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Glossary of Chromatographic Terms

If you are a beginner looking for a quick explanation or a more seasoned scientist who wants a quick refresher, the following Glossary will be helpful to you. Click on the appropriate method to move to the terms on interest. The terms are listed in alphabetical and crossed-referenced where appropriate.

Liquid Chromatography

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

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

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

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

Backpressure
The pressure required to pump the mobile phase through the column. It is related to mobile phase viscosity (h), flow rate (F), column length (L) and diameter (dc), and particle size (dp) by the following equation:

Band Broadening
The dilution of the chromatographic band as it moves down the column. The measure of band broadening is band width, tw, or more correctly, the number of theoretical plates in the column, N.

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

Capacity Factor (k)
Expression that measures the degree of retention of an analyte relative to an unretained peak, where tR is the retention time for the sample peak and to is the retention time for an unretained peak. A measurement of capacity will help determine whether retention shifts are due to the column (capacity factor is changing with retention time changes) or the system (capacity factor remains constant with retention time changes).

Cation Exchange Chromatography
The ion exchange procedure used for the separation of cations. Both resins and bonded phases are available for this mode. The sulfonic acid group is a typical strong cation exchange functional group. A carboxylic acid group on a bonded or coated stationary phase would be an example of a weak cation exchanger.

Chain Length
The length of carbon chain bonded to a reversed phase packing. It is expressed as the number of carbon atoms (e.g., C8, C18).

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

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

Coverage
Refers to the amount of bonded phase on a silica support. Coverage is usually described in µmol/m2 or % Carbon.

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

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).

Efficiency (N)
Also number of theoretical plates. A measure of peak band spreading determined by various methods, some of which are sensitive to peak asymmetry. The most common are shown here, with the ones most sensitive to peak shape shown first:

4-sigma / tangental

Half height

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

Eluent
Mobile phase used to carry out a separation.

Eluotropic Series
A series of solvents with an increasing or decreasing 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 Volume (VR)
Refers to the volume of mobile phase required to elute a solute from the column at maximum concentration (apex). VR = F o tR, where F is flow rate in volume/time and tR is the retention time for the peak of interest.

Exclusion Limit
In SEC, the upper limit of molecular weight (or size) beyond which molecules will elute at the same retention volume, called the exclusion volume. Many SEC packings are referred to by their exclusion limit.

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

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

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

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

Gradient Elution
Technique for decreasing separation time by increasing mobile phase strength over time during the chromatographic separation. Other gradients include temperature, pH, salt, and flow rates.

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

where L is column length in millimeters and N is the number of theoretical plates.

Hydrophilic
"Water loving": refers both to stationary phases that are compatible with water and to water soluble molecules in general.

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

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 (pH, ionic strength, counterion type, etc).

Ion Exchange Capacity
The number of ionic sites on the packing that can take part in the exchange process. Exchange capacity is expressed in mEq/g.

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

Linear Velocity
The flow rate normalized by the column cross section. This effects column performance and is directly related to column pressure. Linear velocity is given by the following equation where L is column length and to is the breakthrough time of an unretained peak:

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

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

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

N (Number of Theoretical Plates)
See Efficiency.

Octadecylsilane (ODS or C18)
The most popular reversed phase packing in HPLC. Octadecylsilane phases are bonded to silica or polymeric supports. Both monomeric and polymeric phases are available.

Overload
The mass of sample injected onto the column at which efficiency and resolution begin to be affected if the sample size is further increased.

Partition Coefficient (K)
The amount of solute in the stationary phase relative to the amount of solute in the mobile phase.

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

Pore Volume
The total volume of the pores in a porous packing; usually expressed in mL/g.

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

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

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

Resolution (Rs)
Ability of a column to separate chromatographic peaks. Resolution can be improved by increasing column length, decreasing particle size, increasing temperature, changing the eluent or stationary phase. It can also be expressed in terms of the separation of the apex of two peaks divided by the tangental width average of the peaks:

Retention Time (tR)
The time between injection and the appearance of the peak maximum.

Retention Volume (VR)
The volume of mobile phase required to elute a substance from the column: VR = F o tR.

Reversed Phase Chromatography (RPC)
The most common HPLC mode. Uses hydrophobic packings such as octadecyl- or octylsilane phases bonded to silica or neutral polymeric beads. Mobile phase is usually water and a water-miscible organic solvent such as methanol or acetonitrile. There are many variations of RPC in which various mobile phase additives are used to impart a different selectivity.

SAX
Strong anion exchanger.

SCX
Strong cation exchanger.

Selectivity (alpha)
A thermodynamic factor that is a measure of relative retention of two substances, fixed by a certain stationary phase and mobile phase composition. Where k1 and k2 are the respective capacity factors.

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

Siloxane
The Si-O-Si bond. A principal bond found in silica gel or for attachment of a silylated compound or bonded phase. Stable except at high pH value.
Size Exclusion Chromatography (SEC)
A noninteractive technique which separates solutes according to their molecular size in solution.

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

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

Solvent Strength
Refers to the ability of a solvent to elute a particular solute or compound from a column. See eluotropic series.

Stationary Phase
The immobile phase involved in the chromatographic process.

Tailing
The phenomenon in which the normal Gaussian peak has an asymmetry factor > 1. Tailing is most often caused by sites on the packing that have a stronger than normal retention for the solute.

Tailing Factor (T)
A measure of the symmetry of a peak, given by the following equation where W0.05 is the peak width at 5% height and f is the distance from peak front to apex point at 5% height. Ideally, peaks should be Gaussian in shape or totally symmetrical.
T = W0.05/2f

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

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

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

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

Void Volume (V0)
The total volume of mobile phase in the column: the remainder of the column is taken up by packing material. Can be determined by injecting an unretained substance that measures void volume plus extra column volume.

WAX
Weak anion exchanger.

WCX
Weak cation exchanger.s and Equations

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Gas Chromatography

Adjusted Retention Time (tR')
An analyte's retention time (tR) minus the elution time of an unretained peak (tm).
tR'= tR-tm

Adjusted retention time is also equivalent to the time the analyte spends in the stationary phase.

Capacity Factor (k)
Expression that measures the degree of retention of an analyte relative to an unretained peak, where tR is the retention time for the sample peak and tm is the retention time for an unretained peak. A measurement of capacity will help determine whether retention shifts are due to the column (capacity factor is changing with retention time changes) or the system (capacity factor remains constant with retention time changes).

Thus the higher the capacity factor, the longer the retention time.

Column Efficiency (N)
See Theoretical Plate Number.

Column Evaluation
An application on Thermo GC instruments that measure a column's resistance to flow, accurately controlling gas linear flow rates to ensure consistent retention times.

Detectors
See ECD, FID, FPD, NPD, PID and TCD.

Distribution Constant
A ratio of concentration of solute in the stationary phase versus the mobile phase. Also known as partition co-efficient.

Effective Theoretical Plates (Neff)
A measure of a column performance that accounts for the effects of unretained elution time, where t'R is the adjusted retention time and s is the standard deviation of the peak.

This value also remains constant as retention gaps and guards are used. Depending on the method of peak width calculation, different efficiencies can be reported. This leads to two popular measures:

Where W is the tangential peak width (13.4% peak height).

Where W is the width measured at half height (50% peak height).

ECD
Electron Capture Detector. Uses electron emitting source to ionize the carrier gas. Any electron-deficient analyte will reduce this level of ionization. The ECD detector is sensitive to any analyte with electronegative functionality (e.g. Cl-).

FID
Flame Ionization Detector. A "universal" detector for all carbon-containing compounds. A high temperature hydrogen flame ionizes the sample, causing an increase in current through an electrode, proportional to the amount of carbon passing through the flame.

FPD
Flame Photometric Detector. Analytes are passed through a hydrogen rich flame which is monitored by photocells. FPDs are usually used as a specific detector for sulphur- or phosphorous-containing compounds.

Flow Rate
The volumetric flow in mL/min of the carrier gas; this is different from the linear velocity.

Fronting
Distorted peak where the asymmetry of the peak is towards the front; the peak tailing factor is < 1.

Heartcutting (2D GC)
A method of using two columns of different selectivity to gain more information on a sample. In heartcutting, only a selected portion of eluant from the first column is passed onto the second.

HEEP (Heff)
Height Equivalent to an Effective Plate.

Where L is the column length. The smaller the Neff, the more efficient the column's performance.

HETP (H)
Height Equivalent to a Theoretical Plate is a measure of column efficiency where L is the column length and N is the number of theoretical plates:

HETP is based on actual (tR) rather than adjusted retention times (t'R).

Hold Up Time (tm)
The time for an injected substance, which is not retained on the stationary phase, to pass through the column and reach the detector. Hold Up Time is usually measured by injection of a compound such as methane.

Kovat's Index
A value that expresses the retention of a sample compared to two standards eluting before and after it. The Kovat's Index uses simple alkanes. The value is derived by allotting the alkanes a value of 100 times their carbon number and giving the sample a value equivalent to a hypothetical alkane eluting at the same time.

Leak Test
Process to establish the gas tight nature of all connections. This is an automated test on Thermo GC instruments.

Linear Velocity (u)
Mobile phase flow rate expressed in cm/s and is expressed as

Where L is the column length and to is the breakthrough time of an unretained peak

Mass Distribution Ratio [k(Dm)]
Alternative to Capacity Factor. Described as the ratio between the fraction of an analyte in the stationary phase and mobile phase.

NPD
Nitrogen Phosphorus Detector. Selective to the presence of nitrogen or phosphorus in the sample. NPD is often used in environmental studies.

On-Column Injection
Method of injection where the syringe needle enters and delivers the sample onto the top of the column.

Partition Co-efficient
See Distribution Constant.

Peak Width (W)
There are a number of ways to measure peak width. Most common are:
1. Tangential width: measures the baseline between two tangents taken from the peak inflection points and the baseline.
2. Width at half-height: measures width between the peak measured at 50% peak height
Peak width can be expressed in time or volume.

Phase Ratio (beta)
The ratio of a column's volume of stationary phase to mobile phase. An important value when changing the column dimensions of a method.

PID
Photo Ionization Detector. Photons from the detector UV source ionize solutes passing through the detector. The presence of an analyte increases the signal.

PLOT
Porous Layer Open Tubular column. Capilliary column that has a fine adsorbent bonded to it. PLOT is most often used for very volatile liquids or permanent gases.

Purged Splitless Injection
This is the most common splitless method where the injector is set in split mode for a set time but then the split vent is opened to flush any remaining sample.

Resolution
A measure of the separation of two peaks taking into account both the difference in elution time and the peak widths.

Where t2 and t1 are the two retention times, and W1 and W2 are baseline peak widths.

Retention Index
A value that expresses the retention of a sample compared to two standards that elute before and after it. See Kovat's Index.

Retention Time (tR)
Total time from injection to elution. This takes no account of dead volume or tm.

Retention Volume
Total volume from injection to elution.

SCOT
Support Coated Open Tubular column. A capilliary column where the liquid stationary phase is supported on a solid substrate coating the capillary column walls.

Selectivity (alpha)
The relative retention of two adjacent peaks. Selectivity can be calculated using capacity factor or retention volumes.

Separation Factor
See Selectivity.

Sensitivity
An estimation of the smallest analyte signal that can be detected by the method. Sensitivity is usually specified as a signal-to-noise ratio of 2.

Septum
A small disc usually made of rubber or silicone material that is used to seal the injector from the atmosphere. The syringe needle passes through the septum.

Septum Bleed
Compounds generated form the septum by the effect of temperature on the septum material. The compounds released lead to contamination of the injector and noisy baselines.

Split Injection
Method of introducing a sample onto the column. In split injection most of the sample, once vaporized, passes out of the injector to waste with only a small portion entering the column head.

Splitless Injection
Method of introducing a sample onto the column where the purge valve is closed for a period of time allowing all sample to enter the column. The purge valve is then opened to flush the injector.

Theoretical Plate Number
A measure of efficiency of the whole system. There are a number of ways to calculate efficiency because the calculation includes a term for peak width. Depending on the method of peak width calculation used, different efficiencies can be reported. Also known as Column Efficiency.

TCD
Thermal Conductivity Detector. Heated elements form the arms of a wheatstone bridge. Analytes passing through one chamber change the temperature and therefore the resistance which is monitored. It is a universal, non-destructive detector.

Trennzahl Number
A value to describe a separation. The Trenzahl number is calculated from the resolution between two consecutive homologous hydrocarbons. The Trennzahl number represents the number of peaks that can be included between the two hydrocarbon peaks.

van Deemter Equation
This is a relationship that considers the effect of linear velocity on the HETP or h, where A accounts for eddy currents, B describes the diffusion in the mobile phase term, C refers to the resistance to transfer from the stationary to mobile phase and u is the velocity of the mobile phase.

WCOT
Wall Coated Open Tubular column. Column where the stationary phase is bonded to the inside wall of the capillary column. WCOT is the most commonly used column.

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Solid Phase Extraction

Analyte
Compound to be isolated and measured in a sample preparation scheme

Column Volume
The sum of the interstitial volume plus the pore volume of the sorbent within the column

Bonded Phase
An organic  functionality covalently bonded to a chromatoghraphic  support.

Breakthrough
Lack of analyte retention which occurs when the total mass of the solutes (analytes + interferences) exceeds the capacity of the sorbent or the solute is weakly retained

Buffer
A solution of a weak acid and its salt or a weak base and its salt which can resist pH change upon addition of small amounts of strong acid or base

Capacity
Total quantity of solutes  (analytes & interferences) which can be retained from a specific sample matrix solution by a given mass of sorbent

Conditioning
The preparation of the extraction column to receive the sample. For reversed phase columns, this involves the application of a solvent such as methanol, followed by a similar volume of water. Normal phase columns are conditioned with a solvent similar to the sample solvent. Ion exchange columns are conditioned with a buffer of appropriate pH and ionic strength.

Counter Ion
The ionic species which pairs or associates with the ionic functional group of opposite charge on an ion-exchange sorbent. To be retained, a charged analyte must displace the counter ion associated with the ion exchanger group.

Eluent
Solvent or solvent mixture used for the removal of the solutes from the sorbent bed.

Elution Volume
The volume of solvent required to elute an analyte quantitatively.

Endcapping
A technique used to remove unwanted silanol groups on the surface of the silica which might otherwise undergo secondary interactions with the solutes.

Extraction
Transfer of the analytes from one phase to another

Frit
The porous element which contains the media within an SPE column. The frits are typically manufactured  from porous polyethylene or polypropylene

Hydrophilic
"Water loving": Refers both to stationary phases that are compatible with water and to water soluble molecules in general

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

Interference
Substance in the sample or separation system which may influence retention of the analyte on the sorbent or may co-elute with the analyte and influence analytical determination

Ion Exchange Chromatography
A chromatographic mode in which ions are retained by oppositely charged groups covalently bonded to a solid support. The analyte ions are retained by displacing counterions associated with the bonded functional group.

Liquid / Liquid Extraction (LLE)
A purification technique whereby the sample is first dissolved in a solvent and then agitated with a second immiscible solvent .  Solutes that partition preferentially with the second solvent are extracted and effectively purified.

Matrix
All components of the sample including the solvent, but excluding the analytes

Mobile Phase
The solvent that moves the solute through the column

Non-Polar Molecule
Molecule with a symmetric distribution of charge

Normal Phase
A mode of chromatography whereby retention on a sorbent bed increases with the polarity of the sorbent.  Sorbents used in this mode include silica, florisil and aminopropyl.

pKa
The pH value at which fifty percent of the ionisable groups of an ionic analyte are ionized and fifty percent are in the molecular form

Polar Molecule
Molecule with an unsymmetric distribution of charge

Pore Size
The average diameter of the porous openings on the surface of a sorbent particle

Reversed Phase
A chromatographic mode in which non-polar to moderately polar analytes are extracted from a polar solution using a non-polar sorbent

SAX
Strong anion exchanger

SCX
Strong cation exchanger

Solid Phase Extraction
Solid Phase Extraction (SPE) is a broad term used to describe the separation technique where liquids contact modified solid surfaces and a component of the liquid adheres to the solid.  In a separate step, the solid releases the component.  The solid usually consists of an inert core covered with unique "hooks" that remove the targeted material from the starting liquid.These active solids are packed into containment devices such as plastic columns through which the matrix and subsequent wash & elution solvents are passed to produce a purified material.

Sorbent
Bonded phase silica or adsorbent used as the stationary phase in SPE

Surface Area
The surface area on the surface and wthin the pores of a chromatographic sorbent.  This is normally expressed as m²/g