Selection of mobile phases

The mobile phase must obviously be chosen for its chromatographic properties it must interact with a suitable stationary phase to separate a mixture as fast and as efficiently as possible. As a general mle, a range of solvents is potentially able to solve any particular problem, so selection must be based on different criteria  

The (dynamic) viscosity of a solvent is expressed in millipascal seconds (mPas) (formerly in centipoise (cP) the values remain the same). 

Practical High-Performance Liquid Chromatography, Fourth edition Veronika R. Meyer 2004 John Wiley Sons, Ltd ISBN 0-470-09377-3 (Hardback) 0-470-09378-1 (Paperback) 

As a general rule the mobile phase should not be detector-active, i.e. it should not have a property which is used for detection (exception indirect detection, see Section 6.9). Otherwise it is very possible that unwanted baseline effects and extra peaks will show up in the chromatogram. However, this recommendation 

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For preparative separation, the mobile phase can be selected by performing preliminary analytieal TLC experiments. In PLC, the chromatographic chamber has to be saturated within 2 h beeause the development of preparative plates is much slower than the analytical development. In the analytical preassay during the selection of mobile phase composition, the chromatographic chamber must be hned with a sheet of filter paper to obtain a saturated atmosphere with mobile phase vapor. Then, the optimized analytical mobile phase can be transferred imchanged to preparative separations in the saturated developing chamber. 

Variations in retention and selectivity have been studied in cyano, phenyl, and octyl reversed bonded phase HPLC columns. The retention of toluene, phenol, aniline, and nitrobenzene in these columns has been measured using binary mixtures of water and methanol, acetonitrile, or tetrahydrofuran mobile phases in order to determine the relative contributions of proton donor-proton acceptor and dipole-dipole interactions in the retention process. Retention and selectivity in these columns were correlated with polar group selectivities of mobile-phase organic modifiers and the polarity of the bonded stationary phases. In spite of the prominent role of bonded phase volume and residual silanols in the retention process, each column exhibited some unique selectivities when used with different organic modifiers [84],... 

E. Forgacs, T. Cserhati, S. Balogh, R. Kaliszan, R Haber and A. Nasal, Separation of strength and selectivity of mobile phase by spectral mapping technique. Biomed Chromatogr., 15 (2001) 348-355. 

If there is no or little information on the method s performance characteristics, it is recommended that the method s suitability for its intended use in initial experiments be proven. These studies should include the approximate precision, working range, and detection limits. If the preliminary validation data appear to be inappropriate, the method itself, the equipment, the analysis technique, or the acceptance limits should be changed. In this way method development and validation is an iterative process. For example, in liquid chromatography selectivity is achieved through selection of mobile-phase composition. For quantitative measurements the resolution factor between two peaks should be 2.5 or higher. If this value is not achieved, the mobile phase composition needs further optimization. 

The following table provides guidance in the selection of mobile phases that are to be used in conjunction with ultraviolet spectrophotometric detection.1-2 The data in this table differ from the data in the other solvent tables in this volume in that the wavelength dependence of absorbance is provided here. Moreover, common mixed mobile phases are considered here. The percentages that are given are on the basis of v/v. 

The following table provides guidance in the selection of mobile phases that are to be used in conjunction with ultraviolet spectrophotometric detection.1... 

A fluorescence monitor can conveniently confirm and support data obtained with an absorbance detector. However, any comparison of the relative sensitivity/selectivity of the absorbance mode vs the fluorescence mode depends on the spectrochemical nature of both the pesticide itself, and the co-extracted co-elutants found in the agricultural product extracted. A judicious selection of mobile phase is required to optimize separation of the pesticide and co-extractives on a CN-bonded polar stationary phase. 

The ratio of retention volumes in the two solvents depends on their difference in eluent strength (e° - e ), since a and As are constant. Thus, if one has tabulated values for the eluent strength parameter e°, one can predict the effect on retention of changing e°. This concept forms one basis for the selection of mobile phases in LSC. 

In a similar way, the use of formic acid/ammonium formate was shown to be superior to the use of acetic acid/ammonium acetate in the RP HPLC-ESI MS/MS analysis of a number of heterocyclic aromatic amines [7]. It is clear from numerous other examples in the literature that HPLC resolution and MS sensitivity can be dramatically influenced by the correct selection of mobile phases, organic modifiers, and ion-pairing additives. 

Make initial selection of mobile phase (pH, buffer, salt concentration) and method of delivery (isocratic or gradient elution). 

The selection of mobile phases to be used in conjunction with these columns is dependent on the mode of chromatography (reversed phase versus normal phase) and on the physicochemical properties of the API (and related substances). However, in all instances, it is important to ensure that the API and all related substances can be monitored and are separated. Accordingly, it is of paramount importance that the initial chromatographic conditions elute all the individual components from the column and that no components are eluted at the solvent front (i.e., all compounds of interest are retained). In practice, this dictates the use of broad (5-90% strong solvent in 60 min) gradients. 

One of the key issues in LC-MS involves separation of the analytes of interest from each other and from the matrix in which they are present. The introduction of the high-performance liquid chromatography (HPLC) column prior to the ionization source accomplished this task. The use of different stationary phases coupled with an appropriate selection of mobile phases and gradient conditions allowed for... 

SELECTION OF MOBILE PHASE AND COLUMN TYPE 149 TABLE 8.1 (CoBdMMd)... 

When aluminum plates precoated with the mixture of silica gel 60 and Kieselguhr F254 ( 1.05567) were used, the selection of mobile phases depended on the kind of bile acids which are being separated. The mobile phase 25 20 8 (v/v/v) allows separation of all pairs of bile acids, with the exception of the pair of LC and DC. Using mobile phases in other volume compositions can separate the pair of LC and DC. In this case— the separation on the silica gel 60 and Kieselguhr F254 mixture—the biggest problem was to... 

The application of G IMD to the analysis of complex mixtures is based on the selection of mobile phases suitable to each group of solutes present in the mixture, e.g., polar, weakly polar, and/or non-polar. In the next step, it is possible to design a gradient program. The profile of the gradient can be adjusted to specific properties of the components of particular groups. The number of steps may be varied depending on the number of components— more components require more steps. 

HPLC has been used increasingly in the analysis of food samples to separate and detect additives and contaminants. HPLC can separate a large number of compoimds both rapidly and at high sensitivity, reduce separation times, and reduce the volume of sample needed. HPLC is ideally suited for compounds of limited thermal stability, but requires sample pretreatment such as extraction and filtration. In addition, HPLC requires careful selection of mobile phase and sample pumping rate [24]. 

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