Method Development Protocols

In the new polar organic mode, the ratio of acid/base in the mobile phase affects the selectivity and the concentration of acid and base controls the retention. It is suggested to start the method development with a medium concentration (0.1 %) for both acid and base. If retention is too long or too short, the concentration can be increased to 1 % or reduced to 0.01 %. If no selectivity is observed in this mode, reversed phase is recommended as the next step in the protocols. 

When analytes lack the selectivity in the new polar organic mode or reversed-phase mode, typical normal phase (hexane with ethanol or isopropanol) can also be tested. Normally, 20 % ethanol will give a reasonable retention time for most analytes on vancomycin and teicoplanin, while 40 % ethanol is more appropriate for ristocetin A CSP. The hexane/alcohol composition is favored on many occasions (preparative scale, for example) and offers better selectivity for some less polar compounds. Those compounds with a carbonyl group in the a or (3 position to the chiral center have an excellent chance to be resolved in this mode. The simplified method development protocols are illustrated in Fig. 2-6. The optimization will be discussed in detail later in this chapter. 

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The first consideration when investigating HPLC method development protocols is the chemical structure of the analyte, in particular, the presence of functional groups capable of interacting with the stationary phase and containing or in the vicinity of the stereogenic elements [79]. Since the natural target of macrocyclic antibiotics is the A-acyl-D-alanyl-D-alanine terminus (see Section 2.1), the early choice of suitable substrates for this kind of CSPs was that of amino acids [45]. However, it turned out that the macrocyclic CSPs were very successful not only in amino acids enantioresolution, but also in the separation of a wide variety of different structures. 

For acidic compounds, the pKa should be established the buffer should be adjusted so that it is at least one pH unit above the pKa of the compound. Small neutral compounds are best separated using CDs, but bile acids may be better suited to the separation of the larger, more hydrophobic ones. Figure 5.16 shows methods development protocols for different classes of compounds. 

The step-by-step approach recommended in the ISO Guide and the top down approach have been seen as alternative and substantially different ways of evaluating uncertainty, but the comparison between method development protocols and ISO approach above shows that they are more similar than appears at first sight. In particular, both require a careful consideration and study of the main effects on the result to obtain robust results accounting properly for each contribution to overall uncertainty. However, the top down approach relies on that study being carried out during method development to make use of the data in ISO GUM estimations, the detailed data from the study must be available. 

A Reverse Phase Method Development Protocol for CYCLOBOND 1 2000... 

A graph of retention ratio against the reciprocal of the absolute temperature can also be helpful in the manner previously discussed. The previous chart gives a method development protocol for CYCLOBOND 1 2000 operated in the reverse phase mode. The chart is self explanatory, the first solvent being acetonitrile/bufifer 35/65 v/v. A method development protocol for CYCLOBOND 1 2000 operated in the polar... 

Table 4. Systematic method development protocol for Daicel stationary phases.

In industrial practice a systematic method development protocol using different stationary and mobile phases is often used [12,13]. An example of such a protocol is outlined in Table 4. 

Notes Useful for the separation of hydrocarbons, pesticides, PCBs, phenols, sulfur compounds, flavors and fragrances, and some amines columns are typically stable and low bleed a good all-purpose column used to begin method development protocols. 

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