glossary a to z chromatography

8/8/2019 Glossary a to z Chromatography

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Activity

In adsorption chromatography, this is the relative strength of thesurface of the packing. For silica gel, the more exposed the silanogroups, the more active the surface. Activity can be controlled byadding water or another polar modifier, which is hydrogen bonded tothe 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 anadsorbent. The forces involved can be strong such as hydrogen bondsor weak such as van der Waals forces. For silica gel, the silanolgroup is the driving force for adsorption, and any solute functionagroup 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 toeffect the separation. Silica gel and alumina are the mostfrequently used supports. The molecules are retained by theinteraction of their polar functional groups with the surfacefunctional groups such as silanols of silica.

Adsorption isotherm

In adsorption chromatography, this is a plot of the equilibriumconcentration of sample in the mobile phase per unit volume verses

the concentration in the stationary phase per unit weight. The shapeof the adsorption isotherm can determine the chromatographicbehavior of the solute such as tailing, fronting, and sampleoverload.

Affinity chromatography

A technique in which a biospecific adsorbent is prepared by couplinga specific ligand (such as an enzyme, antigen, or hormone) for the


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macromolecule of interest to a solid support (or carrier). Thisimmobilized ligand will interact only with molecules that canselectively bind to it. Molecules that will not bind eluteunretained. The retained compound can later be released in apurified state. Affinity chromatography is not a chromatographictechnique but selective filtration.

Alumina

An adsorbent sometimes used in adsorption chromatography. Aluminumoxide (AI203) is a porous adsorbent that is available with aslightly basic surface. For this reason, it can have advantages ovesilica, 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 ormobile phase additive that can react with amines. The amino phasehas found some applications as a weak anion exchanger.

Asymmetry

A factor describing the shape of a chromatographic peak. Theoryassumes a Gaussian shape peak that is symmetrical. The peakasymmetry factor is the ratio (at 10 percent of the peak height) ofthe distance between the peak apex and the back side of thechromatographic curve to the distance between the peak apex and the

front side of the chromatographic curve. A value >1 is a tailingpeak, while a value


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broadening, each separated component would elute as a narrow slug opure compound. The measure of band broadening is band width, t w, omore correctly, the number of theoretical plates in the column, N.

Baseline

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

Baseline noise

Interferences to the detector and data system caused by electricalnoise or environmental effects. This interference keeps the baselinefrom being perfectly flat.

Baseline-resolved peaks

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

BET method

A method developed by Bruner, Emmett, and Teller for measuringsurface area by using nitrogen adsorption condensation in pores atliquid nitrogen temperature. Pore volume and pore size distributioncan also be obtained by this method.

Bonded-phase chromatography (BPC)

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

Breakthrough volume

The volume at which a particular solute pumped continuously througha column begins to elute. The breakthrough volume is useful in

determining the total sample capacity of the column for a particulasolute.

Calibration standards

These are standards of known quantities and substances which assistin peak identification or quantitation. Calibration standards can beexternal standards or internal standards.


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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 tothe support that is used to carry the active ligand, usually by acovalent bond. It can also refer to the support in otherchromatographic modes.

Cartridge column

A type of column with no endfittings which is held in a cartridgeholder. The column is an open tube with the packing contained byfrits 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 reversedphase packing. It is expressed as the number of carbon atoms such aC8 and C18.

Channeling

This occurs when voids are created in the packing material of acolumn. It results in the mobile phase and accompanying solutesmoving more rapidly than the average flow velocity producing bandbroadening. These voids are created by poor packing or by erosion othe packed bed.

Chemisorption

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

Chiral stationary phases


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

Chromatography

A chemical separation technique based on the differentialdistribution 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 aparticular result. It allows another analyst following the methodand conditions to reproduce the separation, and achieve the sameresults.

Chromatogram

The electronic result of a chromatographic separation which is aplot of detector signal output versus time or elution volume. It isrepresented 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 phasedifferentially interacts with the sample's constituent compounds asthey are carried along in the mobile phase.

Column backpressure

See Backpressure.

Column chromatography

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

Column-dependent chemical factors


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

Column performance

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

Column switching

The use of multiple columns connected by switching valves. Fractionfrom a primary column can be switched to two or more secondarycolumns, which in turn can be further diverted to additional columnor to the detector. This process is used for better chromatographicseparations or for sample cleanup.

Conductivity detectors

These detectors identify changes in the conductivity of the mobilephase as it passes through a flow cell. They are used to detect awide 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 notcontribute to the separation.

Counterion

In an ion-exchange process, this is the ion in solution which isused to displace the ion of interest from the ionic site. In ionpairing, it is the ion of opposite charge added to the mobile phaseto form a neutral ion pair in solution.

Coupled columns

A form of column switching. This uses a primary column connected totwo secondary columns via a selector valve. Fractions from the first

column can be selectively transferred to the other two columns foradditional separation. This term is also used to describe two ormore columns connected in series to provide increased plate numbers

Coverage


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

Cross-linking

During the process of copolymerization of resins to form a threedimensional matrix, and a difunctional monomer is added to formcross-linkages between adjacent polymer chains. The degree of crosslinking is determined by the amount of this monomer added to thereaction. For example, divinylbenzene is a typical cross-linkingagent for polystyrene ion-exchange resins. The swelling anddiffusion characteristics of a resin are governed by its degree ofcross-linking.

Cyano phase

A stationary phase that usually consists of cyanopropylsilyl groupsIt is used in both normal and reversed-phase chromatography.

Dead volume (Vd)

The volume outside of the column packing itself. The interstitialvolume (intraparticle and interparticle volume) plus extracolumnvolume (contributed by injector, detector, connecting tubing, andendfittings) all combine to create the dead volume. This volume can

be determined by injecting an inert compound. For example, acompound that does not interact with the column packing. It is alsoabbreviated V o or Vm.

Degassing

The process of removing dissolved gas from the mobile phase beforeor during use. Dissolved gas may come out of solution in thedetector cell and cause baseline spikes and noise. Dissolved air canaffect electrochemical detectors by reaction or fluorescencedetectors by quenching. Degassing is carried out by: heating the

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

Desalting


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

Detector

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

Detection limits

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

Detection threshold

The point at which the software determines the beginning and the endof the peak will shift depending on the threshold. The thresholdshould be set as low as possible. If it is set too low, the softwarewill 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 notquantitated.

Detector Linearity

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

Detector Sensitivity

The sensitivity setting is the line between normal background noiseand a true peak. Perturbations from the baseline that fall below thesensitivity 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 athe software tries to integrate peaks out of the noise.

Differential Refractive Index (RI)


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

Diffusion along the flow path

The coefficient for molecular diffusion along the flow path. It isrepresented as the B term in the van Deemter equation. The greaterthe diffusion coefficient of the component in the mobile phase, themore the narrow band of separated component diffuses into thesurrounding mobile phase before going to the detector. This resultsin band broadening. As linear velocity increases, the effect of

axial diffusion on column efficiency decreases. The van Deemterequation reflects this effect by dividing B by u.

Diffusion coefficient(DM or DS)

A fundamental parameter of a molecule in solution (Dm) or stationaryphase (Ds) expressed in cm2/sec. Dmdepends on molecular weight ofthe solute, temperature, solvent viscosity, and molar volume of thesolute. A typical value of a small molecule in RPC at roomtemperature would be 5 x 10 6 cm2/sec.

Diol phase

A stationary phase useful in both normal and reversed phasechromatography. It consists of a diol structure (two OH groups onadjacent carbon atoms in an aliphatic chain). It is less polar thansilica stationary phases used in normal-phase chromatography but ithas been used for the reversed-phase separation of proteins andpolypeptides.

Displacement chromatography

A chromatographic process in which the sample is placed onto thehead of the column and is then displaced by a compound that is morestrongly sorbed than the compounds of the original mixture. Samplemolecules are displaced by each other and by the more stronglysorbed compound. The result is that the eluting sample solute zonesmay be sharpened. Displacement techniques have been used mainly inpreparative EPLC applications.


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Distribution coefficient(D or KD)

See Partition coefficient

Eddy diffusion term

This is represented by the A term in the van Deemter equation. It ithe contribution to plate height that is due to molecules travelingalong different paths through the column and it depends on theparticle size and geometry of the packing. It is also called themultipath term.

See van Deemter equation.

Effective theoretical plates (Neff)

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

Efficiency (N or H)

A measure determined by the number of theoretical plates calculatedfrom 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 reactionbetween 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 electroactivecompounds.

Eluate

Combination of mobile phase and solute exiting column.

Eluent

Mobile phase used to carry out a separation.


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Eluotropic series

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

Elution

The process of passing mobile phase through the column to transportsolutes.

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 isinjected at the head of the column, and the individual compounds areseparated and eluted at the end of the column.

Elution volume (VR)

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

Endcapping

A column is said to be endcapped when a small silylating agent, suchas trimethylchlorosilane, is used to bond residual silanol groups ona packing surface. It is most often used with reversed-phasepackings and may cut down on undesirable adsorption of basic orionic compounds.

Endfitting

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

Exchange capacity

See Ion-exchange capacity.


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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. ManySEC packings are referred to by their exclusion limit. For example,a 105 column of porous silica gel will exclude any compounds with amolecular weight higher than 100,000, based on a polystyrenecalibration standard.

Exclusion volume (VC)

The retention volume of a molecule on SEC packing. All molecules

larger than the size of the largest pore are totally excluded andelute at the interstitial volume of the column.

Extracolumn effects

The band broadening effects of parts of the chromatographic systemoutside of the column itself. Areas of band broadening can includethe injector, connecting tubing, endfittings, frits, detector cellvolume, and internal detector tubing. The variances of all of thesecontributions are additive. These extracolumn effects should beminimized in order to maintain the efficiency of the column.

External standards

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

Fast LC

The use in BPLC of short columns (3 to 7 cm in length) withconventional internal diameters (2 to 6 mm) packed with smallparticles (3 or 5 mm dp). 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 ratesare 1 to 2 mL/min.


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Fluorescence detectors

Fluorescence detectors project a specified wavelength of light intothe sample, causing the component of interest to fluoresce and theemitted light is detected. Fluorescence detectors are commonly usedwith derivatization methods. Fluorescence detectors are veryselective and very sensitive.

Fractionation range

This is the range in which the packing can separate molecules basedon their size. Molecules that are too large to diffuse into thepores are excluded. Molecules that can diffuse into all of the poretotally permeate the packing, eluting (unseparated) at thepermeation volume. In SEC, it is the operating range of a gel orpacking.

Frit

The porous component at either end of a column that serves tocontain the column packing. It is placed at the very ends of thecolumn tube or, more commonly, in the end fitting. Frits are madefrom stainless steel or other inert metal or plastic, such as porouPTFE or polypropylene.

Frontal analysis

A chromatographic technique that involves continuous addition ofsample to the column. The result is that only the least sorbedcompound, which moves at the fastest rate, is obtained in a purestate. The second least sorbed compound elutes with the firsteluting compound, the third least sorbed compound with the first andsecond compound and so forth. This continues until the originalsample is eluting at the column exit. Frontal analysis is seldomused and is mainly a preparative technique.

Fronting

This is an asymmetrical peak shape in which the front partof a peak (before the apex) in a chromatogram tapers inadvance of the remainder of the peak. There is anasymmetric distribution with a leading edge. The asymmetryfactor for a fronting peak has a value


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Gaussian curve

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

Gel

The solid packing used in gel permeation chromatography. A gelactually consists of two parts: the dispersed medium (solid portionand the dispersing medium (the solvent).

Gel filtration chromatography (GFC)

This is size-exclusion chromatography carried out with aqueousmobile phases, also known as aqueous GPC. It generally refers toseparations carried out on soft gels such as polydextrans. Most gelfiltration separations involve biopolymers and are used for theseparation and characterization of polymers.

Gel permeation chromatography (GPC)

See GFC.

Gradient elution

A technique for decreasing separation time by increasing mobilephase strength over time during a chromatographic separation. It isalso known as solvent programming. Gradients can be continuous orstep-wise. Binary, ternary, and quaternary solvent gradients areused routinely in HPLC.

Guard column

A small column placed between the injector and the analytical columIt protects the analytical column against contamination by sampleparticulates, and by strongly retained species. The guard column isusually packed with the same material as the analytical column and

is often of the same i.d. It is much shorter, costs less, and isusually discarded when it becomes contaminated.

HETP

The height equivalent of a theoretical plate. It is a carryover fromdistillation theory which is a measure of a column's efficiency. Foa typical HPLC column packed with 5 m particles, HETP (or H) value


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are usually between 0.01 and 0.03 mm. HETP = L/N, where L is columnlength, and N is the number of theoretical plates.

Hydrophilic

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

Hydrophobic

It is often referred to as water hating. It adverts both tostationary phases not compatible with water and molecules withlittle affinity for water. Hydrophobic molecules have few polarfunctional groups and are mostly hydrocarbons or have highhydrocarbon content.

Hydrophobic interaction chromatography

A technique in which reversed-phase packings are used to separatemolecules by the interactions between their hydrophobic moieties andthe hydrophobic sites on the surface. High salt concentrations areused in the mobile phase and separations are effected by changingthe salt concentration. The technique is analogous to "salting out"molecules from solution. Gradients are run by decreasing the saltconcentration over time.

Injector

A mechanism for accurately injecting a predetermined amount ofsample into the mobile phase stream. The injector can be a simplemanual device, or a sophisticated autosampler that permits automatedinjections of many different samples for unattended operation.

In-line filter

A device that prevents particulate matter from damaging the columnby filtration. Modem in-line filters can be placed between theinjector and the column without contributing to band broadening. A

filter in this position is used to prevent sample particulates fromentering 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.


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Internal standards

Internal standards consist of a specific quantity of a compound thatis known not to be in the sample, but that exhibits the samecharacteristics under the separation conditions as the samplecomponents. Internal standards are used primarily to calibratequantitation, especially for methods susceptible to volumetric erroresulting from sample preparation.

Interstitial volume (VO)

The total volume of mobile phase within the length of the column. Itis made up of the intraparticle volume (inside the packing itself)and interparticle volume (between the packing particles). Same asVoid volume. It is also abbreviated V i or Vm.

Ion chromatography (IC)

An ion-exchange technique in which low concentrations of anions orcations are determined using low capacity ion exchangers with weakbuffers. Conductivity detectors are often used. Ion chromatographyis practiced in two forms: suppressed IC, and non-suppressed IC.

Ion-exchange chromatography (IEC)

A mode of chromatography in which ionic substances are separated oncationic or anionic sites of the packing. The sample ion (andusually a counterion) will exchange with ions already on theionogenic group of the packing. Retention is based on the affinityof different ions for the site and on a number of other solutionparameters such as, pH, ionic strength, and counterion type.

Ion-exchange capacity

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

Ion exclusion

A process in which ionized solutes can be separated from un-ionizedor partially ionized solutes using ion-exchange resins. Separationresults from Donnan membrane potential. This is where ionic solutesexist at a higher concentration in solution than in the resin butnonionic solutes are evenly distributed between the mobile phase andresin. Therefore, ionic solutes will elute faster from the columnthan the nonionic solutes.


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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-phasecolumn. Ion-pairing agents are usually ionic compounds that containa hydrocarbon chain which imparts a certain hydrophobicity so thatthe ion pair can be retained on a reversed-phase column.Ion-pairingcan also occur in normal-phase chromatography when one part of thepair is loaded onto a sorbent, but this technique is not as popularas the RPC technique.

Ion suppression

This involves buffering in an aqueous mobile phase at a particularpH to suppress solute ionization. For example, the ionization ofweak carboxylic acids can be suppressed by adjusting the pH below

their ionization constant. This technique is useful for improvingthe peak shape of weak acids and bases in RPC.

Irregular packing

The shape of a silica gel-based packing. Irregular silicas areavailable in microparticulate sizes. The packings are made bygrinding silica gel into small particles, sizing them into smallparticles, and into narrow fractions using classification machineryWhile spherical packings are now used more often than irregularpackings in HPLC, the less expensive irregular packings are stillwidely used in prep LC.

Irreversible adsorption

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

Isocratic

A constant composition mobile phase used in liquid chromatography.

k or k'

The capacity factor. It can be calculated from the equation, wheretR is retention time for the sample peak, and to is the retentiontime for an unretained peak.


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Ligand

In ligand-exchange chromatography, it is the molecule added to themobile phase that acts as the chelating agent. In affinitychromatography, it is the biospecific material (enzyme, antigen, orhormone) coupled to the support (carrier) to form the affinitycolumn.

Ligand-exchange chromatography

A technique in which chelating ligands are added to the mobile phaseand undergo sorption onto a packing. These sorbed molecules, can actas chelating agents with solutes. For example, using of copper aminechelates for the separation of amino acids. Chelating resinsfunction in a similar manner. The chelating groups are chemicallybonded to the polystyrene backbone.

Linearity

A measurement process which ensures accurate quantitation. Inchromatography it is important that the detector provide a linearresponse over the range of sample concentrations it encounters.

Linear elution adsorption chromatography (LEAC)

A term coined by Lloyd Snyder, which refers to adsorptionchromatography carried out in the linear portion of an adsorptionisotherm.

Linear velocity

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

Liquid-liquid chromatography (LLC)

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

Liquid-solid chromatography (LSC)

Same as Adsorption chromatography.


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Loading

The amount of stationary phase coated or bonded onto a solid supporIn liquid-liquid chromatography, it is the milligram amount ofliquid phase per gram of packing. In BPC, the loading may beexpressed in mmol/m 2 or in percent C. See Coverage.

Macroporous resin

Cross-linked ion-exchange resins that have both micropores ofmolecular dimensions and macropores several hundred wide. Theseare highly porous resins with large internal surface areasaccessible 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 Deemterequation and is referred to as the mass transfer term. The fasterthe process of mass transfer, the better the efficiency of thecolumn. In HPLC, mass transfer is the most important factoraffecting column efficiency. It is increased by the use of smallparticle packings, thin layers of stationary phase, low viscositymobile 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 sothey can interact with the stationary phase. In SEC, the packingshave different pore diameters, allowing molecules of different sizeto be separated. For a typical adsorbent such as silica gel, 60 and 100 pore diameters are most popular. For packings used for theseparation of biomolecules, pore diameters > 300 are used.

Method-dependent chemical factors

The variables in the chromatographic method which include the choiceof 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 provideanother parameter that can be used to change selectivity.

Microbore


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

Microparticulate

Small particles used as HPLC stationary phases. These packingsgenerally have a particle diameter


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The distribution of molecular weight of molecules in a polymersample. Distribution can be defined as weight average and numberaverage.

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 analkyl or arylrnonochlorosilane. Polymeric phases are generallyprepared from a di or trichlorosilane reactant.

Multidimensional chromatography

The use of two or more columns or chromatographic techniques whichenable 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 oon-line by the use of a switching valve. It also called coupledcolumn chromatography, column switching, multicolumn chromatographyor boxcar chromatography .

N

The number of theoretical plates. A measure of the efficiency of acolumn. , where tR is retention time, and wb is the base width ofthe peak. Sometimes it is measured as , where wl/2 is the peak widthat 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 bondedphase.

Non-suppressed Ic

A type of ion chromatography were weakly conducting buffers at lowconcentration are carefully selected, and the entire effluent ispassed through the detector. The ions are detected above thebackground signal.

Normal-phase chromatography


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

Octadecylsilane (ODS)

The most popular reversed phase in HPLC. Octadecylsilane phases arebonded to silica or polymeric packings. Both monomeric and polymeriphases are available.

Open-tubular columns

Columns of small internal diameter. Stationary phases can be bondedon the internal walls of these columns. The most common type is thefused silica tubing made for capillary GC. These columns are

currently being investigated for HPLC, SFC, and capillaryelectrophoresis.

Organic modifier

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

Overload

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

PEEK

Packed bed qualityThe coefficient that reflects the quality ofpacked bed. It is represented as the A term in the expanded vanDeemter equation. This term is determined by the size anddistribution of voids, channels, and other nonuniformities in thepacked bed. It represents a practical lower limit for H, and can bechanged 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 baselineresolved.


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Particle size (dp)

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 LCcolumn. In HPLC, a narrow particle size distribution is desirable. Aparticle size distribution of dp 10 percent would mean that 90percent of the particles fall between 9 and 11 mm for an average 10mm d p packing.

Partition chromatography

A separation process in which one of the liquid phases is heldstationary on a solid support while the other is allowed to flowfreely down the column. Solutes partition themselves between the twophases 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 amountof solute in the mobile phase. It can also be the distributioncoefficient, KD.

Peak

When the detector registers the presence of a compound, the normalbaseline signal it sends to the data system changes, resulting in adeflection from the baseline called a peak. Well resolved peaks aresymmetrical, touch the baseline, and do not interfere with otherpeaks.

Peak Identification

Peak identification is usually performed by comparing the sample

chromatogram to a chromatogram of a standard solution separatedunder the same conditions. Peaks that appear at the same elutiontime as peaks in the standard are identified as the same component.

Peak Quantitation

Correctly detecting the beginning and end of a peak.

Peak shape


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

Peak tailing

Same as tailing peaks.

Peak width (wb)

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 ofdimethylphenylchlorosilane with silica gel. It is claimed to have anaffinity 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, columndimensions, leaks in the fluid path, system bandspreading, anddetector cell design.

Plates

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

Polyacrylamide gel

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

Polymeric packings


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Packings based on polymeric materials, usually in the form ofspherical 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. Ionicgroups are incorporated by various chemical reactions. Neutral PS-DVB beads are used in reversed-phase chromatography. Porosity andmechanical stability can be altered by varying the cross-linkingthrough 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 usuallyexpressed in mL/g. It is measured by the BET method of nitrogenadsorption or by mercury intrusion, where Hg is pumped into thepores under high pressure.

Porosity

The ratio of the volume of the interstices, to the volume of thesolid particles. The pore volume is also used as a measure ofporosity.

Precolumn

A small column placed between the pump and the injector. It removesparticulate matter which could be in the mobile phase, chemicallysorbs substances that might interfere with the separation, or actsas a saturator column presaturating the mobile phase with stationaryphase to prevent stripping of the column. Its volume has littleeffect on isocratic elution but contributes a delay to the gradientelution.

Preparative chromatography

The process of using liquid chromatography to isolate a sufficientamount of material for other experimental or functional purposes.For pharmaceutical or biotechnological purifications, columnsseveral feet in diameter can be used for multiple grams of materialFor isolating just a few micrograms of a valuable natural product,


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an analytical column can be used. Both are preparativechromatographic approaches.

Pulsating flow

Flow originating from a reciprocating pump. Normally, the pulses aredampened out by pulse damper, by an electronic pressure feedbackcircuit, or by an active damper pump head. Some detectors, such aselectrochemical, are affected by flow pulsations.

Qualitation

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

Quantitation

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

Radial compression

The use of radial pressure applied to a flexible wall column tolessen 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 notonly in a longitudinal direction as it flows, but radially as well.

Recovery

The amount of solute (sample) that elutes from a column relative tothe amount injected. This is most often used with proteinseparations in which proteins "hang up" on active sites of thepacking in certain columns.

Recycling

A technique in which the column effluent is recirculated onto thehead of the column in an attempt to gain the advantage of anextended column length. It can be carried out on a single column bypassing the effluent back through the pump. An alternative techniqueuses two columns connected by a switching valve. This technique isvery seldom used in HPLC, and only in size-exclusion chromatography


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Reduced plate height (h)

A calculated value used to measure efficiencies of columns. A BPLCcolumn with an h value


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

Retention factor

Retention factor is how long a compound is retained by thestationary phase relative to the time it resides in the mobile phasIn IUPAC nomenclature, the retention factor is represented by theletter k and is dimensionless. In former usage, k was oftenrepresented as k', and was termed the capacity factor.

Retention time (tR')

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

Retention volume (VR)

The volume of mobile phase required to elute a substance from thecolumn. This is where Vm is the void volume, KD the distributioncoefficient, and Vs the stationary phase volume.

Reversed-phase chromatography (RPC)

The most common HPLC mode. Uses hydrophobic packings such asoctadecyl or octysilane phases bonded to silica or neutral polymeribeads. The mobile phase used is usually water and a water miscibleorganic solvent such as methanol or acetonitrile. There are manyvariations of RPC in which various mobile phase additives are usedto impart a different selectivity. For example, in the RPC of anionthe addition of a buffer and tetraalkylammonium salt would allow ionpairing to occur, resulting in separations that rival ion-exchangechromatography.

Sample capacity

The amount of sample that can be injected onto an LC column withoutoverload. It is often expressed as grams of sample per gram ofpacking. Overload is defined as the sample mass injected at whichthe 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 itto broaden proportionally to the square root of the distance it hastraveled.

Sampling rate


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The frequency with which the detector checks the flow cell. Samplingrate is often called time constant. If it is set too fast too muchraw data will be generated. If it is set too slow, a narrow peakcould be missed.

Selectivity (a)

The same as Separation factor or Relative retention ratio. Athermodynamic factor that is a measure of relative retention of twosubstances. Fixed by a certain stationary phase and mobile phasecomposition.

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. Normalinjection size is in the milligram to low gram range.

Separation factor

The separation factor, sometimes called selectivity, is the relativeretention measured for two adjacent peaks. The separation factor isrepresented by the Greek letter a, and is reported in dimensionlessunits.

The serparation factor for two compounds is calculated using theformula .

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

Silanol

The SiOH group found on the surface of silica gel. There aredifferent strengths of silanols depending on their location andrelationship to each other. The strongest silanols are acidic and

often lead to undesirable interactions with basic compounds duringchromatography.

Siloxane

The Si-O-Si bond. A principal bond found in silica gel, forattachment of a silylated compound, or bonded phase. It is stableexcept at high pH values.


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Silylation

The reaction of an organochloro- or organoalkoxysilane with acompound containing a reactive group. In liquid chromatography, itrefers to the process of derivatizing the solute beforechromatography in order to make it detectable or to prevent unwantedstationary phase interactions. It can also refer to the process ofadding a chemically bonded phase to a solid support or todeactivating 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 withmicroparticles. The packing is suspended in a slurry (10 percentwt/vol) and is rapidly pumped into the empty column. Special highpressure pumps are used.

Soap chromatography

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

Solid-phase extraction (SPE)

A sample preparation technique that uses a solid phase packingcontained in a small plastic cartridge. The solid stationary phasesare the same as HPLC packings but the principle is different fromHPLC. It is sometimes referred to as digital chromatography. Thisprocess as it is most often practiced, requires four steps:conditioning the sorbent, adding the sample, washing away theimpurities, and eluting the sample in as small a volume as possiblewith a strong solvent.

Solid support

Same as Support.

Solute

The dissolved component of a mixture that is to be separated in thechromatographic column.

Solvent strength


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The ability of a solvent to elute a particular solute or compoundfrom a column. Lloyd Snyder described it for LEAC (LSC) adsorptionchromatography on alumina and solvents were quantitatively rated inan eluotropic series. No eluotropic series exists for other modes.

Sorbent

An adsorption packing used in liquid chromatography. A commonsorbent is silica gel.

Specific surface area

The surface area of an LC packing based on a measurement by anaccepted technique, such as the BET method that uses nitrogenadsorption.

Spherical packing

A spherical solid packing material. Spherical packings are generallypreferred over irregular particles.

Stationary phase

The immobile phase involved in the chromatographic process. Thestationary phase in liquid chromatography can be a solid, a bondedor coated phase on a solid support, or a wall coated phase. Thestationary phase used often characterizes the LC mode. For example,silica gel is used in adsorption chromatography, an octadecylsilanebonded phase in reversed-phase chromatography, etc.

Stepwise elution

This process uses eluents of different compositions during thechromatographic run. These eluents are added in a stepwise mannerwith a pump, or by a selector valve. Gradient elution incorporatescontinuous changing of solvent composition.

Steric exclusion chromatography (SEC)

A major LC mode in which samples are separated by virtue of theirsize in solution. Also known as size-exclusion, gel permeation, gelfiltration, or gel chromatography.

Straight-phase chromatography

Same as Normal-phase chromatography.


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Supercritical fluid chromatography (SFC)

A technique that uses a supercritical fluid as the mobile phase. Thetechnique has been applied to the separation of substances thatcannot be handled effectively by liquid chromatography (because ofdetection problems) or gas chromatography (because of the lack ofvolatility). Examples are separations of triglycerides, hydrocarbonand fatty acids. GC detectors and HPLC pumps have been used togethein 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 sampleions can be more easily detected; membrane separator is sometimesused.

Suppressor column

In ion chromatography, it is the column placed after the ion-exchange column. Its purpose is to remove or suppress the ionizationof buffer ions so that sample ions can be observed in a weaklyconducting background with a conductivity detector.

Surface area

In an adsorbent, it is the total area of the solid surface asdetermined by an accepted measurement technique such as the BETmethod using nitrogen adsorption. The surface area of a typicalporous adsorbent such as silica gel can vary from 100 to 600 m 2/g.

Surface coverage

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

Swelling

A process in which resins and gels increase their volume because oftheir solvent environment. The solvent enters the ion-exchange resinto dilute ions, where in gels the solvent penetrates the pores. Ifswelling occurs in packed columns, blockage or increased backpressure 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 isthe result of a component that is excessively retarded in eluting.Tailing is caused by sites on the packing that have a stronger thannormal retention for the solute. A typical example of a tailingphenomenon is the strong adsorption of amines on the residualsilanol groups of a low coverage reversed-phase packing.

Temperature programming

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

Tortuosity

A property of a packed column that indicates the degree ofunevenness of the path followed by the solute molecule as it passesdown the column. The A term in the van Deemter equation takes thisinto consideration.

Total permeation volume (Vp)

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

Trace enrichment

A technique in which trace amounts of compounds from a weak mobilephase or solution are retained on an HPLC packing. They are theneluted by the addition of a stronger mobile phase in a concentratedform. The technique has been most successfully applied in theconcentration of trace amounts of hydrophobic compounds, such aspolynuclear aromatic hydrocarbons, out of water using a reversed-phase column. A strong solvent such as acetonitrile serves to elutethe enriched compounds.

Ultraviolet/Visible Light (UV/Vis)

The tunable or variable wavelength UV/Vis detector is the mostpopular form of detector. For methods involving organic compounds inaqueous mobile phases, the UV/Vis detector takes advantage ofcompounds' varying absorptivities of ultraviolet and visible light.

Unretained compounds


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

Vacancy chromatography

A technique in which a mobile phase additive causes a positivedetector signal output. When a solute elutes from the column, itdilutes the signal and gives a negative peak or a vacancy. Thetechnique has been most recently applied to single column ionchromatography, in which mobile phases that absorb in the UV regionsuch as citrate and phthalate buffers, are used. When a nonabsorbinganion elutes, it dilutes the UV absorbing background and causes anegative peak. The detector output leads are usually reversed tomake the chromatogram look normal.

van Deemter equation

An equation used to explain band broadening in chromatography. Thisequation represents the height equivalent of a theoretical plate andhas three terms. The A term is used to describe eddy diffusion,which considers the different paths a solute may follow when passingover particles of different sizes. The B term is for thecontribution caused by molecular diffusion or longitudinal diffusionof the solute while passing through the column. The C term is thecontribution of mass transfer and allows for the finite rate oftransfer of the solute between the stationary phase and mobile phas

Velocity (u)

Same as Linear velocity.

Void

The formation of a space, usually at the head of the column, causedby a settling or dissolution of the packing. A void in the columnleads to decreased efficiency and loss of resolution. Even a smallvoid can be disastrous for small microparticulate columns. The voidcan sometimes be removed by filling it with glass beads or porouspacking.

Void time (tm or to)

The time for elution of an unretained peak.

Void volume (Vi)The total volume of mobile phase in the column with the remainder othe column taken up by packing material. It can be determined byinjecting an unretained substance that measures void volume plus


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extracolumn volume. It also is referred to as interstitial volume. Vo or Vm are sometimes used as symbols.

Wall effect

The consequence of the looser packing density near the walls of therigid HPLC column. Mobile phase has a tendency to flow slightlyfaster near the wall because of the decreased permeability. Thesolute molecules that happen to be near the wall are carried alongfaster than the average of the solute band and band spreadingresults.

Waste container

At the end of the fluid path, the mobile phase and separated samplecomponents are collected into a waste container. This container issuitable for safely collecting and disposing of the solvents used inthe separation.

Xerogels

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

X-axis

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

Y-axis

The Y-axis of the chromatogram records the strength of the detectorsignal, which is usually proportional to the concentration of samplein the eluent passing through the detector. The units depend on thetype of detector being used.

ZwitterionsCompounds that carry both positive and negative charges in solution

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