Frontal analysis competitive

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]. 

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

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. 

In the elution mode when the mobile phase contains a competitive additive, in gradient elution when the initial concentration of the strong solvent is different from zero, or in frontal analysis when the elution of successive concentration steps is carried out, the column contains a constant concentration of a component interacting with the stationary phase at the beginning of the experiment. Thus, the initial condition is... 

Figure 4.2 illustrates the best competitive adsorption isotherm model for benzyl alcohol and 2-phenylethanol [16]. The whole set of competitive adsorption data obtained using Frontal Analysis was fitted to obtain the Langmuir parameters column saturation capacity qs =146 g/1), equilibrium constant for benzyl alcohol bsA = 0.0143) and the equilibrium constant for 2-phenylethanol (bpE = 0.0254 1/g). The quality of the fit obtained with this simple model is in part explained by the small variation of the activity coefficients of the two solutes in the mobile phase when the solute concentrations increased from 0 to 50 g/1. The Langmuir competitive adsorption isotherm simplifies also in the case where activity coefficients are of constant value in both phases over the whole concentration range [17]. 

Few multicomponent competitive isotherms have been measured so far although the progress in the development of methods and the pressure arising from the development of preparative chromatography and the need better to understand competitive isotherms combine to render such investigations attractive. The experimental data of two ternary isotherms were measured by frontal analysis [17, 23] while those of a quaternary isotherm were determined by the perturbation method [24]. 

Lisec et al. [23] measured by frontal analysis the ternary isotherm data for phenol, 2-phenylethanol and 3-phenyl-l-propanol on Kromasd-Cig, with a water/methanol (1/1) solution. The data were fitted to the model equations of the competitive Langmuir and competitive bi-Langmuir models and to the LAS and RAS models derived from the Langmuir model. No substantial improvements were observed with the more complex models. Satisfactory agreement was observed between experimental band profiles and the profiles calculated from the ternary Langmuir isotherm. 

This isotherm model has been used successfully to accoimt for the adsorption behavior of numerous compounds, particularly (but not only) pairs of enantiomers on different chiral stationary phases. For example, Zhou et ah [28] foimd that the competitive isotherms of two homologous peptides, kallidin and bradyki-nine are well described by the bi-Langmuir model (see Figure 4.3). However, most examples of applications of the bi-Langmuir isotherm are found with enantiomers. lire N-benzoyl derivatives of several amino acids were separated on bovine serum albumin immobilized on silica [26]. Figure 4.25c compares the competitive isotherms measured by frontal analysis with the racemic (1 1) mixture of N-benzoyl-D and L-alanine, and with the single-component isotherms of these compounds determined by ECP [29]. Charton et al. foimd that the competitive adsorption isotherms of the enantiomers of ketoprofen on cellulose tris-(4-methyl benzoate) are well accounted for by a bi-Langmuir isotherm [30]. Fornstedt et al. obtained the same results for several jS-blockers (amino-alcohols) on immobilized Cel-7A, a protein [31,32]. 

Jandera et al. [35] measured by frontal analysis the competitive isotherms of the enantiomers of mandeHc acid, phenyl-glycine and tryptophan on the glyco-peptide Teicoplanin, in water/methanol or ethanol solutions. The less retained L enantiomers of the two amino acids follow Langmuir isotherm behavior while the D isomers foUow bi-Langmuir behavior. The enantiomeric separation factors increase with increasing alcohol concentration while the solubilities of these com-poimds decrease. Similar results were reported by Loukih et al. [36] for the separation of the enantiomers of tryptophan on a teicoplanin- based CSR The authors insisted on the importance of the nature of the ions in a supporting salt. Optimization of the experimental conditions for maximum production rate must take this effect into account. 

Figure 4.16 Experimental isotherm data (symbols) of R and S-l-indanol obtained by competitive frontal analysis on a narrow bore column packed with Chiracel OB. The data were fitted to the Toth (dash-dot), the bi-Langmuir (dash), the Langmuir-Freundlich (dot), and the Langmuir (solid) models. The inset shows low concentration data. Reproduced with permission from D. Zhou, K. Kaczmarski, A. Cavazzini, X. Liu, G. Guiochon, J. Chromatogr. A, 1020 (2003) 199 (Fig 4).

There is a dearth of competitive adsorption data, in a large part because they are difficult to measme, but also because little interest has been devoted to them, as, until recently, there were few problems of importance whose solution depended on their understanding. Besides the static methods, which are extremely long and tedious and require a large amoimt of material, the main methods of measurement of competitive isotherms use column chromatography. Frontal analysis can be extended to competitive binary isotherms [14,73,93-99], as well as pulse techniques [100-104]. The hodograph transform is a powerful method that permits an approach similar to FACP for competitive binary isotherms [105,106]. 

Frontal analysis can easily be extended to binary mixtures. The shape of the breakthrough profiles and the effect of axial dispersion on these shapes have been studied theoretically [93,94] and experimentally [14,73,95-99]. These profiles are characterized by the successive elution of two steep fronts (shock layers) for a binary mixture. The use of these profiles for the determination of the competitive isotherms of two components has been developed by Jacobson et al. [14]. 

Figure 4.21 [14] shows the breakthrough ciuves obtained in two-component frontal analysis with competitive Langmuir isotherms, with four successive concentration steps, and with a column efficiency of 5000 theoretical plates. The thin and thick solid lines correspond to the first and the second components, respectively. The first step gives a different profile from all the following steps because the column is initially equilibrated with the pure mobile phase only [initial condition, Q(x, f = 0) = 0]. For this first step, two shock layers signal the successive exit of the lesser and the more retained components. The first component subplateau is more concentrated than the feed there is no subplateau for the second... 

Figure 4.21 Schematic of the determination of competitive binary isotherms by frontal analysis. Main figure Typical experimental chromatogram in two-component frontal analysis. Thin line, concentration profile of the first component thick line, concentration profile of the second component. Inset Expansion of one step in the main figure. "Sub" indicates the intermediate subplateau during the breakthrough of the binary mixture echelon.

For each successive step, the profile of the second component exhibits an intermediate plateau at a concentration that is intermediate between the initial plateau concentration and the feed concentration in the new step, while, simultaneously, the first component profile exhibits an intermediate plateau with a concentration that is greater than the feed step. This is the result in frontal analysis of the displacement effect, itself the result of competition. Upon arrival at the column exit of the second component front (whose plateau concentration is equal to the feed concentration, the first component concentration undergoes a drop to the feed concentration. Note that the concentrations of both components converge simultaneously to the feed composition. The first step of a two-component frontal analysis has been studied experimentally and theoretically by Carta et al. [107], for the breakthrough of two amino acids, and by Zhu et al. [73] for the breakthrough of 2-phenylethanol and 3-phenylpropanol (Figure 4.22). 

Figure 4.22 Experimental concentration profiles in the column effluent for adsorption isotherm determination by binary frontal analysis. (Left) Bottom trace solid line, experimental UV profile dotted line reconstructed profile of 3-phenylpropanol (P) dashed line reconstructed profile of 2-phenylethanol (E). Arrows 1-5 indicate the time when the eluent sample was taken for on-line analysis. Top trace On-line analysis of the sampled eluent. Reproduced with permission from J. Zhu, A. Katti and G. Guiochon, J. Chromatogr. 552 (1991) 71 (Fig. 1). (Right) Examples of one-step binary frontal analyses for the determination of the competitive isotherms of N-benzoyl-D,L-alanine. Injection of large volumes (5 mL) of solutions of increasing concentrations of racemic mixture. Reproduced with permission from S.C. Jacobson, A. Felinger and G. Guiochon, Biotechnol. Progr., 8 (1992) 533 (Fig. 1), 1992 American Chemical Society.

Figure 4.23 Schematic of an equipment designed for the experimental determination of competitive isotherms by binary frontal analysis.

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. 

As an example, we show in Figure 4.25 the competitive isotherms of the mixture of p-cresol (Figure 4.25a) and phenol (Figure 4.25b) on octadecyl silica [14], and those of N-benzoyl-D- and L-alanine on BSA immobilized on silica [29]. The isotherms in Figure 4.25 were measured by binary frontal analysis (Section 4.2.1). 

Figure 4.26 Comparison of the competitive adsorption isotherm measured by FA and calculated by two different methods. p-Cresol (Left) and phenol (Right), Top Data from the mass balance method (MMB, binary frontal analysis) at molar ratios of 3 1 (Q)/ Id ( ) and 1 3 (A). Solid hnes calculated by the method of composition velocity (MMC). Bottom Comparison of the competitive isotherms obtained by MMB (Q) and HBBM (square s)nnbol) (n) for p-ciesol and phenol in three concentration regimes. Reproduced with permission from J. Jacobson and J. Frenz, ]. Chromatogr., 499 (1990) 5 (Figs. 2 and 5).

In all these figures, we used the competitive Langmuir isotherm model to calculate the band profiles. However, the coefficients of the isotherms used for Figures 11.21 are the coefficients of the single-component isotherms determined by frontal analysis, while the coefficients of the isotherms used to calculate the profiles in Figure 11.22 are measured by the simple wave method (Chapter 4, Section 4.2.4). These latter coefficients are certainly empirical coefficients, and their use would not permit an accurate prediction of single-component bands. However, they permit the calculation of band profiles that are in much better agreement... 

This value is in agreement with the one derived from band profiles calculated with the equilibrium-dispersive model [9]. The time given by Eq. 16.20 provides useful information regarding the specifications for the experimental conditions under which staircase binary frontal analysis must be carried out to give correct results in the determination of competitive isotherms. The concentration of the intermediate plateau is needed to calculate the integral mass balances of the two components, a critical step in the application of the method (Chapter 4). This does not apply to single-pulse frontal analysis in which series of wide rectangular pulses are injected into the column which is washed of solute between successive pulses. 

Piqtkowski el al. measured the single-component and the competitive equilibrium isotherms of phenetole (ethoxy-benzene) and n-propyl benzoate on a 150 x 3.9 mm S3onmetry -Cig (endcapped) column (Waters), using a methanol/water (65 35, v/v) as the mobile phase [26]. The adsorption equilibrium data of the single-component systems were acquired by frontal analysis. For both compoimds. 

Competitive frontal analysis Frontal analysis carried out with multicomponent mixtures, for the determination of competitive isotherms or the enrichment of certain feed components. 

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