Frontal analysis method

In general, the frontal analysis is a less cotnnonly applied mode of chronatography, in v iich the semple is fed into the column continuously. The feed of the seniple can be interrupted and reserved at intervals to produce break-through curves or frontalograns 

F1 pure 11. Ideal frontalograms for interacting compounds M+L = ML. The curves A and B refer to total (constituent) concentrations of the components, respectively. The shaded areas describe the fractions v4iere complex ML is found. The binding ratio is calculable from the concentration of free L and from its (known) total concentration and thus molar absorptivity of complexed L is not needed when eluent is monitored by light absorption. 

The data for binding constants of the systan are directly available without knowing molar absorptivity of complexed L or possible absorption by M, since the total ligand concentration in the original sample is independently known. By repeating frontal analysis in different concentrations of L, the binding ratio and stoichiometry is obtained (see equation 2). 

When no complexation between M and L or when adsorption of L on the support takes place, the elution volume of L (Vl) will be V(,+V-j and the front is sharp. When the association constants increase, the advancing side of the frontalogram should become more skewed and Vl approaches Vg. Slow attainment of equilibria between M and L or slow diffusions within the gel matrix should yield similar effects on the frontalo-grams. If these effects can be excluded,the slope and breakthrough volume of L also indicate the magnitude of equilibrium constants. Similar discussion holds for the trailing side. The frontalogram is conveniently expressed as its time derivative, which shows minute differences more clearly. 

Certain conditions must be satisfied in frontal analysis of interacting systems. Naturally, the fronts of two components must separate from each other and a true steady state must be attained. This is shown by presence of concentration plateaus of both constituents and by the attainment of a limiting value (concentration same as with the sample) for the latter (ref. 18). In addition, the elution volume of constituents should not be dependent on their concentration. If it is, false elevations of the slower plateau ma(y occur (ref. 18). 

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O. Lisec, P. Hugo and A. Seidel-Morgenstein, Frontal analysis method to determine competitive adsorption isotherms. J. Chromatogr.A 908 (2001) 19-34. 

Fig. 6.1 A breakthrough curve generated by the frontal analysis method [31], The analysis represents a high-volume injection of caffeine through a reversed-phase column, at a concentration representative of the linear region of the binding isotherm. Adapted with permission from Elsevier.

Fig. 1 Schematic representation of the experimental setups of the mobility-shift method and the Hummel-Dreyer method (A) the vacancy peak method and the vacancy affinity capillary electrophoresis method (B) the equilibrium-mixture method and the frontal analysis method (C) for drug-protein binding analysis. drug protein gg drug-protein complex Q buffer. (Reprinted with permission from Ref. 38. Copyright 1992 Elsevier Science.)...

Shibukawa et al. (40) discussed the frontal analysis method, also called high-performance frontal analysis (HPFA) or high-performance capillary electrophoresis/frontal analysis (HPCE/FA), compared it to conventional methods, and focused on the application to stereoselective protein binding. The affinity of the drugs warfarin, verapamil, and carbamazepine and the drug candidate BOF-4272 to HSA was investigated. 

Finally, a few characteristics of the frontal analysis method are summarized ... 

The method is advantageously combined with the frontal analysis method, which also requires a concentration plateau and thus shares the disadvantage of high sample consumption if operated in open mode. As indicated in Fig. 6.24, the measurement procedure starts at maximum concentration. This concentration plateau is reduced step-by-step by diluting the solution. To reduce the amount of samples needed for the isotherm determination the experiments can be done in a closed loop arrangement (Fig. 6.17). It is also possible to automate this procedure. 

It has been shown in many investigations that ECP, FA, and FACP give the same experimental isotherm as the static method. An example of such results is given in Figure 3.45a [8], which shows that data obtained by a static method and data derived by a dynamic (i.e., frontal analysis) method compare very well. 

The major drawbacks of the frontal analysis method are the important number of measurements to be made, the considerable amount of time that it takes to determine a set of competitive isotherms and the large amount of sample required. The competitive isotherms are sets of n surfaces in an n -b 1 space where n is the number of components. For a binary mixture, we have two surfaces, /i(Ci, C2) and /2(Ci, C2). These surfaces depend minimally on four parameters, often on more, depending on the isotherm model selected. 

Accurate determination of the isotherm parameters requires a rather large number of data points, corresponding to a wide range of absolute and relative concentrations. The acquisition of pure component data as well as equilibrium data for 1 3, 1 1, and 3 1 mixtures seems to be a bare minimtun [14]. Compared to other methods of isotherm determination, the frontal analysis method has the advantage of being nearly independent of the kinetics of mass transfer and axial dispersion, i.e., of the column efficiency. On the other hand, the results achieved can be very reproducible if proper experimental care is taken. Figure 4.24 [9] illustrates, for cis- and fraus-androsterone, the reproducibility of two runs before and... 

Experiments Sorption equihbria are measured using apparatuses and methods classified as volumetric, gravimetric, flow-through (frontal analysis), and chromatographic. Apparatuses are discussed by Yang (gen. refs.). Heats of adsorption can be determined from isotherms measured at different temperatures or measured independently by calorimetric methods. 

This type of chromatographic development will only be briefly described as it is rarely used and probably is of academic interest only. This method of development can only be effectively employed in a column distribution system. The sample is fed continuously onto the column, usually as a dilute solution in the mobile phase. This is in contrast to displacement development and elution development, where discrete samples are placed on the system and the separation is subsequently processed. Frontal analysis only separates part of the first compound in a relatively pure state, each subsequent component being mixed with those previously eluted. Consider a three component mixture, containing solutes (A), (B) and (C) as a dilute solution in the mobile phase that is fed continuously onto a column. The first component to elute, (A), will be that solute held least strongly in the stationary phase. Then the... 

The precise measurement of competitive adsorption isotherms not only of theoretical importance but may help the optimization of chromatographic processes in both analytical and preparative separation modes. The methods applied for the experimental determination of such isotherms have been recently reviewed [90], Frontal analysis using various flow rates can be successfully applied for the determination of competitive adsorption isotherms [91]. 

Using this methodology via measurement of adsorption isotherms, Guiochon and coworkers investigated site-selectively the thermodynamics of TFAE [51] and 3CPP [54] on a tBuCQD-CSP under NP conditions using the pulse method [51], the inverse method with the equilibrium-dispersive model [51, 54], and frontal analysis [54]. 

Several variants of separation methods based on dialysis, ultrafiltration, and size exclusion chromatography have been developed that work under equilibrium conditions. Size exclusion chromatography especially has become the method of choice for binding measurements. The Hummel-Dreyer method, the vacancy peak method, and frontal analysis are variants that also apply to capillary electrophoresis. In comparison to chromatographic methods, capillary electrophoresis is faster, needs only minimal amounts of substances, and contains no stationary phase that may absorb parts of the equilibrium mixture or must be pre-equilibrated. 

Plasma protein binding is also an important parameter in the pharmacokinetic field. Frontal analysis combined with capillary zone electrophoresis (CZE-FA) (67-69) is a powerful technique for high-throughput assay, because it is relatively rapid and easy to automate, in comparison with conventional methods such as dialysis, ultrafiltration, and ultracentrifugation. Recently, we introduced the EKC approach with ionic CDs to frontal analysis for anionic drugs that cannot be analyzed by conventional CZE-FA (70). In this approach, ionic CDs work as an EKC pseudostationary not for proteins but for small solutes. 

Kraak et al. (38) reported the first ACE application to study drug binding to a plasma protein. They used the model system warfarin-human serum albumin (HSA) to compare the suitability of the Hummel-Dreyer, frontal analysis, and vacancy peak methods. A more methodologically intended paper from Erim and Kraak (39) used VACE to study the displacement of warfarin from bovine serum albumin (BSA) by furosemide and phenylbutazone. They concluded that VACE is especially suited to examining competitive properties of simultaneously administered compounds toward a given protein-drug system. 

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