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.