Mobile phase classical approach

Additional modes of HPTC include normal phase, where the stationary phase is relatively polar and the mobile phase is relatively nonpolar. Silica, diol, cyano, or amino bonded phases are typically used as the stationary phase and hexane (weak solvent) in combination with ethyl acetate, propanol, or butanol (strong solvent) as the mobile phase. The retention and separation of solutes are achieved through adsorp-tion/desorption. Normal phase systems usually show better selectivity for positional isomers and can provide orthogonal selectivity compared with classical RPLC. Hydrophilic interaction chromatography (HILIC), first reported by Alpert in 1990, is potentially another viable approach for developing separations that are orthogonal to RPLC. In the HILIC mode, an aqueous-organic mobile phase is used with a polar stationary phase to provide normal phase retention behavior. Typical stationary phases include silica, diol, or amino phases. Diluted acid or a buffer usually is needed in the mobile phase to control the pH and ensure the reproducibility of retention times. The use of HILIC is currently limited to the separation of very polar small molecules. Examples of applications... 

One of the classical approaches of liquid chromatography, paper chromatography, was used for chiral resolution about 50 years ago but is not part of modem practice. In paper chromatography, the stationary phase is water bonded to cellulose (paper material), which is of course chiral and hence provides a chiral surface for the enantiomers. However, some workers used chiral mobile phase additives also in paper chromatography [73,74]. In 1951 some research groups independently [73,75-77] resolved the enantiomers of amino acids. Simultaneously, numerous interesting publications on chiral resolution by paper chromatography appeared [70]. 

For the assessment of the extent of change of the phase ratio of a HPLC column system with temperature or another experimental condition, several different experimental approaches can be employed. Classical volumetric or gravimetric methods have proved to be unsuitable for the measurement of the values of the stationary phase volume Vs or mobile phase volume Vm, and thus the phase ratio ( = Vs/Vm). The tracer pulse method266,267 with isotopically labeled solutes as probes represents a convenient experimental procedure to determine Vs and V0, where V0 is the thermodynamic dead volume of the column packed with a defined chromatographic sorbent. The value of Vm can be the calculated in the usual manner from the expression Vm = Eo — Vs. In addition, the true value of Vm can be independently measured using an analyte that is not adsorbed to the sorbent and resides exclusively in the mobile phase. As a further independent measure, the extent of change of 4> with T can be assessed with weakly interacting neutral or... 

This is more or less the classical approach to selectivity optimization, except that in the past binary-solvent mobile phases were used most commonly. 

Classical liquid chromatography is typically practiced in what is referred to as the normal-phase mode that is, the stationary phase is usually a polar sorbent such as silica and alumina and the mobile phase consists of a nonpolar constituent such as hexane modified with a somewhat more polar solvent such as chloroform or ethyl acetate. In this mode, the more polar compounds are preferentially retained. The reversed-phase (RP) mode utilizes the opposite approach for the separation of nonpolar analytes or compounds that have some hydrophobic character. In this case, the stationary phase must consist of sorbent that is nonpolar in nature and the mobile phase is composed of a primary polar solvent, usually water, that is modified by a more nonpolar constituent such as methanol, acetonitrile, or tetrahydrofuran. 

This chapter provides an overview of modern HPLC method development and discusses approaches for initial method development (column, detector, and mobile phase selection), method optimization to improve resolution, and emerging method development trends. The focus is on reversed-phase methods for quantitative analysis of small organic molecules since RPLC accounts for 60-80% of these applications. Several case studies on pharmaceutical impurity testing are presented to illustrate the method development process. For a detailed treatment of this subject and examples of other sample types, the reader is referred to the classic book on general HPLC method development by L. Snyder et al.1 and book chapters2,3 on pharmaceutical method development by H. Rasmussen et al. Other resources include computer-based training4 and training courses.5... 

LC uses mostly packed columns, as the use of open tubular columns in this method is not practical because of the extremely small column diameters required for good separation. In gas chromatography, both packed and open tubular columns can be used, but the latter are far more popular because of their vastly superior properties. The mobile phase is usually forced through the stationary phase at elevated pressure, although other approaches are also possible (e.g., electrically driven flow in electrochromatography (EC), gravity driven flow in classical LC or flow driven by capillary forces in TLC). 

For the separation of baizoic acids, planar electrochromatography was used. In this approach, an electro-osmotic flow is used to drive the mobile phase in TLC. Planar electrochromatography has several advantages over classical TLC, especially substantially faster separation. For example, separation by planar electrochromatography can be 10 times faster than that using ordinary TLC. 

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