it may cause damage to cells due to the formation of ice during freezing.

Other Routine Sample Manipulation

Besides the routine operations previously listed, other ones are common in the laboratories. Among such operations can be listed: grinding, filtration, centrifugation, evaporation, dissolution, digestion, pH adjustment, various forms of homogenization, taking sample aliquots, cell disruption, sample reconstitution. These operations may have as a result a simplification of the sample matrix, or concentration of the analytes, and are further discussed in this chapter.

Preservation of Sample Integrity during Processing

A special aspect of sample preparation, regardless of the type of sample manipulation, is the attention that must be paid for not altering inadvertently the content of the analytes in the sample. Sample instability can be of physical or chemical nature. Some samples may lose their initial state due to physical modifications that occur in time. Part of these modifications may take place during storage, and the problem must be addressed immediately after the sample collection (see Section 1.3). However, some samples may change their physical state during the sample preparation or during analysis. For example, during an evaporation process intended to reduce the amount of solvent, some volatile analytes also may be lost. Special sample modifications are required for samples that are reactive or that can degrade (by chemical modifications or biological processes). The addition of preservatives in biological samples is a common practice for preserving them. Samples that are too basic or too acidic can also suffer changes, for example, by getting contaminated by reaction with the containers or with CO2 from the air. A number of reports discuss in detail the sample preparation requirements for the preservation and analysis of samples with reactivity problems.2,3 Samples that present health risk or environmental problems also must be prepared using special procedures. A discussion on the requirements related to the processing of hazardous samples is beyond the purpose of this book. Besides reactivity, special attention must be paid, for example, to samples that form colloidal solutions. Some substances such as proteins, polysaccharides, and Maillard browning polymers may form stable colloidal solutions with the appearance of a true solution. However, the colloid may adsorb irreversibly on a stationary phase, for example when using a solid-phase cleanup. During sample preparation this type of problem may go easily unnoticed.

2.2. Preliminary Processing of the Sample

General Comments

Depending on the nature of the sample, the preliminary processing may be very different. The choice of the planned chromatographic analysis typically influences the initial steps. However, some preliminary operations are common for most samples. These operations may include: (1) homogenization of the raw sample, (2) adjustment of sample content in water (moisture), (3) sample dissolution, and (4) resampling for further reduction of sample size.

Sample Homogenization

Initial sampling from the bulk material generates in many cases a larger amount than the one further subjected to analysis. Although this is not always the case, a resampling for further reduction of the sample size is a common operation. The requirement for the initial raw sample to be representative can be achieved when the sample is not homogeneous (e.g., is solid or mixed phase), as discussed in Section 1.3, by collecting a larger amount of material. When this material must be resampled to further reduce the sample amount, the raw sample must be initially homogenized. This homogenization for solid samples is achieved by...