Reverse phase method development experiment

The selection of starting conditions is the first step in HPLC method development. Instead of starting with arbitrary conditions or conditions derived from the chromatographer s experience or intuition, the software can predict suitable initial conditions for reversed-phase methods from analyte structures and properties of the sorbent/solvent system [1-3]. If structures are known, the theoretical approach has the potential to save time and effort, since in this way the experimental method development process will start under the theoretically predicted optimum conditions. 

The PRISMA model was developed by Nyiredy for solvent optimization in TLC and HPLC [142,168-171]. The PRISMA model consists of three parts the selection of the chromatographic system, optimization of the selected mobile phases, and the selection of the development method. Since silica is the most widely used stationary phase in TLC, the optimization procedure always starts with this phase, although the method is equally applicable to all chemically bonded phases in the normal or reversed-phase mode. For the selection of suitable solvents the first experiments are carried out on TLC plates in unsaturated... 

Successful separations can be carried out only by planning and careful experimentation, the details of which are discussed extensively in Chapter 5. In one sense there are too many ways to achieve a separation in LC. But while this makes the first choice of where to start difficult, the good news is that there are many ways to achieve success. By looking at Figure 4-1 it is obvious that the use of the reverse-phase mode in LC has broad applicability and is, in fact, the most used mode of LC. Reverse phase is used for 80-85% of the separation problems encountered by users of HPLC. For this reason, the majority of Chapter 5, on developing methods, is devoted to reverse-phase examples. Additionally, Chapter 11 is a useful experiment to experience the method development aspect of this mode. Chapter 9 is a useful experiment to experience method development in the normal phase mode. 

By examining this brief history of the development of instrumentation and methods, it should be clear what parameters define the instrument of choice for analysis of pharmaceutically important molecules. Such an instrument is one capable of performing LC-MS, typically using a reverse-phase HPLC separation and one of the API techniques. MS-MS capability is desirable—using an ion trap, a triple quadrupole, or other tandem mass analyzer instrument. Accurate mass measurement capability is also desirable. These attributes make up a prioritized list of capabilities for the ideal full-purpose instrument to be used in a pharmaceutical research environment. As one progresses through this list, the expense of the instrument increases, the sophistication of the instrument increases, and the intellectual and technological commitment required to do these experiments increases. 

With nonaqueous samples (part B of Fig. 3.8), the decisions for sorbents are somewhat reversed. For example, an analyte that is polar and ionic is best recovered with ion exchange. This example is similar to that of an aqueous sample. If the analyte is polar but nonionic, then the sorbent choice could be reversed phase or normal phase. The choice depends on the organic solvent, either polar or nonpolar, respectively. Finally, if the analyte is nonpolar, the sorbent choice is reversed phase. The second step in methods development is to execute the SPE experiment. Lastly, one has to optimize and troubleshoot the SPE method. 

Normal-phase, bonded-phase columns are likely underutilized for separations where they should be the method of choice. This is due both to the ease of use of reversed-phase, bonded-phase columns, discussed next, and also to the many problems inherent in the use of bare silica and alumina. Very straightforward method development in normal-phase chromatography can be performed by combining the solvent and stationary-phase selectivity triangles. The three columns, each used with the three recommended modifiers, should provide the maximum difference in selectivity available. These nine experiments, used in conjunction with chemometric optimization schemes, should then provide a ratio-... 

HPLC techniques have also been used in the determination of log P values. Lambert et al. (1990), for example, have described the development of a preformulation lipophilicity screen utilizing a C18 derivatized HPLC column. They appeared to prefer this column to the traditional reverse-phase HPLC columns, which may yield a poor correlation between log P and the capacity factor (k ). A potential problem with the use of HPLC retention data is that it is not a direct method and thus requires calibration. Futhermore, there may be problems with performing experiments above pH 8. 

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