Analyte interactions, with the stationary phase

Indicate for each of the chromatographic techniques below the term which best expresses the analyte interaction with the stationary phase. 

The most popular classification scheme stems from the manner in which the analyte interacts with the stationary phase. With this approach, chromatography may be divided into five separation mechanisms adsorption, partition, size exclusion, affinity, and ion exchange, as illustrated in Figure 1.1. 

Adsorption chromatography is based on competition for neutral analytes between the liquid mobile phase and a neutral, solid stationary phase. The analytes interact with the stationary phase according to the premise like likes like polar solutes will be retained longest by polar stationary phases, and nonpolar solutes will be retained best by nonpolar stationary phases. In adsorption chromatography the solute molecules are in contact with both the stationary phase and the mobile phase, simultaneously. Under these conditions, the solutes are said to be in an anisotropic environment. 

Specific and essentially stand-alone mode of liquid chromatography is associated with the absence or suppression of any analyte interactions with the stationary phase, which is called size-exclusion chromatography (SEC). In SEC the eluent is selected in such a manner that it will suppress any possible analyte interactions with the surface, and the separation of the analyte molecules in this mode is primarily based on their physical dimensions (size). The larger the analyte molecules, the lower the possibility for them to penetrate into the porous space of the column packing material, and consequently the faster they will move through the column. The schematic of this classification is shown in Figure 1-1. 

The fourth type of HPLC technique, size-exclusion HPLC (see Chapter 6), is based on the absence of any specific analyte interactions with the stationary phase (no force employed in this technique). 

Modern HPLC is a routine tool in any analytical laboratory. Standard HPLC system represents a separation output in the form of chromatogram (typical modern chromatogram is shown in Figure 1-6). Each specific analyte in the chromatogram is represented by a peak. In the absence of the strong specific analyte interactions with the stationary phase and at relatively low analyte concentration, peaks are symmetrical and resemble a typical Gaussian (normal distribution) curve. 

The equilibrium constant, K, thermodynamically could be described as the exponent of the Gibbs free energy of the analyte s competitive interactions with the stationary phase. In hquid chromatography the analyte competes with the eluent for the place on the stationary phase, and resulting energy responsible for the analyte retention is actually the difference between the analyte interaction with the stationary phase and the eluent interactions for the stationary phase as shown in equation (1-5)... 

Equation (1-7) shows that in an ideal case the selectivity of the system is only dependent on the difference in the analytes interaction with the stationary phase. It is important to note that the energetic term responsible for the eluent interactions was canceled out, and this means that the eluent type and the eluent composition in an ideal case does not have any influence on the separation selectivity. In a real situation, eluent type and composition may influence the analyte ionization, solvation, and other secondary equilibria effects that will have effect on the selectivity, but this is only secondary effect. 

In Section 2.1 the main chromatographic descriptors generally used in routine HPLC work were briefly discussed. Retention factor and selectivity are the parameters related to the analyte interaction with the stationary phase and reflect the thermodynamic properties of chromatographic system. Retention factor is calculated using expression (2-1) from the analyte retention time or retention volume and the total volume of the Uquid in the column. Retention... 

TYPES OF ANALYTE INTERACTIONS WITH THE STATIONARY PHASE... 

The LSER theory allows the association of the analyte retention behavior with the energetic components of the analyte interactions with the stationary phase. On the other hand, this theory is based on the assumption of the applicability of equation (2-43) to the analyte retention process, and this equation only allows the existence of one single partitioning retention mechanism. Coexistence of several different retention mechanisms, or the presence of secondary equilibria in a chromatographic system, effectively does not allow the applicability of this theory to the description of the analyte retention [59]. 

In everyday method development practice, it is important to ensure the separation of target compounds, matrix components, and other impurities. The elution of the analyte at the void volume means that it did not interact with the stationary phase and thus could not be separated from other components that do not interact with the surface either. To ensure the analyte interaction with the stationary phase, it is usually recommended to choose chromatographic conditions when any component of interest elutes with at least 1.5 void volume values or even greater. The error of the void volume determination for these purposes could be 20% or even greater (insofar as these void volume values are not used for any calculations but just to estimate where the least retained analye elutes). The use of uracil, thiourea, or allantoin as analytical void volume markers is most common in practical analytical work. 

Nevertheless, the history of supports for HPLC does not stop at this point, and there are several further developments aimed at either a kinetic optimization of the materials (see Chapter 3), an extension of the usable pH range, or the pressure stability of the silicas. In the course of kinetic optimization of the materials, essentially the diffusion paths of the analytes interacting with the stationary phase are shortened. This is done primarily by reducing the particle diameter of the supports. 

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