Retention mechanism

Many attempts have been made to explain polymer fractionation in GPC (see the reviews of Audebert [22] and Hagnauer [23]), and the size exclusion mechanism is now widely accepted rmd demonstrated [24-28]. In actual experiments, however, several additional mechanisms based on interactions between the solute and the stationary phase may occur in the column, such as adsorption and liquid-liquid partition. 

Adsorption Mechanism. Adsorption mechanism mainly occurs with mineral porous gel particles well-known for their surface activity. This is the classical mechruiism of solid-liquid adsorption chromatography. The elution volume takes the form  

Size Exclusion Mechanism. Steric exclusion (or size exclusion) is the main process of polymer fractionation by GPC. This mechanism is based on a thermodynamic equilibrium between two phases the interstitial solvent in the dead volume V and the solvent filling the porous volume Vp. If is the partition coefficient, the elution volume of a macromolecule is defined by  

The evolution of the partition coefficient as a function of chain size and topology on the one hand, and of pore size distributions on the other, has a purely entropic origin, and it is well predicted by the theory of Casassa [29-35] The partition function for a chain of a given size N trapped in a pore with volume Vp and inpenetrable walls, Zp ,e, can be obtained by an enumeration of all possible random paths of N steps which start in the pore volume and never cross the wall (e.g. such as the full line and unlike the dotted line in Fig. 4). 

The partition function of the same chain in an equivalent element of the dead volume V , is given by the same enumeration without the condition of not crossing a wall It is larger than the Zp , by an amount representing all conformations such as the dotted line in Fig. 4. Therefore, entering the pore 

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Chiral separations present special problems for vaUdation. Typically, in the absence of spectroscopic confirmation (eg, mass spectral or infrared data), conventional separations are vaUdated by analysing "pure" samples under identical chromatographic conditions. Often, two or more chromatographic stationary phases, which are known to interact with the analyte through different retention mechanisms, are used. If the pure sample and the unknown have identical retention times under each set of conditions, the identity of the unknown is assumed to be the same as the pure sample. However, often the chiral separation that is obtained with one type of column may not be achievable with any other type of chiral stationary phase. In addition, "pure" enantiomers are generally not available. 

Direct interception refers to a sieve-type mechanism in which contaminants larger than the filter pore size are directly trapped by the filter. This sieve retention mechanism of particle arrest is the mechanism of choice and occurs owing to geometric or spatial restraint. This type of particle arrest is considered to be absolute, that is, it is independent of filtration conditions. 

Retention, too, is highly tissue-specific. Sometimes, the extraction mechanism is also the retention mechanism, as for Tc-sestamibi, which is retained in mitochondria as long as transmembrane potentials remain intact. Others are separate. F-2-Fluorodeoxyglucose enters the cell by the same pathway as glucose, but is trapped because it is not a substrate for hexokinase, preventing further intracellular metabohsm. 

Hydrophobic interactions and trapping of molecules in a molecular sieve formed by humic materials have been hypothesized as retention mechanisms for prometryn. It has been shown that fluridone, fluazifop, and bipyridyhum herbicides penetrate into interlamellar spaces of smectites and can become trapped. 

The retention efficiency of membranes is dependent on particle size and concentration, pore size and length, porosity, and flow rate. Large particles that are smaller than the pore size have sufficient inertial mass to be captured by inertial impaction. In liquids the same mechanisms are at work. Increased velocity, however, diminishes the effects of inertial impaction and diffusion. With interception being the primary retention mechanism, conditions are more favorable for fractionating particles in liquid suspension. 

The amounts of material released from a damaged plant are usually expressed in fractions of the isotopic quantities in the core. These source terms (meaning source for the ex plant transport) depend on accident physics, amount of core damage, time at elevated temperatures, retention mechanisms, and plate-out deposition of material as it transports from the damaged core to release from containment. This section gives an outline of early source term assessments, computer codes used in calculations, and some comparisons of result.s. 

J. R K. Huber and G. Lamprecht, Assay of neopterin in serum by means of two-dimensional high-performance liquid chromatography with automated column switching using tliree retention mechanism , 7. Chromatogr. B 666 223-232(1995). 

In the elucidation of retention mechanisms, an advantage of using enantiomers as templates is that nonspecific binding, which affects both enantiomers equally, cancels out. Therefore the separation factor (a) uniquely reflects the contribution to binding from the enantioselectively imprinted sites. As an additional comparison the retention on the imprinted phase is compared with the retention on a nonimprinted reference phase. The efficiency of the separations is routinely characterized by estimating a number of theoretical plates (N), a resolution factor (R ) and a peak asymmetry factor (A ) [19]. These quantities are affected by the quality of the packing and mass transfer limitations, as well as of the amount and distribution of the binding sites. 

Due to mechanistic requirements, most of these enzymes are quite specific for the nucleophilic component, which most often is dihydroxyacetone phosphate (DHAP, 3-hydroxy-2-ox-opropyl phosphate) or pyruvate (2-oxopropanoate), while they allow a reasonable variation of the electrophile, which usually is an aldehyde. Activation of the donor substrate by stereospecific deprotonation is either achieved via imine/enamine formation (type 1 aldolases) or via transition metal ion induced enolization (type 2 aldolases mostly Zn2 )2. The approach of the aldol acceptor occurs stereospecifically following an overall retention mechanism, while facial differentiation of the aldehyde is responsible for the relative stereoselectivity. 

Equation (12) can also be used to identify the type of retention mechanism that is taking place in a particular separation by measuring the retention volume of the solute over a range of temperatures. 

Complete resolution was not achieved due to the carryover of interfering substances which frequently occurs when separating the components of biological samples. The column carried a reverse phase, but as the mobile phase contained low concentrations of lauryl sulfate, some would have adsorbed on the surface of the stationary phase and significantly modified its interacting properties. The retention mechanism is likely to have involved both ionic interactions with the adsorbed ion exchanger together with dispersive interactions with any exposed areas of the reverse phase. 

The mixed retention mechanism described above has a parallel in the effect of exclusion on retention when using porous stationary phases. The smaller molecules can enter more pores and thus interact with more stationary phase than the larger molecules that are excluded from many pores and, consequently, interact with less stationary phase. Assuming the smaller molecules are more strongly retained, the exclusion of large molecules augments the difference in retention between molecules of different size. 

A further example of mixed retention mechanisms on the resolution of geometric isomers is afforded by the separation shown in figure 6. 

Typically, lyases are quite specific for the nucleophilic donor component owing to mechanistic requirements. Usually, approach of the aldol acceptor to the enzyme-bound nucleophile occurs stereospedfically following an overall retention mechanism, while the facial differentiation of the aldehyde carbonyl is responsible for the relative stereoselectivity. In this manner, the stereochemistry of the C—C bond formation is completely controlled by the enzymes, in general irrespective of the constitution or configuration of the substrate, which renders the enzymes highly predictable. On the other hand, most of the lyases allow a reasonably broad variation of the electrophilic acceptor component that is usually an aldehyde. This feature... 

A number of different retention mechanisms operate in HPLC and interested readers may find further details elsewhere [2-4]. It is sufficient to say here that the interaction may be considered in terms of the relative polarities of the species involved. As indicated in Section 2.1 above, there are two extremes of interaction, neither of which is desirable if separation is to be achieved. 

General Description. Liquid chromatography encompasses any chromatographic method in which the mobile phase is a liquid (c.f. gas chromatography). A variety of stationary phases and retention mechanisms are available such that a broad range of modes of separation are possible. It is worthwhile to briefly describe the important modes that find use in clinical chemistry. 

Use of densitometric detection provides an insight into the concentration profiles of chromatographic bands, thus furnishing an indispensable prerequisite, needed for proper assessment of the retention mechanisms in the preparative adsorption TLC. Figure 2.4 shows three types of the band eoncentration profiles. The Gaussian peak (a) in this figure represents the linear isotherm of adsorption of a given species, peak... 

The elaboration of the most efficient chromatographic systems for the optimization of velocity and resolution of the chromatographic process is necessary for solving different analytical problems. The most important factor in the TLC optimization is the mobile phase composition. Taking into consideration the similarity in the retention mechanism between TLC and PLC, the optimized TLC mobile phase can be transferred to the preparative chromatographic system. There are different accepted models and theories for the separation and optimization of chromatographic systems [19,20,61]. 

P. A., Testa, B. Solvatochromic analysis of the retention mechanism of two novel stationary phases used for measuring lipophilicity by RP-HPLC./. Liquid Chromatogr. 1992, 35, 2133-2151. 

Stella, C., Galland, A., liu, X., Testa, B., Rudaz, S., Veuthey, J. L, Carrupt, P. A. Novel RPLC stationary phases for lipophilicity measurement solvatochromic analysis of retention mechanisms for neutral and basic compounds. /. Sep. Sci. 2005, 28, 2350-2362. 

M., La Rotonda, M. L, Testa, B. Structural properties governing retention mechanisms on immobilized artificial membrane (lAM) HPLC columns. Helv. Chim. Acta 2002, 85, 519-532. 

The variables that control the extent of a chromatographic separation are conveniently divided into kinetic and thermodynamic factors. The thermodynamic variables control relative retention and are embodied in the selectivity factor in the resolution equation. For any optimization strategy the selectivity factor should be maximized (see section 1.6). Since this depends on an understandino of the appropriate retention mechanism further discussion. .Jll be deferred to the appropriate sections of Chapters 2 and 4. 

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