Mobile phases maximizing retention

In the former case [32], the production rate of 99% pme enantiomers from the racemic mixture of R- and S-2-phenylbutyric acid was maximized as a function of the sample size and the mobile phase composition. The calculations were based on the column performance and the equilibrium isotherms of the two components (bi-Langmuir isotherms. Chapter 3). The separation was performed on immobilized bovine serum albumin, a chiral stationary phase, using water-methanol solution as the mobile phase. The retention times decrease with increasing methanol content, but so does the separation factor. For this reason, the optimum retention factor is around 3. Calculated production rates agree well with those measured (Table 18.4). The recovery yield is lower than predicted. 

A particularly compelling argument for dynamic ion-exchange has put forward the observation that retention of anionic and cationic sample components increases and decreases with increasing concentration of a cationic hetaeron, respectively. Whereas anionic hetaerons are expected to promote the elution of anionic eluites and to enhance the retention of cationic eluites, the quantitative data presented in this regard (226) are not wholly consistent with the model since the hetaeron concentration at which the effect is half-maximal is different for anionic and cationic eluites. If the observed phenomena were due to the presence of bound hetaeron in both cases, the two effects would have identical dependence on the hetaeron concentration in the mobile phase. 

This relationship has been experimentally verified for numerous mobile phases (and different solvents) and a wide variety of solutes, with both alumina (IS) and silica (18) as adsorbents. Some examples of the applicability of Eq. (31a) in these LSC systems are given in Fig. 15a (alumina) and Fig. 15b (silica). The use of 3-solvent or 4-solvent mobile phases (18, 20) allows the continuous variation ofm while holding e° constant, which greatly facilitates retention optimization by maximizing a without changing c°. 

When this procedure is repeated for every possible pair of overlapping bands in the sample, and the/ s plots for each band pair are superimposed, the ORM plot of Fig. 25b results. Now it is seen that the white area for R > 1.0 is very much reduced. Also indicated (x) is the optimum composition for maximizing the resolution of the most poorly separated band pair. The actual separation based on this optimum mobile-phase composition is shown in Fig. 26 for several nominally similar 25-cm silica columns. The desired resolution (Rg > 1.0) is indeed observed for all three columns. This is an important point when retention optimization is applied to complex mixtures if column-to-column variability in retention is significant, an optimum separation on one column may not be transferable to... 

The present model predicts how solvent selectivity will vary with mobile-phase composition, and this allows the selection of extreme solvents for maximum differences in selectivity. This information plus the ability to calculate solvent strength versus composition of the mobile phase then allows development of a general strategy for optimizing retention of any sample, so as to maximize resolution. This four-solvent approach can be further refined by use of computer-assisted procedures, such as the overlapping-resolution-mapping technique. 

In order to maximize such interactions, these stationary phases can be used in conjunction with mobile phases containing mixtures of hexane/alcohols. It was found that linear alcohols such as ethanol and n-propanol produced a longer retention time than the branched alcohols. The reason for such a phenomenon is attributed to the behavior of linear alcohols, which self-associates in the presence of a nonpolar solvent such as hexane. Such association is at the expense of the interaction of these alcohols with the stationary phase. 

The cellulosic CSPs are most often used with nonpolar mobile phases composed of hexane and a polar modifier such as 2-propanol. These mobile phases are used to maximize the attractive interactions between solute and CSE Since these interactions contain a polar component, an increase in the polarity of the mobile phase, for example, a lager alcohol content, will reduce the retention and vice versa. These phases have also been used with aqueous mobile phases and a new leversed-phase version of the OD CSP is currrently being marketed (56). 

The concentration of organic solvent should be low enough to make the existence of micelles possible. Such maximal amount depends on the type of surfactant and organic solvent, and is usually unknown. For SDS, the maximal volume fractions of acetonitrile, propanol, butanol, and pentanol that seem to guarantee the presence of micelles are 20%, 15%, 10%, and 7% (v/v), respectively. However, analytical reports where authors claim the use of hybrid micellar mobile phases and these maximal values are exceeded—micelles do not exist— are not unusual. In such conditions, the system bears closer resemblance to an aqueous-organic system, although the surfactant monomers still affect the retention and efficiencies. 

Resolution can be increased if one of the three terms in Eq. 4.14 is increased. As mentioned in Chapter 2.4.3 the first term describes the influence of selectivity. This term should be maximized by maximizing a in a selectivity screening with different adsorbents and mobile phases. The second term should be kept in a certain range and not be maximized, because the maximum value of 1 is reached for an infinite retention factor. At infinite retention the productivity would decrease due to the high cycle time. The last term of Eq. 4.14 describes the efficiency of the column in terms of the number of plates. Resolution can be increased by selecting efficient adsorbents with small particle size and appropriate narrow particle size distribution. For these adsorbents fluid dynamic and mass transfer resistances are minimized. Con-... 

The choice of reverse phase packing material will depend on the amount of information available on the component of interest and on other sample components. Initial tests such as solvent partitioning behavior, solubility m various solvents, and others see Chapter 1) can be used to estimate polarity and hence be of use in initial column/mobile phase selection. The most retentive of the silica-based reverse phase supports, Cl8 and C8, are a sensible first choice, as the retention of polar compounds is maximized, while the retention of nonpolar materials can be easily modulated by choice of eluent. If the compound of interest is very nonpolar (or the sample contains components that bind very strongly to retentive phases such as C8/C18), a shorter chain alkyl-bonded phase such as C6 or C4 may be more suitable. 

Additionally, the retention and selectivity in IP-RP systems can be controlled by the change of type and concentration of the organic modifier in the aqueous mobile phase and the pH of the mobile phase, " which should be selected to obtain maximal ionization of solute molecules and ion-pairing reagent molecules for the possibility of forming an ion pair. For the basic solutes, analyses of the pH range 7.0-7.5 are often applied. 

In common with other application areas of chromatographic separation, a considerable amount of effort has been expended recently on the development of different elution conditions and types of stationary phases for peptide separations in attempts to maximize column selectivities without adversely affecting column efficiences. Peptide retention will invariably be mediated by the participation of electrostatic, hydrogen bonding, and hydrophobic interactions in the distribution phenomenon. The nature of the predominant distribution mechanism will be dependent on the physical and chemical characteristics of the stationary phase as well as the nature of the molecular forces which hold the solute molecules within the mobile and stationary zones. The retention of the solute in all HPLC modes can be described by the equation... 

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