Links:

A-term. The first term in the van Deemter equation. See Eddy diffusion term and van Deemter equation.

Active Moiety. Portion of a molecule responsible for the physiological or pharmacological action of a drug substance.

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.

Adjusted retention time (t 'R). A measure of the retention time adjusted for the void volume. t'R = tR - tm (or t0).

Adjusted retention volume (V'R). Adjusts the retention volume for the dead volume. V'R = VR - Vm, where Vm, is the dead, or void, volume.

Adsorbent. Packing used in adsorption chromatography. Silica gel and alumina are the most frequently used adsorbents in HPLC.

Adsorption. The process of interaction between the solute and the surface of an adsorbent. The forces involved can be strong (for example, hydrogen bonds) or weak (van der Waals forces). For silica gel, the silanol group is the driving force for adsorption, and any solute functional group that can interact with this group can be retained by liquid-solid chromatography on silica.

Adsorption chromatography. One of the basic LC modes. Relies on the adsorption process to effect the separation. Silica gel and alumina are the most frequently used supports; molecules are retained by the interaction of their polar functional groups with the surface functional groups (e.g., silanols of silica).

Adsorption isotherm. In adsorption, a plot of the equilibrium concentration of sample in the mobile phase per unit volume vs. the concentration in the stationary phase per unit weight. The shape of the adsorption isotherm can determine the chromatographic behavior of the solute: tailing. fronting, overload, etc.

Aerogel. A packing that can be prepared if the dispersing agent can be removed from a gel system without collapsing the gel structure. Silica gels and glass beads used for size-exclusion chromatography (SEC) are examples of aerogels, which can retain their structures even at the high pressures used in HPLC .

Affinity chromatography. A technique in which a biospecific adsorbent is prepared by coupling a specific ligand (such as an enzyme, antigen. or hormone) for the macromolecule of interest 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 not a chromatographic technique as such, but is actually selective filtration.

Agarose. High-molecular-weight polysaccharide used as a separation medium in biochromatography. It is used in bead form, often in gel-filtration chromatography with aqueous mobile phases.

Alkoxysilane. A reactant used for the preparation of chemically bonded phases. It will react with silica gel as follows: R3SiOR + =SiOH - =Si-O-SiR3 + ROH

Alumina. An adsorbent sometimes used in adsorption chromatography. Aluminum oxide (Al2O3) is a porous adsorbent that is available with a slightly basic surface. For this reason, it can have advantages over silica, which is considered to have an acidic surface.

Amino phase. A propylamino stationary phase used mostly in normal bonded-phase chromatography. It is somewhat reactive for any solute molecule or mobile phase additive that can react with amines. The amino phase has found some applications as a weak anion exchanger.

Amphoteric ion-exchange resin. Ion-exchange resins that have both positive and negative ionic groups. Are most useful for ion retardation in which all ionic materials can be removed from solution; the anionic and cationic functionalities coexist on the same material .

Anion-exchange chromatography. The ion-exchange procedure used for the separation of anions. Both resins and bonded phases are available for this mode. The tetralkylammonium group is a typical strong anion-exchange functional group. An amino group on a bonded or coated stationary phase would be an example of a weak anion exchanger.

Asymmetry. Factor describing the shape of a chromatographic peak. Theory assumes a Gaussian shape and that peaks are symmetrical. The peak asymmetry factor is the ratio (at 10% of the peak height) of the distance between the peak apex and the back side of the chromatographic curve to the distance between the peak apex and the front side of the chromatographic curve. A value > 1 is a tailing peak, while a value <1 is a fronting peak.

B-term. Same as Molecular diffusion term.

Backflushing. A column-switching technique in which a four-way valve placed between the injector and the column allows for mobile phase flow in either direction. Backflushing is used to elute strongly held compounds at the head of the column. It can be used to analyze these compounds or merely to remove them from the column.

Band broadening. The dilution of the chromatographic band as it moves down the column. The peak is injected as a slug, and, if not for the process of band broadening, each separated component would elute as a narrow slug of pure compound. The measure of band broadening is band width, tw, or more correctly, the number of theoretical plates in the column. N.

Band dispersion. See Band broadening

Band spreading. See Band broadening.

Band width. The width of the chromatographic band during elution from the column. It is usually measured at the baseline by drawing tangents to the sides of the Gaussian curve representing the peak. Small band widths usually represent efficient separations. Also referred to as peak width.

Batch. A specific quantity of a drug or other material that is intended to have uniform character and quality, within specified limits, and is produced according to a single manufacturing order during the same cycle of manufacture.

Batch Formula. The complete listing of all the ingredients and their amounts to be used for the manufacture of a representative batch of a drug product. A separate batch formula is submitted for each formulation of a drug product. All ingredients must be listed in the batch formula, whether or not they remain in the finished product.

BET method. A method developed by Bruner, Emmett, and Teller for measuring surface area by using nitrogen adsorption condensation in pores at liquid nitrogen temperature. Pore volume and pore-size distribution can also be obtained by this method.

Bioavailability. The rate and extent to which an active drug ingredient or therapeutic moiety is absorbed from a drug product and becomes available at the site of drug action.

Biocompatible. Term to indicate that the column or instrument component will not irreversibly or strongly adsorb or deactivate biomolecules such as proteins.

Bonded-phase chromatography (BPC). The most popular LC mode. A stationary phase chemically bonded to a support is used for the separation. The most popular Support is microparticulate silica gel. and the most popular type of bonded phase is the organosilane, such as octadecyl (for reversed-phase chromatography). Approximately 70% of all HPLC is carried out on chemically bonded phases.

Bioequivalent Drug Products. Pharmaceutical equivalents whose rate and extent of absortion do not show a significant difference when administered at the same therapeutic moiety molar dose under single or multiple dose.

Breakthrough volume. The volume at which a particular solute pumped continuously through a column will begin to elute The breakthrough volume is useful in determining the total sample capacity of the column for a particular solute.

C4, C8, C18, etc. Refers to the alkyl chain length of a reversed bonded phase.

C-term. The mass transfer term of the van Deemter equation. See Mass transfer and van Deemter equation .

Capacity. See Sample capacity.

Capacity factor. A chromatographic parameter that measures the degree of retention . See k ' for calculation method.

Capillary tubing. Tubing for connecting various parts of the chromatograph. Most capillary tubing used in HPLC is <0.020 in. i d. The smallest useful i d. is about 0.004 in.

Capping. Same as Endcapping.

Carbon Loading. The amount of stationary phase coated or bonded onto a solid support. In liquid-liquid chromatography, the milligram amount of liquid phase per gram of packing. In RPC. the loading may be expressed in µmol/m2 or in %C.

Carrier. A term used most often in affinity chromatography. Refers to the support that is used to carry the active ligand, usually by a covalent bond. Can also refer to the Support in other chromatographic modes .

Cartridge column. A type of column that has no end fittings and is held in a cartridge holder. The column is a tube; the packing is contained by frits in each end of the tube. Cartridges are easy to change and are less expensive and more convenient than conventional columns with end fittings.

Cation-exchange chromatography. The form of ion exchange chromatography that uses resins or packings with functional groups that can separate cations. A sulfonic acid would be an example of a strong cation-exchange group; a carboxylic acid would be a weak cation-exchange group.

Chain length. The length of carbon chain in the hydrocarbon portion of a reversed-phase packing. It is expressed as the number of carbon atoms (e.g., C8. C18).

Challenge Condition. A purposely generated deviation in a process stream condition, intended to test the ability of the finished product to meet specifications.

Channeling. Occurs when voids created in the packing material of a column may cause mobile phase 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.

Chemisorption. Sorption caused by a chemical reaction with the packing. Most such interactions are irreversible; usually occur on packings with reactive functional groups such as silanol or bonded amino phases .

Chiral stationary phases (CSP). Stationary phases that are designed to separate enantiomeric compounds. They can be bonded to solid supports or created in situ on the surface of the solid support, or they can be surface cavities that allow specific interactions with one enantiomeric form.

Chromatogram. A plot of detector signal output versus time or elution volume during the chromatographic process.

Column back pressure. See Head pressure.

Column chromatography. Any form of chromatography that uses a column or tube to hold the stationary phase. Open-column chromatography, HPLC, and open-tubular capillary chromatography are all examples .

Column performance. Refers to the efficiency of a column; measured as the number of theoretical plates for a given test compound.

Column switching. The use of multiple columns connected by switching valves to effect better chromatographic separations or for sample cleanup. Fractions from a primary column can be switched to two or more secondary columns, which in turn can be further diverted to additional columns or to the detector(s) .

Controlled surface porosity support. Same as Porous-layer bead.

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 mobile phase to form a neutral ion pair in solution.

Coupled columns. A form of column switching. Uses a primary column connected to two secondary columns via a selector valve. Fractions from the first column can be selectively transferred to the other two columns for additional separation. Term also used to describe two or more columns connected in series to provide increased plate numbers.

Cross-linking. During the process of copolymerization of resins to form a three dimensional matrix, a difunctional monomer is added to form cross-linkages between adjacent polymer chains. The degree of cross-linking is determined by the amount of this monomer added to the reaction. For example, divinylbenzene is a typical cross-linking agent for polystyrene ion-exchange resins. The swelling and diffusion characteristics of a resin are governed by its degree of cross-linking.

Cyano phase. A stationary phase that usually consists of cyanopropylsilyl groups; used in both normal and reversed-phase chromatography.

Dead volume (Vd). Also known as Void Volume (V0). The volume outside of the column packing itself. The interstitial volume (intraparticle volume + interparticle volume) plus extra column volume (contributed by injector, detector, connecting tubing, and end fittings) all combine to create the dead volume. This volume can be determined by injecting an inert compound (i.e.. a compound that does not interact with the column packing).

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 electrochemical detectors (by reaction) or fluorescence detectors (by quenching). Degassing is carried out by heating the solvent or by vacuum (in a vacuum flask), or on-line using evacuation of a tube made from a gas-permeable substance such as PTFE, or by helium sparging.

Desalting. Technique in which low-molecular-weight salts and other compounds are removed from nonionic and high-molecular-weight compounds. An example is the use of a reversed-phase packing to retain sample compounds by hydrophobic effects but to allow salts to pass through unretained. Use of an SEC column to exclude large molecules and retain lower-molecular-weight salts is another example.

Diffusion coefficient (Dm or Ds). A fundamental parameter of a molecule in solution (Dm) or in stationary phase (Ds). Expressed in cm2/s. Dm depends on molecular weight of the solute, on temperature, on solvent viscosity, and on molar volume of the solute. A typical value of a small molecule in RPC at room temperature would be S x 10 cm2/s.

Diol phase. A stationary phase useful in both normal and reversed-phase chromatography. Consists of a diol structure (two -OH groups on adjacent carbon atoms in an aliphatic chain). Less polar than silica stationary phases used in normal-phase chromatography; has been used for the reversed-phase separation of proteins and polypeptides.

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

Distribution coefficient (D or KD). See Partition coefficient.

Eddy diffusion term. The A-term in the van Deemter equation. It is the contribution to plate height that is due to molecules traveling along different paths through the column: depends on the particle size and geometry of the packing. Also called the multipath term. A = 2 (lambda)dp. See van Deemter equation.

Effective theoretical plates (Neff). The true number of plates in a column; corrects theoretical plates for dead volume. Neff = 16(tR -tm)2 , where tm is the void time.

Efficiency (N or H). A measure determined by the number of theoretical plates calculated from the equation shown for H (see HETP).

Effluent. Same as Eluate.

Eluate. Combination of mobile phase and solute exiting column; also called effluent.

Eluent. Mobile phase used to carry out a separation.

Eluotropic series. A series of solvents with an increasing degree of polarity, generally used to explain solvent strength in liquid-solid or adsorption chromatography. A nonpolar solvent such as pentane would be at one end of the scale; dichloromethane would be an intermediate solvent; a strongly polar solvent, such as water, would be at the other end of the scale. Thus, when developing a method or running a gradient, an eluotropic series is useful for selecting solvents .

Elution. The process of passing mobile phase through the column to transport solutes.

Elution chromatography. The most commonly used chromatographic method. The sample is applied to the head of the column, and individual molecules are separated and eluted at the end of the column. Compare Displacement chromatography and Frontal analysis.

Elution volume ( VR). Refers to the volume of mobile phase required to elute a solute from the column at maximum concentration (apex). VR = F o tR.

Elutriation. A technique used to fractionate packing particles by size. Most often used for the separation of ion-exchange resins, such as amino acid resins. that must have a particularly narrow size range. The technique involves the upward flow of water into a large tube. The unsized beads are added to the moving water, and the particles seek their own level, depending on their density and particle size. They are then removed at certain levels in the tube.

Endcapping. A column is said to be end capped when a small silylating agent (e.g., trimethylchlorosilane) is used to bond residual silanol groups on a packing surface. Most often used with reversed-phase packings. May cut down on undesirable adsorption of basic or ionic compounds.

End fitting. The fining at the end of the column that connects it to the injector or detector. Most HPLC end fittings contain a frit to hold the packing and have a low dead volume for minimum band spreading. Usually made of stainless steel.

Exchange capacity. See Ion-exchange capacity.

Exclusion chromatography. See Steric-exclusion chromatography.

Exclusion limit. In SEC, the upper limit of molecular weight (or size), beyond which molecules will elute at the same retention volume, called the exclusion volume. Many SEC packings are referred to by their exclusion limit. For example, a 10 column of porous silica gel will exclude any compounds with a molecular weight higher than 100,000, based on a polystyrene calibration standard.

Exclusion volume (Ve). The retention volume of a molecule on an SEC packing; all molecules larger than the size of the largest pore are totally excluded and elute at the interstitial volume of the column.

Extra column effects. The band-broadening effects of pans of the chromatographic system outside of the column itself. Extra column effects must be minimized in order to maintain the efficiency of the column. Areas of band broadening can include the injector, connecting tubing, end fittings, frits, detector cell volume, and internal detector tubing. The variances of all of these contributions are additive.

Fast LC. The use in HPLC of short columns (3-7 cm in length) with conventional internal diameters (2-6 mm), packed with small particles (3- or 5- µm dp). Separation times in minutes, sometimes seconds. are common .

Flow rate (F). The volumetric rate of flow of mobile phase through an LC column. For a conventional HPLC column of 4.6-mm i.d.. typical flow rates are 1 to 2 ml/min.

Fractionation range. In SEC, refers to the operating range of a gel or packing This is 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.

Frit. The porous element at either end of a column that serves to contain the column packing. It is placed at the very ends of the column tube or, more commonly, in the end fitting. Frits are made from stainless steel or other inert metal or plastic, such as porous PTFE or polypropylene.

Frontal analysis. Chromatographic technique that involves continuous addition of sample to the column. with the result that only the least-sorbed compound, which moves at the fastest rate. is obtained in a pure state. The second least-sorbed compound elutes with the first-eluting compound, the third least-sorbed compound with the first and second compound, etc., until the original sample is eluting at the column exit . Frontal analysis is seldom used and is mainly a preparative technique.

Fronting. Peak shape in which the front pan of a peak (before the apex) in a chromatogram tapers in advance of the remainder of the peak. There is an asymmetric distribution with a leading edge. The asymmetry factor for a fronting peak has a value < 1. The opposite effect is tailing. Fronting is related to the shape of the sorption isotherm.

Gaussian curve. A standard error curve, based on a mathematical function. that is a symmetrical, bell shaped band or peak. Most chromatographic theory assumes a Gaussian peak.

Gel. The solid packing (media) used in gel permeation chromatography. A gel actually consists of two parts: the dispersed medium (solid portion) and the dispersing medium (the solvent).

Gel filtration chromatography (GFC). Size-exclusion chromatography carried out with aqueous mobile phases. Generally refers to separations carried out on soft gels such as polydextrans. Most gel filtration separations involve biopolymers.

Gel permeation chromatography (GPC). SEC carried out with organic mobile phases. Used for the separation and characterization of polymers SEC with aqueous mobile phases is referred to as aqueous GPC, or GFC.

Gradient elution. Technique for decreasing separation time by increasing mobile phase strength over time during the chromatographic separation. Also known as solvent programming. Gradients can be continuous or stepwise. Binary, ternary, and quaternary solvent gradients have been used routinely in HPLC .

Guard column. A small column placed between the injector and the analytical column. Protects the analytical column against contamination by sample particulate and, perhaps. by strongly retained species. The guard column is usually packed with the same material as the analytical column and is often of the same i.d. It is much shorter, costs less, and is usually discarded when it becomes contaminated.

H. Same as HETP.

Head pressure. The pressure above gravity at the head of the column. Expressed in pig. bar. ATM, or Map

Heart cutting. In preparative LC, refers to collection of the center of the peak, where purity should be maximum. Also used in column switching.

HETP. Height equivalent to a theoretical plate. A carryover from distillation theory: a measure of a column's efficiency. For a typical HPLC column well-packed with 5 µm particles, HETP (or H) values are usually between 0.01 and 0.03 mm. HETP = L/N, where L is column length and N is the number of theoretical plates.

Hydrophilic. ''Water-loving'': refers both to stationary phases that are compatible with water and to water soluble molecules in general. Most columns used to separate proteins are hydrophilic in nature and should not sorb or denature protein in the aqueous environment.

Hydrophobic. 'Water-hating'': refers both to stationary phases that are not compatible with water and to molecules in general that have little affinity for water. Hydrophobic molecules have few polar functional groups: most are hydrocarbons or have high hydrocarbon content.

Hydrophobic interaction chromatography. A technique in which reversed-phase packings are used to separate molecules by virtue of the interactions between their hydrophobic moieties and the hydrophobic sites on the surface. High salt concentrations are used in the mobile phase; separations are effected by changing the salt concentration. The technique is analogous to salting out'' molecules from solution. Gradients are run by decreasing the salt concentration over time.

In-line filter. A device that prevents particulate matter from damaging the column. Modern in-line filters can be placed between the injector and the column without contributing to band broadening. A filter in this position is used to prevent sample particulate from entering the packed bed or the inlet frit.

Infinite diameter effect. Name given by John Knox to the following phenomenon: At a certain column length, a sample injected into the center of the packed bed spreads by radial diffusion but never reaches the column wall, where wall effects can cause band broadening. Knox showed that a sample peak collected in the exact center of the column exit displayed a higher efficiency than a sample peak collected near the wall. The infinite diameter effect depends on column length, internal diameter, particle size, and mobile phase properties. Very seldom applied in HPLC.

Inlet. The initial part of the column, where the solvent and sample enter. There is usually an inlet frit that holds the packing in place and, in some cases, protects the packed bed.

Interstitial volume (V0). The total volume of mobile phase within the length of the column. It is made up of the intraparticle volume (inside the packing itself) and interparticle volume (between the packing particles). Same as Void volume. Also abbreviated V or Vm.

Ion chromatography (IC). An ion-exchange technique in which low concentrations of anions or cations are determined using low capacity ion exchangers with weak buffers. Conductivity detectors are often used. Ion chromatography is practiced in two forms. In suppressed IC, a second column is used to remove the buffer ions so that sample ions can be more easily detected: a membrane separator is sometimes used. In non suppressed IC, weakly conducting buffers at low concentration are carefully selected. and the entire eluent is passed through the detector: ions are detected above the background signal .

Ion-exchange chromatography (IEC). A mode of chromatography in which ionic substances are separated on cationic or anionic sites of the packing. The sample ion (and usually a counterion) will exchange with ions already on the ionic group of the packing. Retention is based on the affinity of different ions for the site and on a number of other solution parameters (pH, ionic strength, counterion type, etc. ).

Ion-exchange capacity. The number of ionic sites on the packing that can take part in the exchange process. Exchange capacity is expressed in mequiv/g: typical strong anion-exchange resin may have 3-5 mequiv/g capacity.

Ion exclusion. The process in which ionized solutes can be separated from un-ionized or partially ionized solutes using ion-exchange resins. Separation results from Donnan membrane potential, where ionic solutes exist at a higher concentration in solution than in the resin whereas nonionic solutes are evenly distributed between the mobile phase and resin. Therefore. ionic solutes will move faster down the column than the nonionic solutes.

Ion-pair chomatography. Form of chromatography in which ions in solution can be paired" or neutralized and separated as an ion pair on a reversed-phase column. Ion-pairing agents are usually ionic compounds that contain a hydrocarbon chain that imparts a certain hydrophobicity so that the ion pair can be retained on a reversed-phase column. Ion-pairing can also occur in normal-phase chromatography when one pan of the pair is loaded onto a sorbent. but this technique is not as popular as the RPC technique .

Ion retardation. Refers to the use of amphoteric ion exchange resins that are used to retard ionic molecules and allow nonionic molecules or nonelectrolytes to elute preferentially.

Ion suppression. Buffering in an aqueous mobile phase at a particular pH to suppress solute ionization For example. one can suppress the ionization of weak carboxylic acids by adjusting the pH below their ionization constant. Useful for improving peak shape of weak acids and bases in RPC.

Irregular packing. Refers to the shape of a silica gel based packing. Irregular silicas are available in microparticulate sizes. The packings are made by grinding silica gel into small particles and then sizing them into narrow fractions using classification machinery. Spherical packings are now used more often than irregular packings in HPLC, but less-expensive irregular packings are still widely used in prep LC.

Irreversible adsorption. When a compound that has a very strong affinity for the adsorbent is injected onto a column, it can be adsorbed so strongly that it cannot be eluted from the column. A chemical reaction between the sample and the surface of the adsorbent is an example of irreversible adsorption.

Isocratic. Use of a constant-composition mobile phase in liquid chromatography.

k'. Capacity factor; k' = (mass in stationary phase) (mass in mobile phase). Can be calculated from the equation k ' = (tR - t0)/t0, where tR is retention time for the sample peak, and t0 is the retention time for an unretained peak.

Kieselguhr. A diatomaceous earth used both in column chromatography and as a sample cleanup medium. Only weakly adsorptive, it is used as a support in liquid liquid chromatography. Rarely used in HPLC.

Knox equation. A modification of the van Deemter equation developed by John Knox

Ligand. In ligand-exchange chromatography. refers to the molecule added to the mobile phase that acts as the chelating agent. In affinity chromatography, refers to the biospecific material (enzyme. antigen, or hormone) coupled to the Support (carrier) to form the affinity column.

Ligand-exchange chromatography. A technique in which chelating ligands are added to the mobile phase and undergo sorption onto a packing. These sorbed molecules can act as chelating agents with solutes. An example would be the use of copper amine chelates for the separation of amino acids Chelating resins function in a similar manner: chelating groups are chemically bonded to the polystyrene backbone.

Linear elution adsorption chromatography (LEAC). A term coined by Lloyd Snyder. Refers to adsorption chromatography carried out in the linear portion of an adsorption isotherm.

Linear velocity (u). The velocity of the mobile phase moving through the column. Expressed in cm/s. Related to flow rate by the cross-sectional area of the column. Sometimes expressed as v.

Liquid-liquid chromatography (LLC). Same as Partition chromatography. The earliest form of HPLC, it gave way to chemically bonded phases in the early 1970s.

Liquid-solid chromatography (LSC). Same as Adsorption chromatography.

Longitudinal diffusion. Same as Molecular diffusion term; B-term in van Deemter equation . See van Deemter equation.

Lot. A batch, or a specific intended portion of a batch, having uniform character and quality within specified limits; or, in the case of a drug product produced by a continuous process, a specific amount produced in a unit of time or quantity in a manner that assures having uniform character and quality within specified limits.

Macroporous resin. Cross linked ion-exchange resins that have both micropores of molecular dimensions and macropores several hundred Å wide. These are highly porous resins with large internal surface areas accessible to large molecules.

Mass transfer. The process of solute movement into and out of the stationary phase or mobile phase. The C-term of the van Deemter equation is referred to as the mass transfer term. The faster the process of mass transfer, the better the efficiency of the column. In HPLC, mass transfer is the most important factor affecting column efficiency. It is increased by the use of small-particle packings, thin layers of stationary phase, low viscosity mobile phases, and high temperatures.

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. 60-Å and 100-Å pore diameters are most popular. For packings used for the separation of biomolecules, pore diameters > 300 Å are used.

Micellar chromatography. The addition of micelles to the mobile phase to effect separations. The micelles act as displacing or partitioning agents and provide another parameter that can be used to change selectivity,

Micro LC. Refers collectively to techniques in which a column of smaller-than-usual internal diameter (i.d.) is used for separation. In micro HPLC. columns of <0.5-mm i.d. are used.

Microbore. Refers to columns with smaller-than usual internal diameters (<2 mm) used in HPLC.

Microparticulate. Refers to small particles used as HPLC stationary phases. Generally, packings with a particle diameter < 10 um that are totally porous are considered microparticles.

Microporous resin. Same as Micro reticular resin.

Micro reticular resin. Cross-linked synthetic ion exchange resins with pore openings that correspond to molecular sizes. Diffusion into the narrow pores can be impaired, and low exchange rates, as well as poor performance, can occur, especially for large molecules .

Minimum plate height. The minimum of the curve that results from a plot of H vs. u. This value represents the most theoretical plates that can be obtained for a certain column and mobile phase system. Usually occurs at very low flow rates.

Mixed-bed column. Combination of two or more stationary phases in the same column. used most often in IEC and SEC. Advantage in IEC is the total removal of ionic compounds: useful in SEC because a wider molecular-weight range can be accommodated by the column.

Mobile phase. The solvent that moves the solute through the column.

Modifier. Additive that changes the character of the mobile phase. For example. in reversed phase, water is the weak solvent; methanol, the strong solvent, is sometimes called the modifier.

Molecular diffusion term. Refers to the B-term (second term) of the van Deemter equation. Also called longitudinal or axial diffusion term. Important in band broadening only at very low flow rates below the minimum plate height where the diffusion of individual solutes can occur in a longitudinal (lengthwise) direction on the column. See van Deemter equation.

Molecular weight distribution. The distribution of molecular weight of molecules in a polymer sample. Distribution can be defined as weight average and number average.

Monomeric phase. Refers to a bonded phase in which single molecules are bonded to a Support. For silica gel, monomeric phases are prepared by the reaction of an alkyl or aryl monochlorosilane. Polymeric phases are generally prepared from a di- or trichlorosilane reactant.

Multidimensionai chromatography. The use of two or more columns or chromatographic techniques to effect a better separation. Useful for sample cleanup, increased resolution, and increased throughput. It can be used off-line by collecting fractions and reinjecting onto a second column or on-line by the use of a switching valve. Also called coupled-column chromatography, column switching, multicolumn chromatography, or box-car chromatography.

N, The number of theoretical plates. N = 16(tR/Wb)2, where tR is retention time, and Wb is the base width of the peak. A measure of the efficiency of a column. Sometimes measured as N = 5.54(tR/W 1/2)2 where w1/2 is the peak width at half height.

Narrow-bore column. Columns of <0.5-mm i.d. used in HPLC. At this time. few columns of this type are commercially available.

Nonporous particle. Refers to a solid particle used as a Support for a porous-coated or bonded phase.

Normal-phase chromatography. A mode of chromatography carried out with a polar stationary phase and a nonpolar mobile phase. Adsorption on silica gel using hexane as a mobile phase would be a typical normal-phase system. Also; refers to the use of polar bonded phases, such as CN or NH2. Sometimes referred to as straight-phase chromatography.

Octadecylsilane (ODS = RP-18). The most popular reversed phase in HPLC. Octadecylsilane phases are bonded to silica or polymeric packings. Both monomeric and polymeric phases are available.

Open-tubular columns. Columns of small internal diameter, currently being investigated for HPLC. SFC, and capillary electrophoresis. Stationary phases can be bonded on the internal walls of these columns. The most common type is the fused-silica tubing made for capillary GC.

Optically active resin. Incorporation of optically active groups into an ion-exchange resin to allow separation of optically active isomers. Not many are commercially available in HPLC.

Organic modifier. Water-miscible organic solvent added to an aqueous mobile phase to effect separations in reversed-phase HPLC.

Overload. In preparative chromatography, the overload condition is defined as the mass of sample injected onto the column at which efficiency and resolution begin to be affected if the sample size is further increased. See Sample capacity.

Packing. The adsorbent, gel, or solid Support used in the HPLC column. Most HPLC packings are <10 um in average diameter.

Paired-ion chromatography. Same as Ion-pair chromatography.

Particle size (dp). The average particle size of the packing in an LC column. A 5-um column would be packed with particles having definite particle size distribution; packings are never monodisperse. See Particle-size distribution.

Particle-size distribution. A measure of the distribution of the particles used to pack the LC column. In HPLC, a narrow particle-size distribution is desirable. A particle-size distribution of dp +/- 10% would mean that 90% of the particles fall between 9 and 11 µm for an average 10-µm dp packing.

Partition chromatography. Separation process in which one of the liquid phases is held stationary on a solid support while the other is allowed to flow freely down the column. Solutes partition themselves between the two phases based on their individual partition coefficients. Liquid-liquid chromatography is an example.

Partition coefficient (K). The amount of solute in the stationary phase relative to the amount of solute in the mobile phase. Can be distribution coefficient, KD

Peak shape. Describes the profile of a chromatographic peak. Theory assumes a Gaussian peak shape (perfectly symmetrical); peak asymmetry factor describes shape as a ratio. See Asymmetry.

Peak width . Same as Band width.

PEI. Polyethyleneimine, an anionic polymeric phase used to coat silica. Most often used for the separation of proteins and peptides.

Pellicular. See Porous-layer bead.

Permeation. In SEC, refers to the process in which a solute can enter a mobile-phase-filled pore of the packing .

Phenyl phase. A nonpolar bonded phase prepared by the reaction of dimethylphenylchlorosilane with silica gel. Claimed to have affinity for aromatic compounds .

Pirkle columns. Chiral ''brush-type'' stationary phases, based on 3 ,5-dinitrobenzoylphenylglycine silica, used in the separation of a wide variety of enantiomers. Named after the developer, Dr. William Pirkle, University of lllinois.

Plate height (H). See HETP,

Plates. Refers to theoretical plates in a packed column See Theoretical plate.

Polyacrylamide gel. Neutral hydrophilic polymeric packings used in aqueous SEC. Prepared by the copolymerization of acrylamide with (N,N'-methylene)bisacrylamide .

Polymeric packings. Packings based on polymeric materials, usually in the form of spherical beads. Common polymers used in LC are polystyrene-divinylbenzene. polyacrylamide, polymethylacrylate, polyethyleneoxide, polydextran, and polysaccharide .

Polystyrene-divinylbenzene resin (PS-DVB). The most common polymer base for ion-exchange chromatography. Ionic groups are incorporated by various chemical reactions. Neutral PS-DVB beads are used in reversed-phase chromatography. Porosity and mechanical stability can be altered by varying the cross-linking through the variation of the DVB content .

Pore diameter. Same as Mean pore diameter.

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, where Hg is pumped into the pores under high pressure.

Porosity. For a porous adsorbent, the ratio of the volume of the interstices to the volume of the solid particles. The pore volume is also used as a measure of porosity.

Porous-layer bead. A small glass bead coated with a thin layer of stationary phase. The thin layer can be an adsorbent, a resin, or a phase chemically bonded onto the adsorbent. These were the first packings to be used in HPLC. They were larger (20-40 µm) than the microparticulate packings of today, but were easy to pack into columns and gave adequate efficiency. Also referred to as controlled surface porosity supports and pellicular packings.

Precolumn. A small column placed between the pump and the injector. It removes particulate matter that may be in the mobile phase, chemically sorbs substances that might interfere with the separation. or, as a saturator column, presaturates the mobile phase with stationary phase to prevent stripping of the column. Its volume has little effect on isocratic elution but contributes a delay to the gradient in gradient elution .

Preparative chromatography. The process of using liquid chromatography to isolate a sufficient amount of material for other experimental or functional purposes. For pharmaceutical or biotechnological purifications, columns several feet in diameter can be used for multiple grams of material. For isolating just a few micrograms of a valuable natural product, an analytical column can be used. Both are preparative chromatographic approaches.

Pulsating flow. Flow originating from a reciprocating pump. Normally, the pulses are dampened out by a pulse damper, by an electronic pressure feedback circuit, or by an active damper pump head. Some detectors (e.g., electrochemical) are affected by flow pulsations.

Radial compression. The use of radial pressure applied to a flexible-wall column to lessen wall effects.

Radial diffusion. Diffusion across the LC column in a radial direction. If the sample is injected into the exact center of a column, it will spread not only in a longitudinal direction as it flows, but radially as well .

Recovery. The amount of solute (sample) that elutes from a column relative to the amount injected. Most often used with protein separations in which proteins hang up'' on active sites of the packing in certain columns .

Recycling. A technique in which the column effluent is recirculated onto the head of the column in an attempt to gain the advantage of extended column length. Can be carried out on a single column by passing the eluent back through the pump. An alternative technique uses two columns connected by a switching valve. Very seldom used in HPLC, and then only in size-exclusion chromatography.

Reduced plate height (h). Used to measure efficiencies of columns. An HPLC column with an h value <2 is considered to be well-packed. h = H/dp.

Reduced velocity (v). Along with the reduced plate height, is used to compare different chromatographic columns. It relates the solute diffusion coefficient (Dm ) in the mobile phase to the particle size of the column packing (dp).V = dp/Dm.

Regeneration. Returning the packing in the column to its initial state after gradient elution. Mobile phase is passed through the column stepwise or in a gradient. The stationary phase is solvated to its original condition. In ion-exchange chromatography, regeneration involves replacing ions taken up in the exchange process with the original ions that occupied the exchange sites. Regeneration can also refer to bringing back any column to its original state (e.g., the removal of impurities with a strong solvent).

Relative retention ratio. Same as Separation factor or Selectivity.

Residual silanols. The silanol (-Si-OH) groups that remain on the surface of a packing after a phase is chemically bonded onto its surface. These silanol groups may not be accessible to the reacting bulky organosilane (e.g., octadecyldimethylchlorosilane) but may be accessible to small polar compounds. Often they are removed by endcapping with a small organosilane such as trimethylchlorosilane. See Endcapping.

Resin. A solid packing used in ion-exchange separations. The most popular resins are polystyrene-divinylbenzene copolymers of small particle size (<10 m). Ionic functionality is incorporated into the resin .

Resolution (Rs). Ability of a column to separate chromatographic peaks. It is usually expressed in terms of the separation of two peaks. One attempts to achieve the best resolution possible. Resolution can be calculated in two ways:  Rs = 2(tR.2 - tR.1)/(Wb.1 + Wb.2) or Rs = 1/4(a-1/a) (k'/1+k')N1/2  A value of 1 is considered to be the minimum for a measurable separation to occur and to allow good quantitation . Values of 1.7 or larger are generally desirable for rugged methods

Retention time (tR). The time between injection and the appearance of the peak maximum. The adjusted retention time t'R adjusts for the column void volume. t 'R = tR - t0 (or tm).

Retention volume ( VR) . The volume of mobile phase required to elute a substance from the column VR = F x tR or VR = Vm - KD Vs, where Vm is the void volume, KD the distribution coefficient. and Vs the stationary phase volume.

Reversed-phase chromatography (RPC). The most common HPLC mode. Uses hydrophobic packings such as octadecyl- or octylsilane phases bonded to silica or neutral polymeric beads. Mobile phase is usually water and a water-miscible organic solvent such as methanol or acetonitrile. There are many variations of RPC in which various mobile phase additives are used to impart a different selectivity. For example, for the RPC of anions, the addition of a buffer and tetralkylammonium salt would allow ion pairing to occur and effect separations that rival ion exchange chromatography.

Sample capacity. Refers to the amount of sample that can be injected onto an LC column without overload. Often expressed as grams of sample per gram of packing. Overload is defined as the sample mass injected at which the column efficiency falls to 90% of its normal value.

Saturator column. See Precolumn.

SAX. Strong anion exchanger. A typical strong anion exchange functional group would be tetraalkylammonium .

SCX. Strong cation exchanger. A typical strong cation exchange functional group would be a sulfonic acid.

Sedimentation. A technique used to size resins for ion-exchange chromatography. Particles of different size and density will settle at different velocities into a gradient. Very narrow cuts of particle size can be obtained .

Selectivity (a). Same as Separation factor or Relative retention ratio. A thermodynamic factor that is a measure of relative retention of two substances. Fixed by a certain stationary phase and mobile phase composition. a = k'2/k'1.

Semipreparative chromatography. Refers to preparative liquid chromatography carried out on an analytical size (4-5 mm i.d.) or slightly larger (6-10 mm i.d. ) column. Normal injection size is in the milligram to low-gram range. (A somewhat subjective definition . )

Separation factor. Same as Selectivity.

Silanol. The Si-OH group found on the surface of silica gel. There are different strengths of silanols, depending on their location and relationship to each other. The strongest silanols are acidic and often lead to undesirable interactions with basic compounds during chromatography.

Silica gel. The most commonly used packing in liquid chromatography. It has an amorphous structure, is porous, and consists of siloxane and silanol groups. It is used as a bare packing for adsorption, as the support in liquid-liquid chromatography or for chemically bonded phases, and. with various pore sizes. as packing in size-exclusion chromatography. Microparticulate silicas of 5- and 10-um average particle diameter are used in HPLC.

Siloxane. The Si-O Si bond. A principal bond found in silica gel or for attachment of a silylated compound or bonded phase. Stable except at high pH values.

Silylation. The reaction of an organochloro- or organoalkoxysilane with a compound containing a reactive group. In liquid chromatography, it refers to the process of derivatizing the solute before chromatography in order to make it detectable or to prevent unwanted stationary phase interactions. It can also refer to the process of adding a chemically bonded phase to a solid support or to deactivating the packing to cut down on surface activity.

Size-exclusion chromatography (SEC). Same as Steric exclusion chromatography.

Slurry packing. The technique most often used to pack HPLC columns with microparticles. The packing is suspended in a slurry (10% wt/vol) and is rapidly pumped into the empty column. Special high pressure pumps are used.

Soap chromatography. An early name for ion-pair chromatography. Long-chain soaps or detergents were used as mobile phase additives.

Solid-phase extraction (SPE). A sample-preparation technique that uses a solid-phase packing contained in a small plastic cartridge. The solid stationary phases are the same as HPLC packings; however. the principle is different from HPLC. Sometimes referred to as digital chromatography. The process as most often practiced requires four steps: conditioning the sorbent, adding the sample, washing away the impurities, and eluting the sample in as small a volume as possible with a strong solvent.

Solid support. Same as Support.

Solute. The dissolved component of a mixture that is to be separated in the chromatographic column.

Solvent strength. Refers to the ability of a solvent to elute a particular solute or compound from a column. Described by Lloyd Snyder for LEAC (LSC) adsorption chromatography on alumina: solvents were quantitatively rated in an eluotropic series. No eluotropic series exists for other modes.

Sorbent. Refers to an adsorption packing used in liquid chromatography. A common sorbent is silica gel .

Specific surface area. The surface area of an LC packing based on measurement by an accepted technique, such as the BET method using nitrogen adsorption .

Spherical packing. Refers to spherical solid packing materials. Spherical packings are generally preferred over irregular particles.

Stationary phase. The immobile phase involved in the chromatographic process. The stationary phase in liquid chromatography can be a solid, a bonded or coated phase on a solid Support, or a wall-coated phase. The stationary phase used often characterizes the LC mode. For example. silica gel is used in adsorption chromatography, an octadecylsilane bonded phase in reversed-phase chromatography, etc.

Stepwise elution. Use of eluents of different compositions during the chromatographic run. These eluents are added in a stepwise manner with a pump, or by a selector valve. Gradient elution incorporates continuous changing of solvent composition.

Steric exclusion chromatography (SEC). A major LC mode in which samples are separated by virtue of their size in solution. Also known as size-exclusion. gel permeation, gel filtration, or gel chromatography.

Straight-phase chromatography. Same as Normal phase chromatography.

Supercritical fluid chromatography (SFC). A technique that uses a supercritical fluid as the mobile phase. The technique has been applied to the separation of substances that cannot be handled effectively by liquid chromatography (because of detection problems) or gas chromatography (because of the lack of volatility). Examples are separations of triglycerides, hydrocarbons, and fatty acids. GC detectors and HPLC pumps have been used together in SFC.

Superficially porous packing. See Porous-layer bead .

Suppressor column. In ion chromatography. refers to the column placed after the ion-exchange column Its purpose is to remove or suppress the ionization of buffer ions so that sample ions can be observed in a weakly conducting background with a conductivity detector.

Surface area. In an adsorbent, refers to the total area of the solid surface as determined by an accepted measurement technique such as the BET method using nitrogen adsorption. The surface area of a typical porous adsorbent such as silica gel can vary from 100 to 600 m2/g.

Surface coverage. Usually refers to the mass of stationary phase per unit area of an LC support. Sometimes the %C is given as an indicator of surface coverage.

Swelling. Process in which resins and gels increase their volume because of their solvent environment. Solvent enters ion-exchange resin to dilute ions: in gels, solvent penetrates pores. If swelling occurs in packed columns. blockage or increased back pressure can occur. In addition. column efficiency can be affected .

Tailing. The phenomenon in which the normal Gaussian peak has an asymmetry factor > 1. The peak will have a skewed or trailing edge. Tailing is caused by sites on the packing that have a stronger-than normal retention for the solute. A typical example of a tailing phenomenon is the strong adsorption of amines on the residual silanol groups of a low-coverage reversed-phase packing.

Temperature programming. Changing column temperature as a function of time during the separation. Rarely used in HPLC: if so, usually in a stepwise manner

Theoretical plate. A concept described by Manin and Synge. Relates chromatographic separation to the theory of distillation. Measure of column efficiency. Length of column relating to this concept is called height equivalent to a theoretical plate (HETP). See HETP.

Titania. TiO2, an uncommon adsorbent used in ad sorption chromatography.

Tortuosity. A property of a packed column that indicates the degree of unevenness of the path followed by the solute molecule as it passes down the column. Covered in the A-term of the van Deemter equation.

Total permeation volume ( Vp). The retention volume on an SEC packing in which all molecules smaller than the smallest pore will elute. In other words, at Vp, all molecules totally permeate all of the pores and elute together.

Trace enrichment. Technique in which trace amounts of compounds from a weak mobile phase or solution are retained on an HPLC packing and then are eluted by the addition of a stronger mobile phase in a concentrated form. The technique has been most successfully applied in the concentration of trace amounts of hydrophobic compounds (e.g.. polynuclear aromatic hydrocarbons) out of water using a reversed-phase column. A strong Solvent such as acetonitrile serves to elute the enriched compounds.

Vacancy chromatography. Technique in which a mobile phase additive causes a positive detector signal output. When a solute elutes from the column, it dilutes the signal and gives a negative peak (a vacancy). The technique has been most recently applied to single-column ion chromatography, in which mobile phases that absorb in the UV region, such as citrate and phthalate buffers, are used. When a non absorbing anion elutes, it dilutes the UV-absorbing background and causes a negative peak the detector output leads are usually reversed so that the chromatogram looks normal.

Validation. Establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes. A validated manufacturing process is one that has been proved to do what it purports or is represented to do. The proof of validation is obtained through collection and evaluation of data, begining at the process development phase and continuing through the production phase. Process validation includes process qualification (the qualification of materials, equipment, systems, facilities, and personnel), and also includes the documented control of all the processes required for repeated batches.

van Deemter equation. An equation used to explain band broadening in chromatography. The equation represents the height equivalent of a theoretical plate and has three terms. The A-term is used to describe eddy diffusion, which allows for the different paths a solute may follow when passing over particles of different sizes. The B-term is for the contribution caused by molecular diffusion or longitudinal diffusion of the solute while passing through the column. The C term is the contribution of mass transfer and allows for the finite rate of transfer of the solute between the stationary phase and mobile phase. h = A + B/v + Cv.

Velocity (u). Same as Linear velocity.

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. Even a small void can be disastrous for small microparticulate columns. The void can sometimes be removed by filling it with glass beads or porous packing

Void time (tm or t0). The time for elution of an unretained peak.

Void volume ( Vi). The total volume of mobile phase in the column: the remainder of the column is taken up by packing material. Can be determined by injecting an unretained substance that measures void volume plus extra column volume. Also referred to as interstitial volume. V0 or Vm are sometimes used as symbols.

Wall effect. The consequence of the looser packing density near the walls of the rigid HPLC column. Mobile phase has a tendency to flow slightly faster near the wall because of the decreased permeability The solute molecules that happen to be near the wall are carried along faster than the average of the solute band and. consequently. band spreading results.

WAX. Weak anion exchanger. Ionizable groups such as primary, secondary, or tertiary amino groups on a packing are considered to be weak.

WCX. Weak cation exchanger. Carboxylic groups on a packing are typical of a weak cation exchanger.

Xerogels. Gels used in size-exclusion chromatography that swell and shrink in different solvents.

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