Selection of the mobile phase

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 rule, a range of solvents is potentially able to solve any particular problem, so selection must be based on different criteria  

Practical High-Performance Liquid Chromatography, Fifth edition Veronika R. Meyer 

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 cannot be followed in the case of bulk-property detectors such as the refractive index detector. 

---

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

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 principle of 2-D TLC separation is illustrated schematically in Figure 8.4. The multiplicative law for 2-D peak capacity emphasizes the tremendous increase in resolving power which can be achieved in theory, this method has a separating capacity of n, where n is the one-dimensional peak capacity (9). If this peak capacity is to be achieved, the selectivity of the mobile phases used in the two different directions must be complementary. 

All the advantages of these methods for the optimization of the mobile phase by means of preassays in TLC can be exploited at the preparative scale. Finally, the separated zones may be easily removed from PLC plate and eluted in order to isolate quantities of the expected compounds. In PLC selection of the mobile phase, the subsequent recovery of the separated zone should be taken into consideration also. 

FIGURE 5.17 Analytical preselection of a suitable selectivity of the mobile phase on silica gel using the CAMAG HPTLC Vario Chamber. 

With regard to quantitative measurements of APG surfactants in, e.g. environmental samples, the authors stressed that it was of crucial importance to promote the formation of the desired molecular (or adduct) ion in order to obtain reproducible mass spectra. If tuning of the ESI interface parameters did not suffice to yield abundant ions of the selected species, acquisitions of the mass spectrometric detector after negative ionisation in conjunction with appropriate selection of the mobile phase composition were used as an alternative despite the lower sensitivity in this mode [1,2],... 

In GC, the mobile phase acts only as a carrier. In LC, solute undergoes interaction with liquids or mixtures of liquids used as the mobile phase. Selection of the mobile phase is critical. The most useful criteria are the solubility parameter concept, Snyder s selectivity triangle, and solva-tochromic parameters. 

The selection of the mobile phase is the key aspect in chiral resolution. The mobile phase is selected according to the solubility and the structure of the drugs to be resolved. In the normal phase mode, the use of pure ethanol or 2-propanol is recommended. To decrease the polarity of the mobile phase and increase the retention times of the enantiomers, investigators use hexane, cyclohexane, pentane, or heptane as one of the main constituents of the mobile phase. However, other solvents (e.g., alcohols, acetonitrile) are also used in the mobile phase. Normally, if pure ethanol or 2-propanol is not well suited for the mobile phase, pure ethanol and hexane, 2-propanol, or ethanol in the ratio of 80 20 is used as... 

Figure 3.7 Illustration of the selection of phase systems for LC according to eqn.(3.30). A solute with a polarity of 12.5 (middle scale) can be eluted from silica (Ss= 16 top scale) with a non-polar mobile phase (Sm=9 bottom scale) or with a polar solvent in a reversed phase system. The shaded areas indicate the latitude with respect to the selection of the mobile phase. Figure taken from ref. [311]. Reprinted with permission.

C. A rule of thumb in small-molecule GPC is that it is possible to analyze two compounds whose molecular weight differs by 10% or more by the judicious selection of the mobile phase. For instance, the separation of tolnaftate (the active ingredient of an antifungal preparation) (MW = 302) and BHT (which is present as an antioxidant) (MW = 220) can be accomplished using methylene chloride, a nonhydrogen bonding solvent as shown in Figure 11-4. For this assay, sample prep-... 

In many forms of secondary equilibria separations, the concentration of the equilibrant, or the mobile phase component which participates in the secondary equilibria, controls, at least partially, the strength and selectivity of the mobile phase. In micellar chromatography the concentration of micelles plays this role, which means that for all separations carried out with micellar mobile phases, the strength of the mobile phase can be changed while maintaining an unchanging bulk solvent composition. This unique aspect of micellar mobile phases does indeed allow the solution to "problems that cannot be solved by other means . 

Chemical Stability. Hydrolytic stability of base material is the most important parameter because most reversed-phase HPLC separations are performed in water/organic eluents with controlled pH. Selection of the mobile phase pH is mainly dictated by the properties of the ionizable analytes to ensure that they are in one predominate ionization state. 

The requirements for HPLC methods include careful selection of the mobile phases to avoid sample precipitation or emulsification. At the same time, chromatographic conditions should provide positive retention of the drug substance so it won t elute with the void volume. 

The teicoplanin CSP (Chirobiotic T) exhibits enantioselectivity for underivatized and Mderivatized (FMOC or Z) amino acids, hydroxy carboxylic acids and other chiral acids including chiral phenols, small peptides, neutral aromatic analytes and cyclic aromatic and aliphatic amines [285] (see also Table 9.11). Selection of the mobile phase mcxle (reversed-phase, normal-phase, or polar-organic phase mode) follows the same criteria as described for vancomycin CSP. 

Selection of the mobile phase composition can be based on the enormous amount of information given in Stahl s book [17] and in a wide range of other publications, and also by using optimisation models (see e.g., Nyiredy et al. 42j). 

Adsorption TLC selection of the mobile phase is conditioned by sample and stationary-phase polarities. The following polarity scale is valid for various compound classes in NPTLC in decreasing order of K values carboxylic acids>amides>amines>alcohols>aldehydes > ketones > esthers > nitro compounds > ethers > hal-ogenated compounds > aromatics >olefins > saturated hydrocarbons > fluorocarbons. For example, retention on silica gel is controlled by the number and functional groups present in the sample and their spatial locations. Proton donor/acceptor functional groups show the greatest retention, followed by dipolar molecules, and, finally, nonpolar groups. 

Most RP-HPLC separations are done in the iso-cratic mode (i.e., where the composition of the mobile phase is held constant during the analysis). This approach is suitable when the sample consists of analytes having similar properties or where their hydrophobic-ities encompass a small or moderate range. Under these conditions, all solutes in the sample will be eluted over a reasonable time span (i.e., not too short to prevent resolution of individual analytes and not too long to result in an inconvenient analysis period). Therefore, proper selection of the mobile-phase composition is essential in the development of any re-versed-phase separation method. Fortunately, due to the decades of long practice of RP-HPLC, there exists in the literature and from commercial sources, a wealth of information on suitable mobile-phase compositions for particular types of sample, especially for the Cig stationary phase. In addition, the retention of solutes on hydrophobic phases has been modeled mathematically and there exist computer programs for assisting in the optimization of mobile-phase composition in the solution of various separation problems. 

Since the functionality of most silica stationary phases is the same, the selection of the mobile phase is very important during the screening and method development of a normal-phase silica separation. As mentioned above, typical mobile phases involve a nonpolar solvent like heptane and a polar solvent like ethanol, but the combination of solvents is almost endless. A good screening (using TLC or analytical HPLC) should examine numerous solvent combinations utilizing different types of solvents, such as THF instead of alcohols, ethyl acetate or methylene chloride instead of heptane and should also examine the use of additives, e.g., acetic acid or triethylamine, to adjust the pH to either suppress or enhance interactions with the stationary phase. More information on the method development is provided in Section V. 

The first step of system development is the choice of the mobile phase. While the use of neat solvents is preferred, very often a mixture of solvents must be used to obtain good results. The selection of the mobile phase should be based on ... 

The most important criterion for solvent selection is throughput, which mainly depends on a sufficient solubility of the solutes and the corresponding selectivity of the separation. Because solubility and selectivity depend on the interaction between the three elements of the chromatographic system, the selection of the mobile phase dependent on these parameters is further discussed in Section 4.3. 

In designing of 2D LC x LC systems, the selection of the mobile phase for each chromatographic dimension is of fundamental importance, in order to achieve maximal utilization of the 2D separation space. In contrast to off-line 2D LC procedures, where the collected fraction can be subjected to evaporation, dilution, or extraction, before injection onto the column of the second dimension, the compatibility of the mobile phases in online 2D LC x LC in terms of miscibility, solubility, viscosity, and eluotropic strength is much more important. The mobile phases used in SEC x RPC, SEC x NPC, RP x CEX, RP x AEX RP x CEX, NPC x HILIC, NPC x CEX, and NPC x AEX are compatible (see Figure 16). 

Careful selection of the mobile phase should be followed by just as careful preparation. 

Selection of the mobile phase is critical in the characterization of silica sols by SEC. As with the other separation methods, pH should be slightly basic, and low ionic strength must be used to prevent particle aggregation. In addition, the mobile phase must interact with the surface of the packing-particle pores to neutralize undesirable charge effects. Negatively charged surfaces within the pore can result in ion-exclusion effects whereby... 

The utility of HDC is somewhat limited by the relatively poor resolution and particle-size discrimination of the method, which restrict the precision of HDC in silica sol characterization. In principle, accurate particle-size distributions of silica sols also are possible with the HDC method. However, for such characterizations special software with corrections for the extensive band dispersion in HDC is required, along with a suitable band deconvolution method (28). Commercial HDC apparatus with this sophisticated software package apparently is no longer available. Standards are generally required, although quantitative retention relationships have been reported for capillary HDC systems in characterizing polymers (37). As with all of the other separation methods, careful selection of the mobile phase is required in HDC. Mobile phases generally are the same as those used for the FFF methods and SEC. 

Detection in HPTLC, unlike that in HPLC, is a static process, being completely separated from chromatographic development. Consequently, the selection of the mobile phase does not limit the choice of the detector. For example, UV absorbing solvents cannot be used with UV detectors in HPLC. In HPTLC the solvent is completely evaporated between development and measurement so that it does not influence the detection process. 

---