Separation efficiency and resolution

Efficiency and resolution of an electrophoretic separation are influenced by the electrophoretic flow as well as the EOF. The apparent mobility, Papp, of an analyte is determined by the sum of its electrophoretic mobility, pep, and the electroosmotic mobility, peof - 

If the EOF dominates over the electrophoretic motion, then all analyte components move towards the cathode. They are separated from each other due to their difference in apparent mobilities (Table 3.1, Fig. 3.5). Cations have the highest Papp, because their pep is in the same direction as the peof- Neutral species have no Pep. Hence, they are dragged along at the velocity of the bulk solution. Anions reach the cathode last, as their pep opposes the direction of the peof- 

The migration velocity, v, of an analyte is defined as the product of its apparent mobility, Papp, and the applied electric field strength, E, (equation 3.8). The field strength, E, is the ratio of applied voltage, V, over capillary length, L. Hence, v can be expressed as  

The migration time, t, of an analyte, is defined as the capillary length, L, over the migration velocity, v. Substituting v for the expression in equation 3.9 leads to  

the larger the applied field strength E, the faster the velocity and the shorter the migration time. This, however, is restricted by Joule heating (section 3.1.2). 

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Figure 1.8 shows the influence of the polymerization time on the separation efficiency and resolution of MS/BVPE columns toward biomolecules (e.g., oligonucleotides) and small molecules... 

Because the electroosmotic flow affects the amount of time a solute resides in the capillary, both the separation efficiency and resolution are related to the direction and flow of the EOF. The EOF flow profile, as shown in Figure 4.7, is comparatively pluglike. Unlike the laminar flow that is characteristic of pressure-driven fluids,5 the EOF has minimal effect on resistance to mass transfer. As a result, the plate count in a capillary is far larger than that of a chromatography column of comparable length. 

The development of the first CE-MS was prompted by the early reports on electrospray ionization (ESI-MS) by Fenn and co-workers in the mid-1980s [1], when it was recognized that CE would provide an optimal flow rate of polar and ionic species to the ESI source. In this initial CE-MS report, a metal coating on the tip of the CE capillary made contact with a metal sheath capillary to which the ESI voltage was applied [5]. In this way, the sheath capillary acted as both the CE cathode, closing the CE electrical circuit, and the ESI source (emitter). Ideally, the interface between CE and MS should maintain separation efficiency and resolution, be sensitive, precise, linear in response, maintain electrical continuity across the separation capillary so as to define the CE field gradient, be able to cope with all eluents presented by the CE separation step, and be able to provide efficient ionization from low flow rates for mass analysis. 

This impressive effect of temperature on efficiency and resolution is not common and improvements of this order of magnitude are not always realized by raising the column temperature. Nevertheless, temperature is a variable that needs to be considered depending on the type of mixture being separated. 

Diffusion and mass transfer effects cause the dimensions of the separated spots to increase in all directions as elution proceeds, in much the same way as concentration profiles become Gaussian in column separations (p. 86). Multiple path, molecular diffusion and mass transfer effects all contribute to spreading along the direction of flow but only the first two cause lateral spreading. Consequently, the initially circular spots become progressively elliptical in the direction of flow. Efficiency and resolution are thus impaired. Elution must be halted before the solvent front reaches the opposite edge of the plate as the distance it has moved must be measured in order to calculate the retardation factors (Rf values) of separated components (p. 86). 

Typical NP conditions involve mixtures of n-hexane or -heptane with alcohols (EtOH and 2-propanol). In many cases, the addition of small amounts (<0.1%) of acid and/or base is necessary to improve peak efficiency and selectivity. Usually, the concentration of alcohols tunes the retention and selectivity the highest values are reached when the mobile phase consists mainly of the nonpolar component (i.e., n-hexane). Consequently, optimization in NP mode simply consists of finding the ratio n-hexane/alcohol that gives an adequate separation with the shortest possible analysis time [30]. Normally, 20% EtOH gives a reasonable retention factor for most analytes on vancomycin and TE CSPs, while 40% is more appropriate for ristocetin A-based CSPs. Ethanol normally gives the best efficiency and resolution with reasonable backpressures. Other combinations of organic solvents (ACN, dioxane, methyl tert-butyl ether) have successfully been used in the separation of chiral sulfoxides on five differenf glycopepfide CSPs, namely, ristocetin A, teicoplanin, TAG, vancomycin, and VAG CSPs [46]. 

In chromatographic and related separation techniques the basic requirements for the resolution (Rs) of two peaks are a column with a high number of plate counts and a factor to induce some selectivity for the separation. Basically resolution is the product of separation efficiency and selectivity ... 

In preparative GC one is not primarily concerned with resolution or theoretical plates. The basic reasons for the technique is its high separation efficiency and high speed. 

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