Mechanism of separation

There are many theories which describe the mechanisms of SEC separation, a good review being provided by Janca (1984). The simplest way to 

The stationary phase is represented as a porous packing material containing different size pores (cross-linked inert gel). The mobile phase is normally a single solvent capable of dissolving the sample. 

Represents the SEC chromatogram of monodispersed standards of diffenng molecular weight 

As flow is applied to the system, solvent will flow only in the interstitial spaces around the gel particles as the channel produced between the stationary phase particles in the column is much larger than the pores inside the gels. These pores ultimately will contain stagnant mobile phase. 

On injection of the sample molecules onto the column, molecules too large to penetrate and hence diffuse into the pores will be excluded and elute from the column first. Medium size molecules may be excluded from the denser parts of the stationary phase pore structure, but diffuse freely in the larger passages of the pores selective permeation . Molecules of comparable size to that of the solvent will distribute themselves through- 

There are few differences between the separation in gas chromatography [14-16] and the separation in liquid chromatography (LC), because it is assumed that the differential solvation of the diastereomeric compounds during the LC separation does not play a very important role [17], Helmchen et al. [18] explained the separation of diastereomeric amides using LC with a silica gel stationary phase under normal-phase conditions. In order to explain their separation, the authors made some assumptions  

Secondary amides adopt essentially the same conformation in polar solutions and in the adsorbed state (on silica gel). 

In the adsorbed state, a parallel alignment of the planar amide group and the surface of silica gel is preferred. 

Apolar groups (i.e., alkyl, aryl) outside the amide plane cause a disturbance of this preferred arrangement in proportion to their steric bulk in a direction perpendicular to the amide plane. Such groups are classified as large and small by indices L and S, respectively. 

That member of a diastereomeric pair in which both faces of the amide plane are more shielded than the least shielded face in the other member is eluted first. 

For electrically neutral molecules eluting between f0 and fM (Fig. 5.7), the capacity factor, k, is given by17 

To calculate the capacity factor, it is necessary to know the migration time not only of the analyte but also of the micelle and the EOF. Although there is no ideal marker in MECC, a very hydrophobic molecule, such as Sudan III, will spend most of its time partitioned in the micellar phase and 

As with the capacity factor, as the velocity of the micellar phase approaches zero, the equation for resolution approaches that of classic chromatography. 

In MECC anionic surfactants are most frequently used, but cationic surfactants are also very popular. In addition, chiral surfactants, nonionic surfactants, zwitterionic surfactants, biological surfactants, or mixtures of each are finding increasing use. In all categories, variations in alkyl chain length will affect resolution or selectivity, as will changes in buffer concentration, pH, and temperature or the use of additives such as metal ions or organic modifiers. Typical surfactant systems used in MECC are shown in Table 5.3. 

Surfactants suitable for MECC should (1) have enough solubility in the buffer to form micelles, (2) form a low-viscosity solution, and (3) form a solution that is homogeneous and UV transparent. The surfactant concentration should be kept below 200 vaM because the viscosity of the buffer above that concentration, and therefore the current, becomes too high. The buffer concentration should be greater than 10 mM and higher than that of the surfactant in order to maintain a constant pH throughout the run. 

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The mechanism of separation is the same for Zorbax PSM columns as it is for other types of SEC columns. As the mobile phase flows through the column, large molecules are forced down the column at faster rates than small molecules because the large molecules have less access to the column volume inside the pores. Consequently, molecules that are too large to permeate any of the pore... 

Electrodriven techniques are useful as components in multidimensional separation systems due to their unique mechanisms of separation, high efficiency and speed. The work carried out by Jorgenson and co-workers has demonstrated the high efficiencies and peak capacities that are possible with comprehensive multidimensional electrodriven separations. The speed and efficiency of CZE makes it possibly the best technique to use for the final dimension in a liquid phase multidimensional separation. It can be envisaged that multidimensional electrodriven techniques will eventually be applied to the analysis of complex mixtures of all types. The peak capacities that can result from these techniques make them extraordinarily powerful tools. When the limitations of one-dimensional separations are finally realized, and the simplicity of multidimensional methods is enhanced, the use of multidimensional electrodriven separations may become more widespread. 

Cause-and-effect analysis reveals that steam purity and quality are both reduced by the degree of carryover taking place in a boiler, and carryover is itself a function of the effectiveness of steam-water separation. In turn, the mechanics of separation are a function of three areas, each with its own variables ... 

Considering that each stationary phase has a different mechanism of separation, the analyst should apply the most suitable phase for a particular carotenoid separation, not forgetting that it is very useful to look for previous experiences among the several examples available in the literature and that it is also possible to find different solutions for the same problem. 

Because the HPLC mobile phase is a liquid, there are some very obvious differences between HPLC and GC. First, the mechanism of separation in HPLC involves the specific interaction of the mixture components with a specific mobile phase composition, while in GC the vapor pressure of the components,... 

Compare HPLC with GC in terms of (a) the force that moves the mobile phase through the stationary phase, (b) the nature of the mobile phase, (c) how the stationary phase is held in place, (d) what types of chromatography are applicable, (e) application of vapor pressure concepts, (f) sample injection, (g) mechanisms of separation, (h) detection systems, (i) recording systems, and (j) data obtained. 

Replacement of the hydrophilic acrylamide by the more hydrophobic N-iso-propylacrylamide, in combination with the pre-functionalization of the capillary with (3-methacryloyloxypropyl) trimethoxysilane, afforded a monolithic gel covalently attached to the capillary wall. A substantial improvement in the separations of aromatic ketones and steroids was observed using these fritless hydrogel columns, as seen by the column efficiencies of 160,000 found for hydrocortisone and testosterone [92]. The separations exhibited many of the attributes typical of reversed-phase chromatography and led to the conclusion that, in contrast to the original polyacrylamide-based gels, size-exclusion mechanism was no longer the primary mechanism of separation. 

There are four different mechanisms of separation utilized in HPLC adsorption, partition, ion-exchange, and size exclusion chromatography. 

The mechanism of separation of biological molecules such as proteins and amino acids, and the parameters that affect the extraction distribution coefficient and the kinetics of extraction have been studied more extensively than the extraction of inorganic solutes. This is mainly due to the variety of size and structure of these molecules and, furthermore, to the fact that their characteristics may be adversely affected by their contact with solvents and surfactants. 

The interaction between particle and applied field determines the concentration profile distribution inside the channel. Theory assumes that the particles do not interact with each other, that is, the particle concentration is so low that they can be considered in a condition of infinite dilution and thus with an ideal behavior. Under these assumptions, the field-particle interaction has been classified into three major elution modes Brownian (or normal), steric, and hyperlayer (or focusing). These elution modes, which are limiting cases, correspond to different mechanisms of separation and, theoretically, they can explain FFF retention on the basis of particle properties such as the... 

ILs have been used to separate and determine the purity of anthraqui-nones. Rapid and sensitive determination of anthraquinones in Chinese herb using l-butyl-3-methylimidazolium-based IL with p-cyclodextrin (p-CD) as a modifier in CZE was provided by Qi et al. [49]. Successful separation and identification of four anthraquinones of Paedicalyx attopevensis Pierre ex Pitard extracts has been achieved. In the running electrolyte the anthraquinones may associate with the imidazolium ions or with the p-CDs. They may be entirely or partly embedded in the cavity of the p-CDs, so the association with the free imidazolium ions in the bulk solution was weak and those analytes, that were not embedded in the cavity of the p-CDs had rather stronger association with the imidazolium ions in the system. The mechanism of separation is illustrated in Figure 6.6. 

Figure 6.6 Mechanism of separation of anthraquinones using [C4CjIm]-based IL and P CD as BGE additives. (Adapted from Yu, L., Qin, W., and Li, S. R Y.,Anal. Chim. Acta, 547, 165-171, 2005. With permission.)...

Mechanism of Separation. There are several requirements for chiral recognition. (/) Formation of an inclusion complex between the solute and the cydodextrin cavity is needed (4,10). This has been demonstrated by performing a normal-phase separation, eg, using hexane—isopropanol mobile phase, on a J3-CD column. The enantiomeric solute is then restricted to the outside surface of the cydodextrin cavity because the hydrophobic solvent occupies the interior of the cydodextrin. (2) The inclusion complex formed should provide a rdatively "tight fit" between the hydrophobic species and the cydodextrin cavity. This is evident by the fact that J3-CD exhibits better enantioselectivity for molecules the size of biphenyl or naphthalene than it does for smaller molecules. Smaller compounds are not as rigidly held and appear to be able to move in such a manner that they experience the same average environment. (5) The chiral center, or a substituent attached to the chiral center, must be near to and interact with the mouth of the cydodextrin cavity. When these three requirements are fulfilled the possibility of chiral recognition is favorable. 

Gravity settling is by far the most widely used mechanism of separation. This results primarily from the simplicity of the equipment necessary and the readily available force of gravity. 

A second type of impingement separation device is a knitted wire mesh pad. The primary mechanism of separation in the knitted wire mesh is impingement. It also utilizes centrifugal and gravitational force in the collection of small liquid particles. 

Viscosity affects the various mechanisms of separation in accordance with the appropriate settling law. Tor instance, viscosity has no effect on terminal velocities in the range where Newton s law applies except as it affects the Reynolds Number which determines which settling law applies. Viscosity does affect the terminal velocity in both the Intermediate law range and Stokes law range as well as help determine the Reynolds Number. As the pressure increases or the temperature decreases the viscosity of the gas increases. Viscosity becomes a large factor in very small particle separation (Intermediate and Stokes law range). 

It is uninformative to refer to liquid phase as being selective, since all liquid phases are selective to varying degrees. Selectivity refers to the relative retention of two components and gives no information regarding the mechanism of separation. Most separations depend upon boiling point differences, variations in molecular weights of the components, and/or the structure of the components. 

The mechanism of separation between large and small molecules is what distinguishes TFLC from other methods of sample cleanup. Protein precipitation is often used to remove much of the sample matrix. While much of the proteins present in a given matrix can be crashed out of solution by the addition of an... 

The mechanism of separation in NCE is based on the difference in the electrophoretic mobility of the separated species. Under NCE conditions, the migration of the separated species is controlled by the sum of the intrinsic electrophoretic mobility (fxe/)) and the electroosmotic mobility (fxeo), due to the action of electroosmotic flow (EOF). The observed mobility 0bs) of the species is related to xeo and juep by the following equation ... 

In addition to HPLC, microchips have also been used in other modalities of liquid chromatography, including capillary electrochromatography and mi cel -lar electrokinetic chromatography. Many workers have attempted to achieve nano separations at high speed of different molecules with high efficiency, reproducibility, and low detection limits. The state of the art of separation in these modalities is discussed in this chapter, with special emphasis on their applications, optimization, and mechanisms of separation. 

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