Chromatographic systems adsorbents

The optimization of preparative and even micropreparative chromatography depends on the choice of an appropriate chromatographic system (adsorbent and eluent), sample application and development mode to ensure high purity, and yield of desirable compounds isolated from the layer. For the so-called difficult separations, it is necessary to perform rechromatography by using a system with a different selectivity. But it should be taken into account that achievement of satisfactory results frequently depends on a compromise between yield and the purity of the mixture component that is being isolated. 

Adsorption is the process of analyte accumulation on the surface under the influence of the surface forces. Determination of the total amount of the analyte adsorbed on the surface requires the definition of the volume where this accumulation is observed, usually called the adsorbed layer volume (U ). In chromatographic systems, adsorbents have large surface area, and even very small variation in the adsorbed layer thickness lead to a significant variation on the adsorbed layer volume. There is no uniform approach to the definition of this volume or adsorbed layer thickness in the literature [14,21,22]. 

The enthalpy of the H-bonds among the majority of the organic compounds is relatively low (usually within the range of about 20 kJ per one mol of hydrogen bonds) and therefore they can easily be disrupted. In order to demonstrate the presence of lateral interactions in chromatographic system, low-activity adsorbents are most advisable (i.e., those having relatively low specific surface area, low density of active sites on its surface, and low energy of intermolecular analyte-adsorbent interactions, which obviously compete with lateral interactions). For the same reason, the most convenient experimental demonstration of lateral interactions can be achieved in presence of the low-polar solvents (basically those from the class N e.g., n-hexane, decalin, 1,4-dioxane, etc.) as mobile phases. 

In the case of two analytes able to participate in the mixed lateral interactions (i.e., able to form the hydrogen bonds of the AB. .. A,AB. .. B, or AB,... ABj type) and chromatographed in mild chromatographic systems (i.e., those composed of a low-active adsorbent and a low-polar mobile phase), mixed lateral interactions can even prevent a given pair of analytes from a successful separation (whereas under the slightly more drastic separation conditions, resolntion of a given pair of analytes can be perceptibly worsened, at the least). 

Hence, in order to obtain a successful separation of a mixture of the H-bonded analytes, the most recommended are such chromatographic systems in which lateral interactions either are entirely eliminated or manifest themselves to a rather weak extent. Thus, it is evident that in such cases, the most recommended are the chromatographic systems composed of an active enough adsorbent and a polar enough mobile phase. 

Solvent selectivity refers to the ability of a chromatographic system to separate two substances of a mixture. It depends on the chemistry of the adsorbent surface, such as the layer activity and type of chemical modihcation. The separation power or resolution is given by Equation 4.8 [27] ... 

Snyder and Soczewinski created and published, at the same time, another model called the S-S model describing the adsorption chromatographic process [19,61]. This model takes into account the role of the mobile phase in the chromatographic separation of the mixture. It assumes that in the chromatographic system the whole surface of the adsorbent is covered by a monolayer of adsorbed molecules of the mobile phase and of the solute and that the molecules of the mobile phase components occupy sites of identical size. It is supposed that under chromatographic process conditions the solute concentrations are very low, and the adsorption layer consists mainly of molecules of the mobile phase solvents. According to the S-S model, intermolecular interactions are reduced in the mobile phase but only for the... 

As was reported by Soczewinski, a three-component mixture containing 5 mg of each of the ingredients can be completely separated using a 90 X 100 X 0.5 mm layer of silica [26]. This indicates that the capacity of the chromatographic system can be considerably increased by the apphcation of wider and thicker layers of adsorbent on the chromatoplate [44]. 

Mixing the additive in the eluent used as a mobile phase can also modify the chromatographic system (dynamic modification), but the use of modified adsorbents has led to an improvement of resolution. Example works include that by Armstrong and Zhou [11], who used a macrocyclic antibiotic as the chiral selector for enantiomeric separations of acids, racemic drugs, and dansyl amino acid on biphenyl-bonded silica. 

FIGURE 5.24 (See color insert following page 280.) Schematic comparison of the adsorption mechanisms of a solute from aqueous solutions of (a) methanol and (b) acetonitrile onto a RPLC material. Three different phases (bulk mobile phase, the adsorbed mono- or multilayer of organic modifier molecules, and the C is phase) are involved in the chromatographic system. The solute is represented by small ovals. (Reproduced from Gritti, F. and Guiochon, G, Anal. Chem., 77, 4257, 2005. With permission.)... 

It is essential that the eluent is inert and does not react with the sample components or the adsorbent. It must be borne in mind that aliphatic ketones like acetone or butanone easily undergo condensation reactions on active adsorbents and thus change the elution behavior ol he chromatographic system. r... 

Another way of decreasing the retention volume of functional macromolecules can be found in raising the column temperature. In this case, it should be borne in mind that a change in temperature causes a number of changes in the chromatographic system, mainly associated with the shift in the equilibrium between the components of the adsorbed mobile phase and water, which results in a change in the eab of the mixture and the adsorbent activity. 

An analagous process for isolating and concentrating portions of a sample is the use of thin layer chromatography. Here, however, it is common practice, in the treatment of an unknown sample, to place a known standard on the same plate and to run the two systems parallel so that differences in performance of an adsorbing system from one laboratory to another are accounted for by basing isolation of the desired components from the plate on the determined position of the known standard, run on the same plate. The normally limited capacity of thin layer chromatographic systems tends to exclude their wide use for the combined isolation-concentration steps of trace analysis, but circumstances in which this technique may be useful should not be overlooked. ... 

Chromatographic systems are inexpensive, and are generally rugged enough to be operated on a research vessel or could be modified relatively easily for this purpose. It is possible to pass large volumes of water through an adsorbent in a device that is the size of an double-A battery, for preconcentration and separation. 

Sophisticated mathematical models based on the numerical simulation of the chromatographic process consider different kinetic and thermodynamic mechanisms [19], The theoretical approaches describe the biospecific adsorption of monovalent and multivalent adsorbates. They also account for the film mass transfer and pore diffusion contributions to the adsorption process and can be applied to analyze various complex experimental situations. Thus, ideally, the appropriate model will have to be selected to describe the actual chromatographic system. 

The basis for the analyte retention in reversed-phase chromatography is the competitive interactions of the analyte and eluent components with the adsorbent surface. The stronger the interactions of the analyte with the surface, the longer its retention. Selectivity or the ability of chromatographic system to discriminate between different analytes is also dependent on differences in the surface interactions of the analytes. 

While dynamic distribution of the analyte between the mobile phase and adsorbent surface is a primary process, there are many secondary processes in the chromatographic system that significantly alter the overall analyte retention and selectivity. Detailed theoretical discussion of the influence of secondary equilibria on the chromatographic retention is also given in Chapter 2. 

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