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Chemical Analysis by Francis Rouessac in pdf


Download this PDF book: Chemical Analysis: Modern Instrumentation Methods and Techniques 2nd Edition by Francis Rouessac, Annick Rouessac.

Completely revised and updated, Chemical Analysis: Second Edition is an essential introduction to a wide range of analytical techniques and instruments.

Assuming little in the way of prior knowledge, this text carefully guides the reader through the more widely used and important techniques, whilst avoiding excessive technical detail.

Provides a thorough introduction to a wide range of the most important and widely used instrumental techniques

Maintains a careful balance between depth and breadth of coverage

Includes examples, problems and their solutions

Includes coverage of latest developments including supercritical fluid chromatography and capillary electrophoresis

Chapter One

General aspects of chromatography

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 in the gaseous or liquid state. The basic principle is based 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 components of the sample have differing solubilities in each phase. The differential migration of compounds lead to their separation. Of all the instrumental analytical techniques this hydrodynamic procedure is the one with the broadest application. Chromatography occupies a dominant position that all laboratories involved in molecular analysis can confirm.

1.1 General concepts of analytical chromatography

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

A basic chromatographic process may be described as follows (Figure 1.1):

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 mixture to be separated into individual components.

3. The sample is then taken up by continuous addition of the mobile phase, which goes through the column by gravity, carrying the various constituents of the mixture along with it. This process is called elution. If the components migrate at different velocities, they will become separated from each other and can be recovered, mixed with the mobile phase.

This basic procedure, carried out in a column, has been used since its discovery on a large scale for the separation or purification of numerous compounds (preparative column chromatography), but it has also progressed into a stand-alone analytical technique, particularly once the idea of measuring the migration times of the different compounds as a mean to identify them had been conceived, without the need for their collection. To do that, an optical device was placed at the column exit, which indicated the variation of the composition of the eluting phase with time. This form of chromatography, whose goal is not simply to recover the components but to control their migration, first appeared around 1940 though its development since has been relatively slow.

The identification of a compound by chromatography is achieved by comparison: To identify a compound which may be A or B, a solution of this unknown is run on a column. Next, its retention time is compared with those for the two reference compounds A and B previously recorded using the same apparatus and the same experimental conditions. The choice between A and B for the unknown is done by comparison of the retention times.

In this experiment a true separation had not been effected (A and B were pure products) but only a comparison of their times of migration was performed. In such an experiment there are, however, three unfavourable points to note: the procedure is fairly slow; absolute identification is unattainable; and the physical contact between the sample and the stationary phase could modify its properties, therefore its retention times and finally the conclusion.

This method of separation, using two immiscible phases in contact with each other, was first undertaken at the beginning of the 20th century and is credited to botanist Michael Tswett to whom is equally attributed the invention of the terms chromatography and chromatogram.

The technique has improved considerably since its beginnings. Nowadays chromatographic techniques are piloted by computer software, which operate highly efficient miniature columns able to separate nano-quantities of sample. These instruments comprise a complete range of accessories designed to assure reproducibility of successive experiments by the perfect control of the different parameters of separation. Thus it is possible to obtain, during successive analyses of the same sample conducted within a few hours, recordings that are reproducible to within a second (Figure 1.2).

The essential recording that is obtained for each separation is called a chromatogram. It corresponds to a two-dimensional diagram traced on a chart paper or a screen that reveals the variations of composition of the eluting mobile phase as it exits the column. To obtain this document, a sensor, of which there exists a great variety, needs to be placed at the outlet of the column. The detector signal appears as the ordinate of the chromatogram while time or alternatively elution volume appears on the abscissa.

* The identification of a molecular compound only by its retention time is somewhat arbitrary. A better method consists of associating two different complementary methods, for example, a chromatograph and a second instrument on-line, such as a mass spectrometer or an infrared spectrometer. These hyphenated techniques enable the independent collating of two different types of information that are independent (time of migration and `the spectrum'). Therefore, it is possible to determine without ambiguity the composition and concentration of complex mixtures in which the concentration of compounds can be of the order of nanograms.

1.2 The chromatogram

The chromatogram is the representation of the variation, with time (rarely volume), of the amount of the analyte in the mobile phase exiting the chromatographic column. It is a curve that has a baseline which corresponds to the trace obtained in the absence of a compound being eluted. The separation is complete when the chromatogram shows as many chromatographic peaks as there are components in the mixture to be analysed (Figure 1.3).

A constituent is characterized by its retention time [t.sub.R], which represents the time elapsed from the sample introduction to the detection of the peak maximum on the chromatogram. In an ideal case, [t.sub.R] is independent of the quantity injected.

A constituent which is not retained will elute out of the column at time [t.sub.M], called the hold-up time or dead time (formerly designated [t.sub.0]). It is the time required for the mobile phase to pass through the column.

The difference between the retention time and the hold-up time is designated by the adjusted retention time of the compound, [t'.sub.R].

If the signal sent by the sensor varies linearly with the concentration of a compound, then the same variation will occur for the area under the corresponding peak on the chromatogram. This is a basic condition to perform quantitative analysis from a chromatogram.

1.3 Gaussian-shaped elution peaks

On a chromatogram the perfect elution peak would have the same form as the graphical representation of the law of Normal distribution of random errors (Gaussian curve 1.1, cf. Section 22.3). In keeping with the classic notation, ? would correspond to the retention time of the eluting peak while [sigma] to the standard deviation of the peak ([[sigma].sup.2] represents the variance). y represents the signal as a function of time x, from the detector located at the outlet of the column (Figure 1.3).

This is why ideal elution peaks are usually described by the probability density function (1.2).


y = 1 / [square root of 2[pi]] . exp [- [x.sup.2] / 2] (1.2)

This function is characterized by a symmetrical curve (maximum for x = 0, y = 0 3999) possessing two inflection points at x = +/ - 1 (Figure 1.3), for which the ordinate value is 0.242 (being 60.6 per cent of the maximum value). The width of the curve at the inflection points is equal to 2[sigma], ([sigma] = 1 .

In chromatography, [w.sub.1/2] represents the width of the peak at half-height [w.sub.1/2] = 2.35[sigma] and [[sigma].sup.2] the variance of the peak. The width of the peak `at the base' is labelled w and is measured at 13.5 per cent of the height. At this position, for the Gaussian curve, w = 4[sigma] by definition.

Real chromatographic peaks often deviate significantly from the Gaussian ideal aspect. There are several reasons for this. In particular, there are irregularities of concentration in the injection zone, at the head of the column. Moreover, the speed of the mobile phase is zero at the wall of the column and maximum in the centre of the column.

The observed asymmetry of a peak is measured by two parameters, the skewing factor a measured at 10 per cent of its height and the tailing factor TF measured at 5 per cent (for the definition of these terms, see Figure 1.4):

a = b / f (1.3)

TF = b + f / 2f (1.4)

1.4 The plate theory

For half a century different theories have been and continue to be proposed to model chromatography and to explain the migration and separation of analytes in the column. The best known are those employing a statistical approach (stochastic theory), the theoretical plate model or a molecular dynamics approach.

To explain the mechanism of migration and separation of compounds on the column, the oldest model, known as Craig's theoretical plate model is a static approach now judged to be obsolete, but which once offered a simple description of the separation of constituents.

Although chromatography is a dynamic phenomenon, Craig's model considered that each solute moves progressively along a sequence of distinct static steps. In liquid-solid chromatography this elementary process is represented by a cycle of adsorption/desorption. The continuity of these steps reproduces the migration of the compounds on the column, in a similar fashion to that achieved by a cartoon which gives the illusion of movement through a sequence of fixed images. Each step corresponds to a new state of equilibrium for the entire column.

These successive equilibria provide the basis of plate theory according to which a column of length L is sliced horizontally into N fictitious, small plate-like discs of same height H and numbered from 1 to n. For each of them, the concentration of the solute in the mobile phase is in equilibrium with the concentration of this solute in the stationary phase. At each new equilibrium, the solute has progressed through the column by a distance of one disc (or plate), hence the name theoretical plate theory.

The height equivalent to a theoretical plate (HETP or H) will be given by equation (1.5):

H = L / N (1.5)

This employs the polynomial approach to calculate, for a given plate, the mass distributed between the two phases present. At instant I, plate J contains a total mass of analyte [m.sub.T] which is composed of the quantity [m.sub.M] of the analyte that has just arrived from plate J - 1 carried by the mobile phase formerly in equilibrium at instant I - 1, to which is added the quantity [m.sub.S] already present in the stationary phase of plate J at time I - 1 (Figure 1.5).

[m.sub.T] (I, J) = [m.sub.M] (I - 1, J - 1) + [m.sub.S] (I - 1, J)

If it is assumed for each theoretical plate that: [m.sub.S] = K[m.sub.M] and [m.sub.T] = [m.sub.M] + [m.sub.S], then by a recursive formula, [m.sub.T] (as well as [m.sub.M] and [m.sub.S]), can be calculated. Given that for each plate the analyte is in a concentration equilibrium between the two phases, the total mass of analyte in solution in the volume of the mobile phase [V.sub.M] of the column remains constant, so long as the analyte has not reached the column outlet. So, the chromatogram corresponds to the mass in transit carried by the mobile phase at the (N + 1)th plate (Figure 1.6) during successive equilibria. This theory has a major fault in that it does not take into account the dispersion in the column due to the diffusion of the compounds.

* The plate theory comes from an early approach by Martin and Synge (Nobel laureates in Chemistry, 1952), to describe chromatography by analogy with distillation and counter current extraction as models. This term, used for historical reasons, has no physical significance, in contrast to its homonym which serves to measure the performances of a distillation column.

The retention time [t.sub.R], of the solute on the column can be sub-divided into two terms: [t.sub.M] (hold-up time), which cumulates the times during which it is dissolved in the mobile phase and travels at the same speed as this phase, and [t.sub.S] the cumulative times spent in the stationary phase, during which it is immobile. Between two successive transfers from one phase to the other, it is accepted that the concentrations have the time to re-equilibrate.

* In a chromatographic phase system, there are at least three sets of equilibria: solute/mobile phase, solute/stationary phase and mobile phase/stationary phase. In a more recent theory of chromatography, no consideration is given to the idea of molecules immobilized by the stationary phase but rather that were simply slowed down when passing in close proximity.

1.5 Nernst partition coefficient (K)

The fundamental physico-chemical parameter of chromatography is the equilibrium constant K, termed the partition coefficient, quantifying the ratio of the concentrations of each compound within the two phases.

K = [ITLITL.sub.S] / CM = Molar concentration of the solute in the stationary phase / Molar concentration of the solute in the mobile phase (1.6)

Values of K are very variable since they can be large (e.g. 1000), when the mobile phase is a gas or small (e.g. 2) when the two phases are in the condensed state. Each compound occupies only a limited space on the column, with a variable concentration in each place, therefore the true values of [ITLITL.sub.M] and [ITLITL.sub.S] vary in the column, but their ratio is constant.

Chromatography and thermodynamics. Thermodynamic relationships can be applied to the distribution equilibria defined above. K, ([ITLITL.sub.S]/[ITLITL.sub.M]), the equilibrium constant relative to the concentrations ITLITL of the compound in the mobile phase (M) and stationary phase (S) can be calculated from chromatography experiments. Thus, knowing the temperature of the experiment, the variation of the standard free energy [DELTA]Go for this transformation can be deduced:

[ITLITL.sub.M] [??] [ITLITL.sub.S] [DELTA]Go = -RT ln K

In gas chromatography, where K can be easily determined at two different temperatures, it is possible to obtain the variations in standard enthalpy [DELTA]Ho and entropy [DELTA]So (if it is accepted that the entropy and the enthalpy have not changed):


The values of these three parameters are all negative, indicating a spontaneous transformation. It is to be expected that the entropy is decreased when the compound moves from the mobile phase to the stationary phase where it is fixed. 

In the same way the Van't Hoff equation can be used in a fairly rigorous way to predict the effect of temperature on the retention time of a compound. From this it is clear that for detailed studies in chromatography, classic thermodynamics are applicable.

d ln K / dT = [DELTA]H / R[T.sup.2]


PART 1: Separation Methods

General aspects of chromatography

Gas chromatography

High-performance liquid chromatography

Ion chromatography

Thin layer chromatography

Supercritical fluid chromatography

Size exclusion chromatography

Capillary electrophoresis and electrochromatography.

PART 2: Spectroscopic Methods

Ultraviolet and visible absorption spectroscopy

Infrared spectroscopy

Fluorimetry and chemiluminescence

X-ray fluorescence spectrometry

Atomic absorption and flame emission spectroscopy

Atomic emission spectroscopy

Nuclear magnetic resonance spectroscopy

PART 3: Other Methods

Mass spectrometry

Labelling methods

Elemental analysis

Potentiometric methods

Voltammetric and coulometric methods

Sample treatment

Basic statistical parameters

About the Author

Francis Rouessac and Annick Rouessacis the authors of Chemical Analysis: Modern Instrumentation Methods and Techniques, 2nd Edition, published by Wiley.

About the book:

Publisher ‏ : ‎ Wiley; 2nd edition (May 6, 2013)

Publication date ‏ : ‎ May 6, 2013

Language ‏ : ‎ English

Pages ‏ : ‎ 821

File‏ : ‎ PDF, 19 MB


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