Chromatographic Migration

Chromatographic separation is a result of the differential migration of components. Differential migration can be understood only if the basic migration process is well characterized. This section and several to follow will be devoted to the fundamentals of migration, emphasizing the physicochemical processes that control the motion of each solute component down the column or bed. The treatment here expands on the introduction to this subject in Section 9.3. 

As a first approximation we assume that the distribution of solute between phases quickly reaches equilibrium, at least at the zone center. When one introduces a narrow sample zone at the head of the column, an 

As pointed out earlier, zone migration is slow for strongly sorbed solutes, that is, those with a low R value. This can be shown quantitatively as follows. Of the total solute in the zone, the fraction R is carried along with the mobile phase at an average velocity u. (For simplicity we henceforth use v instead of (v) for the cross-sectional average velocity.) The remaining fraction, 1 - / , is held stationary this fraction has zero velocity. The velocity of the zone as a whole is the average velocity of its solute (fraction in mobile phase, R ) x v + (fraction in stationary phase, 1 - R ) x O = R v. Thus zone velocity is directly proportional to R  

Equation 10.1 shows that R is a measure of the retardation or slowing of the zone or peak with respect to mobile phase velocity. A peak that experiences no retardation because its solute does not partition into the stationary phase (R = 1) is termed a nonretained peak or void peak such a peak travels at mobile phase velocity v. Solute retained to some extent by the stationary phase migrates as a retained peak, for which R 1. (The smaller R, the greater the retention.) Because of its key role in specifying retention, R is termed the retention ratio. 

The capacity factor k to be discussed shortly, is an alternate measure of retention. While k is used more often than R in chromatography, the use of R is advantageous because (i) it is directly proportional to peak migration velocity and is thus a more direct measure of retention than k (ii) most equations describing chromatography are simpler when expressed in terms of R rather than k and (iii) R is a more universal measure of retention R but not k applies to other perpendicular flow methods such as field-flow fractionation. 

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Fig. 3. Example of an immunochromatographic assay for a Streptococcal antigen (Sa) using antibody to the antigen (ctSa ) linked to a blue-colored latex bead ( ). The downward pointing triangles represent chromatographic migration. Formation of a sandwich linking the antigen between the latex bead and...

Figure 1. Schematic of the effects of brief treatment with mild alkali on the thin-layer chromatographic migrations of three types of glycolipids. 1, 2 control and alkali-treated neutral glycosphingolipid 3, 4 control and alkali-treated galactosyl-alkylacylglycerol 5, 6 control and alkali-treated galactosyldiacylglycerol. OR origin FR solvent front.

Following the separation of GSL by thin-layer chromatography, the GSLs were stained with iodine vapour and gel zones that corresponded in chromatographic migration with authentic human GSL of known chemical structure were scraped, transferred into scintillation vials and counted in 10 ml of "Liquiscint"... 

Carbetidine Would be expected to chromatographically migrate on silica HPLC. CH3OH (l%NH4OH)-CH2C12 (1 9), similar to piperdolate [10]... 

Since chromatographic migration rates depend on the amount of liquid phase and on the liquid velocity, the above noted gradients, established by the actions of capillarity, have important chromatograhic effects [14]. More details on the chromatographic consequences of capillary flow can be found in the literature (12) and in subsequent chapters. 

A solute undergoing chromatographic migration partitions between the stationary and mobile phases, a process driven by thermodynamic equilibrium. At equilibrium (established fully only at the zone center), the concentration in the stationary phase (c,) relative to that in the mobile phase (cm) is given by the thermodynamic distribution constant K, as shown by comparing Eqs. 2.18 and 2.19. Thus... 

The erratic motion of a chromatographically migrating molecule resembles a random-walk process. In order to apply random-walk ideas to chromatography, we must identify the effective step lengths and step numbers associated with the molecular migration. This is the main task to follow. 

Chromatographic Migration of Lipids Iodinated in a Lactoperoxidase-Catalyzed Reaction... 

Thin-layer chromatographic migration rates (Rf) of sialic acids on 0.1 mm cellulose plates using 1-propanol/l-... 

For separations where both selectivity and high column efficiency are needed (e.g. resolution of certain isomeric substances) it may be of advantage that the interaction of solute and mobile phase molecules can be sensitively adjusted by pressure. Different fluids and pressure regions can be explored to achieve chromatographic migration of various non-volatile and unstable molecules. 

Band broadening in time is the result of slow introduction of a gaseous sample or of sample vapors into the column for all solutes, bands have the same width in terms of time, because the first material has the same lead over the last in terms of chromatographic migration time. A band is considered to be sharp... 

Reconcentration of bands broadened in time requires that the first solute material entering the system is hindered in its gas chromatographic migration until the last one entered, i.e., a temporary increase of the retention power. 

Solute material spread into the GC separation column by the flow of liquid can be reconcentrated by placing the flooded zone into an uncoated (but deactivated) precolumn. This precolumn represents a zone of low retention power ( retention gap ) the solute material moves through it at a low temperature and is accumulated in the inlet of the separation column because the temperature is still too low to enable noticeable chromatographic migration. The uncoated precolumn must be at least as long as the flooded zone. The retention gap technique shortens initial bands by a factor of 100-1000, enabling the acceptance of flooded zones as long as 50 m. 

Solvent molecules adsorbed in the dry layer do not mix with liquid solvent ascending in the layer. Unless these are extremely polar like water, methanol, acids, and bases, the preadsorbed solvent molecules are pushed ahead by the front. This leads to the effect that the apparent solvent front, i.e. the boundary between wet and dry area, moves ahead of the real front, i.e. the liquid that has contributed to the chromatographic migration of the analyte fractions. The difference between apparent front, to which observed Rf relate, and real front relating to corrected Rf (R/), can easily amount up to 25%, depending on the type of solvent, the shape of the developing device, and the duration of pre-equilibration. 

As a general conclusion, one can assert that the isomeric and stereoisomeric constitutions influence on the chromatographic migration thus, the TLC method is very suitable for the separation of enantiomers via diastereoisomers. 

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