Adsorption chromatography mobile phase

With binary and ternary supercritical mixtures as chromatographic mobile phases, solute retention mechanisms are unclear. Polar modifiers produce a nonlinear relationship between the log of solute partition ratios (k ) and the percentage of modifier in the mobile phase. The only form of liquid chromatography (LC) that produces non-linear retention is liquid-solid adsorption chromatography (LSC) where the retention of solutes follows the adsorption isotherm of the polar modifier (6). Recent measurements confirm that extensive adsorption of both carbon dioxide (7,8) and methanol (8,9) occurs from supercritical methanol/carbon dioxide mixtures. Although extensive adsorption of mobile phase components clearly occurs, a classic adsorption mechanism does not appear to describe chromatographic behavior of polar solutes in packed column SFC. 

The mode of separation in the HPLC depends on the selection of the stationary and mobile phases. In HPLC of lipids, normal- and reversed-phase modes are primarily used, with the reverse phase being more common than the normal phase. Separation in the re-versed-phase mode is mainly by partition chromatography, whereas separation in the normal phase mode is primarily by adsorption chromatography. Normal-phase HPLC is used for the separation of the lipids into classes of Upids [1,F]. Reversed-phase HPLC (RP-HPLC), on the other hand, is mainly used to separate each lipid class into individual species [2,B1]. For example, several triglycerides were separated from each other via nonaqueous reversed-phase HPLC, involving an octadecyl (ODS) column and a nonpolar (non-aqueous) mobile phase. RP-HPLC alone can be used to separate the fat molecules into classes and species [2,B1]. 

Typical thin-layer separations are performed on a glass plate that is coated with a thin and adherent layer of finely divided particles this layer constitutes the stationary phase. The particles are similar to those described in the discussion of adsorption, normal- and reversed-phase partition, ion-exchange, and size-exclusion column chromatography. Mobile phases are also similar to those employed in high-performance liquid chromatography. 

The principle of adsorption chromatography (normal-phase chromatography) is known from classical column and thin-layer chromatography. A relatively polar material with a high specific surface area is used as the stationary phase, silica being the most popular, but alumina and magnesium oxide are also often used. The mobile phase is relatively nonpolar (heptane to tetrahydrofuran). The different extents to which the various types of molecules in the mixture are adsorbed on the stationary phase provide the separation effect. A nonpolar solvent such as hexane elutes more slowly than a medium-polar solvent such as ether. 

The terms normal phase and reverse phase are used to describe adsorption and many bonded phase separations (but not in connection with ion-exchange or exclusion). Normal phase means that the polarity of the stationary phase is higher than that of the mobile phase, which is what happens, for example, when silica is used in adsorption chromatography. Reverse phase means that the polarity of the stationary phase is less than that of the mobile phase, which is the case with hydrocarbon-type bonded phases. Polar bonded phases can be used in either normal or reverse phase modes. With both techniques, solutes are eluted in order of polarity (increasing or decreasing), and we can change the retention times of solutes by changing the polarity of the stationary phase or (more easily) of the mobile phase. These facts are summarised in Fig. 3,1c. 

Poor reproducibility in adsorption chromatography (normal-phase chromatography) is, aside from the limited solubility of polar compounds in organic solvents, the main problem of these systems. Almost always, the problem is with the (uncontrolled) water content in the total system, i.e. the HPLC instrument, the sample, the mobile phase and the column. 

In liquid-solid adsorption chromatography (LSC) the column packing also serves as the stationary phase. In Tswett s original work the stationary phase was finely divided CaCOa, but modern columns employ porous 3-10-)J,m particles of silica or alumina. Since the stationary phase is polar, the mobile phase is usually a nonpolar or moderately polar solvent. Typical mobile phases include hexane, isooctane, and methylene chloride. The usual order of elution, from shorter to longer retention times, is... 

Kovat s retention index (p. 575) liquid-solid adsorption chromatography (p. 590) longitudinal diffusion (p. 560) loop injector (p. 584) mass spectrum (p. 571) mass transfer (p. 561) micellar electrokinetic capillary chromatography (p. 606) micelle (p. 606) mobile phase (p. 546) normal-phase chromatography (p. 580) on-column injection (p. 568) open tubular column (p. 564) packed column (p. 564) peak capacity (p. 554)... 

In contrast to vapour phase chromatography, the mobile phase in liquid chromatography is a liquid. In general, there are four main types of liquid chromatography adsorption, partition, ion-chromatography, and gel filtration. 

SERS has also been applied as a sensitive, molecule-specific detection method in chromatography, e.g. thin layer, liquid, and gas chromatography. SERS-active colloids were deposited on the thin layer plates or mixed continuously with the liquid mobile phases. After adsorption of the analytes, characteristic spectra of the fractions were obtained and enabled unambiguous identification of very small amounts of substance. 

The great leap forward for chromatography was the seminal work of Martin and Synge (7) who in 1941 replaced countercurrent liquid-liquid extraction by partition chromatography for the analysis of amino acids from wool. Martin also realized that the mobile phase could be a gas rather than a liquid, and with James first developed (8) gas chromatography (GC) in 1951, following the gas-phase adsorption-chromatographic separations of Phillips (9). 

The same concepts apply to column chromatography, where the stationary phase is normally small particles of silica, Si02, or alumina, A1,0 . These substances are not very reactive and have specially prepared surfaces to increase their adsorption ability. The column is saturated with solvent, and a small volume of solution containing the solutes is poured onto the top. As soon as it has soaked in, more solvent is added. The solutes travel slowly down the column and are eluted (removed as fractions) at the bottom (Fig. 2). If the mobile phase is less polar than the stationary phase, the less polar solutes will be eluted first and the more polar ones last. 

In contrast to straight phase or adsorption chromatography, partition chromatography involves the separation of sample molecules owing to their different partition coefficients between the liquid stationary and mobile phases. The liquid stationary phase is located in the pores of a sorbent, ideally only acting as a support, making no contribution to the retention of the sample molecules. 

The recommended mobile phase must assure a proper adsorption isotherm for a given stationary phase. Chapter 2 discusses the adsorption planar chromatography in the nonhnear region. 

The adsorption mechanism in chromatography on alumina differs from that on silica gel because of the structural differences between these adsorbents. Relationships between the values of solutes and the adsorption data for the mobile phase components on sihca gel G and alumina G have been investigated by Rozylo [64,65]. The theoretical and experimental results obtained by the relation 2 = /( 1) show a good agreement for the two adsorbents. 

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