Selection of the HPLC Method in Chemical Analysis

Selection of the HPLC Method in Chemical Analysis

Serban C. Moldoveanu

Victor David

Table of Contents

Cover image

Title page

Copyright

Preface

Chapter 1. Start of the Implementation of a New HPLC Method

1.1. Collection of Information and Planning for a New Method

1.2. Overview of an Analytical Technique

1.3. Statistical Evaluation of Data and Criteria for Method Validation

Chapter 2. Short Overviews of Analytical Techniques Not Containing an Independent Separation Step

2.1. Summary Classification of Analytical Techniques Not Containing an Independent Separation

2.2. Optical Techniques

2.3. Mass Spectrometry

2.4. Electrochemical Methods

2.5. Other Analytical Techniques Not Including the Separation of Sample Components

2.6. Selection of a Nonhyphenated Method of Analysis versus one Containing a Separation Step

Chapter 3. Short Overviews of the Main Analytical Techniques Containing a Separation Step

3.1. Separation Types Used in the Core Analytical Techniques

3.2. Gas Chromatography

3.3. Supercritical Fluid Chromatography

3.4. High-Performance Liquid Chromatography

3.5. Electrophoresis and Electrochromatography

3.6. Selection of GC, SFC, CZE, or HPLC

Chapter 4. Basic Information Regarding the HPLC Techniques

4.1. Basic Information About Instrumentation in HPLC

4.2. Parameters Describing the Chromatographic Process

4.3. Retention and Elution Mechanisms in Different Types of HPLC

4.4. The Influence of pH, Temperature, and Additives on Retention Equilibria

Chapter 5. Properties of Analytes and Matrices Determining HPLC Selection

5.1. Physicochemical Properties Related to Separation

5.2. Physicochemical Properties Related to Detection

5.3. Selection of the HPLC Separation Based on Sample Properties

5.4. Selection of the HPLC Separation Based on Analyte Properties

5.5. Selection of the HPLC Separation Depending on the Matrix

5.6. Review of Sample Properties with the Goal of Selection of a Detector in HPLC

Chapter 6. General Aspects Regarding the HPLC Analytical Column

6.1. Construction of the High-Performance Liquid Chromatography (HPLC) Column

6.2. Column Properties Affecting Separation

6.3. General Rules for Column Selection, Utilization, and Care

Chapter 7. RP-HPLC Analytical Columns

7.1. Types of Reversed Phases and Their Preparation

7.2. Properties of Reversed-Phase HPLC Columns

7.3. Parameters Used for the Characterization of Reversed-Phase HPLC Columns

7.4. Common and Special RP Columns

7.5. Selection of an RP-HPLC Column

Chapter 8. Polar Analytical Columns

8.1. Types of Polar HPLC Phases and Their Preparation

8.2. Properties and Characterization of Polar HPLC Phases

8.3. Columns With a Polar Stationary Phase

8.4. Selection of a Polar Column

Chapter 9. Stationary Phases and Columns for Ion Exchange, Ion-Moderated, and Ligand Exchange Chromatography

9.1. Types of Phases and their Preparation

9.2. Characterization of Ion Exchange Phases

9.3. Common Columns with Ion Exchange Phases

9.4. Selection of an Ion Exchange Phase

Chapter 10. Stationary Phases and Columns for Chiral Chromatography

10.1. Types of Chiral Phases and Their Preparation

10.2. Characterization of Chiral Phases

10.3. Selection of a Chiral Phase

Chapter 11. Stationary Phases and Columns for Size Exclusion

11.1. Types of Phases and Their Preparation

11.2. Characterization of Size-Exclusion Phases and Columns

11.3. Selection of Columns Used in SEC

Chapter 12. Stationary Phases and Columns for Immunoaffinity Type Separations

12.1. Types of Phases and Their Preparation

Chapter 13. Solvents, Buffers, and Additives Used in the Mobile Phase

13.1. Characterization of Liquids as Solvents

13.2. Additional Properties of Liquids Affecting Separation

13.3. Properties of Solvents of Importance for Detection

13.4. Buffers and Additives

13.5. Selection of Mobile Phase in HPLC

13.6. Selection of a Solvent for Sample Injection

13.7. Selection of a Solvent for the Needle Wash

Chapter 14. Gradient Elution

14.1. The Use of Gradient in HPLC

14.2. Parameters Characterizing the Gradient Separation

14.3. Selection of Gradient in Different Chromatographic Types

Chapter 15. The Practice of HPLC

15.1. The Development of an HPLC Method

15.2. Special HPLC Techniques

Appendix 1. USP Classification of HPLC Columns

Appendix 2. Hydrophobic Stationary Phases

Appendix 3. HILIC and NPC Stationary Phases

Appendix 4. Ion Exchange and Ion-Moderated Stationary Phases

Appendix 5. Chiral Stationary Phases

Appendix 6. Size-Exclusion Stationary Phases

Appendix 7. Properties of Mobile Phase Components

Index

Copyright

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Preface

High-performance liquid chromatography (HPLC) is very likely the most utilized technique in analytical chemistry. An enormous amount of information about HPLC is available in peer-reviewed journals, in books, in manufacturer catalogs, and on the Internet. The selection of the most appropriate HPLC method for a given objective from this multitude of sources is a challenging task. In addition, new possibilities for more selective and sensitive HPLC methods are continuously emerging based on the progress made in instrumentation and new chromatographic materials. Although numerous scattered publications exist, a unified and coherent presentation of the procedures for the selection and development of an HPLC method is still poorly represented in the literature. This book intends to cover this gap as well as possible. It provides criteria for the selection of an appropriate method for a particular task when such method is available in the literature, and it offers guidance for the development of a new method when the literature does not describe an adequate one for a specific objective. The book intends to provide a systematic and up-to-date material regarding the selection of an HPLC method. However, because continuous progress is made in HPLC, only the latest published literature can capture the most novel developments. The book is basically segmented into four parts. The first part presents aspects related to the collection of information necessary before the selection of a new analytical method. Comments on statistical analysis of data and validation criteria and a general discussion about alternative techniques utilized in chemical analysis are also included in this part. The first part also presents the advantages and disadvantages of selecting HPLC as a preferred method of analysis versus other analytical techniques. The second part describes various aspects of HPLC, such as instrumentation, parameters characterizing a chromatographic separation, and retention mechanism in several HPLC types. The third part of the book presents in some detail the characteristics of HPLC columns and criteria for the selection of an appropriate column for a specific analytical task. This part also includes a detailed description of possibilities to select and use a mobile phase. The last part of the book is a collection of tables with detailed information about chromatographic columns, solvents, and additives. These data provide an important source of information necessary for selecting or developing a new HPLC method. Valuable help for improving the presentation of the material from this book was provided by Owen Bussey, Crystal Byrd, Chelsea Cooke, Garry Dull, Anthony Gerardi, Christopher Junker, Carol Moldoveanu, and Wayne Scott, for which the authors are most thankful. The authors also wish to thank the editorial team from Elsevier, Amy Clark, Maria Bernard, and Kathryn Morrissey for their contribution to the publication of this book.

Chapter 1

Start of the Implementation of a New HPLC Method

Abstract

An overview of the main steps of analytical methodology based on HPLC is given, starting with the collection of information about the analyzed samples and ending with the evaluation of the quality of analytical results obtained from the chromatographic analysis. In this chapter the roles of sample collection, of sample preparation, and of the selection of core analytical operation for the analysis are reviewed. Also, a presentation is made for the general steps required in preparation for developing a new analytical method of analysis. Common procedures for the analytical data evaluation using statistical concepts and general criteria for the validation of an HPLC method are also described. Quantitative information is discussed in relation to precision, accuracy, sensitivity, detection limits, and linearity of the detector response.

Keywords

Analytical information; Data evaluation; Decision process; Method validation; Quantitation; Sample; Statistics

1.1. Collection of Information and Planning for a New Method

For good results, the collection of as much information as possible before starting the implementation of a new HPLC method of analysis is very important. It is also important to assess how reliable the various items of the collected information are. The information collected before starting a selection for an analytical method should cover a number of aspects that include the following: (1) the purpose of analysis, (2) general information about the samples, (3) the sample's constituents, (4) the required quality of the results, (5) instrumentation availability, expertise in the laboratory, and funding, (6) information about various methods of analysis, and (7) new developments in instrumentation.

The collection of information regarding the method of analysis should continue even after the method development has started. Any additional findings obtained after the development has started and the feedback from the initial trials of a specific method are valuable data for the improvement and final selection of an adequate method. For this reason, the collection of information should be considered a continuous process that may even have an iterative path, the results obtained from the first set of runs lead to the need for more starting information and so on until the method is well established.

The Purpose of Analysis

The purpose of analysis should be the first type of information obtained before starting the implementation of a new analytical method. Sources of information about the purpose can be very diverse, the most common being the direct request from a customer (including self-imposed requests). A number of items can be listed as important to know regarding the purpose of a chemical analysis. This information should describe why the analysis is performed, what kind of results are expected from the analysis, and possibly the utility of the analysis. A wide range of requests can be made for an analysis, and samples must be analyzed for numerous reasons. In industrial environments such purposes may include official or legal requirements, assessing the quality of raw materials, process control or troubleshooting, assuring the quality of finished products, research, reverse engineering, and development purposes. Samples are frequently analyzed for health-related purposes (e.g., medical analyses, analysis of pharmaceuticals, analysis of metabolites), for evaluating environmental issues, for forensic purposes, for exploratory reasons, and for fundamental research. Depending on the analysis reason, specific decisions are made about the analytical process. The purpose of analysis should describe if the analysis is related to a material, a process, or both. It is important to know if the whole sample must be analyzed (all constituents are in this case analytes) or only a specific part of the sample.

Important information must be collected indicating the required type of analysis regarding qualitative, quantitative, semiquantitative, or both qualitative and quantitative. It should be indicated if a special analysis is to be performed such as separation of enantiomers or regarding structural elucidation. The purpose should specify if the analysis has a specific target, if it is only exploratory, or the goal of performing the analysis is vague. Also, it should be known if there is a plan to perform the analysis on a repeated basis in the future or only at one time. The number of samples to be analyzed at one point or in an extended period of time should be known. The rapidity with which the results must be delivered should also be known before starting the development or implementation of a new method.

General information must specify whether a specific protocol must be followed during the analysis or that no regulations are imposed. Some analyses are required to be nondestructive, and in certain cases the analysis is done in conjunction with preparative purposes, which also should be known. There are important issues related to analyses associated with preparatory purposes since some of them modify the nature of the sample. If the biological activity of the samples must be preserved, this aspect should also be known.

The level of accuracy and precision of the analysis should be described, indicating if the analysis is geared toward major constituents of the sample, minor constituents, or traces. Also, information should be collected regarding the novelty of the planned analysis or if it was previously performed. Knowledge regarding other analyses already performed on the samples is always important, and occasionally it is useful to have information about analyses that are planned to be performed on the samples in the future.

The general information regarding the purpose of analysis allows early planning of the type of analysis to be performed on the samples. In case that very little information is available about the samples, preliminary analyses should sometimes be performed. This preliminary analysis can be qualitative or semiquantitative. If only qualitative analysis is required, high-performance liquid chromatography (HPLC) is not usually the best choice as the method of analysis. When information exists that the sample contains unique compounds or it is a mixture of very few components, nonchromatographic techniques may be considered for obtaining qualitative information. Very useful are the spectroscopic procedures such as ultraviolet (UV), infrared (IR), or nuclear magnetic resonance (NMR). When the samples have more components, chromatographic techniques are typically necessary for an initial separation of sample components. Scan type chromatographic techniques associated with detection by mass spectrometry such as gas chromatography–mass spectrometry (GC–MS) are probably the best tools for compound identification, provided the samples are amenable for this type of analysis (contain volatile analytes). In some cases, when the samples have a limited number of components which are known to be potentially present, HPLC or thin-layer chromatography (TLC) can be useful for identifying sample composition. Mass spectrometry hyphenated with liquid chromatography (LC) such as LC-MS or LC-MS/MS may provide qualitative information on unknown samples, although the identification of unknown compounds by LC-MS or LC-MS/MS techniques has limitations. For quantitative analysis, chromatography is typically preferred, including GC or LC (HPLC). Also, an array of techniques must be necessary for the analysis of certain samples. For large numbers of samples to be analyzed, automation must be considered.

General Information About the Samples

The information about the material(s) to be analyzed (indicated as sample) should cover chemical aspects of the sample, physical properties information, as well as any other contingent information. Two parts of a sample are to be considered in a chemical analysis, the analytes and the matrix. The analytes are the molecular or ionic species of interest in the sample and the matrix indicates the rest of the sample components. Similar to the case of information about the purpose of analysis, as much information as possible about the samples is desirable, although in many practical cases this information remains incomplete. A critical piece of information is related to the samples' complexity. This type of information is an early criterion related to the choice of the need for a hyphenated technique (separation  +  measurement) for the analysis. Information about the sampling process is also very useful, in particular indicating the samples' homogeneity, the age of the samples, and potential of contamination. If nonhomogeneous samples need to be analyzed it should be known if the analysis must be performed in a specific way (e.g., after homogenization, or selecting parts of the sample). Also, it must be known if the whole sample should be analyzed, or only a specific part (surface, soluble component, selected points, etc.). Information about the samples' physical state should be collected, indicating if the samples are gas, liquid, solid, solution, semisolid, or mixed phases. Also, knowledge should be collected whether the samples are inorganic, organic, composite, of biological origin, environmental, or of a special source. Other data about the samples should indicate the amount available (large quantity, small quantity, readily available, uniqueness, etc.) and also the value of the samples. In some cases the samples must be returned to the provider after a small amount has been used for analysis and this should also be known. Other useful information includes the samples' thermal stability and perishability, as well as any safety concerns. The information about the samples should also indicate if the samples are of a new type or if similar samples were previously analyzed.

The general information about the samples is closely related to the sampling process if the samples were not already collected. It should guide the precautions regarding sampling to avoid, for example, contamination, amount of necessary sample, the requirements of samples transportation, and the safety measures related to sampling. Nonhomogeneous samples typically need careful sampling followed by the separation of phases. Sample preparation is frequently needed. Besides the sampling process, the general information on the samples has input regarding the initial sample processing, such as physical processing (grinding, drying, homogenization), and also samples storage and preservation. Inorganic samples are typically processed differently than organic or complex samples. Sometimes, dissolution of inorganic samples is an important sample preparation step. Thermally labile samples may need special storage and handling, and HPLC type techniques are frequently used for this type of analysis. Perishability may be modified using preservatives, and the addition of such compounds must be known. The analysis of complex samples may also require sample preparation. Sample complexity also determines the use of a separation technique hyphenated with the measurement. The most common such technique is chromatography. For this reason, chromatography is the technique preferred in many organic, biological, and environmental analyses. Gases and the compounds that are thermally stable at their boiling point are preferably analyzed by GC, and sample preparation may not be necessary. Many organic compounds can be analyzed by HPLC, this technique being necessary for molecules that cannot be volatilized, but can also be used as a choice for volatile and semivolatile molecules. Appropriate measures must be taken if hazard concerns are present.

The Sample Constituents

The information about sample constituents can be placed into two categories: (1) analytical and (2) general properties. Analytical information includes data about the chemical nature of the constituents, the strength of bonding of the analytes to the matrix, range of concentrations of the analytes, etc. General properties include physicochemical data such as volatility of analytes and matrix, solubility in various solvents, acid–base character, hydrophilic–hydrophobic properties, reactivity, etc. The knowledge about the chemical nature of the analytes is essential. It is preferable to know the list of compounds (single or multiple component analysis) that must be analyzed or at least the class of the analytes (inorganic, organic, functional groups in organic compounds, ionic character, etc.). When the analytes of interest are known, it is useful to know if the analytes are volatile, nonvolatile, ionic, polymeric, weak interaction compounds, or a mixture of species of the same compound. If this information is missing, it must be known at least if the analytes are small molecules or polymeric. In the case of small analyte molecules, data regarding volatility, solubility, and reactivity are very useful. For macromolecules, a general characterization is typically necessary. Other data regarding the analytes are helpful, such as information on the estimated level of analytes in the samples (ultra-low trace, trace, minor constituent, medium levels, or major constituent). The level of analytes may be totally unknown and then preliminary analyses may be necessary. From the general information about the samples it may be possible to estimate the range of concentration for the analytes in the samples. The iterative aspect of collecting information about the samples allows for the improvement of the information about the sample composition, and as the analytical process starts to be implemented, additional information, as well as additional questions, may emerge.

Similar information to that about the analytes should be examined for the matrix. It is important to know if the matrix is homogeneous or not, if it is inorganic, organic, has an ionic character, if it is polymeric, or if it is of biological origin. Data regarding the volatility and stability of the sample matrix are also very useful. The interaction between the matrix and the analytes may play an important role in the analysis. The matrix may form stable molecular associations with the analytes (e.g., salts, clathrates including host–guest complexes, inclusion compounds), or may generate artifact analytes in specific conditions (e.g., generation of free amino acids from proteins).

This information is related to planning of different operations in the analysis and can go back to instructions about sampling (if not restricted to a sample already provided). It includes the choice of preliminary sample processing (physical processing), and the selection of a sample preparation process, such as sample cleanup and analyte concentration. The information about the chemical nature of the analytes and the matrix are the main data needed in planning a specific type of analysis. Inorganic analytes can be analyzed using chromatographic methods such as ion chromatography (IC), but techniques such as inductively coupled plasma (ICP) or atomic absorption (AA) are more frequently applied. For most organic and complex samples chromatography is the preferred technique. Samples with small volatile molecules are typically analyzed using GC (with or without sample preparation). Since GC can be easily coupled with MS as a detector, and because of the existence of large libraries of MS spectra, GC–MS is the most powerful technique for compound identification, and is also very useful for quantitation. HPLC is the most common analytical technique for the quantitation of a wide range of organic molecules. The utility of HPLC for qualitative analysis is somewhat limited even when it is coupled with MS detection. However, for quantitation purposes, HPLC is the most appropriate and commonly utilized analytical technique for organic molecules. Polymers can be analyzed by nonchromatographic methods such as IR, or they can be separated and analyzed using size exclusion chromatography (gel permeation [GPC] or gel filtration chromatography [GFC]). Sometimes the polymers are also analyzed after preliminary hydrolysis or pyrolysis that reduces the initial macromolecules to smaller molecules.

As part of the analytical information the knowledge about the range of the analytes contained in the samples allows for the selection of the appropriate sensitivity for the method of analysis. Since specific sensitivities are characteristic for different analytical techniques the information about the range of the analyte levels may be necessary for selecting the type of analytical technique and analytical instrumentation, and not only for adjusting method sensitivity. Data about the level of analytes may also guide the need for sample preparation such as analyte concentration procedures.

The Required Quality of the Results

The results of the chemical analysis are typically expected to answer specific questions related to the purpose of analysis. In order to have appropriate answers, the results must satisfy specific requirements regarding accuracy, precision, and the number of samples characterized. Also, it is important to know if the results are part of a larger study, if they can be compared with the results from other laboratories or with results on standard materials.

The type of necessary results has implications on various aspects of planning a new method of analysis. One starts with sampling. A specifically designed sampling protocol may be necessary when specific statistical analysis of the data is required. Also, the level of precision of the required data influences the planning for specific sensitivity of the analytical method. The use of the results in specific collaborative studies may also impose a given protocol for the method of analysis.

Instrumentation Availability, Expertise in the Laboratory, and Funding

The selection of an analytical procedure is frequently determined by various external factors besides the purpose of analysis and type of samples. Among such factors are the instrument availability, the expertise in the laboratory, the time available for developing or implementing a specific analytical procedure, and also funding. In addition to instruments, information should be collected about the materials available, indicating for example the need for preliminary purchases of chromatographic columns, reagents, solvents, and standards. Related to the safety concerns, appropriate laboratory conditions must also be assured for the analysis of certain samples. Information about all these aspects is of primary importance such that the analysis can be performed.

The information about the resources available for performing a specific analysis in a selected manner is critical for successful results. Missing the necessary instrument, for example, is an obvious barrier for planning the development of a new method analysis that would use that instrument. Potential purchase of new instrumentation must be considered in specific cases. The expertise available in the laboratory to perform the analysis is also an important factor, including information about the need for training. The performance of some analyses is a very simple task, while other analyses require considerable resources. Among the external factors that could also be considered are the effects of used chemicals from the laboratory on the environment. For this reason the utilization of environment-friendly solvents and reagents is recommended, as well as of analytical procedures that minimize the consumption of dangerous unavoidable chemicals. For example, organic solvents that could be considered green [1] are desirable in chromatographic analysis because their use avoids toxicity and environmental problems [2,3].

Information About Various Methods of Analysis

A large body of information is available regarding methods of analysis. This information is available in the form of articles published in peer-reviewed journals, books, company application notes and user guides, various forms of Internet information, as well as numerous courses at universities and private organizations (all further indicated as literature). Before starting the development or implementation of a new method of analysis a critical step is the reviewing of the available information related to the analysis of interest. For almost any analyte, there are methods of analysis that are described in the literature. The already-reported methods may not be intended for the specific type of sample on which the new method should be applied or may not satisfy certain requirements for the new method. It is also possible that the method described in the literature cannot be implemented, for example, due to the lack of instrumentation. However, this literature information always offers a valuable starting point in a new method development. When a method is available and can be utilized, it is frequently beneficial to implement the method and directly evaluate its adequacy for the new purpose. In certain cases, where no method is available in the literature, procedures for the analysis of similar compounds as those planned for new analysis are helpful.

The choice of a method of analysis requires knowledge about all the aspects previously described. When a method is already reported for the same type of sample, or for similar samples, it is typically advisable to implement that method as is, even if modifications and adjustments are to be made afterward. In most situations, multiple choices for an analytical method are available, even when the analysis has been previously practiced on the same type of analytes and samples. It is common that more than one choice can give good results, when most of the required conditions for an appropriate analysis are fulfilled. The method of analysis with all its aspects including the necessary sample processing, the type of analysis, and data processing may be more or less convenient depending on particular choices. Different analytical techniques for the same analytes and the same type of sample can offer advantages and may have disadvantages. Also, some may offer excellent qualities although not necessary needed. For example, the use of an LC-MS/MS method versus an HPLC method with UV detection may offer higher sensitivity and positive identification of the analytes, although this may not be necessary for a specific analysis. Arbitrary choices can be made for some analyses, and the quality of the result may or may not be affected. Although an optimization of the analytical method is sometimes possible, a balance must always be made between the effort of improving the method and using it as is, if the results are still satisfactory. The choice of a specific analytical method can be critical, but it is also possible that a number of alternatives lead to the same desired analytical goal and one choice or another is not critical. The selection of a method of analysis may be determined by many reasons, for example the instrument availability, personnel training, limited funding, and may be the subject of various constraints that do not allow the selection of the best possible alternative. Nevertheless, the goal of this book is to describe the best choices for selecting an HPLC method of analysis.

For HPLC analysis, a wide range of choices can be made with the end result of successfully performing a specific analysis. This book discusses various aspects of HPLC analysis that must be taken into consideration for the selection, development, and implementation of a successful method using this technique.

New Developments in Instrumentation

The improvements of analytical instrumentation, of accessories, of consumables, and the development of new technologies, and new materials is a continuous and very active process. All of these new developments may offer new possibilities for the analysis either through a variety of improvements in sensitivity of analytical instruments, media for better separations, simplifications in the necessary operations for specific analyses, cost reduction of analyses, speeding the analytical process, enhancing reliability of the analyses, etc. A variety of sources are available for providing information regarding all these new developments such as scientific meetings and conferences, literature provided by manufacturers, various Internet sites, etc.

Knowledge about all the new developments related to the analytical capabilities is very important for adopting the best path for a specific analytical task. However, the implementation in the laboratory of such new developments is not always possible. The cost of new instruments may be prohibitive, the training of the personnel may be lacking, and the new developments do not always represent better ways to solve a specific analytical problem. Nevertheless, it is always useful to be informed about novel alternatives for a chemical analysis.

1.2. Overview of an Analytical Technique

Chemical analysis can be qualitative, semiquantitative, quantitative, or addressing structural problems, can be designed for the analysis of materials or for process control, analysis of gases, liquids, solids, or mixed phases, and can be designed for organic materials, inorganic ones, or mixed organic–inorganic, etc. It can be performed using a wide variety of procedures. Before the development of modern instruments for analysis, wet chemistry was widely used for qualitative analysis, and gravimetric and volumetric methods were used for quantitative analysis. Although these older procedures can still be utilized, this is done much less frequently, and other methods much more sensitive and selective based on various instruments are now practiced. A more systematic path for the chemical analysis is also usually implemented. An overall path for a modern chemical analysis can be schematically described by the diagram in Fig. 1.2.1. The scheme for the path of a chemical analysis can be much more complicated than the simplified scheme indicated in Fig. 1.2.1. However, the scheme describes the basic operations in a modern chemical analysis.

Figure 1.2.1  Simplified schematics for the path of a chemical analysis.

The decisions regarding various selections for the whole analysis are highly interrelated in the sequence: sample collection → sample preparation → core analytical procedure → data analysis. Depending on the sample, a specific sample preparation must be selected with the goal of a specific core analytical procedure. At the same time, the goal of using a specific core analytical operation is critical for the selection of the sample preparation step. An iterative selection process of sample preparation ↔ core analytical procedure is frequently necessary for the best results. Regarding sample collection, this is not always optional for the analyst, but in some instances sample collection can be adjusted to match the sample preparation and core analytical procedure.

Sample Collection

Sample collection (sampling) is the operation of obtaining the raw sample and delivering it in an appropriate form to the analytical process. Sampling is a very important process and a large body of information presents this subject in detail (see, e.g., [4–7]). The implications of sample collection on the selection of the method of analysis and in particular on the selection of HPLC technique are significant. For example, the collection may include a specific matrix, or may be associated with some type of concentration of the analytes which influence the further selection of the method of analysis. However, the subject of sample collection is beyond the purpose of this chapter. Starting with a given sample, various decisions must be made regarding the analysis, and such decisions will be further discussed only related to HPLC analysis.

Sample Preparation Step

The raw sample is frequently subjected to a sample preparation step. Sample preparation has the role of modifying the raw sample to make it more amenable for the core analytical operation. The operation of sample preparation can range from a minor step to very complex processing of the raw sample (sample as obtained from sampling). Among the potential operations in sample preparation are the routine manipulations of the sample such as homogenization, reduction of the sample size, drying, weighing, volume measuring, and sample dissolution. Also these operations may include cleanup procedures when the matrix of the sample is simplified, fractionation, concentration of the analytes, and chemical modifications. Various separation operations can be applied to the raw sample such as mechanical (e.g., filtration), phase transformation of the mixture of components (e.g., distillation, dissolution, crystallization), extraction with solvents, separations using solid phase extraction, migration in a field for separation (gravitational, magnetic, electric), etc. The initial raw sample can also be subjected to various chemical modifications in order to make it more amenable for the core analysis. Chemical modification may include reactions with one or more reagents (derivatizations), chemical degradations (applied for example to polymers), and pyrolysis (thermal degradation). All these operations will deliver a processed sample that is more amenable for further operations of separation and detection that compose the core analytical operation. Sample preparation is always performed considering the planned core analytical processes that follow in the analysis (further component separation and detection). Since sample preparation is performed with the goal of rendering the raw sample more amenable for the core analytical process, the characteristics of the core analytical operation are always taken into consideration during sample preparation.

Besides the preparation of the raw sample in order to deliver a processed sample, other operations must be considered in this stage of the analysis. These include, for example, the preparation of standards for the generation of calibrations or for obtaining preliminary information on the levels of materials that are further used in the analysis. Most analytical methods include a sample preparation step such that sample preparation is an important part of the whole analysis. For this reason, the subject of sample preparation is extensively presented in the literature and dedicated books, and other materials present as a standalone subject the methodologies for sample preparation (see, e.g., [8–11]). Sample preparation cannot be disconnected from the selection of the core analytical procedure, and depending on the extent and nature of sample preparation, an adequate core analytical method is selected. However, this book presents the specifics for the selection of an HPLC analysis when a given sample must be analyzed after preliminary processing. For this reason, the subject of sample preparation is not further discussed, although within a complete strategy for a chemical analysis sample preparation is an integral part of the analytical process.

Sample preparation is frequently a time- and manpower-consuming step, although it renders to the core analytical method a cleaner and possibly more concentrated processed sample. For this reason, the tendency to use simpler and shorter sample preparation techniques is common. However, these simpler techniques may result in a processed sample having a less simplified matrix and with a modest or no increase in the analyte concentration. As a result, the core analytical technique (e.g., HPLC) has a more difficult task in providing accurate results, and the development of a new and better core analytical technique is a common task.

The Core Analytical Operation

The core analytical operation receiving the processed sample has the role of delivering the analytical results that are further evaluated in the data analysis step for providing the necessary information about the sample. The core analytical operation is usually performed using instrumental techniques that may involve spectroscopic, electrochemical, radiochemical, thermal, or other instrumental procedures. When applied on pure compounds, or when a compound in a mixture has unique physicochemical properties, the analysis can be performed without having a separation step added to the core analytical operation (sample preparation should be considered different from a core analytical procedure). Such techniques can be indicated as nonhyphenated (separation for different ions could be imbedded in the technique itself). Fig. 1.2.2 shows the schematic descriptions of the flow of operations in a nonhyphenated analytical operation.

Figure 1.2.2  Schematic descriptions of the flow of operations in a nonhyphenated core analytical operation.

Because of the necessity to analyze more and more complex samples, many modern analytical techniques that are able to address such analyses have a separation step imbedded in the core analytical operation. These techniques can be indicated as hyphenated. Sample preparation may use separation techniques for sample processing (see, e.g., [8,9]), but they are not online and are not part of the core analytical step. The processed sample may have a less complex matrix than the raw sample or may have a higher concentration of analytes, but it is common that further separations are needed for analytical purposes. When such a separatory step is necessary and it is imbedded (online) in the core analytical procedure, the technique can be indicated as hyphenated. Fig. 1.2.3 shows the schematic descriptions of the flow of operations in an analytical technique that contains the core analytical operations in a separation step.

The separation can be performed by several procedures, but the most common is chromatography. Chromatography designates several similar techniques that allow the separation of different molecular species from a mixture. For the chromatographic separation, the sample is placed in a fluid called the mobile phase, which carries the sample through a material called the stationary phase. The separation in chromatography is achieved because the stationary phase retains stronger or weaker different molecular species from the flowing mobile phase and releases them separately in time back into the mobile phase (different molecular species will have different retention times). The process of moving the analytes through the stationary phase is known as elution. The detection and measurement in analytical chromatography is usually based on a certain physicochemical property of the separated molecule (UV-absorption, refractive index, fluorescence, molecular mass and fragmentation in a mass spectrometer), a property which is different from that of the mobile phase. For analyte detection, many instrumental techniques developed independent of a chromatographic separation step were adapted to be used in analytical chromatography. As a result, numerous chromatographic detectors are based on UV-absorption, refractive index, fluorescence, molecular mass, and fragmentation in a mass spectrometer, etc.

Figure 1.2.3  Schematic descriptions of the flow of operations in an analytical technique that contains in the core analytical operations a separation step ( hyphenated technique).

Besides chromatography, other separation techniques can be connected online with an instrumental technique for detection. Among these electro-separations and membrane separations can be mentioned. Also, more complicated patterns of operations can be encountered, such as combinations of hyphenated techniques (e.g., in bidimensional separations).

Important decisions are made regarding the selection of the type of core analytical procedure. Depending on several factors, such as properties of the analytes, properties of matrix, requested turnaround time of the analysis, available equipment, etc., a technique including a separation step (a hyphenated technique) is or is not selected. The subject is further discussed in this book.

Data Evaluation

Data evaluation, also a part of a chemical analysis, is discussed in many published materials. Data evaluation (or analysis) typically involves statistical evaluations (with numerous chemometrics tools), but includes more general operations such as inspecting, cleaning, transforming, and modeling of the data with the goal of discovering useful information. Data analysis is a much wider field than applied to chemical analysis, but even restricted to this subject, data analysis will be only briefly presented in this book (see Section 1.3). Detailed information regarding statistical data processing can be found in many publications (see, e.g., [12–15]. For details regarding data analysis, the dedicated literature for this subject is recommended.

Qualitative and Quantitative Analysis

The information typically generated in a chemical analysis can be related to the finding of the nature of the analytes (qualitative analysis), level of the analyte (quantitative analysis), or both. Qualitative analysis can refer to various degrees of knowledge beginning with a brute formula, indications of the compound class, presence of specific functional groups, up to a true identification of the compound (possibly including stereo-isomeric structure, detailed structure of the molecule, etc.). Quantitative analysis describes how much analyte is present in a specific sample, and this information may also range from a semiquantitative analysis to a very precise one. The capability of different analytical techniques to provide qualitative and/or quantitative information about the samples is further discussed in Chapters 2 and 3.

For the quantitative analysis, the ideal case is when the signal of the analytical instrument depends linearly on the analyte concentration in a given volume (or amount) of sample. The concentrations C of interest can be determined from the signal y using the relation:

(1.2.1)

The value b is the slope for the dependence and can be obtained using calibrations with standards of known concentration. The standards are typically used as pure compounds and the calibrations are done independently of the sample. The calibration standards can be considered external standards. (An external standard is analyzed in a different run from the sample, while an internal standard is added and analyzed together with the sample.) In many practical applications it is preferable to make the calibrations by adding different levels of the calibration compound to a blank sample that does not contain the analyte. This procedure makes the analysis of the samples containing the calibration standards as close as possible to the analysis of a real sample and allows the subtraction of the overall influence of the matrix in the analysis. For compounds that have similar structures, the calibration curve for only one of the compounds is utilized sometimes, and different compounds are quantitated based on the same calibration. However, this is not a recommended practice and calibration curves are usually necessary for each analyte that must be quantitated.

In order to better mimic the influence of the matrix on the analyte response, it is common to use in the analyses an internal standard at constant concentration that will generate a signal yi.s. for any standard or sample. The calibrations are then generated using the formula:

(1.2.2)

The use of expression 1.2.2 for calibration instead of expression 1.2.1 is expecting that any influence of the matrix on the value of y will also affect yi.s. such that the matrix effects will be compensated.

Some linear calibrations do not pass through the origin, and the calculation of the concentration from the response is done using a relation of the form:

(1.2.3)

This type of dependence may indicate some problems with the particular analytical method, such as sample decomposition or loss of sample in the measurement process when a is negative, or a background interference when a is positive.

Besides linear dependencies between the concentration of the analyte and the signal y, other types of dependencies are also encountered. For example, quadratic dependence is rather common for signal vs. concentration dependence, mainly for wide ranges of concentrations of the analyte.

A different quantitation technique besides the external calibration is that of standard addition. The standard addition method can be used to analyze an unknown sample without the use of a calibration curve obtained in separate runs. The sample to be analyzed has an unknown concentration C0  =  q0/V0, where q0 is the unknown amount in the sample and V0 is the known volume of the sample to be analyzed. It must be assumed, however, that the dependence of the signal on the concentration follows expression 1.2.1 (and not expression 1.2.3). A set of known amounts of analyte {qi} i  =  1, 2…n are added to the unknown sample, leading to the concentrations Ci  =  (q0  +  qi)/(V0  +  Vi), where Vi is the volume of the added solution with the standard and C0  =  q0/V0. The relation between the concentration Ci and the signal yi is in this case given by the relation:

(1.2.4)

The values for C0 and b (as unknown parameters) can be obtained from the added amounts {qi}i  =  1…n and the signal measurements {yi}i  =  0, 1…n using, for example, least-squares fitting. The standard addition method can be used even with a single added amount to the unknown sample. If one single addition q1 is made to the sample, two signals y0 and y1 are generated corresponding to C0 and C1. The two equations of the form 1.2.4 are as follows:

(1.2.5)

The value for qx can be obtained from expression 1.2.5 and the value for Cx = C0 is given by the formula:

(1.2.6)

When the addition of the standard does not dilute the sample (V1  =  0), expression 1.2.6 can be written in the form:

(1.2.7)

The accuracy of standard addition is significantly better when more than one standard addition is performed. The calculation of concentration using standard addition using only one addition is in some respects equivalent to using a calibration curve with only two points.

Other procedures can also be used for quantitation (see, e.g., [8]). One of these procedures uses a response factor for quantitation. This response factor is calculated as the ratio of the responses yA and yB of two compounds A and B at the same concentration, A being the analyte and B the internal standard. The ratio of the two signals is usually obtained as an average of several measurements giving the response factor:

(1.2.8)

Ideally, the value for fA remains constant for an interval of values for the pairs of concentrations of the standard and the analyte. The unknown concentration CA,x of compound A is then obtained by measuring in the same run the signal of the compound A to be analyzed (at unknown concentration) and the signal of the standard B (at known concentration CB) using the formula:

(1.2.9)

Typically, it is recommended that the analyte A and the internal standard B are chemically similar or even identical but isotopically labeled.

The selection of a specific quantitation procedure depends on numerous factors such as properties of the matrix, concentration of the analyte in the sample, and availability of isotopically labeled standards. Although ideally all procedures should generate the same result, some differences exist between different procedures. The calibration with pure standards has the disadvantage that the matrix may influence the instrument response, and for real samples the response to the same concentration of the analyte may be different than the response for pure solutions. The use of an internal standard and use of expression 1.2.2 for calibration requires that the internal standard response must be constant in every sample. For the case of analyzing multiple analytes and one internal standard, any error in the internal standard measurement will affect all the other measurements, which can be a disadvantage. The use of standard addition takes into account the influence of sample matrix, and when used with multiple standard additions provides sufficient reliability of the procedure. However, conditions such as the linearity of the dependence and zero value for the response in the absence of the analyte (dependence of the form 1.2.1) must be satisfied for its applicability. The use of a response factor for quantitation requires identical behavior of the compound used as internal standard B and of the analyte A.

Figure 1.2.4  Diagram suggesting the iterative process of establishing a method for analysis.

Decision Process in Selecting a Method of Analysis

The path for selection of a method of analysis is an iterative process. Each part of the analysis has its own characteristics and the sample characteristics, decisions on sample preparation and each component of the core analytical method must be taken into consideration with implications from one phase to another. This process is schematically illustrated in Fig. 1.2.4.

The cycle indicated in Fig. 1.2.4 can be followed only once, but it is possible to be repeated as decisions at one step are influenced by those from previous steps. In the decision process all the preliminary information should be included, but also any additional information gathered along the decision process and possibly results from preliminary experiments. The time factor is also to be taken into consideration in the decision process, the implementation of an analytical method usually has a deadline.

1.3. Statistical Evaluation of Data and Criteria for Method Validation

The results from both qualitative and quantitative analysis can be processed using statistical concepts (see, e.g., [8]), although statistics is more frequently applied for the evaluation of the quantitative results. Accuracy (nearness to the true value) and precision (repeatability of the results) are the most common parameters generated using statistical evaluations. However, other aspects related to analytical data characterization have a statistical interpretation.

Precision and Accuracy in Quantitative Chemical Analysis

In most quantitative analytical determinations the measurement of the amount or of the concentration of an analyte is performed using the dependence of a measured signal y on the concentration (or quantity of the analyte) x:

(1.3.1)

The amount or the concentration x can be calculated based on the dependence described by relation 1.3.1 using a number of procedures that generate a calibration function:

(1.3.2)

When more than one measurement is performed, the results for x are typically scattered around a specific value, the measurements being always affected by errors. The errors of measurements are classified as systematic (determinate) or random (see, e.g., [16]). Systematic errors are generated by a specific cause, and they affect the accuracy of the results. Random errors do not have an assigned cause, and they affect the precision of the measurement. Precision refers to the reproducibility of measurement within a set, indicating the scatter or dispersion of a set of measurements about their central value [12]. For example, repeated determination of the amount or concentration of an analyte generates for each measurement j a value xj. If the number of measurements is n, they will generate a set of measurements also indicated in statistics as a sample {x1, x2…xn} of measurements (not to be confused with sample  =  specimen in a chemical analysis). For this set, an average (or the mean) m, and a standard deviation (SD) s can be obtained using the following formulas:

(1.3.3)

and:

(1.3.4)

The standard deviation s shows the distribution of measurements about the mean and characterizes precision. A small standard deviation indicates good precision for the set of measurements. Both m and s are expressed in the same units. A relative standard deviation % (RSD%) is frequently used instead of standard deviation s for the characterization of precision, and it is expressed by the formula:

(1.3.5)

Instead of standard deviation s, it is also common to use for precision characterization the value s² called variance.

In statistics, any obtained set of measurements {x1, x2…xn} represents a sample of values taken by a random variable x. This sample is a part of an infinite set of values that variable x may have. This infinite set of values is known as a population. Similar to the average m and standard deviation s for one sample, the population is characterized by the mean μ of the population and the standard deviation σ of the population which are given by the formulas:

(1.3.6)

(1.3.7)

Since an infinite number of measurements cannot be performed, the true values for μ and σ are not known, and they are approximated with m and s for large number of measurements n. The true (ideal) value for μ represents the ideal result for all the measurements, but does not necessarily represent the true value of the measured quantity. The difference between μ and the true value (if known) of a quantity is called the bias. This true value can be, for example, the level of an analyte in a standard (regardless of the fact that making of the standard can also be affected by errors). Another convention for a true value is the assumption that it is the one generally accepted. Accepting m0 as a true value of the measurement and taking the population mean μ  ≈  m, the bias is approximated by the following formula:

(1.3.8)

A method is considered accurate when the bias is very small (or even zero).

For the same quantity, more than one set of measurements can be performed (e.g., sets of measurements of the same sample performed in different laboratories), generating a number of samples of data, each one with its own mean m1, m2, … mk. Although expected to be close to each other, these means are not necessarily identical. A total mean m can be obtained from these partial means, m being equal with the average of all measurements (addition is associative). The standard deviation of the mean m is known a standard error of the mean sm and it is expressed by the formula:

(1.3.9)

One question regarding the distribution of measurements about their mean is the expected frequency of occurrence of an error as a function of the error magnitude. It is expected that larger random errors will be less frequent than small errors. The most commonly utilized function, which describes well the relative frequency of occurrence of random errors in large sets of measurements, is given by the Gauss formula:

(1.3.10)

This frequency function f(x) (known as Gaussian density function) shows that the point of maximum frequency is obtained for the mean (when x  =  μ), the distribution of positive and negative errors is symmetrical, and as the magnitude of the deviation from the mean increases, an exponential decrease in the frequency takes place. The errors with the relative frequency of occurrence given by relation 1.3.10 have a so-called normal distribution N(μ,σ). Besides Gaussian density functions, other frequency functions are known (e.g., Student frequency function).

For x with a normal distribution N(μ,σ), the following substitution:

(1.3.11)

leads to a variable y with a normal distribution N(0,1).

The value (x  –  μ) is so-called mean-centered value, and by division with σ, y is expressed in σ units (or it is standardized). Mean-centered standardized variables y (standardized variates) are commonly used in statistical data processing.

The area under the curve f(x) for x  <  α, with f(x) given by formula 1.3.10, will give a cumulative frequency distribution expressed by the formula:

(1.3.12)

The cumulative frequency distribution is equal to the probability P for x to have a value below α in any measurement. The integral of f(x) over the whole space gives P  =  1. The values of function F(α) (for f(x) a Gaussian function) are known and tabulated (e.g., [14]) or given in computer statistical packages (e.g., [15]).

The mean mk of a sample of n values {x1, x2…xn. Assuming that x has a normal distribution N(μ,σ(m) takes a continuous range of values with a normal distribution N(μ,σ/n¹/²).

A mean-centered standardized variable defined by the formula:

will have an N(0,1) distribution. With the help of variable z it is possible to evaluate how close the values of μ and mk are for a certain population. For the variable z given by relation 1.3.13, two values –zα/2 and z1−α/2 can be found such that the probability for z of being outside the interval (-zα/2, z1−α/2) is equal to α (areas α/2 indicated in Fig. 1.3.1). The interval (–zα/2, z1−α/2) is indicated as confidence interval. For z being inside the interval (–zα/2, z1−α/2), or:

the value for probability will be P  =  1  –  α.

Expression 1.3.14 can indicate the limits for mk with the probability P  =  1  –  α. For this purpose, it should be noted that zα/2  =  z1–α/2, and rearranging expression 1.3.14 the result is as follows:

(1.3.15)

The values for z1–α/2 for a selected probability P and Gaussian distribution are available in the literature (see, e.g., [14,17]). Such values are given for P  =  1  –  α (two-sided normal distribution) or for P  =  (1  –  α)/2 (one-sided normal distribution). Fig. 1.3.1 shows the curve N(0,1) on which are indicated two values zα/2 and z1–α/2 such that the probability for z of being outside the confidence interval (–zα/2, z1−α/2) is equal to α (area under the curve). Larger values for α have correspondingly smaller values for P and smaller values for z1−α/2.

Sensitivity and Limit of Detection

The sensitivity of a quantitative analytical method can be defined as the slope of the curve that is obtained when the result of a series of measurements is plotted against the amount (or concentration) that is to be determined. For the dependence described by or:

Figure 1.3.1  Gaussian curve N(0,1), showing two values, – z α/2 and z 1 − α/2 , such that the probability for z of being outside the interval (– z α/2 , z 1 − α/2 ) is equal to α (and area under the curve is P   =   1 – α showing probability inside the same interval).

(1.3.16)

It is common that the dependence described by expression 1.3.16 is a linear function (see expression 1.2.3) and has the expression:

(1.3.17)

is equal to the constant b. Sensitivity can therefore be determined from the calibration curve for the method. For nonlinear dependencies the definition still can be applied, but the sensitivity is not