Method development in reversed-phase chromatography

In reversed-phase separations, the first step in method development is usually a solvent gradient from 10 to 100% solvent B. This approach is explained in Section 18.2. What follows here are proposals with a recommended sequence of changes of the various parameters. It is, e.g., more convenient to try another B solvent before changing the stationary phase. 

Use a Cg or Cig stationary phase with unbuffered water-acetonitrile at ca. 40 °C if temperature adjustment is possible, otherwise at ambient temperature. 

Neue et al.. Method Development in RP Chromatography, in I.D. Wilson, ed., Bioanalytical Separations, Elsevier, Amsterdam, 2003, pp. 185-214. 

Adjust %B or gradient range for retention factors between 1 and 10 (or 1 and 20 for difficult separations). If the separation is inadequate, adjust selectivity in the following order  

Change the stationary phase (preferably to a type which has markedly different properties, see Section 10.4). It is probably necessary to start at step 1 again. 

Use a Cg or Ci8 stationary phase with unbuffered water-acetonitrile at ca. 40°C 

Optimize the physical parameters such as column dimensions, particle size or flow rate. 

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J. R. Mazzeo, K. Tran, D. M. Wagrowski-DieW, Method development in reversed-phase chromatography . Handbook of Analytical Separations Vol. 4 (Ed. I. D. Wilson), Elsevier Science, Amsterdam, 2003. 

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-... 

The solvation parameter model has been used as the basis of a structure-driven retention model for method development in reversed-phase thin-layer chromatography [95,151,161-164]. The model should be applicable to normal-phase separations on chemically bonded layers as well, but not silica gel layers [164]. Solute size differences and site-specific interactions on silica gel are not adequately accounted for by the model, which results in poor predictions of retention. The solvation parameter model is described in section 1.4.3. The Rm value (section 6.3.1) is used as the dependent variable. 

Galushko, S.V. Kamenchuk, A.A. Pit, G.L. Software for method development in reversed-phase liquid-chromatography. Amer. Lah. 1995, 27, G33-J33. 

There are no standardized pretreatment procedures for commercially available HPLC silicas. As a consequence, one can observe significant differences in the retention behavior of a set of standards on different silicas (12). Therefore, one cannot a priori expect that a chromatographic method developed on one silica can be transferred to another silica without difficulties. However, anoe the mobile-phase composition plays such an important role in normal-phase chromatography, the conversion of a method from one silica to another is easier in normal-phase chromatography than in reversed-phase chromatography. 

With all these tools, you can usually find a way to separate the components of a mixture if it does not contain too many compounds. If reversed-phase chromatography fails, normal-phase chromatography or one of the methods in Chapter 26 could be appropriate. Method development is part science, part art, and part luck. 

Based on the fact that aromatic sulfonic and carboxylic acids were successfully separated by reversed-phase chromatography in the presence of organic electrolytes, Chaytor and Heal (158) developed a method for the separation of 15 synthetic colors using a mobile phase containing o-phosphoric acid (Table 7). The presence of the electrolyte provided lower variation in response and retention over a period of time. Furthermore, eluted peaks were sharper than those seen in ion-pair chromatography. 

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