Column factors affecting selection

Three separate factors affect resolution (1) a column selectivity factor that varies with a, (2) a capacity factor that varies with k (taken usually as fej). and (3) an efficiency factor that depends on the theoretical plate number. 

The selectivity of a column determines the degree by which the maxima of the two peaks are separated as in Figure 3.1c. The following factors affect the selectivity of a column ... 

Pore size was also found to be the main factor affecting separation selectivity of C18 columns from different manufacturers, compared to evaluate the applicability of sequence-specific retention calculator peptide retention prediction algorithms. Differences in end capping chemistry did not play a major role while the introduction of embedded polar groups to the C18 functionality enhanced the retention of peptides containing hydrophobic amino acid residues with polar groups [6]. 

Experimental work of Kalasz et al. resulted in the statement of the characteristics and basic rules of displacement chromatography. They conceived properties of the fully developed displacement train, factors affecting displacement development, efficacy of separation, analysis of displaced fractions, determination of displacement diagrams from Langmuirian isotherms, as well as selection of the column, carrier, and displacer for displacement chromatography. Concentration of the sample is a particular feature of displacement chromatography. However, the displacer in the carrier is also definitely concentrated through the development of the displacement train. 

These ions, such as calcium for amino acids, impart a specific selectivity (see the entry Ion-Exchange, Mechanism and Factors Affecting Separation). Alternatively, a layer of counterions is applied by the user by conditioning a column with the salt of interest. An intermediate step of washing with a weak acid may accelerate the equilibration process. 

The chemical structure and the stereoisomerism of a stationary phase are not the only factors that control chiral selectivity. There are two other major factors that also affect selectivity and they are the stationary phase loading on the column and the operating temperature. The effect of these two variables on the retention ratio of a pair of enantiomers has been reported by Supelco and their data is shown in figure 5.9. 

The method finds increasing application for the determination of amino acids. Factors affecting the retention of the isoindole derivatives by reversed phase columns have been investigated [335, 337]. Usually columns with Ci8 brushes are used [350]. Numerous gradient elution systems suitable for the separation of amino acid-derived isoindoles have been published their number is still increasing with the availability of new column types. The various references [184, 337, 339-365] are a representative selection of those available, most of which involve minor variations of the basic method. 

Among the factors that affect column efficiency and selectivity are the structure of the mesogen (in relation to the solutes of interest), the phase type(s) and degree of ordering, temperature, column loading and coverage, concentration (solubility) effects, and the surface structure and specialized treatment of the support. The column surface can dramatically affect the orientation of the liquid crystalline molecules (especially for monomeric mesogens) and thus alter separation efficiency. For polymeric liquid... 

Temperature variation may also be a relevant factor in flowrate stability. Since the viscosity of the solvent is temperature dependent, wide swings in the ambient temperature can directly affect pump performance. The direct effects of temperature on pump performance usually are far smaller, however, than the effects on retention and selectivity therefore, control of column temperature is generally sufficient to obtain high reproducibility. 

First, we will explore the three fundamental factors in HPLC retention, selectivity, and efficiency. These three factors ultimately control the separation (resolution) of the analyte(s). We will then discuss the van Deemter equation and demonstrate how the particle diameter of the packing material and flow rate affect column efficiencies. 

Resolution is a term used to describe the degree of separation between neighboring solute bands or peaks. It is affected by the selectivity (a), efficiency (N) and capacity (k ) of the column. The resolution equation [Eq. (1.3)] describes the relationship between those factors and indicates how they can be manipulated in order to improve the resolution between two peaks. 

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