Efficiency and resolution

Typical normal-phase operations involved combinations of alcohols and hexane or heptane. 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 polar solvents such as alcohol determines the retention and selectivity (Fig. 2-18). Since flow rate has no impact on selectivity (see Fig. 2-11), the most productive flow rate was determined to be 2 mL miiT. Ethanol normally gives the best efficiency and resolution with reasonable back-pressures. It has been reported that halogenated solvents have also been used successfully on these stationary phases as well as acetonitrile, dioxane and methyl tert-butyl ether, or combinations of the these. The optimization parameters under three different mobile phase modes on glycopeptide CSPs are summarized in Table 2-7. 

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). 

This chapter provides an overview of essential concepts in HPLC including retention, selectivity, efficiency, and resolution as well as their relationships with key column and mobile phase parameters such as particle size, column length and diameter, mobile phase strength, pH, and flow rate. The significance of several concepts important in pharmaceutical analysis such as peak capacity, gradient time, void volume, and limit of quantitation are discussed. 

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... 

The effects of temperature on a CE separation are severalfold. With increasing temperature, the viscosity of the running electrolyte decreases and analysis times are shorter. The high currents associated with elevated temperatures generates additional heat thus, the efficiency and resolution may be altered. Changes in selectivity are often observed with different temperatures because solute mobilities are a function of diffusion coefficients, which are, in turn, dependent on temperature. Changes in selectivity may result from alteration of solute pKa values with temperature changes. 

Chromatography is described and measured in terms of four major concepts capacity, efficiency, selectivity, and resolution. The capacity and selectivity of the column are variables that are controlled largely by the column manufacturer, whereas efficiency and resolution can be controlled,... 

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. 

If the sample is dissolved in a solvent that is weaker than the mobile phase, then the sample can be enriched on the head of the column without penetrating into the column bed. This compression effect is particularly important for capillary LC applications, since it permits significantly larger injection volumes. A substantial increase in sensitivity results, and conventional autosamplers with 20-jnl loops can be used.16 However, sample solubility and recovery, miscibility of the sample with the mobile phase, and the maximum tolerable loss in column efficiency and resolution must all be assessed experimentally for optimum on-column focusing.16... 

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