Solvent mobile phase selection based

In the mobile phase selection for the separation of compounds on thin silica gel layers, it is necessary to use not only eluotropic series based on the eluting capacity of the solvent but also eluotropic series of compounds established according to their interaction with silica gel. 

Neutral Solutes. In the reversed-phase mode, water is used as the weak solvent and acetonitrile, methanol, or THF (where applicable) is used as the strong solvent. (It is notable that the addition of acid or base to the mobile phase used for neutral molecules does not preclude separation, and, as such, the approach outlined later for ionizable components is equally viable.) In normal-phase HPLC, hexane is used as the weak solvent and isopropanol is used as the strong solvent. To change selectivity based on the strong solvent, isopropanol may be replaced (in part) with methylene chloride, methyl t-butyl ether, or ethyl acetate. However, note should be made of the relatively high UV cutoffs of these solvents when UV detection is to be used and precautions should be taken to ensure solvent miscibility across the range of the gradient. 

The choice of reverse phase packing material will depend on the amount of information available on the component of interest and on other sample components. Initial tests such as solvent partitioning behavior, solubility m various solvents, and others see Chapter 1) can be used to estimate polarity and hence be of use in initial column/mobile phase selection. The most retentive of the silica-based reverse phase supports, Cl8 and C8, are a sensible first choice, as the retention of polar compounds is maximized, while the retention of nonpolar materials can be easily modulated by choice of eluent. If the compound of interest is very nonpolar (or the sample contains components that bind very strongly to retentive phases such as C8/C18), a shorter chain alkyl-bonded phase such as C6 or C4 may be more suitable. 

In LC, a means of detection is employed for identification and quantification. While detection is covered elsewhere in this volume, mobile phase selection can play an important role in the detectability of the compounds of interest, and vice versa. A solvents lowest usable (cutoff) wavelength is important for UV detectors, solvent refractive index (RI) effects the sensitivity of RI detection, and solvent volatility is an important consideration for evaporative light scattering and mass spectrophotometric based detectors. Table 4 lists some common LC mobile phase spectral data. 

Typical normal-phase operations involved combinations of alcohols and hexane or heptane. In many cases, the addition of small amounts (< 0.1 %) of acid and/or base is necessary to improve peak efficiency and selectivity. Usually, the concentration of polar solvents such as alcohol determines the retention and selectivity (Fig. 2-18). Since flow rate has no impact on selectivity (see Fig. 2-11), the most productive flow rate was determined to be 2 mL miiT. Ethanol normally gives the best efficiency and resolution with reasonable back-pressures. It has been reported that halogenated solvents have also been used successfully on these stationary phases as well as acetonitrile, dioxane and methyl tert-butyl ether, or combinations of the these. The optimization parameters under three different mobile phase modes on glycopeptide CSPs are summarized in Table 2-7. 

The selection of proper mobile phase in TLC exerts a decisive influence on the separation of inorganic ions. With a particular stationary phase, the possibility of separation of a complex mixture is greatly improved by the selection of an appropriate mobile phase system. In general, the mixed aqueous-organic solvent systems containing an acid, a base, or a buffer have been the most favored mobile phases for the separation of ionic species. The mobile phases used as developers in inorganic PLC include ... 

THF and methanol employed as organic modifiers of mobile phase provided a considerable difference in selectivity based on the polar interactions between solutes and the organic solvent molecules in the stationary phase. Acidic compounds, phenols and nitroaromatics, were preferentially retained in the THF-based mobile phase, whereas esters and ketones were preferentially retained in the methanol (a hydrogen-bond donor) containing mobile phase. The system presented here seems to be very practical because any laboratory possessing two sets of HPLC equipment and two C j g columns can attempt similar 2D HPLC by simply changing the mobile phase for the two dimensions. 

---