Injectors

The injector, which is the sample’s entrance to the chromatograph, has different functions. Besides its role as an inlet for the sample, it must vaporize, mix with the carrier gas and bring about the sample at the head of the column. The characteristics of the injectors, as well as the modes of injection, differ according to column type. The use of an automatic injection system can significantly enhance measurement precision.
Direct vaporization injector

Sample introduction and the injection chamber

Sample introduction
The most common injection method is where a microsyringe is used (Figure 2.3) to inject a very small quantity of sample in solution (e.g. 05 L), through a rubber septum into a flash vaporizer port at the head of the column. For gaseous samples,

Carrier gas and flow regulation

The mobile phase is a gas (helium, hydrogen or nitrogen), either drawn from a commercially available gas cylinder or obtained, in the case of hydrogen or nitrogen, from an on-site generator, which provides gas of very high purity. The carrier gas must be free of all traces of hydrocarbons, water vapour and

Components of a GC installation

   
A gas chromatograph is composed of several components within a special frame. These components include the injector, the column and the detector, associated with a thermostatically controlled oven that enables the column to attain high temperatures (Figure 2.1). The mobile phase that transports the analytes through the column is a gas referred to as the carrier gas. The carrier gas flow, which is precisely controlled, enables reproducibility of the retention times.
Analysis starts when a small quantity of sample is introduced as either liquid or gas into the injector, which has the dual function of vaporizing the sample and mixing it with the gaseous flow at the head of the column. The column is usually a narrow-bore tube which coils around itself with a length that can vary from 1 to over 100 m, depending upon the type and the contents of the stationary phase. The column, which can serve for thousands of successive injections, is housed in a thermostatically controlled oven. At the end of the column, the mobile phase (carrier gas), passes through a detector before it exits to the atmosphere. Some gas chromatographs models of reduced size have their own electrical supply, enabling them to operate in the field (see Figure 2.19).

Gas chromatography

Gas chromatography (GC) is a widely used technique whose first applications date back more than 60 years. Since then, development has continued making the best use of the extreme sensitivity, versatility, the possibilities of automation and the ease with which new analyses can be developed. Because separation of compound mixtures on the column occurs while they are in the gaseous state, solid and liquid samples must first be vaporized. This represents, without hesitation, the greatest constraint of gas phase chromatography and weighs against it,

Supercritical fluid chromatography (SFC)

Here the mobile phase is a fluid in its supercritical state, such as carbon dioxide at about 50

Gas phase chromatography (GC)

The mobile phase is an inert gas and as above this form of chromatography can be sub-divided according to the nature of the phase components:

Liquid phase chromatography (LC)

This type of chromatography, in which the mobile phase is a liquid belongs to the oldest known form of the preparative methods of separation. This very broad category can be sub-divided depending on the retention phenomenon.

Liquid/solid chromatography (or adsorption chromatography)
The stationary phase is a solid medium to which the species adhere through the dual effect of physisorption and chemisorption. The physico-chemical parameter involved here is the adsorption coefficient. Stationary phases have made much progress since the time of Tswett, who used calcium carbonate or inulin (a very finely powdered polymer of ordinary sugar).
Ion chromatography (IC)

Classification of chromatographic techniques

Chromatographic techniques can be classified according to various criteria: as a function of the physical nature of the phases; of the process used; or by the physico-chemical

Optimization of a chromatographic analysis

Analytical chromatography is used essentially in quantitative analysis. In order to achieve this effectively, the areas under the peaks must be determined with precision, which in turn necessitates well-separated analytes to be analysed. A certain experience in chromatography is required when the analysis has to be optimized, employing all available resources in terms of apparatus and software that can simulate the results of temperature modifications, phases and other physical parameters.
  In gas phase chromatography, the separations can be so complex that it can be difficult to determine in advance whether the temperature should be increased or decreased. The choice of column, its length, its diameter, the stationary phase composition and the phase ratio (VM/VS) as well as the parameters of separation (temperature and flow rate), are amongst the factors which interact with each other.

Knox’s equation

Another, more recent equation, the Knox equation, is applicable to various types of liquid chromatography and includes the adjusted height h:

Golay’s equation

A few years after Van Deemter, Golay proposed a modified relationship reserved to capillary columns used in gas phase chromatography. There is no A term because there is no packing in a capillary column (see paragraph 2.5.2).

Van Deemter’s equation

This equation is based on a Gaussian distribution, similar to that of plate theory. Its simplified form, proposed by Van Deemter in 1956, is well known (expression 1.30). The expression links the plate high H to the average linear velocity of the mobile phase ¯u in the column (Figure 1.11).


The three experimental basic coefficients A, B and C are related to diverse physico-chemical parameters of the column and to the experimental conditions. If H is expressed in cm, A will also be in cm, B in cm2/s and C in s (where velocity is measured in cm/s).


This equation reveals that there exists an optimal flow rate for each column, corresponding to the minimum of H, which predicts the curve described by Equation 1.30.
The loss in efficiency as the flow rate increases is obvious, and represents what occurs when an attempt is made to rush the chromatographic separation by increasing the pressure upon the mobile phase.
However, intuition can hardly predict the loss in efficiency that occurs when the flow rate is too slow. To explain this phenomenon, the origins of the terms A, B and C must be recalled . Each of these parameters represents a domain of influence which can be perceived on the graph (Figure 1.11).
The curve that represents the Van Deemter equation is a hyperbola which goes through a minimum H
min
 when:


Term A is related to the flow profile of the mobile phase passing through the stationary phase. The size of the particles (diameter d
), their dimensional distribution and the uniformity of the packing (factor characteristic of packing ) can all be the origin of flow paths of different length which cause broadening of the solute band and improper exchanges between the two phases. This results in turbulent or Eddy diffusion, considered to have little importance in liquid chromatography and absent for WCOT capillary columns in GC (Golay’s equation without term A, cf. paragraph 1.10.2). For a given column, nothing can be done to reduce the A term.

The rate theory of chromatography

In all of the previous discussion and particularly in the plate theory, the velocity of the mobile phase in the column and solute diffusion are, perhaps surprisingly, never taken into account. Of all things, the speed should have an influence upon the progression of the analytes down the column, hence their dispersion and by consequence, upon the quality of the analysis undertaken.

Resolution factor between two peaks

To quantify the separation between two compounds, another measure is provided by the resolution factor R. Contrary to the selectivity factor which does not take into account peak widths, the following expression is used to calculate R between two compounds 1 and 2 (Figure 1.9):

Separation (or selectivity) factor between two solutes

The separation factor , (1.24) enables the comparison of two adjacent peaks 1 and 2 present in the same chromatogram (Figure 1.8). Using Equations 1.20 and 1.19, it can be concluded that the separation factor can be expressed by Equation 1.25.
By definition  is greater than unity (species 1 elutes faster than species 2):

Retention (or capacity) factor k

When a compound of total mass mT
is introduced onto the column, it separates into two quantities:mM, the mass in the mobile phase and m, the mass in the stationary phase. During the solute’s migration down the column, these two quantities remain constant. Their ratio, called the retention factor k, is constant and independent of mT:S

Hold-up volume (or dead volume) VM

The volume of the mobile phase in the column (known as the dead volume), VM
, corresponds to the accessible interstitial volume. It is often calculated from a chromatogram, provided a solute not retained by the stationary phase is present. The dead volume is deduced from tM
and the flow rate F:

Retention volume (or elution volume) VR

The retention volume VR
of an analyte represents the volume of mobile phase necessary to enable its migration throughout the column from the moment of entrance to the moment in which it leaves. To estimate this volume, different methods (direct or indirect) may be used, that depend of the physical state of the mobile phase. On a standard chromatogram with time in abscissa, VR
is calculated from expression 1.16, if the flow rate F is constant,

Retention parameters

Hold-up times or volumes are used in chromatography for various purposes, particularly to access to retention factor k. and thermodynamic parameters. Only basic expressions are given below.

 Retention times

The definition of retention times, hold-up time, t M, retention time, tR and adjusted retention time, t R, have been given previously.

Height equivalent to a theoretical plate (HETP)

The equivalent height of a theoretical plate H, as already defined (expression 1.5), is calculated for reference compounds to permit a comparison of columns of different lengths. H does not behave as a constant, its value depends upon the compound chosen and upon the experimental conditions.
For a long time in gas chromatography an adjustment value called the effective height of a theoretical plate Heff was calculated using the true efficiency. This corresponds to the Equation 1.14;

Effective plates number (real efficiency)

In order to compare the performances of columns of different design for a given compound – or to compare, in gas chromatography, the performances between a capillary column and a packed column – more realistic values are obtained by replacing the total retention time t
, which appears in expressions 1.8–1.10, by the adjusted retention time t RRR
which does not take into account the hold-up time t
spent by any compound in the mobile phase t R=tR−t The three preceding expressions become:

Column efficiency Theoretical efficiency (number of theoretical plates)

Column efficiency
Theoretical efficiency (number of theoretical plates)

As the analyte migrates through column, it occupies a continually expanding zone (Figure 1.6). This linear dispersion measured by the variance
increases with the distance of migration. When this distance becomes L, the total column length, the variance will be:

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.

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 C
M
and C

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):

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  to the standard deviation of the peak (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).

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).

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.

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.

PART 1 Separation methods

PART 1
Separation methods

The invention of chromatography

Who invented chromatography, one of the most widely used laboratory techniques? This question leads to controversies. In the 1850s, Schönbein used filter paper to partially separate substances in solution. He found that not all solutions reach the same height when set to rise in filter paper. Goppelsröder (in Switzerland) found relations between the height to which a solution climbs in paper and its chemical composition. In 1861 he wrote ‘I am convinced that this method will prove to be very practical for the rapid determination of the nature of a mixture of dyes, especially if appropriately chosen and characterised reagents are used’.