Glossary of HPLC Terms

a b c d e f g h i k l m n o p q r s t u v w x y z

Activity

In adsorption chromatography, this is 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.

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 such as hydrogen bonds, or weak such as 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 which relies on the adsorption process to effect the separation. Silica gel and alumina are the most frequently used supports. The molecules are retained by the interaction of their polar functional groups with the surface functional groups such as silanols of silica.

Adsorption isotherm

In adsorption chromatography, this is a plot of the equilibrium concentration of sample in the mobile phase per unit volume verses the concentration in the stationary phase per unit weight. The shape of the adsorption isotherm can determine the chromatographic behavior of the solute such as tailing, fronting, and sample overload.

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 but selective filtration.

Alumina

An adsorbent sometimes used in adsorption chromatography. Aluminum oxide (AI₂0₃) 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.

Asymmetry

A factor describing the shape of a chromatographic peak. Theory assumes a Gaussian shape peak that is symmetrical. The peak asymmetry factor is the ratio (at 10 percent 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.

Backflushing

A column switching technique in which a four-way valve placed between the injector and the column allows the mobile phase to 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.

Backpressure

The pressure above gravity at the head of the column. Expressed in psig, bar, atm., or MPa.

Bandspreading

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, t_w, or more correctly, the number of theoretical plates in the column, N.

Baseline

The baseline is the line drawn by the data system when the only signal from the detector is from the mobile phase.

Baseline noise

Interferences to the detector and data system caused by electrical noise or environmental effects. This interference keeps the baseline from being perfectly flat.

Baseline-resolved peaks

When both sides of a peak reach the baseline without interfering with other peaks.

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.

Bonded-phase chromatography (BPC)

A stationary phase chemically bonded to a support that is used for the separation. It is the most commonly used LC mode. The most popular support used is microparticulate silica gel. An organosilane, such as octadecyl (for reversed-phase chromatography), is the most accepted type of bonded phase. Approximately 70 percent of all HPLC is carried out on chemically bonded phases.

Breakthrough volume

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

C            [back to top]

Calibration standards

These are standards of known quantities and substances which assist in peak identification or quantitation. Calibration standards can be external standards or internal standards.

Capacity factor

A chromatographic parameter that measures the degree of retention. See k’ for calculation method.

Capping

Same as Endcapping.

Carrier

A term used most often in affinity chromatography, which refers to the support that is used to carry the active ligand, usually by a covalent bond. It can also refer to the support in other chromatographic modes.

Cartridge column

A type of column with no endfittings which is held in a cartridge holder. The column is an open tube with the packing contained by frits in each end. Cartridges are easy to change, less expensive, and more convenient than conventional columns with endfittings.

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 such as C₈ and C₁₈.

Channeling

This occurs when voids are created in the packing material of a column. It results in the mobile phase and accompanying solutes moving more rapidly than the average flow velocity producing band broadening. These voids are created by poor packing or by erosion of the packed bed.

Chemisorption

This is sorption caused by a chemical reaction with the packing. Most of these interactions are irreversible. They usually occur on packings with reactive functional groups such as silanol or bonded amino phases.

Chiral stationary phases

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

Chromatography

A chemical separation technique based on the differential distribution of the constituents of a mixture between two phases, one of which moves relative to the other.

Chromatographic methods

A record of the parameters used in a separation that yields a particular result. It allows another analyst following the method and conditions to reproduce the separation, and achieve the same results.

Chromatogram

The electronic result of a chromatographic separation which is a plot of detector signal output versus time or elution volume. It is represented as a series of peaks from the data system.

Chromatographic conditions

Parameters used in an analysis such as column type, mobile phase, and wavelength.

Column

A tube which contains the stationary phase. The stationary phase differentially interacts with the sample’s constituent compounds as they are carried along in the mobile phase.

Column backpressure

See Backpressure.

Column chromatography

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

Column-dependent chemical factors

Chemical factors associated with column packing materials including the nature of the base material, surface activity of the bonded phase, and degree of interference from silanol groups.

Column performance

The efficiency of a column which is measured as the number of theoretical plates for a given test compound.

Column switching

The use of multiple columns connected by switching valves. 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. This process is used for better chromatographic separations or for sample cleanup.

Conductivity detectors

These detectors identify changes in the conductivity of the mobile phase as it passes through a flow cell. They are used to detect a wide variety of ionized species separated by ion chromatography.

Connecting tube

A tube that connects the column to the injector and detector. Diffusion within connecting tubing broadens the peaks, but does not contribute to the separation.

Counterion

In an ion-exchange process, this is the ion in solution which is 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. This 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. This term is also used to describe two or more columns connected in series to provide increased plate numbers.

Coverage

The amount of bonded phase on a silica support in bonded phase chromatography. The coverage is usually described in mmol/m² or in terms of percent C.

Cross-linking

During the process of copolymerization of resins to form a three dimensional matrix, and 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. It is used in both normal and reversed-phase chromatography.

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Dead volume (V_d)

The volume outside of the column packing itself. The interstitial volume (intraparticle and interparticle volume) plus extracolumn volume (contributed by injector, detector, connecting tubing, and endfittings) all combine to create the dead volume. This volume can be determined by injecting an inert compound. For example, a compound that does not interact with the column packing. It is also abbreviated V_o or V_m.

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, by vacuum ( in a vacuum flask), on-line using evacuation of a tube made from a gas permeable substance such as PTFE, or by helium sparging.

Desalting

A technique in which low molecular weight salts and other compounds are removed from non-ionic and high molecular weight compounds.

Detector

An electronic device that quantitatively discerns the presence of the separated components as they elute. There are different types of detectors. Some of the common detector types are: UV/Visible light absorbance, differential refractive index, electrochemical, conductivity, and fluorescence.

Detection limits

The post-column concentration of a compound and not the concentration in the sample. A detector specified to detect a compound at 0.1 ppm may be also acceptable for a method that tests a water sample containing the compound at a much lower concentration. This is because the compound can be concentrated on the column or sample preparation techniques may used to preconcentrate the sample as well.

Detection threshold

The point at which the software determines the beginning and the end of the peak will shift depending on the threshold. The threshold should be set as low as possible. If it is set too low, the software will interpret baseline noise as peaks. If it is set too high, a portion of the bottom of the peak may be cut off and is not quantitated.

Detector Linearity

The linearity of the response over a range of sample concentrations controlled by a response factor setting on the detector, or in the detector controller software.

Detector Sensitivity

The sensitivity setting is the line between normal background noise and a true peak. Perturbations from the baseline that fall below the sensitivity setting are considered noise, and are filtered out. Setting the sensitivity too high may result in missing small peaks, while setting it too low may result in a lot of spurious raw data as the software tries to integrate peaks out of the noise.

Differential Refractive Index (RI)

Detectors that identify the difference in the mobile phase’s refractive index when it contains dissolved sample. RI detectors are universal detectors which respond to the presence of all compounds. Because the RI detector responds to changes in mobile phase composition, they are not suitable for gradient methods. RI detectors are less sensitive than UV/Vis detectors, but are useful when the sample molecule does not have a chromophore.

Diffusion along the flow path

The coefficient for molecular diffusion along the flow path. It is represented as the B term in the van Deemter equation. The greater the diffusion coefficient of the component in the mobile phase, the more the narrow band of separated component diffuses into the surrounding mobile phase before going to the detector. This results in band broadening. As linear velocity increases, the effect of axial diffusion on column efficiency decreases. The van Deemter equation reflects this effect by dividing B by u.

Diffusion coefficient
(D_M or D_S)

A fundamental parameter of a molecule in solution (D_m) or stationary phase (D_s) expressed in cm²/sec. D_mdepends on molecular weight of the solute, temperature, solvent viscosity, and molar volume of the solute. A typical value of a small molecule in RPC at room temperature would be 5 x 10⁶ cm²/sec.

Diol phase

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

Displacement chromatography

A 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 EPLC applications.

Distribution coefficient
(D or K_D)

See Partition coefficient

Eddy diffusion term

This is represented by 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 and it depends on the particle size and geometry of the packing. It is also called the multipath term.

See van Deemter equation.

Effective theoretical plates (N_eff)

The true number of plates in a column that corrects the number of theoretical plates for dead volume. , where t_m is the void time.

Efficiency (N or H)

A measure determined by the number of theoretical plates calculated from the equation:

This can be visually represented by the following figure.

Measure peak width at 4.4% of peak height

Electrochemical detector

Al detector which monitors an oxidation or reduction reaction between the detector’s working electrode and the sample. Electrochemical detectors are characterized by high sensitivity, with detection limits in the low ppb range common for electroactive compounds.

Eluate

Combination of mobile phase and solute exiting column.

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 order

The order in which the separated compounds comes off of the column (elutes) and through the detector.

Elution chromatography

The most commonly used chromatographic method. The sample is injected at the head of the column, and the individual compounds are separated and eluted at the end of the column.

Elution volume (V_R)

The volume of mobile phase required to elute a solute from the column at maximum concentration (apex).

Endcapping

A column is said to be endcapped when a small silylating agent, such as trimethylchlorosilane, is used to bond residual silanol groups on a packing surface. It is most often used with reversed-phase packings and may cut down on undesirable adsorption of basic or ionic compounds.

Endfitting

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

Exchange capacity

See Ion-exchange capacity.

Exclusion chromatography

See Steric-exclusion chromatography (SEC).

Exclusion limit

In SEC, this is the upper limit of molecular weight (or size), beyond which molecules will elute at the same retention 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.

Exclusion volume (V_C)

The retention volume of a molecule on SEC packing. All molecules larger than the size of the largest pore are totally excluded and elute at the interstitial volume of the column.

Extracolumn effects

The band broadening effects of parts of the chromatographic system outside of the column itself. Areas of band broadening can include the injector, connecting tubing, endfittings, frits, detector cell volume, and internal detector tubing. The variances of all of these contributions are additive. These extracolumn effects should be minimized in order to maintain the efficiency of the column.

External standards

A separate sample containing known quantities of the same compounds of interest. External standards are used primarily for peak identification by comparing elution times.

F            [back to top]

Fast LC

The use in BPLC of short columns (3 to 7 cm in length) with conventional internal diameters (2 to 6 mm) packed with small particles (3 or 5 mm d_p). The separation times are commonly in minutes and sometimes seconds.

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.

Fluorescence detectors

Fluorescence detectors project a specified wavelength of light into the sample, causing the component of interest to fluoresce and the emitted light is detected. Fluorescence detectors are commonly used with derivatization methods. Fluorescence detectors are very selective and very sensitive.

Fractionation range

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. In SEC, it is the operating range of a gel or packing.

Frit

The porous component 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 endfitting. Frits are made from stainless steel or other inert metal or plastic, such as porous PTFE or polypropylene.

Frontal analysis

A chromatographic technique that involves continuous addition of sample to the column. The result is 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 and so forth. This continues until the original sample is eluting at the column exit. Frontal analysis is seldom used and is mainly a preparative technique.

Fronting

This is an asymmetrical peak shape in which the front part 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.

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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 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)

This is size-exclusion chromatography carried out with aqueous mobile phases, also known as aqueous GPC. It generally refers to separations carried out on soft gels such as polydextrans. Most gel filtration separations involve biopolymers and are used for the separation and characterization of polymers.

Gel permeation chromatography (GPC)

See GFC.

Gradient elution

A technique for decreasing separation time by increasing mobile phase strength over time during a chromatographic separation. It is also known as solvent programming. Gradients can be continuous or step-wise. Binary, ternary, and quaternary solvent gradients are used routinely in HPLC.

Guard column

A small column placed between the injector and the analytical column. It protects the analytical column against contamination by sample particulates, and 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.

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HETP

The height equivalent of a theoretical plate. It is a carryover from distillation theory which is a measure of a column’s efficiency. For a typical HPLC column 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

It is often referred to as water loving. It adverts both to water compatible stationary phases, and to water soluble molecules. Most columns used to separate proteins are hydrophilic in nature and should not sorb or denature protein in the aqueous environment.

Hydrophobic

It is often referred to as water hating. It adverts both to stationary phases not compatible with water and molecules with little affinity for water. Hydrophobic molecules have few polar functional groups and are mostly hydrocarbons or have high hydrocarbon content.

Hydrophobic interaction chromatography

A technique in which reversed-phase packings are used to separate molecules by the interactions between their hydrophobic moieties and the hydrophobic sites on the surface. High salt concentrations are used in the mobile phase and 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.

I            [back to top]

Injector

A mechanism for accurately injecting a predetermined amount of sample into the mobile phase stream. The injector can be a simple manual device, or a sophisticated autosampler that permits automated injections of many different samples for unattended operation.

In-line filter

A device that prevents particulate matter from damaging the column by filtration. Modem 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 particulates from entering the packed bed or the inlet frit.

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.

Internal standards

Internal standards consist of a specific quantity of a compound that is known not to be in the sample, but that exhibits the same characteristics under the separation conditions as the sample components. Internal standards are used primarily to calibrate quantitation, especially for methods susceptible to volumetric error resulting from sample preparation.

Interstitial volume (V_O)

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. It is also abbreviated V_i or V_m.

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: suppressed IC, and non-suppressed IC.

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 ionogenic group of the packing. Retention is based on the affinity of different ions for the site and on a number of other solution parameters such as, pH, ionic strength, and counterion type.

Ion-exchange capacity

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

Ion exclusion

A 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. This is where ionic solutes exist at a higher concentration in solution than in the resin but nonionic solutes are evenly distributed between the mobile phase and resin. Therefore, ionic solutes will elute faster from the column than the nonionic solutes.

Ion-pair chromatography

A 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 which 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 part of the pair is loaded onto a sorbent, but this technique is not as popular as the RPC technique.

Ion suppression

This involves buffering in an aqueous mobile phase at a particular pH to suppress solute ionization. For example, the ionization of weak carboxylic acids can be suppressed by adjusting the pH below their ionization constant. This technique is useful for improving the peak shape of weak acids and bases in RPC.

Irregular packing

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, sizing them into small particles, and into narrow fractions using classification machinery. While spherical packings are now used more often than irregular packings in HPLC, the less expensive irregular packings are still widely used in prep LC.

Irreversible adsorption

A state when a compound with a very strong affinity for the adsorbent is injected into a column, it is so strongly adsorbed that it cannot be eluded from the column. An example of irreversible adsorption is a chemical reaction between the sample and the surface of the adsorbent.

Isocratic

A constant composition mobile phase used in liquid chromatography.

K            [back to top]

k or k'

The capacity factor. It can be calculated from the equation, where t_R is retention time for the sample peak, and t_o is the retention time for an unretained peak.

L            [back to top]

Ligand

In ligand-exchange chromatography, it is the molecule added to the mobile phase that acts as the chelating agent. In affinity chromatography, it is 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. For example, using of copper amine chelates for the separation of amino acids. Chelating resins function in a similar manner. The chelating groups are chemically bonded to the polystyrene backbone.

Linearity

A measurement process which ensures accurate quantitation. In chromatography it is important that the detector provide a linear response over the range of sample concentrations it encounters.

Linear elution adsorption chromatography (LEAC)

A term coined by Lloyd Snyder, which refers to adsorption chromatography carried out in the linear portion of an adsorption isotherm.

Linear velocity

A measure of the speed with which an unretained compound moves through the column. Linear velocity is represented by the letter u and is reported in cm/min or mm/sec. Linear velocity is used to calculate flow rate based on the cross sectional area of a column. Linear velocity is useful for adapting methods from one column diameter to another.

Liquid-liquid chromatography (LLC)

This is the same as Partition chromatography. It was the earliest form of HPLC, which was replaced with chemically bonded phases in the early 1970s.

Liquid-solid chromatography (LSC)

Same as Adsorption chromatography.

Loading

The amount of stationary phase coated or bonded onto a solid support. In liquid-liquid chromatography, it is the milligram amount of liquid phase per gram of packing. In BPC, the loading may be expressed in mmol/m² or in percent C. See Coverage.

M            [back to top]

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. It is represented by the C term of the van Deemter equation and 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 in a porous packing. The pore diameter is important because it allows free diffusion of solute molecules so they can interact with the stationary phase. In SEC, the packings have different pore diameters, allowing molecules of different sizes to 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.

Method-dependent chemical factors

The variables in the chromatographic method which include the choice of mobile phase, column temperature, sample preparation method, sample size, flow rate, reagent quality, and laboratory technique.

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.

Microbore

Columns with smaller than usual internal diameters ( < 2 mm) used in HPLC.

Microparticulate

Small particles used as HPLC stationary phases. These packings generally have a particle diameter <10 mm are considered microparticles.

Microporous resin

Same as Microreticular resin.

Microreticular 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. It usually occurs at very low flow rates.

Mixed-bed column

Combination of two or more stationary phases in the same column. It is used most often in IEC and SEC. The advantage in IEC is the total removal of ionic compounds. It is 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

An additive that changes the character of the mobile phase. For example, in reversed phase water is the weak solvent and methanol is the strong solvent. Methanol is known then as the modifier.

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

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 arylrnonochlorosilane. Polymeric phases are generally prepared from a di or trichlorosilane reactant.

Multidimensional chromatography

The use of two or more columns or chromatographic techniques which enable a better separation. It is 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. It also called coupled column chromatography, column switching, multicolumn chromatography, or boxcar chromatography.

N            [back to top]

N

The number of theoretical plates. A measure of the efficiency of a column. , where t_R is retention time, and w_b is the base width of the peak. Sometimes it is measured as , where w_l/2 is the peak width at half height.

Narrow-bore column

Columns of < 0.5 mm i.d. used in HPLC.

Nonporous particle

A solid particle used as a support for a porous coated or bonded phase.

Non-suppressed Ic

A type of ion chromatography were weakly conducting buffers at low concentration are carefully selected, and the entire effluent is passed through the detector. The ions are detected above the background signal.

Normal-phase chromatography

A mode of chromatography carried out with a polar stationary phase and a nonpolar mobile phase. Adsorption on silica normal phase system. It also refers to the use of polar bonded phases, such as CN or NH₂. It is sometimes referred to as straight phase chromatography.

O            [back to top]

Octadecylsilane (ODS)

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. 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. These columns are currently being investigated for HPLC, SFC, and capillary electrophoresis.

Organic modifier

A water miscible organic solvent which is added to an aqueous mobile phase to effect separations in reversed-phase HPLC.

Overload

The increased mass of sample injected onto a column which begins to affect efficiency and resolution. See Sample capacity.

P            [back to top]

PEEK

Packed bed qualityThe coefficient that reflects the quality of packed bed. It is represented as the A term in the expanded van Deemter equation. This term is determined by the size and distribution of voids, channels, and other nonuniformities in the packed bed. It represents a practical lower limit for H, and can be changed by using a better packed bed.

Paired-ion chromatography

Same as Ion-pair chromatography.

Partially-resolved peaks

When two or more adjacent peaks run together and are not baseline resolved.

Particle size (d_p)

The average particle size of the packing in an LC column.

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 d_p ±10 percent would mean that 90 percent of the particles fall between 9 and 11 mm for an average 10 mm d_p packing.

Partition chromatography

A 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. It can also be the distribution coefficient, K_D.

Peak

When the detector registers the presence of a compound, the normal baseline signal it sends to the data system changes, resulting in a deflection from the baseline called a peak. Well resolved peaks are symmetrical, touch the baseline, and do not interfere with other peaks.

Peak Identification

Peak identification is usually performed by comparing the sample chromatogram to a chromatogram of a standard solution separated under the same conditions. Peaks that appear at the same elution time as peaks in the standard are identified as the same component.

Peak Quantitation

Correctly detecting the beginning and end of a peak.

Peak shape

The profile of a chromatographic peak. Theory assumes a Gaussian peak shape (perfectly symmetrical). A peak asymmetry factor is used to describe a shape as a ratio. See Asymmetry.

Peak tailing

Same as tailing peaks.

Peak width (w_b)

Same as Band width.

Permeation

In SEC, this is the process in which a solute can enter a mobile phase filled pore of the packing.

Phenyl phase

A non polar bonded phase prepared by the reaction of dimethylphenylchlorosilane with silica gel. It is claimed to have an affinity for aromatic compounds.

Photodiode Array (PDA)

PDA detectors are UV/Vis detectors that record the absorbance of light at many different wavelengths simultaneously.

Physical factors

Variables such as particle size, particle surface area, column dimensions, leaks in the fluid path, system bandspreading, and detector cell design.

Plates

The theoretical plates in a packed column. See Theoretical plate.

Polyacrylamide gel

Neutral hydrophilic polymeric packings used in aqueous SEC. It is 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. The 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 that is 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

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.

Precolumn

A small column placed between the pump and the injector. It removes particulate matter which could be in the mobile phase, chemically sorbs substances that might interfere with the separation, or acts as a saturator column presaturating 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 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 pulse damper, by an electronic pressure feedback circuit, or by an active damper pump head. Some detectors, such as electrochemical, are affected by flow pulsations.

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Qualitation

An analysis process which is designed to identify the components of a substance or mixture.

Quantitation

An analysis process which is designed to determine the amounts or proportion of the components of a substance.

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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. This is 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 an extended column length. It can be carried out on a single column by passing the effluent back through the pump. An alternative technique uses two columns connected by a switching valve. This technique is very seldom used in HPLC, and only in size-exclusion chromatography.

Reduced plate height (h)

A calculated value used to measure efficiencies of columns. A BPLC column with an h value <2 is considered to be well packed.

Reduced velocity (v)

A calculated value used to compare different chromatographic columns. It relates the solute diffusion coefficient (D_m) in the mobile phase to the particle size of the column packing (d_p).

Regeneration

A process of restoring the packing in the column to its initial state after a gradient elution. The mobile phase is passed through the column step-wise or in a gradient solvating the stationary phase 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 by removing impurities with a strong solvent.

Relative retention

Relative retention is a measure of the difference of affinities of two compounds for the stationary phase. Mathematically it is the ratio of the retention factors of two compounds, one of which is usually a standard. It is represented by the letter r, and is reported in dimensionless units. Relative retention is used in quality control, reproducibility, and method validation calculations.

Relative retention ratio

Same as Separation factor or Selectivity.

Residual silanols

The silanol (SiOH) 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 such as octade- cyldimethylchlorosilane, but may be accessible to small polar compounds. Often they are removed by endcapping with a small organosilane such as trimethylchlorosilane. See Endcapping.

Resolution (R_s)

The ability of a column to separate chromatographic peaks. It is usually expressed in terms of the separation of two peaks.

Retention factor

Retention factor is how long a compound is retained by the stationary phase relative to the time it resides in the mobile phase. In IUPAC nomenclature, the retention factor is represented by the letter k and is dimensionless. In former usage, k was often represented as k’, and was termed the capacity factor.

Retention time (t_R’)

The time between injection and the appearance of the peak maximum. The adjusted retention time t_R’ adjusts for the column void volume.

Retention volume (V_R)

The volume of mobile phase required to elute a substance from the column. This is where V_m is the void volume, K_D the distribution coefficient, and V_s the stationary phase volume.

Reversed-phase chromatography (RPC)

The most common HPLC mode. Uses hydrophobic packings such as octadecyl or octysilane phases bonded to silica or neutral polymeric beads. The mobile phase used 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, in the RPC of anions, the addition of a buffer and tetraalkylammonium salt would allow ion pairing to occur, resulting in separations that rival ion-exchange chromatography.

Sample capacity

The amount of sample that can be injected onto an LC column without overload. It is 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 percent of its normal value.

Sample diffusion (within the column)

As a band of sample migrates along the column, diffusion causes it to broaden proportionally to the square root of the distance it has traveled.

Sampling rate

The frequency with which the detector checks the flow cell. Sampling rate is often called time constant. If it is set too fast too much raw data will be generated. If it is set too slow, a narrow peak could be missed.

Selectivity (a)

The 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.

Semipreparative chromatography

Preparative liquid chromatography carried out on an analytical size (4 to5 mm i.d.) or slightly larger (6 to 10 mm i.d.) column. Normal injection size is in the milligram to low gram range.

Separation factor

The separation factor, sometimes called selectivity, is the relative retention measured for two adjacent peaks. The separation factor is represented by the Greek letter a, and is reported in dimensionless units.

The serparation factor for two compounds is calculated using the formula .

Separation can be thought of as measuring either the difference between the centers of each peak, or the distance betwee the tops of the peaks. Two compounds with a high a can appear far apart on the chromatogram, though they are not resolved.

Silanol

The SiOH 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.

Siloxane

The Si-O-Si bond. A principal bond found in silica gel, for attachment of a silylated compound, or bonded phase. It is 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 percent 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 but the principle is different from HPLC. It is sometimes referred to as digital chromatography. This process as it is 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

The ability of a solvent to elute a particular solute or compound from a column. Lloyd Snyder described it for LEAC (LSC) adsorption chromatography on alumina and solvents were quantitatively rated in an eluotropic series. No eluotropic series exists for other modes.

Sorbent

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 a measurement by an accepted technique, such as the BET method that uses nitrogen adsorption.

Spherical packing

A spherical solid packing material. 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

This process uses 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.

Support

The solid particles in a column. The support can be naked, coated, or can have a chemically bonded phase in HPLC.

Suppressed IC

A second column is used to remove the buffer ions so that sample ions can be more easily detected; membrane separator is sometimes used.

Suppressor column

In ion chromatography, it is 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, it is 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

The mass of stationary phase per unit area of an LC support. It is often expressed in mmol/m² of surface. Sometimes the percent C is given as an indicator of surface coverage.

Swelling

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

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Tailing

A peak with an asymmetrical factor of >1. An asymmetrical peak is the result of a component that is excessively retarded in eluting. 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

A technique that changes the column temperature as a function of time during the separation. It is rarely used in HPLC, then it is done in a stepwise manner.

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. The A term in the van Deemter equation takes this into consideration.

Total permeation volume (V_p)

The retention volume on an SEC packing in which all molecules small than the smallest pore will elute. Therefore, at Vp, all molecules totally permeate all of the pores and elute together.

Trace enrichment

A technique in which trace amounts of compounds from a weak mobile phase or solution are retained on an HPLC packing. They are then 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, such as polynuclear aromatic hydrocarbons, out of water using a reversed-phase column. A strong solvent such as acetonitrile serves to elute the enriched compounds.

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Ultraviolet/Visible Light (UV/Vis)

The tunable or variable wavelength UV/Vis detector is the most popular form of detector. For methods involving organic compounds in aqueous mobile phases, the UV/Vis detector takes advantage of compounds’ varying absorptivities of ultraviolet and visible light.

Unretained compounds

These compounds are not retained at all on the column but elute at the beginning of the chromatogram immediately after the void volume.

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Vacancy chromatography

A 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 or 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 nonabsorbing anion elutes, it dilutes the UV absorbing background and causes a negative peak. The detector output leads are usually reversed to make the chromatogram look normal.

van Deemter equation

An equation used to explain band broadening in chromatography. This equation represents the height equivalent of a theoretical plate and has three terms. The A term is used to describe eddy diffusion, which considers 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.

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 (t_m or t_o)

The time for elution of an unretained peak.

Void volume (Vi)

The total volume of mobile phase in the column with the remainder of the column taken up by packing material. It can be determined by injecting an unretained substance that measures void volume plus extracolumn volume. It also is referred to as interstitial volume. V_o or V_m are sometimes used as symbols.

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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 band spreading results.

Waste container

At the end of the fluid path, the mobile phase and separated sample components are collected into a waste container. This container is suitable for safely collecting and disposing of the solvents used in the separation.

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Xerogels

Gels that are used in size-exclusion chromatography. They have the ability to swell and shrink in different solvents.

X-axis

The X-axis of the chromatogram records the time or volume of mobile phase that passes though the detector.

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Y-axis

The Y-axis of the chromatogram records the strength of the detector signal, which is usually proportional to the concentration of sample in the eluent passing through the detector. The units depend on the type of detector being used.

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Zwitterions

Compounds that carry both positive and negative charges in solution.