Use of Solvent Gradients

A good example of this application is the work of Lyons and co-workers [23] on the use of solvent gradient adsorption LC in a study of interchain compositional inhomogeneities in PS-co-ethylene random copolymers. 

Multiple development techniques using stepwise solvent gradients enable a subset of optimal separation conditions to be used to separate a mixture of wide polarity that cannot be separated using a single mobile phase (117,119,120,125). As an example of this approach the separation of 20 common protein amino acid PTH-derivatives is shorn in Figure 7.12 (126). Five... 

In any chromatographic analysis the method of detection is determined by the nature of the analyte and the mobile phase used must not interfere with this system. The use of ultraviolet absorption detection systems is very common but the solvents used must not absorb significantly at the wavelength used. For instance, absorption at 280 nm is frequently used to detect protein but some solvents, e.g. acetone, absorb at this wavelength. Similarly the use of concentration gradients in the mobile phase may present problems with refractive index and electrochemical detection systems. 

FIGURE 9.15 Separation of poly(styrene-comethyl methacrylate)s on a silica gel column with 5 p.m particles using a solvent gradient including using chloroform with 3.5% ethanol added as the desorption promoting solvent and CO2. Flow rates CO2,0.5 mL/min chloroform with ethanol additive (0.25-2.5 mL/min in 30 min) at 333 K and back pressure 20 MPa. (Reprinted from E. Kawai et al., J. Chromatogr. A, 991 197 (2003). With permission.)... 

To an ice-cooled solution of N-methyl-l-naphthalenemethylamine hydrochloride (2.1 g) in methanol (40 ml) and water (10 ml) was added sodium hydroxide powder (2 g) followed by dropwise addition of epichlorohydrin (8 ml). The mixture was heated at 60°C for 3 h, then cooled to room temperature. Volatile materials were removed in vacuo and the residue was taken up in ethyl acetate and washed with water. The organic phase was collected, dried over sodium sulfate, filtered and evaporated to dryness. The crude mixture was purified by flash chromatrography on silica gel (grade 9385, Merck, 230-400 mesh, 60 A) using a solvent gradient of a mixture of hexane and ethyl acetate (95 5, 90 10 and 85 15) as eluent, affording the N-methyl-N-naphthylmethyl-2,3-epoxypropane (1.85 g, 81.5%) as an oil. 

As mentioned earlier, flow programming may be used with UV detection. In Figure 7-32, a sample of Triton X-100 is separated using flow programming and monitoring the UV absorbance. The resolution obtained on this sample compares very favorably with that obtained using a solvent gradient. 

Figure 22.2 LC-MS reconstracted total-ion chromatogram of a separation of a homologous series of oligonucleotides p(dT) with n between 12 and 30 on a micropellicular octadecylated PS-DVB column using a solvent gradient of acetonitrile in 50 mmol/1 TEABC. Reprinted from [9] with permission. 1999, American Chemical Society.

In summary, gradient elution is a powerful method for the separation and analysis of complex mixtures containing components with a wide variety of polarities and hydrophobicities. It can also be used to help establish an isocratic mobile phase for the analysis of simpler mixtures. In either case, utmost care must be taken in the selection and use of solvents of high purity and selectivity. 

The major limitation of thermal FFF occurs in the separation of low-molecular-weight materials. Thus, the technique is not widely applicable to molecular weights below about 10 g/mol. This limit can be reduced somewhat by the use of solvent mixtures. For example, polystyrene components as small as 2500 g/mol were resolved in a mixture of tetrahydrofuran and dodecane [4]. Even lower molecular weights than 2500 g/mol have been retained, but only through the use of special channels, which were highly pressurized in order to increase the temperature gradient without boiling the solvent. 

A fast solution for these separation problems is the use of generic gradients. Table 4.5 lists typical solvents and conditions for gradient runs for RP and NP systems. 

Extraction temperature Solvent power increases with temperamre, but selectivity decreases until feed and solvent become miscible. Clearly, the extreme of complete miscibility must not be allowed. The use of temperature gradients in extraction towers aids selectivity. 

The difficulties encountered in LLC can be overcome by the use of chemically bonded stationary phases or bonded-phases. Most bonded phases consist of organochlorosilanes or organoalkoxysilanes reacted with micro-particulate silica gel to form a stable siloxane bond. The conditions can be controlled to yield monomeric phases or polymeric phases. The former provides better efficiency because of rapid mass transfer of solute, whereas the polymeric phases provides higher sample capacity. BPC can be used in solvent gradient mode since the stationary phase is bonded and will not strip. Both normal-phase BPC (polar stationary, non-polar mobile) and reversed-phase BPC (non-polar stationary, polar mobile) can be performed. The latter is ideal for substances which are insoluble or sparingly soluble in water, but soluble in alcohols. Since many compounds exhibit this behaviour, reversed phase BPC accounts for about 60% of published applications. The main disadvantage of silica bonded phases is that the pH must be kept between 2 to 7.5. However, bonded phases with polymer bases (polystyrene-divinylbenzene) can be used in the pH range of 0 to 14. 

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