Glossary

Asymmetry factor (As)Peak shape

Peak symmetry measure As = b/a, where a and b are the front and back half-widths at 10% of peak height. As = 1 is symmetrical; As > 1 indicates tailing and As < 1 fronting. Acceptable ranges depend on the column, test method and application, so take them from the column instructions.

Bed volume (BV)Column

Geometric volume of the packed resin bed (cross-sectional area × bed height). Usually used interchangeably with column volume.

Column volume (CV)Column

Volume of the packed bed (πr²h), used as the unit for buffer volumes such as equilibration, wash and gradient lengths, so that methods transfer between column sizes. It includes the volume between the particles, the pore volume and the matrix. For a well-packed SEC column, Cytiva™ gives the void volume as roughly 30% of CV; the fraction depends on the medium and packing.

Dead volumeColumn

Extra-column volume in the flow path, for example between injection point and detector (tubing, connectors, detector cell). Contributes to band broadening and lowers measured efficiency. The volume between detector and fraction collector (delay volume) must also be known for correct fraction mapping. Distinct from the void volume (V₀) inside the column.

Dynamic binding capacity (DBC)Column

Amount of target that binds under flow before a defined breakthrough of the target is reached, often reported at 10% breakthrough (Qb10). Depends on the protein, resin, residence time and buffer, and is usually lower than static binding capacity measured at equilibrium.

HETPEfficiency

Height equivalent to a theoretical plate, H = L/N, where L is bed height and N the plate number. Lower H means higher efficiency. H varies with flow velocity, a dependence described by van Deemter-type equations.

Hofmeister seriesChemistry

Ordering of ions by their effect on protein precipitation and hydrophobic interaction. Salting-out ions such as sulfate and phosphate promote HIC binding and precipitation; ions at the other end, such as perchlorate and thiocyanate, weaken these effects. Cytiva™ gives the anion order SO₄²⁻ > HPO₄²⁻ > CH₃COO⁻ > Cl⁻ > NO₃⁻ > Br⁻ > ClO₃⁻ > I⁻ > ClO₄⁻ > SCN⁻. Effects depend on the protein and salt concentration.

Isocratic elutionOperation

Elution at constant mobile-phase composition. Standard for SEC, where species separate by pore access rather than by changing buffer conditions.

KavSEC

SEC elution parameter Kav = (Ve − V₀) / (Vc − V₀), where Vc is the geometric bed volume. Because Vc includes matrix volume that no solute can enter, Kav is not a true distribution coefficient, but for a given resin it is proportional to Kd = (Ve − V₀) / (Vt − V₀), which runs from 0 (excluded) to 1 (full pore access). Some texts write Vt for the bed volume in the Kav expression. Kav versus log Mr of standards gives an apparent molecular weight relative to those standards.

KosmotropeChemistry

Solute that promotes salting-out and generally stabilises protein structure in solution; the opposite of a chaotrope. Ammonium sulfate is the kosmotropic salt most often used in HIC.

Linear velocity (u)Operation

Superficial velocity is volumetric flow divided by column cross-sectional area: u_s = Q/A = Q/(π × r²), usually in cm/h after unit conversion. Interstitial velocity is u_int = Q/(ε_i A), where ε_i is the interparticle void fraction; the efficiency simulator uses this latter convention. Keep the velocity convention and model coefficients consistent. Keeping superficial velocity and bed height constant is the usual way to transfer a method between column diameters.

pI (isoelectric point)Chemistry

pH at which a molecule carries zero net charge. Below the pI a protein is net positive, above it net negative, which is why the pI guides the choice between cation and anion exchangers. IEX retention depends on surface charge and its distribution, so it cannot be predicted with certainty from the pI alone.

Plate count (N)Efficiency

Efficiency measure from a test peak: N = 5.54 × (VR/wh)², where wh is the peak width at half height in the same units as VR. Usually measured with a small non-interacting tracer (for example acetone) under stated conditions, and often reported per metre. Results depend on the system and test conditions, so compare a column with its own earlier results or with the vendor test under matching conditions.

Resolution (Rs)Peak shape

Separation between two peaks: Rs = 2(VR2 − VR1) / (wb1 + wb2), where wb are the peak base widths. Rs ≥ 1.5 is commonly quoted as baseline separation for symmetrical peaks of similar size; unequal or asymmetric peaks need more. A well-resolved peak is not proof of purity.

Retention factor (k)Retention

Dimensionless measure of retention relative to an unretained marker: k = (VR − VM) / VM, where VM is the hold-up volume. Most meaningful for isocratic elution in interactive modes; higher k means stronger retention.

Selectivity (α)Separation

Ratio of retention factors for two adjacent peaks under the same conditions: α = k₂ / k₁, with k₂ ≥ k₁. α = 1 means the two co-elute regardless of efficiency. Cytiva™ notes that good selectivity matters more than high efficiency for resolution.

Theoretical plateEfficiency

Conceptual equilibrium stage used to express column efficiency as N or H. More plates means narrower peaks for a given retention. Plate numbers depend on column length, particle size, packing, flow rate and test solute, so there is no general range.

Total liquid volume (Vt)SEC

In SEC, the elution volume of small molecules with full access to the pores, usually slightly less than one CV. Some texts use Vt for the geometric bed volume, so check the convention before comparing Kav values.

Void volume (V₀)SEC

Elution volume of solutes that neither enter the pores nor interact with the medium, i.e. the liquid between the particles. In SEC, species too large for the pores elute at about V₀. Often measured with a large tracer such as Blue Dextran 2000 or 2000 kDa dextran; labelled dextrans can adsorb to some media, including some anion exchangers, so check the tracer suits the medium.

Sources and scope

Definitions follow Cytiva™’s Size Exclusion Chromatography, Ion Exchange Chromatography, Hydrophobic Interaction and Reversed Phase Chromatography and System and Column Characterization handbooks (official, accessed 2026-09-25). The retention factor and HETP flow-dependence entries use standard textbook definitions; the IUPAC nomenclature could not be opened in this review. Symbols and conventions differ between sources, so check the definition used by the document you compare against.