Mobile phase adsorbents used

The organic solvent used to elute the compound must be adequately strong (polar for the adsorbent silica gel) and a good solvent for the component. Absolute methanol should be avoided as a siugle solvent because silica gel itself and some of its common impurities (Fe, Na, SO4) are soluble iu this solvent and will contaminate the isolated material. Solvent containing less than 30% methanol is recommended, or ethanol, acetone, chloroform, dichloromethane, or the mobile phase originally used for PLC are other frequently nsed choices for solnte recovery. Water is not recommended because it is so difficult to remove by evaporation during the concentration step (removal by lyophilization is necessary). A formula that has been used to calculate the volume of solvent needed when the PLC mobile phase is chosen for elution is ... 

Adsorption chromatography. A solid stationary phase and a liquid or gaseous mobile phase are used. Solute is adsorbed on the surface of the solid particles. The more strongly a solute is adsorbed, the slower it travels through the column. 

Most liquid chromatographic experiments performed with PAD employ alkaline mobile phases or use postcolumn addition of base to get the electrode at the appropriate pH for the formation of the oxide. The exceptions to this are the detection of carbohydrates and alcohols in acidic media and the detection of sulfur compounds. The oxidation of carbohydrates and alcohols is not oxide catalyzed, and since they exhibit a stronger adsorption to piatinum than gold, they can be determined under acidic conditions. Sulfur compounds are adsorbed at oxide-free surfaces, and the kinetics for detection are favorable even at pH values below 7. 

Adsorption chromatography The process can be considered as a competition between the solute and solvent molecules for adsorption sites on the solid surface of adsorbent to effect separation. In normal phase or liquid-solid chromatography, relatively nonpolar organic eluents are used with the polar adsorbent to separate solutes in order of increasing polarity. In reverse-phase chromatography, solute retention is mainly due to hydrophobic interactions between the solutes and the hydrophobic surface of adsorbent. Polar mobile phase is used to elute solutes in order of decreasing polarity. 

Silica and alumina have the highest surface activity when the adsorbents are free of physisorbed water. Addition of water blocks the most active sites on the surface since water, as a polar adsorptive, is preferentially adsorbed. Other polar compounds such as alcohols can also adsorb irreversibly at the surface. Consecutive adsorption of water deactivates the adsorbent surface and the solute retention will decrease concurrently. For this reason the water content of the mobile phase solvents should be controlled carefully if an apolar mobile phase is used (Unger, 1999). The water content only influences retention when apolar eluents are used, e.g. hexane or heptane. When these solvents are mixed with 10% or more of a moderately polar solvent (e.g. acetone, ethyl acetate) the dependency disappears. The influence can be decreased by the addition of a small amount of acetonitrile to the mobile phase. 

Next, a 100% methanol mobile phase is used to remove the adsorbed surfactant in the chromatographic system. A low flow rate (0.5 rnL/min or less) should be used at the beginning because the methanol-water mixture has a high viscosity which is significantly increased by the desorbed surfactant. Once the pressure decreases, the flow rate may be resumed. At least 10 column volumes of pure methanol should be passed through the column. 

As one very striking example of the capabilities of the high-temperature gradient HPLC system, the separation of random ethyleneA inyl acetate copolymers is presented in Fig. 23. On silica gel as the stationary phase and using decaline-cyclo-hexanone as the eluent, full separation of copolymers of different compositions was achieved. In addition, the homopolymers PE and PVAc were well separated from the copolymers. This was the first time that a chromatographic system was available that separates olefin copolymers irrespective of crystallinity and solubiUty over the entire range of compositions. Namely, the mobile phase components used are solvents for both PE and PVAc. The non-polar solvent, decalin, supports adsorption of PVAc on the silica gel, while the polar solvent, cyclohexanone, enables desorption and elution of the adsorbed polymer sample firom the column [155]. 

The complexity of the system increases with the number of solvents used and, of course, their relative concentrations. The process can be simplified considerably by pre-conditioning the plate with solvent vapor from the mobile phase before the separation is started. Unfortunately, this only partly reduces the adsorption effect, as the equilibrium between the solvent vapor and the adsorbent surface will not be the... 

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. 

Cationic samples can be adsorbed on the resin by electrostatic interaction. If the polymer is strongly cationic, a fairly high salt concentration is required to prevent ionic interactions. Figure 4.18 demonstrates the effect of increasing sodium nitrate concentration on peak shapes for a cationic polymer, DEAE-dextran. A mobile phase of 0.5 M acetic acid with 0.3 M Na2S04 can also be used. 

Anionic and neutral polymers are usually analyzed successfully on Syn-Chropak GPC columns because they have minimal interaction with the appropriate mobile-phase selection however, cationic polymers adsorb to these columns, often irreversibly. Mobile-phase selection for hydrophilic polymers is similar to that for proteins but the solubilities are of primary importance. Organic solvents can be added to the mobile phase to increase solubility. In polymer analysis, ionic strength and pH can change the shape of the solute from mostly linear to globular therefore, it is very important to use the same conditions during calibration and analysis of unknowns (8). Many mobile phases have been used, but 0.05-0.2 M sodium sulfate or sodium nitrate is common. 

A variety of chromatographic techniques are now in common use, all of which work on a similar principle. The mixture to be separated is dissolved in a solvent, called the mobile phase, and passed over an adsorbent material, called the stationary phase. Because different compounds adsorb to the stationary phase to different extents, they migrate along the phase at different rates and are separated as they emerge (elute) from the end of the chromatography column. 

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