Retention separation principles

There are two ways to classify liquid chromatographic methods. The first and more common classification is based on the mechanism of retention, and from this the chromatographic modes discussed in Chapter 2 are derived. For example, the normal-phase mode can be performed by taking advantage of either the adsorption mechanism or the partition mechanism. The gel-filtration mode is performed using the mechanism of size exclusion. The second classification discussed below is based on the separation principle and is found mostly in the literature published before the 1990s. 

Fig. 14. S-FFF apparatus designed by Giddings group (A) the separation principle with smaller particles (X), bigger particles (7) and floating particles (Z) with a density smaller than that of the solute [These particles are equally well separated as retention depends on Ap I (B)]. C Fractogram of a separation of polystyrene latexes of different sizes at two different rotational speeds. The ability to shift retention by changing the rotational speed is demonstrated. D The same mixture analyzed by a programmed field run demonstrating that a wider particle size range can be condensed into a reasonable elution span. Reproduced from [14] with kind permission of the American Association for the Advancement of Science...

The particle sizing by field flow fractionation (FFF) is based on the different effect of a perpendicular applied field on particles in a laminar flow [63-66], The separation principle corresponds to the nature of the perpendicular field and may, for example, be based on different mass (sedimentation FFF), size (cross-flow FFF), or charge (electric-field FFF). Cross-flow FFF has been applied recently to investigate nanoemulsions, SLN, and nanostructured lipid carriers (NLC, particles composed of liquid and solid lipids) [58], Although all samples had comparable particle sizes in PCS, their retention in the FFF was very different. Compared to the spherical droplets of the nanoemulsion, SLN and NLC were pushed more efficiently to the bottom of the channel because of their anisotropic shape. Their very different shapes have been confirmed by electron microscopy. 

In the chromatographic method, the injection method and volume must be chosen. The separation principle, the stationary phase, and the mobile phase composition must be chosen to obtain baseline separation of the analyte(s) from other compounds in the sample. Tailing of the chromatographic peak should be avoided, as should overloading of the column. A chromatographic separation with repeatable retention times is required. 

The analytes are detected at their exit from the column. Each molecule is characterized by a retention time that corresponds to the time that passed between the injection of the analyte and its arrival at the detector. Concerning volatility, the separation principle is simple the speed at which a compound migrates in the column relates to its boiling point. The boiling point is a thermodynamic measure that depends mainly on two factors the molecular weight and polarity. A compound is as volatile as its molecular weight and polarity are low. The interaction between analytes and the stationary phase are more complex one must chose the type of chemical film to use according to the nature of the molecules that need to be separated. 

The combination of diafiltration and batch concentration can be used to fractionate two macrosolutes whose retentions differ by as little as 0.2. It is possible in principle to achieve separations that are competitive with chromatography. When tanks and other equipment are considered, as well as the floor space they occupy, the economics of membrane separation of proteins may be attractive [R. van Reis, U.S. Patent 5,256,294 (1993)]. 

It was known for some time that even after the corticoids had been separated from crude extracts of the adrenal cortex, the remaining material, the so-called "amorphous fraction" still possessed considerable mineralocorticoid activity. Aldosterone (250), one of the last steroids to be isolated from this fraction, proved to be the active principle. This compound proved to be an extremely potent agent for the retention of salt, and thus water, in body fluids. An antagonist would be expected to act as a diuretic in those edematous states caused by excess sodium retention. Although aldosterone has been prepared by both total and partial synthesis, the complexity of the molecule discouraged attempts to prepare antagonists based directly on the parent compound. 

The two examples of sample preparation for the analysis of trace material in liquid matrixes are typical of those met in the analytical laboratory. They are dealt with in two quite different ways one uses the now well established cartridge extraction technique which is the most common the other uses a unique type of stationary phase which separates simultaneously on two different principles. Firstly, due to its design it can exclude large molecules from the interacting surface secondly, small molecules that can penetrate to the retentive surface can be separated by dispersive interactions. The two examples given will be the determination of trimethoprim in blood serum and the determination of herbicides in pond water. 

Describe the principle of separation involved in elution chromatography and derive the retention equation ... 

The principles of separation by elution chromatography are considered in Volume 2, Sections 19.1 and 19.2. The derivation of the retention equation is given by equations 19.1-19.8 in Section 19.2.2 of Volume 2. 

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