Basic compounds mobile phase, influence

Classical PLC involves migration of a mobile phase by capillary action through a 0.5- to 2-mm layer of adsorbent for separating compounds in amounts of 10 to 1000 mg. This separation method requires a good knowledge of chromatography, the most basic equipment, and simple operational skills. The main aim of PLC is to obtain a maximum yield of separation, not a maximum peak (spot) capacity [3]. The principal factors that may influence a PLC separation [1 ] are shown in Figure 4.1. 

The pKa of basic compounds as well as the buffered mobile phase were influenced by the organic solvent and depended on its proportion in the soluhon. The results indicated that the stationary phases and mobile phases studied were not suitably optimized for the estimation of lipophilicity of basic compounds. 

As the pH of the mobile phase markedly influences the retention of ionizable compounds, it can be assumed that the separation capacity of RP-HPLC for ionizable analyses can be modified by changing the pH of the mobile phase. The theory of effect of pH gradient on the performance of RP-HPLC systems has been recently elaborated. The basic equation describing the dependence of the retention of the solute on the gradient of pH or organic modifier is ... 

For pharmaceutical compounds, LC-MS has found extremely wide acceptance due to the low-level detection that can be achieved, in addition to the selectivity and specificity that are attained by using HPLC in conjunction with MS detection. LC-MS is also convenient because of its compatibility with reversed-phase HPLC mobile phases. Volatile mobile phase additives such as trifluoroacetic acid, formic acid, and ammonium hydroxide are very common and can be utilized not only to aid in the chromatographic separation but also to influence the ionization state of the molecule (i.e., acid modifiers to protonate [M + H]+1, and basic modifiers to deprotonate [M — H] ). This requirement may require a modification of the potency method if phosphate was utilized however, it is not viewed as a major drawback. 

D. Skyora, E.Tesarova, and M. Popl, Interactions of basic compounds in reversed-phase high-performance liquid chromatography influence of sorbent character, mobile phase composition, and pH on retention of basic compounds, /. chromatogr. A 758 (1997), 37-51. 

L. Pan, R. LoBrutto, Y. V. Kazakevich, and R. Thompson, Influence of inorganic mobile phase additives on the retention, efficiency and peak symmetry of protonated basic compounds in reversed-phase liquid chromatography, J. Chromatogr. A 1049 (2004), 63-73. 

The preparative separations of certain polar (e.g., strongly basic) compounds and of many large molecular compotmds e.g., peptides and proteins) usually involve a complex mass transfer mechanism that is often slower than the mass transfer kinetics of small molecules. This slow kinetics influences strongly the band profiles and its mechanism must be accovmted for quantitatively. The accurate prediction of band profiles for optimization purposes requires a correct mathematical model of the various mass transfer processes involved. The piupose of the general rate model (GRM) is to accormt for the contributions of all the sources of mass transfer resistances to the band profiles [52,62,94,95]. The mass transfer of molecules from the bulk of the mobile phase percolating through the bed to the surface of an adsorbent or the mass of a permeable resin particle involves several steps that must be identified. 

Eluent selectivity is the ability of different mobile phases to change the separation factor a of two or more compounds present in the sample. It has nothing to do with eluent strength but is another means that allows a separation to be influenced. Adsorption chromatographic selectivity has two different aspects, localization and basicity. 

Figure 5-15 shows how the retention of 4-ethylpyridine varies with pH. The greatest increase in retention occurred when trifluoroacetic acid and perchloric acid were employed. The phosphoric modifier did not increase the retention of the 4-ethylpyridine significantly at decreasing pH values. Therefore the trifluoroacetate and perchlorate had a greater influence than phosphate as chaotropic counteranions at any particular pH. It was shown that this chaotropic effect began to occur at pHs less than 3. At all pHs greater than 4 the retention factors of the basic compound were similar and were independent of what type of modifier was used in the mobile phase. 

Si-OH groups are also focal points of attack for water and other reagents in the mobile phase, causing dissolution of underlying silica with gradual deterioration in column performance and eventual reduction in column lifetime (5,6). The pH of the silica surface also varies from acidic, neutral, to basic, and is believed to influence the preparation and properties of bonded phases (7). The inclusion of traces of transition metals in silica matrix can further influence the retention of acids, bases or neutral compounds that can undergo complexation reactions (1). Thus the selectivity and retention characteristics of bonded phase columns also depend on the quality of silica used for the bonding reactions. 

Equations (16.12) and (16.13) are very important, since they easily can be used to predict the influence of any operational parameter on the steepness factor, h, and therefore on the analysis time, efiSciency, and resolution. However, they are based on the validity of Equation (16.10). It has been shown that some deviations occur for some compounds and chromatographic systems (6), especially when retention is not governed solely by hydrophobic interaction. This is, for example, the case when the solutes are strongly basic and the stationary-phase acidity is high. Nevertheless, it is always possible to modify the form of the mobile-phase variation with time in order to maintain the applicability of the linear-solvent-strength theory [Equation (16.1)]. As we have seen above, this type of gradient offers a considerable help in the fundamental understanding of the retention behavior of the solutes and in the optimization of a separation. 

The pH of the buffer should be selected depending on the pKa values of the components in the mixture and the separation requirements. For any given separation system, there is an attraction between the solute (sample compound) and the stationary and mobile phases. This attraction can be influenced through ionisation or ion suppression of the acidic and basic compounds undergoing separation. Using reversed phase HPTC as an example where there is a nonpolar stationary phase and a polar mobile phase, it would be expected that a nonpolar basic compound would have a high affinity for the stationary phase. 

Ionization polarity should be chosen in conjunction with the mobile phase because pH can influence ease of ionization in positive or negative ion modes. Basic or neutral compounds are readily ionized in positive ion mode at a pH below 7. Since many potential drug candidates contain amine moieties, the majority of LC-MS/ MS methods are conducted in positive ion mode. 

While the precise nature of the mechanism of separation of compounds by ion-pair chromatography has been thoroughly examined and is still the subject of considerable debate (146, 362, 369), the basic features can be summarized by a series of equilibria representing the partitioning and/or adsorption of the ions and the complex between the mobile and stationary phases. The equilibria pertinent to reversed phases (Fig. 2.22) account for the possibility of ion-pair formation in either the mobile or stationary phases as well as the partitioning of the ions and the complex between the two phases. When normal phases are considered, equilibria accounting for appropriate interactions with the normal-phase solid support as well as with the stationary aqueous phase bound to the solid support must also be included. The relative influence of these dynamic processes depends upon the nature of the solute, counter ion, mobile phase, and stationary... 

With charged compounds (weak acids or bases), the adjustment of the pH of the aqueous mobile phase has the strongest influence on the change in selectivity of a chromatographic separation. This is due to the fact that organic acids, basic compounds, and hybrid ionic compounds (zwitterions) show very different behavior in response to pH variations of the eluent [7-9]. 

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