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coupled methods, nondestructive testing, and the concept of speciation (distinction between the various structures in which an element may be present in the sample). These methods use small samples that require little or no preparation prior to measurement. Current analytical trends also include an increase in miniaturized, portable instruments, as well as specific or automatic analysers.

      Whatever the tool used to establish the relationship between the signal and the concentration, we also have to determine the limits of quantification. Below that limit, any attempt at quantification is bound to fail, while above it the relationship no longer applies.

      To conduct these studies, analysts must not only be trained in the various techniques, but they must also know the basic concepts of chemistry, particularly since a compound can often be assayed by various methods. Choosing a good method, and if possible the best method, requires knowing many parameters. Therefore, when a new analytical objective has been defined, the problem must be addressed methodically. We must first choose the right method: spectroscopic, electrochemical, separation, etc. Then the choice of technique is made, followed by the choice of process in terms of sampling and prior treatment of the sample. Finally, a protocol, the recipe so to speak, is established. In general, this is a process governed by established national or international standards, which leads to the chosen operating procedure. This approach focuses on standardizing all the steps: from sample preparation all the way to conducting measurements. Finally, results must be established according to existing standards. Additionally, the raw analytical data conserved as computer files cannot be changed. This entire approach is the subject of official texts known as Good Laboratory Practice (GLP).

      To recommend the best method in order to resolve an analytical problem, there is a science called chemometrics. Its purpose is to help the analyst, as a function of specific requirements and aims: minimum sampling plan, appropriate methodology, data processing and interpretation of results. Thanks to the use of IT tools, it seeks to provide a correct answer by exploiting the results with statistical methods in order to reduce the number of tests for long or costly analyses.

      In this way, users may acquire devices that meet the standard of precision and quality necessary to acquire certification or to have a laboratory officially recognized for the quality of its results. These accreditation procedures are now imposed by many control organizations around the world.

      Analytical chemistry is therefore essential in many fields other than the traditional ones of chemistry or chemical engineering. It can be found in fields as varied as medicine, food, biochemistry, the environment (pollution), safety (explosives, drugs and chemicals), and art. Now more widely used in human activities than ever, analytical chemistry can be of benefit to us all.

      INTRODUCTION

      Chromatography, the process by which the components of a mixture can be separated, has become one of the primary analytical methods for the identification and quantification of compounds. The basic principle is founded on the concentration equilibrium of the components of interest between two immiscible phases. One is called the stationary phase, because it is immobilized within a column or fixed upon a support, while the second, called the mobile phase, is forced through the first. The phases are chosen such that the components of the sample have differing solubilities in each phase. The differential migration of compounds leads to their separation. This hydrodynamic process, which has been constantly evolving since its discovery, is an analytical method that no laboratory involved in molecular analysis can ignore, as its applications are so numerous.

      Objectives

      Review the principle of chromatography

      Distinguish between separation and analysis by chromatography

      Explain the protocol of a chromatographic analysis

      Use a chromatogram and model its signals

      Describe the various retention parameters

      Discuss the hydrodynamic aspects of a separation

      Define parameters influencing the efficiency of a separation

      Classify the various chromatography techniques

      Describe the steps of an assay using chromatography

      Chromatography is a physico‐chemical method of separation of components within mixtures, liquid or gaseous, in the same vein as distillation, crystallization or fractionated extraction. The applications of this process are therefore potentially numerous, since many heterogeneous mixtures, or those in solid form, can be dissolved by a suitable solvent (which becomes, of course, a supplementary component of the mixture).

      1 A vertical, hollow glass tube (the column) is filled with a suitable finely powdered solid, the stationary phase.

      2 At the top of this column is placed a small volume of the sample to be separated into individual components.

      3 The sample is then forced through the column from inlet to outlet by continuous addition of the mobile phase, carrying the various constituents of the sample along with it. If the components migrate at different velocities, they will become separated from each other and can be recovered, each in solution with the mobile phase.

      While this use of chromatography has continued since its origins, this process became a method of analysis with the idea of measuring the retention time of compounds through the column in order to identify them. To do so, it became essential to control certain parameters (flow rate, temperature, etc.) and a detector had to be placed at the column’s outlet to identify compositional changes in the mobile phase. This form of chromatography, whose goal is not simply to recover the components but to measure their retention time, has developed slowly.

Schematic illustration of a basic experiment in chromatography.

      In this experiment, there was no true separation (A and B were pure products), only a comparison of the products’ retention times. However, this method does have three weaknesses: the procedure is fairly slow; absolute identification is unattainable; and the physical contact between the sample and the stationary phase could modify the sample’s properties, in particular the retention times.

      This specific method of separation, in its modern

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