Mobile phase preparation example

A typical example of HPLC method development and validation was provided by Boneschans et al. [9]. They developed an HPLC method for piroxicam benzoate and its major hydrolytic degradation products, piroxicam and benzoic acid. The authors utilised a robust stationary phase (Phenomenex Luna, Cig), with an optimised mobile phase comprising of acetonitrile/water/acetic acid (45/7/8 v/v), and a flow rate of 1.5 ml/min. The operating pH of the mobile phase (pH 2.45) was selected on the basis that it is ca. 2 pH units from the pKa of the drug, and hence reasonably insensitive to changes in mobile-phase preparation. The injection volume was 20 pi with a detection wavelength of 254 nm. They utihsed... 

Mixing errors automated mobile phase preparation. Problems encountered with an automated system can still be the result of analyst error. For example, the wrong component may have been dispensed into the HPLC solvent reservoir. Where there is an apparent issue in relation to the retention time of a compound, the mobile phase should always be checked for accuracy of preparation. Proportioning valve errors can also cause a problem with automated systems. When the valve ceases to dispense the correct amount of either solvent, the retention time of the compounds of interest will vary. 

The coupling of supercritical fluid extraction (SEE) with gas chromatography (SEE-GC) provides an excellent example of the application of multidimensional chromatography principles to a sample preparation method. In SEE, the analytical matrix is packed into an extraction vessel and a supercritical fluid, usually carbon dioxide, is passed through it. The analyte matrix may be viewed as the stationary phase, while the supercritical fluid can be viewed as the mobile phase. In order to obtain an effective extraction, the solubility of the analyte in the supercritical fluid mobile phase must be considered, along with its affinity to the matrix stationary phase. The effluent from the extraction is then collected and transferred to a gas chromatograph. In his comprehensive text, Taylor provides an excellent description of the principles and applications of SEE (44), while Pawliszyn presents a description of the supercritical fluid as the mobile phase in his development of a kinetic model for the extraction process (45). 

Another important issue that must be considered in the development of CSPs for preparative separations is the solubility of enantiomers in the mobile phase. For example, the mixtures of hexane and polar solvents such as tetrahydrofuran, ethyl acetate, and 2-propanol typically used for normal-phase HPLC may not dissolve enough compound to overload the column. Since the selectivity of chiral recognition is strongly mobile phase-dependent, the development and optimization of the selector must be carried out in such a solvent that is well suited for the analytes. In contrast to analytical separations, separations on process scale do not require selectivity for a broad variety of racemates, since the unit often separates only a unique mixture of enantiomers. Therefore, a very high key-and-lock type selectivity, well known in the recognition of biosystems, would be most advantageous for the separation of a specific pair of enantiomers in large-scale production. 

The following account, taken from a practical notebook, describes the preparation of the mobile phase used in the example in Section 4.4. 

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