Liquid chromatography mobile phase composition

The development of micellar liquid chromatography and accumulation of numerous experimental data have given rise to the theory of chromatographic retention and optimization methods of mobile phase composition. This task has had some problems because the presence of micelles in mobile phase and its modification by organic solvent provides a great variety of solutes interactions. 

The popularity of reversed-phase liquid chromatography (RPC) is easily explained by its unmatched simplicity, versatility and scope [15,22,50,52,71,149,288-290]. Neutral and ionic solutes can be separated simultaneously and the rapid equilibration of the stationary phase with changes in mobile phase composition allows gradient elution techniques to be used routinely. Secondary chemical equilibria, such as ion suppression, ion-pair formation, metal complexatlon, and micelle formation are easily exploited in RPC to optimize separation selectivity and to augment changes availaple from varying the mobile phase solvent composition. Retention in RPC, at least in the accepted ideal sense, occurs by non-specific hydrophobic interactions of the solute with the... 

Liquid chromatography has a number of different configurations with regard to technical (instrumental) as well as separation modes. The HPLC system can be operated in either isocratic mode, i.e. the same mobile phase composition throughout the chromatographic ran, or by gradient elution (GE), i.e. the mobile phase composition varies with run time. The choice of operation... 

In any liquid chromatography experiment, the composition of the mobile phase is very important in the total separation scheme. In Chapter 11, we discussed the role of a liquid mobile phase in terms of the solubility of the mixture components in both phases. Rapidly eluting components are highly soluble... 

High Performance Liquid Chromatography High Performance Liquid Chromatography (HPLC) is an analytical chemical method which is used on a large scale routinely. If an HPLC method is developed, the question arises whether the analytical results from this method depend critically on small deviations in the mobile phase composition, the selected UV-wavelength for detection etc. 

Liquid chromatography can be operated under mild conditions in terms of pH, ionic strength, polarity of liquid, and temperature. The apparatus used is simple in construction and easily scaled up. Moreover, many types of interaction between the adsorbent (the stationary phase) and solutes to be separated can be utilized, as shown in Table 11.1. Liquid chromatography can be operated isocratically, stepwise, and with gradient changes in the mobile phase composition. Since the performance of chromatography columns was discussed, with use of several models and on the basis of retention time and the width of elution curves, in Chapter 11, we will at this point discuss some of the factors that affect the performance of chromatography columns. 

Depending on the type of chromatography, optimisation can be fairly rapid. Optimisation in gas phase chromatography is easier than in liquid chromatography where the composition of the mobile phase plays a role. Computer software is available that has been specially designed to help determine the correct composition of the mobile phase. 

An overview and discussion is given of literature methods published after 1989 devoted to the ion-interaction chromatographic determination of inorganic anions. Seventy references are quoted. Ion-interaction chromatography makes use of commercial reversed-phase stationary phase and conventional high-performance liquid chromatography instrumentation. The basis of the technique, the modification of the stationary phase surface, the choice of the ion-interaction reagent as well as the dependence of retention on the different variables involved are discussed. Examples of application in the fields of environmental, clinical and food chemistry are presented. The experimental conditions of stationary phase, of mobile phase composition as well as detection mode, detection limit and application are also summarized in tables. 1997 Elsevier Science B.V. 

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. 

Wu, S. T., Xia, Y. Q., and Jemal, M. (2007). High-field asymmetric waveform ion mobility spectrometry coupled with liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-FAIMS-MS/MS) multicomponent bioanalytical method development, performance evaluation and demonstration of the constancy of the compensation voltage with change of mobile phase composition or flow rate. Rapid Commun. Mass Spectrom. 21 3667-3676. 

Xia YQ, Jemal M (2009) Phospholipids in liquid chromatography/mass spectrometry bio-analysis comparison of three tandem mass spectrometric techniques for monitoring plasma phospholipids, the effect of mobile phase composition on phospholipids elution and the association of phospholipids with matrix effects. Rapid Commun Mass Spectrom 23 2125-2138... 

If the stationary phase is asolid surface, then the term adsorption isotherm is more commonly used. However, in some kinds of chromatography (e.g. reversed phase liquid chromatography, see section 3.2) the phase ratio may vary with variations in the mobile phase composition. 

Typically, in gradient elution liquid chromatography, electrochemical detection has been difficult due to base-line shifts that result as a consequence of the altered mobile phase composition. However, a unique property of micelles allows for much improved compatibility of gradients (i.e. gradient in terms of micellar concentration or variation of small amount of additive such as pentanol) with electrochemical detectors. This has been demonstrated by the separation and electrochemical detection of phenols using micellar gradient LC (488). A surfactant (apparently non-micellar) gradient elution with electrochemical detection has also been successfully applied for the assay of some thyroid hormones by LC (491). 

S. Heinisch and J. L. Rocca, Effect of mobile phase composition, pH and buffer type on the retention of ionizable compounds in reversed-phase liquid chromatography Application to method development,/. Chromatogr. A 1048 (2004), 183-193. 

D. Skyora, E.Tesarova, and M. Popl, Interactions of basic compounds in reversed-phase high-performance liquid chromatography influence of sorbent character, mobile phase composition, and pH on retention of basic compounds, /. chromatogr. A 758 (1997), 37-51. 

Both the solvent-interaction model (Scott and Kucera, 1979) and the solvent-competition model (Snyder, 1968, 1983) have been used to describe the effects of mobile-phase composition on retention in normal-phase liquid chromatography. The solvent interaction model on the one hand provides a convenient mathematical model for describing the relationship between retention and mobile phase composition. The solvent competition model on the other hand provides a more complete, quantitative description of the relative strengths of adsorbents and organic solvents used in normal-phase chromatography. 

Jandera, P. Churacek, J. Liquid chromatography with programmed composition of the mobile phase. Adv. Chromatogr. 1981, 19, 125 -260. 

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