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HPLC Terms and Concepts
ELUTION: This term describes the transport of a species through the stationary phase by the continuous flow (addition) of mobile phase.
ELUANT: Mobile phase that carries the sample through the column.
ELUATE or EFFLUENT: Mobile phase with separated components after they emerge from the column.
ISOCRATIC ELUTION: A separation in which the mobile phase composition remains unaltered. The mobile phase may comprise of a single solvent or a pre-mixed mixture of solvents.
GRADIENT ELUTION: HPLC is frequently used for the separation of mixtures that contain compounds with a wide range of polarities. In such situations, isocratic conditions may not provide an acceptable separation (i.e., it is not possible to obtain sufficient resolution or the separation takes an unacceptably long period of time). To solve these problems, the composition of the mobile phase is changed during the separation. Two or three solvents that differ in polarity are employed. After sample introduction, the ratio of these solvents is programmed to vary either continuously or in steps, resulting in enhanced separation efficiency.
The terms ‘binary gradient’, ternary gradient’, and quaternary gradient’ refer to the use of 2, 3, and 4 solvents, respectively, to make up the mobile phase composition in a gradient elution method.
CHROMATOGRAM: When a detector that responds to solute concentration is placed at the column outlet, a plot of the generated signal versus time (or volume of mobile phase) is called a chromatogram. Such a plot, which usually comprises of one or more peaks, may be used for qualitative and quantitative purposes – the location of a peak on the time (or volume) axis serves to identify the component, and the area under the peak provides a quantitative measure of that component.
SENSITIVITY: Minimum limit of detection of a given species. Determined by the smallest ratio of ‘peak height-to-baseline noise’ (signal-to-noise ratio) that allows accurate and reproducible determination of peak height or area. This varies with detection method, instrument used and species being detected.
UNRETAINED COMPOUND: Component of a mixture that moves through a column at the same rate as the mobile phase, i.e., migration is not retarded by physical or chemical interaction with the stationary phase.
VOID VOLUME: Total volume of mobile phase in a fully wet packed column - the space between the particles of the stationary phase (interstitial volume) plus the volume within the particles (pore volume). It is also defined as the volume of mobile phase required to carry an unretained component through a column.
DISTRIBUTION CONSTANT: It describes the equilibrium involving the transfer of an analyte between the mobile and stationary phases. This constant, also called partition ratio or partition coefficient, is defined as K= Cs/Cm, a ratio of the analyte molar concentration in the stationary phase to that in the mobile phase.
RETENTION TIME, t: The time taken by the analyte peak to reach the detector after sample introduction is called the retention time. A more accurate measure of the retention time of an analyte is obtained by subtracting from this value the time taken for an unretained solute to emerge from the column (i.e., the dead time, t0), resulting in the adjusted retention time, t’. The retention time is the most important parameter for component identification under set experimental conditions.
CAPACITY FACTOR (or Retention Factor) k¢ : A measure of the retention volume (or time) of a particular component of the sample with a given combination of stationary phase and mobile phase. It is defined for species A as k¢ A = (tA-t0)/t0, or, k’A = t’A/t0 where t0 is the retention time for an unretained compound and t’A = adjusted retention time of species A.
ALPHA (a ) VALUE: A measure of the separation of any two components under a given set of conditions. It is defined for two components A and B as: a = k¢ A/k¢ B (where k¢ is the respective capacity factor). It is the ratio of the retention volumes of the two components; i.e., their relative retention.
BAND (or Zone): Layer of sample or component moving through the stationary phase, carried by the mobile phase. Represented on the chromatogram as a peak when it emerges from the column.
BAND-BROADENING (or Zone-broadening): Spreading of the sample or component band. Arises due to inefficiency of the column bed or inappropriate choice of mobile phase.
COLUMN EFFICIENCY: Degree to which species flow through the column as "bands", without being spread; less band-broadening implies less likely overlap of peaks in the chromatogram. Efficiency is expressed numerically as Plate Count, N, or as a Height Equivalent to Theoretical Plate (HETP), H. It is a measure of the quality of a filled column.
HETP, H = L/N
number of plates N = 16(t/W)2
where L = column length, t = retention time, and W = peak width at baseline.
The origin of these terms is from the treatment of the chromatographic column as made up of a number of discrete narrow bands called theoretical plates, similar to a distillation column.
RESOLUTION: Measure of the degree of separation between two successively eluting components in a chromatographic run (two adjacent peaks in a chromatogram).
The resolution between species A and B may be expressed as:
R = 2 [tB – tA]/ (WA + WB), where t and W correspond to the retention time and peak width at baseline, respectively. A resolution value of 1.5 implies a complete separation of the two species.
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