Polar organic mobile phase

Nonpolar organic mobile phases, such as hexane with ethanol or 2-propanol as typical polar modifiers, are most commonly used with these types of phases. Under these conditions, retention seems to foUow normal phase-type behavior (eg, increased mobile phase polarity produces decreased retention). The normal mobile-phase components only weakly interact with the stationary phase and are easily displaced by the chiral analytes thereby promoting enantiospecific interactions. Some of the Pirkle-types of phases have also been used, to a lesser extent, in the reversed phase mode. 

In reverse-phase chromatography, the stationary phase is nonpolar (often a hydrocarbon) and the mobile phase is relatively polar (e.g., water, methanol, and acetonitrile). The most polar components elute first, and increasing the mobile phase polarity (i.e., decreasing the organic solvent concentration) increases elution time. 

Different kinds of HPLC exist. Many kinds of column packings and solvents are available. Retention behavior and resolution are affected by column characteristics (C-loading, chain length, porosity, etc.) and by elution scheme characteristics (mobile phase, pH, organic modifier, etc.). The samples can be separated on the basis of solubility and polarity of the sample components. 

NP hydrophilic interaction chromatography gradients—concentration(s) of water (or of water and a more polar organic solvent) in a less polar solvent increase(s) aqueous mobile phases, polar adsorbent, or bonded-phase columns... 

The linear relationship between oq and values [Eq. (4)] is an important characteristic of hpophilicity measurements. The intercept, = Rm, can be considered a measure for the partition of a compound between a polar mobile phase and a nonpolar stationary phase. The physicochemical significance of the slope, fli, is not completely estabhshed. ft was demonstrated experimentally that the slope shows the rate of increasing the compound solubility in the mobile phase. The solubility increases as a consequence of the decreasing of the mobile-phase polarity. The slope, a, depends on the nonpolar surface of the organic compound (i.e., the part of the compound which interacts with the nonpolar stationary phase). 

HILIC Silica (unbonded or bonded) Porous or core-shell silica 3—5-pm particle size Mobile phase high organic solvent concentration Stationary phase attracts H20-enriched layer of stagnant aqueous mobile phase allowing retention of polar compounds Amino acids, monoamines, acetylcholine... 

The mixture to be resolved is spotted onto a paper or thin-layer plate which is placed in a tank containing a shallow reservoir of the solvent. The liquid, which is usually a mixture of water and an organic liquid, rises up the support medium and the molecules to be resolved partition themselves between the stationary and mobile phases. Polar compounds, being more soluble in the polar stationary phase, therefore migrate more slowly than non-polar compounds which are more soluble in the mobile phase. This differential solubility results in their eventual resolution. The separated components can be visualized by spraying with a suitable reagent. 

The most common mobile phase for supercritical fluid chromatography is CO2. Its low critical temperature, 31 °C, and critical pressure, 72.9 atm, are relatively easy to achieve and maintain. Although supercritical CO2 is a good solvent for nonpolar organics, it is less useful for polar solutes. The addition of an organic modifier, such as methanol, improves the mobile phase s elution strength. Other common mobile phases and their critical temperatures and pressures are listed in Table 12.7. 

Select mobile phases for HPSEC based on their ability to dissolve the sample and their compatibility with the column. Zorbax PSM columns are compatible with a wide variety of organic and aqueous mobile phases (Table 3.4), but analysts should avoid aqueous mobile phases with a pH greater than 8.5. As mentioned earlier, select mobile phases that minimize adsorption between samples and silica-based packings. Sample elution from the column after the permeation volume indicates that adsorption has occurred. If adsorption is observed or suspected, select a mobile phase that will be more strongly adsorbed onto the silica surface than the sample. For example, N,N-dimethyl-formamide (DMF) is often used for polyurethanes and polyacrylonitrile because it eliminates adsorption and dissolves the polymers. When aqueous mobile phases are required, highly polar macromolecules such as Carbowax can be used to coat the silica surface and eliminate adsorption. Table 3.5 provides a list of recommended mobile-phase conditions for some common polymers. 

Even with mobile-phase modifiers, however, certain polymer types cannot be run due to their lack of solubility in organic solvents. In order to run aqueous or mixed aqueous/organic mobile phases, Jordi Associates has developed several polar-bonded phase versions of the PDVB gels as discussed earlier. Figures 13.60 thru 13.99 detail examples of some polar and ionic polymers that we have been able to run SEC analysis of using the newer bonded PDVB resins. 

The macrocyclic glycopeptides CSPs arc capable of operating in three different mobile phase systems reversed phase, normal phase, and the new polar organic mode. The new polar organic mode refers to the approach when methanol is used as the mobile phase with small amounts of acid and/or base as the modifier to control... 

In the new polar organic mode, the ratio of acid/base in the mobile phase affects the selectivity and the concentration of acid and base controls the retention. It is suggested to start the method development with a medium concentration (0.1 %) for both acid and base. If retention is too long or too short, the concentration can be increased to 1 % or reduced to 0.01 %. If no selectivity is observed in this mode, reversed phase is recommended as the next step in the protocols. 

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