Glossary Of HPLC-LC Separation Terms – E

The listing should be helpful to those just starting in HPLC but it also can serve as a refresher for long-time users in the field.

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E: See separation impedance.

´See interparticle porosity.Eddy dispersion (diffusion) term (λ): The A term in the van Deemter equation. It is the contribution to plate height from the heterogeneity in axial velocities as a result of the particle size and geometry of the packing, as well as wall effects; A = 2λdp, where λ is an empirical column constant. Typical values of λ for well-packed columns are 0.8–1.0. Some theories of chromatography indicate a velocity-dependent contribution to the height equivalent to a theoretical plate (HETP) from this process. Also known as eddy diffusion, flow-heterogeneity induced broadening, and the multipath term. See also van Deemter equation.

´ e See interstitial porosity.

Effective plate height (H eff ): The column length divided by the effective plate number.

Effective theoretical plates (N eff ): Also called the effective plate number by IUPAC. The true number of plates in a column, because it corrects theoretical plates for dead volume. N eff = 16[(t R'/w b)2 ], where t R' is the adjusted retention time and w b is the bandwidth of the peak (see Figure 2). It is a better figure of merit than simple plate number for comparing devices of very different geometries and phase ratios.

Efficiency (N or H): A measure typically determined by the number of theoretical plates (N) calculated from the equation N = 16(V R/w b)2 = 16 (t R/w b)2, where w b is the peak width measured at the base (see Figure 2). If the peak width is measured at half height, the following equation is used: N = 5.545 (V R/w h)2. The plate height (H) or HETP is determined by H = L/N. The efficiency of asymmetric peaks is better determined from the peak centroid and variance by mathematical analysis of the peak shape. See also Foley–Dorsey equation.

Effluent: The mobile phase leaving the column; same as eluate.

´ i See intraparticle porosity.

Eluate: Combination of mobile phase and solute exiting the column; also called effluent.

Eluent: The mobile phase used to perform a separation.

Eluite: The species being eluted, the analyte, or the sample.

Eluotropic series: A series of solvents (eluents) with an increasing degree of solvent strength generally used in liquid–solid or adsorption chromatography. In normal-phase chromatography, a nonpolar solvent such as pentane would be at the low end of the scale, an intermediate solvent such as methylene chloride would be in the middle of the scale, and a strongly polar solvent such as methanol would be near the upper end of the scale. In reversed-phase chromatography, the reverse order of strength would be observed; water would be weak and acetonitrile strong. Thus, when developing a method or running a gradient, an eluotropic series is useful for selecting solvents. See also Snyder o .

Elute: To chromatograph by elution chromatography. The term elute is preferred over develop, which was used in older nomenclature.

Elution: The process of passing mobile phase through the column to transport solutes down a column.

Elution chromatography: The most commonly used chromatographic method in which a sample is applied to the head of the column as a narrow zone and individual analytes are separated and eluted from the end of the column. Compare with displacement chromatography and frontal analysis.

Elution volume (V R ): Refers to the volume of mobile phase necessary to elute a solute from a column. It is the volume from the point of injection to the volume at maximum concentration (apex) for a symmetrical peak; V R = Ft R, where F is the flow rate and t R is the retention time of the peak of interest.

Elutriation: A technique used to fractionate packing particles by size based on the difference in their Stokes terminal velocities. It most often is used for the separation of ion-exchange resins that require a particularly narrow size range, such as amino acid resins. The technique involves the upward flow of water into a large tube. The unsized beads are added to the moving water, and the particles seek their own level, depending upon their density and particle size. They are removed at certain levels in the tube. High-purity spherical silica gels sometimes are sized by elutriation.

Enantiomeric compound: Chemical compounds that display chiral activity; such compounds will require a separation mechanism that can differentiate between the R- or S-enantiomer and specialty columns are available for this purpose.

Endcapping: A technique used to remove silica gel silanol groups that may remain after reaction with a large silylating agent such as octadecyltrichlorosilane. The column is said to be endcapped when a small silylating reagent (such as trimethyl-chlorosilane or dichlorodimethylsilane) is used to bond residual silanol groups on a silica-gel–based packing surface. Most often used with reversed-phase packings to minimize undesirable adsorption of basic, ionizable, and ionic compounds. Endcapping reactions also are used to remove terminal silanol groups from polymeric phases.

Endfitting: The fitting at the end of the column that permits connection to the injector or detector. Most HPLC endfittings have frits to contain the packing and low dead volumes for minimum band spreading. They usually are constructed of stainless steel, but polyetherether ketone (PEEK) and other polymeric materials also are used.

´ T See total porosity.

Exchange capacity: See ion-exchange capacity.

Excluded volume: See interstitial volume.

Exclusion chromatography: See ion-exclusion chromatography and steric exclusion chromatography.

Exclusion limit: The upper limit of molecular weight (or size) beyond which molecules will be eluted at the same retention volume, called the exclusion volume. Many SEC packings are known by their exclusion limit. For example, a 105 column of porous silica gel will exclude any compounds with a molecular weight greater than 100,000, based on a polystyrene calibration standard.

Exclusion volume (V 0 , V ei ): The minimum retention volume of a molecule on an SEC packing in which all molecules larger than the size of the largest pore are totally excluded. These molecules are incapable of penetrating the pores and are eluted at the interstitial (interparticle) volume of the column.

Exponentially modified Gaussian peak: An asymmetric peak resulting from passing a Gaussian peak through a detector that is excessively slow or has an excessive volume. Frequently used to model peak tailing arising from the column per se. The basis for the Foley–Dorsey equations. See also Foley–Dorsey equation.

Extracolumn effects: The total band broadening effects of all parts of the chromatographic system outside of the column itself. Extracolumn effects must be minimized to maintain the efficiency of a column. Sources of band broadening can include the injector design, injection volume, connecting tubing, endfittings, frits, detector cell volume, and internal detector tubing. The variances of all of these contributions are additive.

Extracolumn volume: The volume between the effective injection point and the effective detection point, excluding the part of the column containing the stationary phase. It comprises the volumes of the injector, connecting lines and frits, and the detector. It determines the extracolumn effects.

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