Initial solvent strength

Step 1. Decrease the initial solvent strength. Make it more polar for reversed phase or less polar for normal phase. Step 2. Increase the time of the gradient. This gives a better selectivity. 

Solvent selectivity is seen as the factor that distinguishes individual solvents that have solvent strengths suitable for separation. In reality, separations result from the competition between the mobile and stationary phases for solutes based on the differences of all intermolecular interactions with the solute in both phases. Solvents can be organized on selectivity scales that are useful for initial solvent selection, but in a chromatographic separation the properties of the stationary phase must be taken into consideration. Methods that attempt to model chromatographic separation need to consider simultaneously mobile and stationary phase properties [38]. 

When the solvent strength of the sample diluent in HPLC does not match well with the solvent strength of the mobile phase at initial conditions, peak deformation is bound to occur. In CE a comparable phenomenon is observed with differences in conductivity between the sample zone and the bulk electrolyte in the capillary.The conductivity (y, Q m ) of a solution is given by the cumulative effect of the contributions of different ions ... 

As shown in Figure 1.2, the solvent strength of supercritical carbon dioxide approaches that of hydrocarbons or halocarbons. As a solvent, C02 is often compared to fluorinated solvents. In general, most nonpolar molecules are soluble in C02, while most polar compounds and polymers are insoluble (Hyatt, 1984). High vapor pressure fluids (e.g., acetone, methanol, ethers), many vinyl monomers (e.g., acrylates, styrenics, and olefins), free-radical initiators (e.g., azo- and peroxy-based initiators), and fluorocarbons are soluble in liquid and supercritical C02. Water and highly ionic compounds, however, are fairly insoluble in C02 (King et al., 1992 Lowry and Erickson, 1927). Only two classes of polymers, siloxane-based polymers and amorphous fluoropolymers, are soluble in C02 at relatively mild conditions (T < 100 °C and P < 350 bar) (DeSimone et al., 1992, 1994 McHugh and Krukonis, 1994). 

After the compounds of interest are eluted from the column, the concentration is linearly increased to 0.1 N acetic acid in 95% (v/v) ethanol/water to remove the more nonpolar components retained on the column. Following an adequate equilibration (greater than 10 column volumes) at maximum solvent strength (95% ethanol), the solvent is linearly programmed to return to the initial conditions of 0.1 N aqueous acetic acid. 

As in analytical liquid chromatography (LC), analyte retention depends on sample concentration, solvent strength, and sorbent characteristics. An empirical approach to methods development initially involves screening the available sorbents. The first step is to determine which sorbents best retain the analyte. The second consideration is to evaluate the solvents needed to elute the compound and the compatibility of those sorbents to the chromatographic testing procedure. The third step is to test the blank sample matrix to evaluate the presence of possible interferents. Finally, recoveries of known quantities of analyte added to the sample matrix must be determined. 

As an initial approximation of the ideal solvent strength for HPLC, the results of TLC studies on acetylated cellulosic plates with various methanol-water mixtures and natural dye extractions were graphed (R/ vs. methanol concentration). An acetylated cellulosic TLC system is not directly comparable to a C-18 HPLC system, but TLC results were nonetheless useful as a rough estimate of solvent strength. In practice, approximately two-thirds of the solvent strength required to elute natural dyes with TLC was necessary to achieve a similar separation of major sample components with HPLC. After individual solvent concentrations were determined, samples of known dyes extracted from wool were eluted in each of the three pairs of solvents (the concentration used for each individual solvent-water system was reduced by one-half) and adjustments were made until each sample eluted with a kf value no larger than 10. Retention times and kf values are equivalent expressions of relative retention of a sample on the column if the flow rate is the same for all trials. With a flow rate of 1.4 mL/min, the maximum time... 

As in normal phase (see section 3.5.3), the first step in mobile-phase optimization is the determination of the solvent strength that will elute the analytes with a A value between 2 and 10 from the chosen stationary phase. It is not important which modifier is chosen to determine the initial conditions, and methanol-water (50 50, v/v) is a convenient starting place. Once the initial conditions have been established, a variety of techniques may be employed to obtain the optimum separation. Most optimization strategies involve the establishment of the isoelutropic concentrations of methanol-water, acetonitrile-water and tetrahydrofuran-water. The isoelutropic concentrations can be determined by experiment or from tables of isoelutropic mixtures (e.g. Table 3.5) (Wells, 1988). The binary solvent systems A, B, C (Table 3.5, Figure 3.7) define the isoelutropic plane, which is then explored to obtain the optimum combination of water, methanol, tetrahydrofuran, and acetonitrile required for the separation. 

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