Chromatography Terms

Activity – In adsorption chromatography, the relative strength of the surface of the packing. For silica gel, the more exposed the silanol groups, the more active the surface. Activity can be controlled by adding water or another polar modifier, which is hydrogen-bonded to the active sites, thereby reducing the surface activity.

Affinity Chromatography – A technique in which, for the macromolecule of interest, a biospecific adsorbent is prepared by coupling a specific ligand (such as an enzyme, antigen or hormone) to a solid support (or carrier). This immobilized ligand will interact only with molecules that can selectively bind to it. Molecules that will not bind elute unretained. The retained compound can later be released in a purified state. Affinity chromatography is a selective filtration rather than a chromatographic technique.

Asymmetry – Asymmetry (skew) is a factor describing the shape of a chromatographic peak. Theoretically it is assumed peaks are symmetrical and of Gaussian shape. Practically, the peak asymmetry factor is measured as shown below.  A value of >1 indicates a tailing peak and <1 a fronting peak.

Bar – A unit of pressure equal to one atmosphere. It is equivalent to 14.5 pounds per square inch (psi) or 0.1 Megapascal.

BET Method – A method developed by Bruner, Emmett and Teller for measuring surface area. The level of liquid nitrogen adsorption within the pores of the phase is measured at very low temperatures. Pore volume and pore size distribution methods can also be obtained by this method.

Biocompatible – It refers to a tubing or fitting material that will not change the biological activity of material coming into contact with it during the HPLC analysis time.

Bonded – Term which implies that the stationary phase is chemically bonded to the surface of the supporting material.

Bonded Phase Coverage – It refers to the amount of bonded phase on a silica support. Coverage is usually described in mmol/m2 or in terms of percentage carbon.

Capacity Factor (k’) – A factor which measures sample retention (tR) independently of eluent flow rate or column
length. Retention times are measured relative to the column dead-time (t0). Its calculation is shown on the end of this page

Channelling – Occurs when voids created in the packing material of a column may cause eluent and accompanying solutes to move more rapidly than the average flow velocity, resulting in band broadening. The voids are created by poor packing or by erosion of the packed bed.

Check Valve – A device built into an HPLC pump which allows the flow of eluent in one direction only.

Column Dead-time (t0) – The time taken for solvent molecules or other non-retained peaks to move through the column.

Column Efficiency (N) – A term used to express the width of a peak produced by a column. Efficiency is measured in terms of the number of plates, a parameter which is inversely related to the square of the peak width.(See above under Assymetry) for the full calculation.

Counterion – In an ion-exchange process, the ion in solution used to displace the ion of interest from the ionic site. In ion-pairing, it is the ion of opposite charge added to the eluent to form a neutral ion pair in solution.

Dead Volume – A measure of solvent accessible volume between injector and detector after the space occupied by the column packing material has been subtracted. Both interstitial (intraparticle and interparticle) column volumes and system (injector, detector, connecting tubing and end fittings) volumes contribute. The dead volume can be determined by injecting an inert compound (ie. a compound that does not interact with the column packing) and measuring its retention volume.

Degassing – The practice of removing dissolved gases in the eluent. It can be achieved by helium sparging, applying vacuum to the eluent, ultrasonification or heating.

Denaturing – The process of destroying the tertiary and quaternary structure of a protein.

Desalting – A technique in which low molecular weight salts and other compounds are removed from non-ionic and high molecular weight compounds. An example is the use of size exclusion columns to exclude large molecules and retain lower molecular weight salts.

Eluotropic Series – A series of solvents with an increasing degree of polarity, generally used to explain solvent strength.

Endcapping – The reaction of a silylating reagent with unreacted accessible silanols remaining on the silica surface after the initial bonding reaction. The process may reduce undesirable adsorption of basic or polar molecules which otherwise may cause peak tailing.

Exclusion Limit – In SEC, the upper limit of molecular weight (or size), beyond which molecules will elute at the same retention volume (exclusion volume). Many SEC packings are referred to by their exclusion limit. For example, a 105 column of porous silica gel will exclude any compounds with a molecular weight higher than 100,000 based on a polystyrene calibration standard.

Flash Chromatography – A very fast form of classic LC used by synthetic organic chemists for rapid purification. Performed primarily in the normal-phase mode, sometimes with reversed-phase chromatography.

Fractionation Range – In SEC, refers to the range in  which the packing can separate molecules based on their size. Molecules that are too large to diffuse into the pores are excluded. Molecules that can diffuse into all of the pores totally permeate the packing, eluting unseparated at the permeation volume.

Fronting – A term describing a peak shape whose front has a leading edge. Gel Filtration Chromatography (GFC). SEC carried out with aqueous eluents. It is sometimes referred to as aqueous GPC. Most gel filtration separations involve biopolymers.

Gel Permeation Chromatography (GPC) – SEC carried out with organic eluents. Used for the separation and characterisation of polymers.

Gradient Elution – The process by which the strength and composition of the eluent is increased during the chromatographic run thereby reducing analysis time.

Guard Column – A short column placed between sample injector and the inlet of the main column. It protects the analytical column against contamination from sample particulates and strongly retained solutes. The guard column is usually of cartridge format and packed with the same material as in the main column.

Helium Sparging – The process of bubbling helium through the eluent to remove dissolved gas.

High Pressure Mixing – A procedure in which two or more solvents are mixed on the high pressure side of the pumping system to form a final eluent. One pump is required per solvent.

Hydrophilic – A description of compounds, solvents or bonded phases that either readily dissolve in water or prefer water to non-polar organic solvents, ie. ‘water-loving’.

Hydrophilic Interaction Chromatography (HILIC) – The use of polar stationary phases and partially aqueous eluents to separate compounds in order of increasing hydrophilicity (polarity).

Hydrophobic – A term describing compounds, solvents or bonded phases that dissolve easily in non-polar organic solvents such as hexane or prefer such solvents to water, ie. ‘water-hating’.

Hydrophobic Interaction Chromatography (HIC) – A protein separation technique in which reversed-phase materials are used with eluents containing high salt concentrations. Gradients are run by decreasing salt concentrations with time.

Injection Solvent – The solvent the sample is dissolved in prior to chromatographic analysis.

Ion-exchange Capacity – A measure of the number of ionic sites that can take part in the exchange process. Exchange capacity is expressed in mequiv/g. Ion Exclusion. The process in which ionised solutes can be separated from non-ionised or partially ionised solutes using ion-exchange resins. Ionised solutes will move faster down the column.

Ion-pair Chromatography – A form of reversed-phase chromatography in which ions in solution can be paired or
neutralised prior to separation as an ion-pair. Ion-pairing reagents are usually ionic compounds that contain a hydrocarbon chain. The latter imparts a certain hydrophobicity to the resultant ion-pair allowing it to be retained on a reversed-phase column.

Isocratic – Chromatographic conditions in which a constant composition eluent is used.

Isoelectric Point – The pH point at which a molecule no longer has a net charge.

Ligand-exchange Chromatography – A technique in which chelating ligands are added to the eluent. On adsorption onto the stationary phase they act as chelating agents. An example is the use of copper amine chelates for the separation of amino acids.

Low Pressure Mixing – A pumping procedure in which two or more solvents are mixed on the low pressure side of the pump. Only one pump is required.

Mean Pore Diameter – A term that refers to the average diameter of the pores within a phase.

MegaPascal (Mpa). A unit of pressure. One Mpa equals about 10 bar (atmospheres) or 145 pounds per square inch (psi).

Modifier – A chemical added to reversed-phase solvent systems designed to optimise the chromatographic separation.

Monomeric Phase – A bonded phase in which individual molecules are bonded to a support. For silica, monomeric phases are typically prepared by the reaction of an alkyl monochlorosilane.

Overload – A saturation of the stationary phase by the solute which is evidenced by band broadening, tailing and
flat edged chromatographic peaks.

Particle Size Distribution – A measure of the distribution of the particles used to manufacture a column. In HPLC a narrow particle size distribution is desirable. For a 10µm size particle, a particle size distribution of dp±10% means that 90% of the particles have a 9-11µm size.

Particle Size (dp) – This term refers to the average particle size of the material packed into a column.

Peak Broadening – The tendency of a chromatographic peak to broaden as it passes through the column. It is also known as peak spreading or peak dispersion. The peak width or the number of theoretical plates in the column (N) is a measure of peak broadening.

Polarity – A measure of the separation of charges within a molecule. Polar molecules interact more strongly with and are best separated on polar stationary phases.

Polymeric Packing – Packings based on polymeric materials, usually in the form of spherical beads. Common polymers include polystyrene-divinylbenzene, polymethacrylate and polyvinylalcohol.

Polymeric Phase – A bonded phase in which typically a di- or trichlorosilane is reacted with more than one reactive silanol group on the surface of silica.

Pore Size (Mean Pore Diameter) – The average pore diameter of the pore in a porous packing. The pore diameter is important in that it must allow free diffusion of solute molecules into and out of the pore so that the solute can interact with the stationary phase. In SEC, the packings have different pore diameters, and therefore molecules of different sizes can be separated. For a typical adsorbent such as silica gel, 60Å and 100Å pore diameters are most popular. For packings used for the separation of biomolecules, pore diameters >300Å are used.

Pore Volume – The total volume of the pores in a porous packing, usually expressed in ml/g. It is measured by the BET method of nitrogen adsorption or by mercury intrusion, in which mercury is pumped into the pores under high pressure.

Precolumn – A column packed with silica placed between the pump and the injector. It presaturates the eluent with stationary phase minimising loss of the latter from the main column. It will also remove particulate material

Pressure Drop – The difference in pressure between the inlet and outlet of a column during flow caused by the hydrodynamic resistance of the packed bed.

Residual Silanols – These are the silanol (-SiOH) groups that remain on the surface of a silica after a bulky phase is chemically bonded to its surface. Their numbers can be reduced by further reacting (endcapping) the silica surface with a small organosilane.

Resolution – A measure of the separation of two adjacent peaks. The higher the resolution value the greater the separation (see HPLC Calulation below)

Retention Time – The elapsed time between sample injection and the appearance of the chromatographic peak apex.

Sample Capacity – The term refers to the amount of sample that can be injected onto a column without overloading it. In preparative applications it is typically expressed as grams of solute per gram of stationary phase.

Selectivity (α) – The ratio of capacity factors of adjacent peaks (see p.342). Also referred to as separation factor or relative retention ratio.

Siloxane Bond – The main -Si-O-Si- bond found in silica.

Size Exclusion Chromatography (SEC) – A mode of HPLC used mainly to separate high molecular weight samples and to determine their molecular weight distribution by virtue of their size in solution. Also known as gel permeation, gel filtration or steric exclusion chromatography.

Surface Area – The total area of the phase’s solid surface. For silica it is typically 100-600 m2/g. Tailing. The phenomenon in which a peak has an asymmetry factor >1. The downside of the peak will be skew.

Theoretical Plate – Measure of column efficiency. Length of column relating to this concept is called height equivalent to a theoretical plate (HETP).

Void – The formation of a space, usually at the head of the column, caused by a settling or dissolution of the packing. A void in the column leads to decreased efficiency and loss of resolution.

Void Volume (V0) – The total volume of eluent in the column, the remainder being taken up by packing material. Can be determined by injecting an unretained substance.

Zero Dead Volume (ZDV) – It refers to a fitting which adds no extra volume to the system. In practice ZDV fittingshave a finite but insignificant volume.

Zwitterions – Compounds that carry both positive and negative charges in solution.