Optimization stationary phases

The relative importance of column efficiency, retention and selectivity is presented with applications to the analysis of complex, volatile samples. Recent trends in the analysis of aroma samples by high resolution gas chromatography include utilizing specific stationary phases to analyze particularly difficult samples. Important considerations in selecting the optimum column for an analysis include the overall efficiency generated by the column, the partition ratio of the solutes to be resolved and the selectivity of the stationary phase towards the compounds of interest. After comparing the relative contributions these three factors, methods of optimizing stationary phase selectivity will be described. 

Kaiser, R. (1968), Support coated (open tubular) glass capillaries with optimal stationary phases. Chromatographia, 1-2,34. 

FIGURE 8.12 Effect of pore diameter on SEC of standards (nondenaturin > mobile phase). Nondenaturing" refers to the effect on the stationary phase. Most iarge proteins were in fact denatured by this mobile phase (which was optimized for use with peptides, not proteins). Accordingly, it was necessary to use polyacrylamide to demonstrate the approximate range and position of Vo under these conditions. The polyacryiamide standards both eiuted at V with the 300-A coiumn (not shown). Columns and flow rate Same as in Fig. 8.11. Mobile phase Same as in Fig. 8.1. Sample key (B) Ovalbumin (43,000 Da) 0) polyacrylamide (1,000,000 Da) (K) polyacrylamide (400,000 IDa) (L) low molecular weight impurity in the polyacrylamide standards. Other samples as in Fig. 8.11. 

In the development of a SE-HPLC method the variables that may be manipulated and optimized are the column (matrix type, particle and pore size, and physical dimension), buffer system (type and ionic strength), pH, and solubility additives (e.g., organic solvents, detergents). Once a column and mobile phase system have been selected the system parameters of protein load (amount of material and volume) and flow rate should also be optimized. A beneficial approach to the development of a SE-HPLC method is to optimize the multiple variables by the use of statistical experimental design. Also, information about the physical and chemical properties such as pH or ionic strength, solubility, and especially conditions that promote aggregation can be applied to the development of a SE-HPLC assay. Typical problems encountered during the development of a SE-HPLC assay are protein insolubility and column stationary phase... 

When multiple development is performed on the same monolayer stationary phase, the development distance and the total solvent strength and selectivity values (16) of the mobile phase (17) can easily be changed at any stage of the development sequence to optimize the separation. These techniques are typically fully off-line modes, because the plates must be dried between consecutive development steps only after this can the next development, with the same or different development distances and/or mobile phases, be started. This method involves the following stages ... 

Method Development and Optimization of Enantiomeric Separations Using Macrocyclic Glycopeptide Chiral Stationary Phases... 

Typical normal-phase operations involved combinations of alcohols and hexane or heptane. In many cases, the addition of small amounts (< 0.1 %) of acid and/or base is necessary to improve peak efficiency and selectivity. Usually, the concentration of polar solvents such as alcohol determines the retention and selectivity (Fig. 2-18). Since flow rate has no impact on selectivity (see Fig. 2-11), the most productive flow rate was determined to be 2 mL miiT. Ethanol normally gives the best efficiency and resolution with reasonable back-pressures. It has been reported that halogenated solvents have also been used successfully on these stationary phases as well as acetonitrile, dioxane and methyl tert-butyl ether, or combinations of the these. The optimization parameters under three different mobile phase modes on glycopeptide CSPs are summarized in Table 2-7. 

There are many advantages of the SMB technology compared to batch preparative chromatography. The process is continuous and the solvent requirement is minimal compared to batch. In SMB, the whole stationary phase is used for the separation while in batch chromatography only a small part of the column is involved in the separation. This allows optimization of productivity with respect to the stationary phase. 

Modifier additives also play a role in method optimization and are typically added to the modifier at concentrations less than 1 % (v/v). Additives can provide increased efficiency by minimizing undesirable interactions between the analyte and the CSP, and may be necessary to elute certain types of compounds. The type of additive (acidic or basic) that will produce the best results depends upon the functionality of the analyte [72]. Certain additives are strongly retained on the stationary phase, and their effect may persist even after they are removed from the eluent [22]. The impact of both modifiers and additives can also be affected by the proximity of the operating conditions to the critical point of the eluent [73]. 

Column chromatographic separations depend on the relative affinity of different proteins for a given stationary phase and for the mobile phase. Association between each protein and the matrix is weak and transient. Proteins that interact more strongly with the stationary phase are retained longer. The length of time that a protein is associated with the stationary phase is a function of the composition of both the stationary and mobile phases. Optimal separation of the protein of interest from other proteins thus can be achieved by careful manipulation of the composition of the two phases. 

Optimized HPLC separation allows most betaxanthins to be separated on a Cl8 reversed phase stationary phase according to their respective polarities. - Considerable progress was achieved by the introduction of a highly polar silica-based column, which allowed major improvement of peak resolution, especially at early... 

In PLC, an inferior separation is always achieved because the particle size and size distribution of adsorbents for preparative purposes are larger, and the preparative plates are much more overloaded with the separated compounds. This means that an optimized mobile phase is necessary for a successful preparative separation. During the optimization process, the volatility of the individual solvents must be considered so as to avoid several problems in subsequent steps such as elution of the compound from the stationary phase and evaporation of the solvents. It is important to use acetic acid as a component of the preparative mobile phase because of the possibility of chemical degradation during concentration of the isolated compounds [1, 7,8]. 

Prus and Kowalska [75] dealt with the optimization of separation quality in adsorption TLC with binary mobile phases of alcohol and hydrocarbons. They used the window diagrams to show the relationships between separation selectivity a and the mobile phase eomposition (volume fraction Xj of 2-propanol) that were caleulated on the basis of equations derived using Soezewiriski and Kowalska approaehes for three solute pairs. At the same time, they eompared the efficiency of the three different approaehes for the optimization of separation selectivity in reversed-phase TLC systems, using RP-2 stationary phase and methanol and water as the binary mobile phase. The window diagrams were performed presenting plots of a vs. volume fraetion Xj derived from the retention models of Snyder, Schoen-makers, and Kowalska [76]. 

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. 

This technique is based on the same separation mechanisms as found in liquid chromatography (LC). In LC, the solubility and the functional group interaction of sample, sorbent, and solvent are optimized to effect separation. In SPE, these interactions are optimized to effect retention or elution. Polar stationary phases, such as silica gel, Florisil and alumina, retain compounds with polar functional group (e.g., phenols, humic acids, and amines). A nonpolar organic solvent (e.g. hexane, dichloromethane) is used to remove nonpolar inferences where the target analyte is a polar compound. Conversely, the same nonpolar solvent may be used to elute a nonpolar analyte, leaving polar inferences adsorbed on the column. 

For optimization of chromatographic separations the ratio of the time spent by the solute in the stationary phase to the time it spends in the mobile phase is more fundiwentally i tortant. This ratio is called the solute capacity factor and is given by equation (1.8)... 

In practice, it is more difficult to optimize resolution as a function of the relative retentlvity than to optimize retention. Thus, unless the mixture is very complex or contains components that are particularly difficult to separate it may be possible to optimize a particular separation using the linear equation (1.72) as demonstrated by Bttre [177]. Figure 1.13 illustrates the relative change in peak position for a polarity test mixture with two identical, serially coupled open tubular columns, coated with a poly(dimethylslloxane) and Carbowax 20 M stationary phases, as a function of their relative retentlvity on the second column. The linear relationship predicted by equation (1.72) effectively predicts the relative peak positions and indicates that a nearly... 

The popularity of reversed-phase liquid chromatography (RPC) is easily explained by its unmatched simplicity, versatility and scope [15,22,50,52,71,149,288-290]. Neutral and ionic solutes can be separated simultaneously and the rapid equilibration of the stationary phase with changes in mobile phase composition allows gradient elution techniques to be used routinely. Secondary chemical equilibria, such as ion suppression, ion-pair formation, metal complexatlon, and micelle formation are easily exploited in RPC to optimize separation selectivity and to augment changes availaple from varying the mobile phase solvent composition. Retention in RPC, at least in the accepted ideal sense, occurs by non-specific hydrophobic interactions of the solute with the... 

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