Developing Isocratic Separations

Lewis, J. A., Snyder, L. R., and Dolan, J. W., Initial experiments in high-performance liquid chromatographic method development. II. Recommended approach and conditions for isocratic separation, ]. Chromatogr. A, 721,15,1996. 

Some LC/MS users adhere to isocratic separation because of the myths around gradient elution (it is complex to develop and transfer between instruments and laboratories, it is inherently slower than isocratic methods because of re-equilibration, and other reasons summarized by Carr and Schelling6). A researcher may have a very good reason to use an isocratic method, for example, for a well defined mixture containing only a few compounds. The isocratic method would certainly not be useful in an open access LC/MS system processing varying samples from injection to injection. 

Hupka et al. [29] developed a method for the determination of morphine and its phase II metabolites, morphine-3-beta-D-glucuronide and morphine-6-beta-D-glucuronide in the blood of heroin victims. The method is based on immunoaffinity SPE, RP-HPLC isocratic separation (mobile phase 90% lOmmol KH2PO4, 2mmol 1-heptanesulfonic acid, adjusted to pH 2.5 with H3PO4 and 10% acetonitrile flow rate 1.5 mL/min), and laser-induced native fluorescence detection. 

What are the general steps in developing an isocratic separation for reversed-phase chromatography with one organic solvent ... 

It is sometimes not possible to develop an isocratic separation for complex mixtures of compounds. Binary gradient methods development starts with a... 

The HPLC assay developed for this activity is based on the isocratic separation of UDP, UDP-Gal, and UMP on an amino-bonded column (/ Bondapak NH2 column) eluted with 0.167 M KH2PO4 (pH 4.0). The separation is shown in Figure 9.67. Detection was at 260 nm. 

Two popular approaches are frequently used in method development (1) mobile-phase optimization based on sequential isocratic runs and (2) mobile-phase optimization based on gradient runs. As specified, a primary goal of late-phase method development is to achieve isocratic operating conditions. Accordingly, the following approaches focus initially on the optimization of isocratic conditions. A discussion of gradient optimization follows for mixtures not amenable to isocratic separation. 

The development of a theory of retention and band broadening for macro-molecular HPLC is intended primarily for improving such separations. We need to relate various separation goals to (1) experimental conditions, (2) the choice of column type and HPLC method (RPLC, SEC, etc.), and (3) the nature of the sample. The present model allows os to do this 1 prediction, rathor than by experiment Our main requirement is usuaOy to achieve adequate separation, or some minimum resolution R, betw adjacent bands of interest. In this section we will show that the separation of macro-molecular samples by either isocratic or gradient elution is understandable and controllable, using the same concepts that we use for optimizing the isocratic separation of small molecules. 

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