Retention partition chromatography

Van Den Dool, H. and P.D. Kratz (1963), Generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography . J. Chromatogr, Vol. 11, p. 463. 

In contrast to straight phase or adsorption chromatography, partition chromatography involves the separation of sample molecules owing to their different partition coefficients between the liquid stationary and mobile phases. The liquid stationary phase is located in the pores of a sorbent, ideally only acting as a support, making no contribution to the retention of the sample molecules. 

At Sirius, a dedicated instmment (Profiler LDA) has been developed for the rapid measurement of log D by liquid-liquid partition chromatography. In this instrument the column is coated with a layer of octanol, and the retention times are therefore tmly related to octanol/water partitioning. Although the method used was first described 25 years ago [29], it has been difficult to apply in an automated system because of the tendency of octanol to be flushed from the column by the eluent, thus requiring frequent renewals of the octanol coating. In our method, the octanol coating is kept in place by reversing the direction of the eluent after each... 

Urushigawa Y, Yonezawa Y. 1979. Chemico-biological interactions in biological purification systems VI. Relation between biodegradation rate constants of di-n-alkyl phthalate esters and their retention times in reverse phase partition chromatography. Chemosphere 5 317-320. 

Centrifugal partition chromatography (CPC) relies on centrifugal force rather than gravitation for the retention of the stationary phase and solvents can be pumped at higher speeds through the instruments. In addition, no need for droplets formation is required. This allows shorter separation times, without loss of resolution, and an infinite choice of solvents with the only requirement of forming two immiscible phases, stable with the time. Chloroform-based systems have been mostly used for the separation of saponins due to their favourable partition coefficient towards such solvents. [116, 119, 120]. 

Solutes undergo retention when weak interaction occurs with the stationary phase. The analyst must select a stationary phase according to the nature of the solute (apolar, polar, ionizable). For convenience we shall consider the following different mechanisms adsorption, partition, chromatography of ionizable substances, chiral separations, exclusion. Table 1 is. a quick selection guide. 

The separation of compounds by their differential partition between two immiscible phases is the basis for partition chromatography. The system consists of a stationary liquid phase coated on an inert solid support, and an immiscible mobile phase. Chromatographic separations are based on the different equilibrium distributions of the samples between these two phases. The greater the quantity of substance in the stationary phase at equilibrium the dower is the migration. For analyses, this equilibrium must remain constant over a suitable concentration range. Thus an increase in the concentration of solute results in a linear increase in the concentration of solute in the mobile and stationary phase, respectively. Under these conditions, the retention time, tR, is independent of the amount of sample chromatographed and a symmetrical peak (gaussian band) is observed. 

Liquid-liquid partition chromatography. The stationary phase consists of 1% tris(2-cyanoethoxy)propane (TCEP) on Zipax support. The mobile phase, hexane, is saturated with TCEP prior to use. A pre-column consisting of 30% TCEP on Gas-Chrom Q is used in order to prevent stripping of the liquid phase from the analytical column. The detector is set at 254 nm for monitoring the column effluent. The retention times of a number of 2,4-dinitrophenylhydrazones obtained with this system are given in Table 4.12. Gradient elution may be made with Permaphase ETH as stationary material and a gradient of hexane-chloroform. The limit of detection is ca. 5 ng per injection. 

Chromatography, as compared to other separation methods based on phase equilibrium, stems from a notoriously heterogeneous physical system. Even in partition chromatography, where supposedly partition between bulk phases is predominant, the different kinds of high-area interfaces often lead to surface effects. In the most general case, we assume that there are several mechanisms of retention, both bulk and interfacial. In this case the numerator of the last equation must be enlarged to incorporate the other mechanisms... 

Columns For most analyses, separation is achieved by partition of compounds in the test solution between the mobile and stationary phases. Systems consisting of polar stationary phases and nonpolar mobile phases are described as normal phase, while the opposite arrangement, polar mobile phases and nonpolar stationary phases, is called reversed-phase chromatography. Partition chromatography is almost always used for hydrocarbon-soluble compounds of a molecular weight that is less than 1000. The affinity of a compound for the stationary phase, and thus its retention time on the column, is controlled by making the mobile phase more or less polar. Mobile phase polarity can be varied by the addition of a second, and sometimes a third or even a fourth, component. 

Retention, expressed by the capacity factor k of nonionic analytes, is a function of the partition coefficient and the volume of pseudostationary phase (micelles), the volume of the mobile phase, the retention time of the analyte, the dead time (corresponding to the migration velocity of the EOF), and the retention time of the micelles. If the micelles were immobilized in the capillary (i.e., if the pseudostationary phase were stationary ), the capacity factor would be similar to the standard equation of retention in chromatography. 

The log Ofj term reflects the differences in capacity ratios of the two peptide solutes Sj and Sj which differ by a functional group and is analogous to the term used to predict selectivity differences for the classical liquid-liquid partition chromatography of peptides. The influence of functional group behavior on the retention of polar solutes in reversed-phase HPLC has been the subject of several recent articles and similar trends are apparent with peptide derivatives (29-31). 

This brief historical overview of RPLC development is far from the full description of all signihcant achievements made in the past however, the primary goal is to show the path of the development, which was, to a larger extent, in the tail of GC development. Consequently, the models and the descriptions of the retention mechanism were essentially transferred from gas-liquid partition chromatography. 

Open-column chromatography with silica gel and alumina is not applicable to the fractionation of tanins because of their strong binding to these adsorbents, which induces extensive loss of tannins. Such losses do not occur with countercurrent chromatography, as it does not use a solid stationary phase. Such molecules are very polar, so butanol-based solvent systems can be used. Centrifugal partition chromatography is more adequate in this case, as compared to hydrodynamic CCC, because of the good retention of the stationary phase of a such solvent system. 

Liquid-liquid partition chromatography systems have been used with or without stationary-phase support. Support-free LLPC has been classified as countercurrent chromatography (CCC). High-speed CCC techniques, based on planetary motion and coaxial orientation of the coiled column, have been developed that achieve both high partition efficiency and excellent retention of the stationary phase, thus circumventing the need for stationary-phase supports [6],... 

Activation by heating at 150-200°C removes the physically bound water. The assumption that one silica is most suitable for adsorption and another for liquid-liquid partition chromatography is questionable and, beyond that, irrelevant because pure adsorption or partition retention mechanisms generally do not occur. 

Homologous series of quaternary alkylamines have been separated on porous microspherical polystyrene-divinylbenzene gel by means of methanol - containing perchloric, sulfuric or hy-drochloric acid as mobile phase (Figs.15.3 and 15.4). The retention behaviour of the alkylamines could be explained in terms of partition chromatography. 

In adsorption chromatography, the only variable that affects the distribution coefficient of analytes is the composition of the mobile phase (in contrast to partition chromatography, where the polarity of the stationary phase can also be varied). Fortunately, enormous variations in retention and thus resolution accompany variations in the solvent system, and only rarely is a suitable mobile phase not available. 

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