Solvent of the mobile phase

The characteristics of the sorbent in the precolumn may lead to problems when coupling the two systems. Therefore, when the analytes are more retained in the precolumn than in the analytical column, peak broadening may appear, even when the analytes are eluted in the backflush mode (40). This has been solved with a special design in which the analytes retained in the precolumn are eluted with only the organic solvent of the mobile phase and the corresponding mobile phase is subsequently formed (40, 41). 

The size-exclusion chromatography (or gel-chromatography) is a means of separation which is exclusively dependent on the exchange of solute molecules between the solvent of the mobile-phase and the same solvent within the pores of the column-packing material. In reality, it is the pore-size-range of the packing material that solely determines the molecular-size-range within which a particular separation can take place effectively. 

There are several systems which can be used to select the solvents of the mobile phase. The number of selected solvents and the solvents which are selected not only depend on the chromatographic problem but also on the method which will be used to optimize the system. With response surface methodology it is appropriate to use a minimum number of solvents. For reasons stated below this minimum number of solvents was four. The second question is, which solvents will be selected, is more difficult to answer when a small number of solvents is used because the consequences of a wrong selection are large. Several approaches are possible to select the solvents. The most simple method is comparison with common solvent systems for the solutes under investigation. A more general approach is to use the selectivity triangle of Snyder [4] in the selection of the solvents. 

The major amount of HPLC separations is performed with re versed-phase columns employing binary or tertiary solvent mixtures with isocratic or gradient elution. The protons of the solvents of the mobile phase cause severe problems for an adequate NMR registration. The receiver of the NMR instrument (either a 12-bit or a 16-bit analog-digital converter (ADC)) is unable to handle the intense solvent signals and the weak substance signals at the same time. 

The model has been further tested in terms of the behavior of solvent molecules isotherms for binary-solvent mixtures A/B adsorption energies (ch values) of the polar solvent B in such mixtures as a function of surface coverage 6b, etc. Again, good agreement of experimental data with calculated values is observed. An important requirement of the model and related experimental correlations is that the solvent molecule must be treated (thermodynamically) in the same manner as solute molecules. Thus, if. solute adsorption energies are measured for a molecule X, the behavior of X as a component (solvent) of the mobile phase should then be predictable. 

The particle beam interface is a system in which the solvent of the mobile phase is evaporated from the sample using reduced pressure and an elevated temperature. This requirement places some restrictions on the HPLC-part of the system. The working range, for the flow rate of the HPLC, is specified (19) at O.l-l.OmL/min. 

Choosing a Mobile Phase Several indices have been developed to assist in selecting a mobile phase, the most useful of which is the polarity index. Table 12.3 provides values for the polarity index, P, of several commonly used mobile phases, in which larger values of P correspond to more polar solvents. Mobile phases of intermediate polarity can be fashioned by mixing together two or more of the mobile phases in Table 12.3. For example, a binary mobile phase made by combining solvents A and B has a polarity index, of... 

The most commonly used gradients are linear gradients where the starting solvent is gradually mixed with a second gradient-forming solvent at the column entrance to yield a volume fraction

mobile phase modulator that increases huearly with time ... 

Concentrations of moderator at or above that which causes the surface of a stationary phase to be completely covered can only govern the interactions that take place in the mobile phase. It follows that retention can be modified by using different mixtures of solvents as the mobile phase, or in GC by using mixed stationary phases. The theory behind solute retention by mixed stationary phases was first examined by Purnell and, at the time, his discoveries were met with considerable criticism and disbelief. Purnell et al. [5], Laub and Purnell [6] and Laub [7], examined the effect of mixed phases on solute retention and concluded that, for a wide range of binary mixtures, the corrected retention volume of a solute was linearly related to the volume fraction of either one of the two phases. This was quite an unexpected relationship, as at that time it was tentatively (although not rationally) assumed that the retention volume would be some form of the exponent of the stationary phase composition. It was also found that certain mixtures did not obey this rule and these will be discussed later. In terms of an expression for solute retention, the results of Purnell and his co-workers can be given as follows,... 

It is seen that the value of (H) is completely dependent on the diffusivity of the solute in the mobile phase, the column radius and the linear velocity of the mobile phase. The simple uncoated open tube can clearly be used to determine the diffusivity of any solute in any given solvent (the mobile phase). This technique for measuring diffusivities will be discussed in a later chapter. 

However, there might be exceptions if the mobile phase consists of a binary mixture of solvents, then a layer of the more polar solvent would be adsorbed on the surface of the silica gel and the mean composition of the solvent in the pores of the silica gel would differ from that of the mobile phase exterior to the pores. Nevertheless, it would still be reasonable to assume that... 

The selection of the mobile phase and the conditions of development. Having chosen the solvent(s) the following are defined from the reduced chromatogram,... 

This is an oversimplified treatment of the concentration effect that can occur on a thin layer plate when using mixed solvents. Nevertheless, despite the complex nature of the surface that is considered, the treatment is sufficiently representative to disclose that a concentration effect does, indeed, take place. The concentration effect arises from the frontal analysis of the mobile phase which not only provides unique and complex modes of solute interaction and, thus, enhanced selectivity, but also causes the solutes to be concentrated as they pass along the TLC plate. This concentration process will oppose the dilution that results from band dispersion and thus, provides greater sensitivity to the spots close to the solvent front. This concealed concentration process, often not recognized, is another property of TLC development that helps make it so practical and generally useful and often provides unexpected sensitivities. 

Controlling for these forces requires variation in the amount of salt, organic solvent, and the pFI of the mobile phase. It is impractical to perform such experiments with 50 mM formic acid an alternative additive must be used that maintains its chaotropic properties independent of salt content or pFI. Fortunately, mobile phases containing 50 mM hexafluoro-2-propanol (HFIP) afford a fractionation range comparable to that of the formic acid (Fig. 8.6), permitting the effects of these variables to be studied systematically. 

---