Glossary Of HPLC-LC Separation Terms – D

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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Data acquisition rate: A term referring to the rate of sampling of a detector output. To characterize a chromatographic peak at least 20–30 data points must be collected. The data acquisition rate, usually measured in hertz, defines how many data points per second are collected while the peak is moving through the detector. For fast chromatography, the data acquisition rate must be sufficiently rapid to characterize a narrow peak. Modern detectors have data rates up to 80 Hz; also known as data rate and sampling rate.

Dead volume (V M ): The column dead volume comprises the entire space accessible to a small molecule that can fully permeate all pores of a packing material. It includes the interstitial volume and the unoccupied pore volume. It is denoted as V M. The system dead volume includes the additional volume in the tubing that connects the injector and detector to the column. The system dead volume usually is approximated by injecting a small, essentially unretained species. Uracil, acetone and thiourea are most commonly used species in reversed-phase chromatography. See also adjusted retention volume, holdup volume, and void volume.DEAE: See diethylaminoethyl.

Degassing: The process of removing dissolved gas from the mobile phase before or during use. Dissolved gas may come out of solution in the detector cell and cause baseline spikes and noise. Dissolved air can affect detectors such as electrochemical (by reaction) or fluorescence (by quenching) detectors. Dissolved gases also can cause pumps to lose their prime. Degassing is performed by heating the solvent, helium sparging, or using vacuum (in a vacuum flask) or on-line evacuation from a tube made of a gas-permeable substance such as polytetrafluoroethylene (PTFE).

Denaturing HPLC: Using reversed-phase HPLC to investigate genetic mutations by the investigation of DNA base pairs.

Desalting: Technique in which low molecular weight salts and other compounds can be removed from nonionic and high molecular weight compounds. An example is using a reversed-phase packing to retain sample compounds by hydrophobic effects yet allowing salts to pass through unretained. Using an SEC column to exclude large molecules and retain lower molecular weight salts is another example.

Dextran: Polydextran-based packing material primarily used for low-pressure biochromatography; an example would be Sephadex (Amersham Pharmacia Biotech, Piscataway, New Jersey).

Diethylaminoethyl (DEAE): A popular weak anion-exchange functionality (typically attached to cellulose or Sepharose [Amersham Pharmacia Biotech]) used for separating biomolecules.

Diffusion coefficient (D M or D S ): A fundamental parameter of a molecule in gas, solution (D M), or the stationary phase (D S). Expressed in square centimeters per second. D M is dependent on the molecular weight of the solute, temperature, solvent viscosity, and molar volume of the solute. A typical value for a 100-Da molecule in reversed-phase chromatography at room temperature is 10—5 cm2/s.

Diol phase: A hydrophilic phase that is useful in normal and reversed phase. It is a diol structure (two –OH groups on adjacent carbon atoms in an aliphatic chain). In normal-phase work, it is less polar than silica. It has been used to separate proteins and polypeptides in reversed-phase chromatography.

Displacement chromatography: A chromatographic process in which the sample is placed onto the column head and then is displaced by a compound that is more strongly sorbed than the compounds of the original mixture. Sample molecules then are displaced by each other and by the more strongly sorbed compound. The result is that the eluted sample solute zones may be sharpened; displacement techniques have been used mainly in preparative-scale HPLC applications.

Distribution constant (coefficient) (K c ): The total equilibrium concentration of a component in all forms or on the stationary phase divided by the total equilibrium concentration of the component in the mobile phase; also called the distribution coefficient or the partition coefficient in partition chromatography. In partition chromatography, K c is used when the concentration in the stationary phase is expressed per unit volume of the phase (V R = V M + K c V S). In a solid stationary phase, K g is used and is expressed per mass (weight) of the dry solid phase. In adsorption chromatography with a well-characterized adsorbent of known surface area, the concentration in the stationary phase is expressed per unit surface area.

D M See diffusion coefficient.

d p See particle size.D S See diffusion coefficient.

Dwell time: The time equivalent to dwell volume; determined by the product of flow rate and the dwell volume.

Dwell volume: The volume between the point of mixing of solvents (usually in the mixing chamber or at the proportioning valves in the liquid chromatograph) and the head of an LC column. Important in gradient elution or in isocratic elution situations when changes in solvent composition are made so that the column experiences the composition change in the shortest possible time. Low-pressure mixing systems generally have larger dwell volumes than high-pressure mixing systems.

Dynamic coating: The formation of in-situ coatings on the packing in HPLC or on capillary walls in CE by adding a substance to the mobile phase that adsorbs onto (or absorbs into) the packing or at the wall surface. The purpose of a dynamic coating is to generate a new stationary phase or to deactivate the packing material or capillary wall to prevent unwanted interactions. One simple example is the adjustment of the mobile phase or running buffer to less than pH 3 to protonate silanols and negate their effect. Another example is coating the phase with a hydrophilic polymeric material to prevent adsorption of proteins.

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