Vocabulary of chromatography

Used for the movable end pieces of columns. It contains a filter and flow distributor, and can be used to connect tubing.

The process of interaction between the solute (e.g., a protein) and the stationary phase.

A group of methods based on various types of specific affinities between target molecule(s) — for example, a protein and a specific ligand coupled to a chromatography resin.

Factor describing the shape of a chromatographic peak. Measured at a defined height of the peak; can be at the base (between tangents drawn) or at 5% or 10% of the actual peak. Peaks are known as tailing when A_s > 1.0 and fronting when A_s < 1.0. Gaussian peaks are defined as A_s = 1.0.

The pressure drop across a column and/or a chromatography system.

The widening of a zone of solute (e.g., a protein) when passing through a column or a chromatography system. Leads to dilution of the solute and reduced resolution. Also often called peak broadening or zone broadening.

Volume of the resin bed that the adapter compresses and locks in place.

The process of interaction between a solute (e.g., a protein) and the stationary phase.

Buffer, solution, or eluent used to equilibrate the column before sample loading.

The maximum amount of material that can bind per milliliter of chromatography resin. See also dynamic binding capacity.

The degree of retention of a solute (e.g., a protein) relative to an unretained peak.

Method that separates proteins based on pI.

A graphic presentation of detector response(s) that indicates the concentration of the solutes coming out of the column during the purification, expressed in volume or time.

From Greek chroma, color, and graphein, to write.

The stationary phase. The chromatography resin is composed of a porous matrix that is usually functionalized by coupling ligands to it. The matrix is in the form of particles or, rarely, a single polymer block (monolith).

Cleaning-in-place (CIP) is a common term for cleaning chromatography columns and/or systems to remove any unwanted or nonspecifically bound material.

Usually refers to column hardware packed with chromatography resin.

Passage of buffer or solution through the chromatography column to establish good binding conditions for the selected sample components. For example, column equilibration can establish the correct pH and ionic strength, and ensure that proper counter ions or counter ligands are present.

The column tube and adapters. Refers to all pieces of the column except for the chromatography resin and the packed bed.

The pressure inside the column. Column hardware pressure that is too high can break the column.

Controlled filling of the column hardware with chromatography resin compressed to a certain, predefined extent to achieve a stable particle bed.

The geometrical volume of the column interior.

Ion of opposite charge that interacts with an ion exchange chromatography resin after the column equilibration. A protein that binds to the ion exchanger displaces the counter ion. If you apply a high concentration of the counter ion, it will compete with the bound protein and elute it from the chromatography column.

Substances that interact with ligands of a chromatography resin and can be displaced by a solute (e.g., protein) binding to the ligand.

The volume in a chromatography system, from the injector to the detector, not including the column volume. Dead volume contributes to band broadening.

Removal of dissolved air from buffers and solutions.

Release or removal of bound substances from the chromatography resin.

Design of experiments (DoE) allows use of a minimum number of experiments, where scientists can vary several experimental parameters simultaneously. Based on the obtained data, you can create a mathematical model of the studied process (e.g., a protein purification protocol or a chromatography step). Then you can use the model to understand the influence of the experimental parameters on the outcome and to find an optimum for the process.

High efficiency means that the band broadening is low, and that you’ll obtain sharp peaks. It’s often given as the number of theoretical plates (N) or as theoretical plates per meter (N/m) to an independent measure of column length in the experiment. Efficiency plays a central role in qualifying and monitoring packed bed performance. The ideal high column efficiency gives low band and peak broadening, and indicates how well packed the column is before starting purification.

Column efficiency is typically defined using two parameters:

• Peak broadening over the column is described by an equivalent number of theoretical plates.
• Peak symmetry is described by a peak asymmetry factor, A_s.

Each Cytiva resin protocol describes the procedure to determine column efficiency. You can also find it in our application note, Column efficiency testing.

Mobile phase leaving the column, eluate.

Mobile phase leaving the column, effluent.

Buffer or solution used during chromatography, mobile phase.

Buffer or solution used for elution (desorption) of bound solutes (e.g., proteins) from a column.

The volume of buffer or solution (eluent) required to elute the solute (e.g., a protein), known as the retention volume.

The time required for elution of a solute (e.g., a protein), known as the retention time.

Volumetric flow (mL/min) or linear flow rate (cm/h). Measures flow through a column and/or chromatography system.

Material that passes through the column during sample loading without binding.

A type of deep filter often used at top and bottom of columns.

Gel filtration (GF) is size-exclusion chromatography. It separates solutes (e.g., proteins) according to size.

Continuous increased or decreased concentration of a substance (in the eluent) that causes elution of bound solutes (e.g., proteins).

Hydrophobic interaction chromatography (HIC) is a method based on the hydrophobic interaction between solutes (e.g., proteins) and the chromatography resin in the presence of high salt concentration.

Mixed-mode ion exchange chromatography method.

Immobilized metal ion affinity chromatography (IMAC) is a method based on the affinity of proteins with His, Cys, or Trp amino acid residues on their surface and metal ions on the chromatography resin.

Ion exchange chromatography (IEX) is a method based on electrostatic interactions between solutes (e.g., proteins) and chromatography resin.

Elution of the solutes without changing the composition of the buffer or solution (eluent).

The specific molecular group that couples to the matrix to functionalize the chromatography resin.

Related to ligand concentration. The distribution of ligands on the surfaces (including the surfaces inside pores) of the chromatography matrix.

The flow rate normalized by the column cross section (cm/h).

Movement of a solute (e.g., a protein) in and out of the stationary phase. Mass transfer is an important factor for column efficiency.

The matrix is the nonfunctional base for the chromatography resin. The matrix has a porous structure and large surface area that you can modify with ligands to enable protein binding.

The fluid (buffer or solution) carrying the solutes during chromatography. Can also be referred to as the eluent.

The mean diameter of the spherical beads.

Same as band broadening.

The number of peaks that you can separate using a chromatography column.

Broadening at the beginning of a peak. Results in decreased asymmetry factor.

Broadening at the end of a peak due to additional delay of a fraction of the solute. Results in increased asymmetry factor.

A measure of chromatography peak broadening and column efficiency. Also referred to as the number of theoretical plates. Scientists use the plate number of a column to measure column efficiency. The more plates a packed column has, the higher the column efficiency.

The plate number is calculated from N = 5.545 × (V_R/W_h)² assuming a Gaussian peak, where:

• V_R = retention (elution) volume
• W_h = peak width at half peak-height.

V_R and W_h must have the same unit (e.g., mL). You can substitute V_R with retention time, t_R, but then the unit of W_h must be time.

Cavity in a chromatography matrix.

The total volume of the pores in a chromatography resin.

The pressure drop across the packed bed when solution passes through the column. Caused by flow resistance in the packed bed.

The amount of target protein retrieved after purification relative to the amount loaded on the column.

Measures the ability of a packed column to separate two solutes (peaks).

The time a buffer or solute spends in the column.

Same as elution volume.

Same as elution time.

Reversed phase chromatography (RPC) is a method based on hydrophobic interactions between solutes (sample components) and ligands coupled to the chromatography resin. Scientists use organic modifiers (e.g., acetonitrile) in the eluent for elution.

The material to be analyzed, or the material loaded on the chromatography column or resin.

Applying and loading sample on the column.

Same as sample application.

The volume of the sample loaded on the chromatography column or resin.

Measures the relative retention of two solutes in a column. Related to the distance between two peaks.

The dissolved substance (e.g., a protein).

Binding capacity determined at equilibrium by batch experiment.

Often called resin, chromatography particles, chromatography material, or chromatography medium or media.

Stepwise increase in concentration of the substance that affects elution of bound solutes.

See plate number.

Theoretical plate numbers per meter. A high value indicates a well packed bed. This value is used to compare columns packed with resins with the same particle size.

The elution volume of solutes that don’t enter the pores or interact with the chromatography resin, and pass between the particles in the packed bed.

Refers to the wash step, when scientists remove any unbound or weakly bound material from a column after the sample loading.

Buffer or solution used for washing the column after sample loading.

Volume of buffer or solution used for the wash step.

Amount of target protein (or other solute) obtained after a purification step, or after the entire purification (multiple steps).

Same as Band broadening.

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