Mobile phase parameters

Recently, Chester has described (21) how a consideration of the phase diagram of the mobile phase shows that a one-phase region (Figure 1.1) is available for the selection of the mobile phase parameters, and that the boundaries separating... 

The effect of the mobile-phase composition on the operation of the different interfaces is an important consideration which will be discussed in the appropriate chapter of this book but mobile-phase parameters which affect the operation of the interface include its boiling point, surface tension and conductivity. The importance of degassing solvents to prevent the formation of bubbles within the LC-MS interface must be stressed. 

Test Mixture Mobile Phase Parameter Reference... 

Mobile Phase Parameter Effect on Fluorescence Emission... 

Mobile-phase parameter Influence on mobile-phase properties Effect on sample retention... 

Mobile phase parameters (solvent strength, pH, ion-pairing reagent, and temperature)... 

This chapter provides an overview of essential concepts in HPLC including retention, selectivity, efficiency, and resolution as well as their relationships with key column and mobile phase parameters such as particle size, column length and diameter, mobile phase strength, pH, and flow rate. The significance of several concepts important in pharmaceutical analysis such as peak capacity, gradient time, void volume, and limit of quantitation are discussed. 

Capillary electrochromatography (CEC) is a miniaturized separation technique that combines aspects of both interactive chromatography and capillary electrophoresis. In this chapter, the theory of CEC and the factors affecting separation such as the stationary phase and mobile phase parameters have been discussed. The chapter focuses on the types and preparation of columns for CEC and describes the progress made in the development of open-tubular, particle-packed, and monolithic columns. The detection techniques in CEC such as the traditional UV detection and improvements made in coupling with more sensitive detectors such as mass spectrometry are also described. The chapter provides a summary of some applications of CEC in the analysis of pharmaceuticals and biotechnology products. 

Similar to HPLC and CE there are a number of important variables that affect the separation process in CEC. These include the electric field strength, mobile phase parameters, and stationary phase surface chemistry that are described as follows. 

Figure 3.15 Variation of the slope with the intercept for linear retention vs. composition curves in RPLC for 32 aromatic solutes on an ODS column using methanol water mixtures as the mobile phase. Parameters S and In fcg correspond to eqn. (3.45). Figure taken from ref. [322]. Reprinted with...

Mobile phase parameters (M) which include the nature and composition of the mobile phase, pH, nature and concentration of additives such as buffers, salts, ion-pairing agents or complexing agents. 

Therefore, the thermodynamic parameters (T), the stationary phase parameters (S) and the mobile phase parameters (M) are the ones we should consider if we wish to select the most relevant parameters for the optimization of chromatographic selectivity. 

In LC we have a choice between optimizing the stationary phase parameters or the mobile phase parameters. Obviously, the latter can be changed more readily. Advantages of mobile phase optimization are ... 

The development of most ion-pairing chromatography (IPC) methods started with optimization of the mobile phase composition after an appropriate column was selected. The theory described in Chapter 3 assists the chromatographer to perform educated guesses. Chapters 7 through 10 discuss the main qualitative and quantitative attributes of tunable mobile phase parameters to illustrate their influence on the global performance of the method. 

P. Kontour, R. Dawson and A. Monjan, Manipulation of mobile phase parameters for HPLC separation of endogenous monoamines in rat brain tissue, J. Neurosci. Methods., 11, 5-18 (1984). 

Mobile Phase Parameters (%B, Buffer, pH, Solvent Type)... 

Mobile phase parameters that have to be optimized include the salt type, concentration, gradient shape, pH, temperature, and possibly the addition of a surfactant or organic solvent [368-372]. The change in free energy on protein binding to the stationary phase is determined mainly by the contact surface area between the protein and stationary phase and by the salt type determined by its ability to increase the surface tension of aqueous solutions. Solvophobic theory predicts that in the absence of specific salt-protein interactions and at sufficient ionic strength the logarithm of the retention factor is linearly dependent on the surface tension of the mobile phase, which in turn, is a linear function of the salt concentration Eq. (4.13)... 

At present, more evidence has been presented in support of the displacement model, although both have been used successfully to predict the effects of various mobile phase parameters. 

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