The Terminology of Chromatography

Absorption
The process where a chemical entity enter the bulk of a liquid, solid or gas phase. In
chromatography the term usually signifies the process by which a solute partitions into a
liquid-like stationary phase.

Additive
A compound added to the mobile phase to improve the chromatographic analysis.

Adjusted Retention Volume, V´R ( or Time, t´R )

The retention volume ( or time ) minus the Hold-up volume ( or time ). Note the difference
between this and the retention volume ( or time )

Adsorbent
The packing used in adsorption chromatography.

Adsorption
The process where a chemical entity is accumulated on a surface.

Adsorption Chromatography
Separation based on differences in adsorption of the components to the stationary phase
surface.

Adsorption isotherm
A plot of the amount of solute per solid phase unit ( weight, volume, area etc) as a function of
its concentration in the bulk phase ( liquid or gas phase ).

Affinity Chromatography
Chromatographic separation based on a specific interaction between the analyte and a ligand
bound to the stationary phase surface.
Agarose
A separation medium for the separation of biomolecules. It is a high molecular weight
polysaccharide.

Alumina
Porous aluminium oxide used as an adsorbent in chromatography .

Amphoteric ion-exchange phase

A stationary phase that has both positively and negatively charged ionic groups bonded to it.

Analyte
The chemical entity to be analyzed. In chromatography the term solute is also frequently
used.

Anion exchange chromatography

The chromatographic process that is used to separate anions by using an ionized positively
charged stationary phase. Tetra alkyl ammonium ions are often used as anion exchange
functional groups.

Baseline
The part of the chromatogram where the detector measures the mobile phase only.

Bed Volume
Synonymous to Column Volume

BET-method
A method for determining the surface area of a solid that was developed by Brunauer, Emmet
and Teller. It uses the known size of the nitrogen molecule in combination with experimental
data of adsorption-condensation of nitrogen to the solid.

Bonded phase
A stationary phase which is covalently bonded to the support particles or the inside wall of a
tube.
Breakthrough volume
When a solute is continuously pumped through a column, it will start to elute at a certain
volume, this is the breakthrough volume.

Capacity factor
See retention factor. IUPAC discourage its usage.

Capillary column
Columns with an inner diameter less than 0.5 mm.

Capillary LC
Liquid chromatography performed by using a capillary column.

Cartridge column
A column type that has no endfittings and is held in a cartridge holder. The column comprises
a tube and packing contained by frits in each end of the tube.

Cation exchange chromatography

The chromatographic process that is used to separate cations by using a ionized negatively
charged stationary phase. A sulfonic acid is an often used cation exchange functional group.

Channeling
Poor packing or erosion creates voids in the packed bed. Channeling occurs because the
mobile phase moves more rapidly in these these voids than in other parts of the bed.

Chemisorption
Adsorption, usually irreversible, accompanied by a chemical reaction with the solid surface.

Chiral stationary phase

Stationary phases that can separate enantiomers.
Chlorosilane
A reagent which is used to create siloxane bonds with silanol groups. Three types are used,
monochlorosilanes;R1R2R3-Si-Cl, dichlorosilanes; R1R2-SiCl2 and trichlorosilanes; R1-
SiCl3 . Ri can have various structures but is often an alkyl group.

Chromatograph (noun)
The instrument which is used to carry out a chromatographic separation

Co-ion
A ion of the same sign of charge as the ionic groups making up the stationary phase.

Column
The tube and the stationary phase through which the mobile phase flow.

Column back pressure

The difference in pressure between the column inlet and outlet.

Column chromatography
The form of chromatography which uses a column or tube to fix the stationary phase.

Column plate number

See Plate number.

Column switching
Two or more columns connected by switching valves. Fractions from one column are
switched to a second column.

Column Volume
The volume of the empty column tube.

Competing base
A basic compound, often a small amine, added to the mobile phase with the intention to
improve the peak shape of a basic solute.
Counterion
When the term is used in ion exchange chromatography it means the ions added to the mobile
phase with charge opposite to the ions bonded to the stationary phase.

Coverage
The concentration of bonded phase on the silica support, usually expressed in mol/m2 or
weight %.

Cross-links
Bonds that connect one polymer chain to another. Cross-linking is important for resins
because it governs its swelling and diffusion characteristics.

Degassing
The removal of dissolved gas from the mobile phase.

Detector
An instrument that measures the change in composition of the eluent.

Displacement chromatography
A form of non linear chromatography where the migration of the solutes is due to a
displacement by an additive that strongly adsorbs to the stationary phase.

Dynamic coating
Modification of the properties of the stationary phase surface by using an additive in the
mobile phase that adsorbs to the surface.

Effluent
The mobile phase that exits the column.

Eluate
The solute - mobile phase mixture which exits the column.

Eluent
Another word for the mobile phase.
Eluite
The eluted solute.

Elute
The use of elution chromatography.

Elution
The passing of mobile phase through the chromatographic bed to transport solutes.

(Size) Exclusion Chromatography

Separation based mainly on differences in molecular size. Differences in shape and/or charge
may also contribute to the separation.

Extracolumn effects
The effect on bandbroadening by all parts in a chromatographic system, except the column.

Extra-column Volume
The volumes of the injector, detector and connecting tubes. ( The term dead-volume is often
used for this volume, IUPAC discourage this term.)

Fast protein LC (FPLC)

HPLC of proteins, generally in glass columns with spherical microbeads and moderate
pressure.

Flow rate
The volume of the mobile phase that passes through the column per unit time.

Frontal chromatography
A chromatographic technique in which the sample is continously added to the column inlet.

Fronting
Asymmetry of a peak such that its rear in a chromatogram is steeper than its front.
Gel filtration chromatography (GFC)
Chromatographic separation according to molecular size usually performed in an aqueous
mobile phase on soft gels such as polydextrans.

Gradient elution
The chromatographic technique by which a mobil phase gradient is used to modulate the
retention times. Usually the mobile phase composition changes. so that its strength increases
with time.

Graphitized carbon packing

A stationary phase consisting of pure graphitic carbon.

Guard column
A small column that protects the analytical column from contamination, it is placed between
the injector and the analytical column.

Heart cutting
A term used in preparative chromatography and column switching for the collection of the
center of a peak.

High performance liquid chromatography (HPLC)

A term coined for the modern and instrumentally developed form of column liquid
chromatography. It is characterised by high flow rates and high back column pressure.

Hold-up Volume, ( VM ) ( or Time ( tM ) )

The volume ( or corresponding time ) of mobile phase required to elute a component that
does not interact with the stationary phase. I.e. the component is not retained by the stationary
phase.

Hydrophobic Interaction Chromatography (HIC)

A chromatographic technique which is primarly used to separate proteins. The technique is
characterised by a hydrophilic solid support with a low coverage of short carbon chains. The
mobile phase is a buffered water solution with a steep gradient of decreasing salt
concentration.
IC
Abbreviation for ion chromatography

Imprinted phases
Stationary phases which are generated in the presence of a template molecule so that a
"footprint" of the molecule is created on the stationary phase. The imprinted phase has a
strong selectivity for the template molecule.

Indirect detection
A detection technique where the solute is indirectly detected by measuring the change in
mobile phase composition at column outlet. A prerequisite for this technique is that the
adsorption isotherm of a component in the mobile phase depends on the concentration of the
solute. E.g. a non-UV absorbing solute is indirectly detected with an UV-detector by adding
an UV absorbing component to the mobile phase. If the adsorption isotherm of this
component depends on the concentration of the solute, its variation in concentration at the
column outlet, caused by the elution of the solute, can be detected with an UV-detector.

(Sample) Injector
A device by which a sample is introduced into the mobile phase.

Inlet
The part of the column where the mobile phase and the solute enter.

In-line filter
A filter that is placed between the column and the injector and which prevents particulate
matter to damage the column.

Interparticle porosity (ee)

The interparticle porosity is; ee = Ve/Vc
Where, Ve is the interstitial volume and Vc the total column volume.

Interstitial volume
In chromatography; the volume between the particles in a packed column.
Intraparticle porosity(ei)
The fraction of the particle volume that is in pores; ei = Vi/Vparticle
Where, Vi is the intraparticle volume and Vparticle the particle volume.

Intraparticle volume
The volume inside the pores of the particles.

Ion Exchange Chromatography

Separation based on differences in the distribution between the mobile phase and a charged
stationary phase.

Ion chromatography (IC)

A technique in which low concentrations of ionic solutes are determined by using ion
exchangers of low exchange capacity and mobile phase with low ionic strength.

Ion exclusion
The exclusion of co-ions from the surface layer. In chromatography the ion exclusion effect
implicates that co-ions migrates faster through the column than a neutral molecule.

Ion moderated partitioning chromatography

A technique used for separating carbohydrates by using a strong cation exchanger.

Ion pair chromatography

A form of reversed phase chromatography in which a charged organic molecule, the ion pair
reagent, is added to the mobile phase. The ion pair reagent adsorbs to the stationary phase
surface and creates a charged surface layer. Ions of opposite charge are attracted to the
charged surface layer and ions of the same charge are repelled . The retention of ions is
modulated by changing the concentration of ion pair reagent in the mobile phase.

Isocratic chromatography
A chromatographic run with a constant mobile phase composition

Linear chromatography
Chromatography performed in the linear range of the adsorption isotherm for the solute.
Linear velocity
The velocity of the mobile phase through the column expressed as m/s. It is estimated as the
column length divided by the time it takes for a non-retained compound to pass the column

Liquid chromatography
Chromatography by using a liquid as mobile phase, usually performed in a column.

Mobile phase
The fluid that flows through the chromatographic column.

Mobile phase velocity

The linear velocity ( u ) of the mobile phase through the column. It is usually estimated from
the time it takes for an unretained compound to pass through the column, tM, and the column
length, L.
u = L / tM

Open-Tubular Column
A column in which the stationary phase coates the inner wall. The column diameter is usually
small, e.g. 0.1 mm.

Partition Chromatography
Separation based mainly on differences in solubilities between the mobile and stationary
phase.

Packed Column
A column containing a solid packing material.

Peak
The part of the chromatogram where the detector response is caused by a solute.

Peak Area
The area of the peak as registered by the detector.
Peak maximum
The point on the peak where detector response is maximum.

Peak-Width
The width of the peak registrered by the detector. It may be represented in the dimension time
or volume. For a Gaussian formed peak, the peak-width is related to the standard deviation
(s) of the peak. The peak width can be estimated by several different methods. For example:
Peak-width at Base, wb = 4s
Peak-width at Half Height, wh = 2.355s
Peak-width at Inflection Point, wi = 2s

Phase ratio
A characteristic constant of a column. It is a measure of the volume ( or area ) of the
stationary phase per unit volume of the mobile phase in the column.

Plate Height ( H)
The column length ( L ) divided by the plate number:
H=L/N

Plate Number ( N )
A dimensionless number that is a measure of the effectivity of a column.
N = ( VR / s)2

Pressure drop
The difference in pressure between the inlet and outlet in a chromatographic system

Reduced mobile phase velocity ( n )

A dimensionless measure of the mobile phase velocity compared to diffusion into the pores.
n = u*dp/DM
where u = linear velocity of the mobile phase; dp = particle diameter and DM = diffusion
coefficient of the solute in the mobile phase.
(Peak ) Resolution ( Rs )
A measure how well two peaks are separated. It is defined as:
Rs = 2(tR2 - tR1) / (wb1 + wb2)
tR = Retention time, wb = peak width at base

Reduced Plate Height ( h )

A dimensionless number defined as the ratio of the plate height divided by the particle
diameter.

Relative retention time( usually denoted RRT)

RRT= tR2/tR1 = VR2/VR1
Note the difference between this and the separation factor. RRT is often used to identify
peaks from system to system.

Retention Factor (k)

The ratio of the adjusted retention volume ( or time ) and the hold-up volume ( or time );
k = V´R/ VM = t´R / tM
The retention factor has for many years also been called the capacity factor, k´. This usage is
not recommended by IUPAC.

Retention Volume ( VR ) ( or Time ( tR )

The volume ( or corresponding time ) of mobile phase that passes through the column
between sample injection and the emergence of the peak maximum. Note the difference
between this and the adjusted retention volume ( or time ).

Separation Factor ( a )
The relative retention values for two adjacent peaks;
a = V´R2/V´R1 = k2 / k1
V´R2 is chosen to be the larger value so that the separation factor becomes larger than unity.

Solid support
The solid that holds the stationary phase.
Solute
A term for the sample components.

Stationary phase
One of the two phases in a chromatographic system. In a chromatographic system the analyte
is distributed between the mobile phase and the stationary phase.

Tailing
Asymmetry of a peak such that its front in a chromatogram is steeper than its rear.

Void Volume
The volume in the column that is filled with the mobile phase. In the ideal case it is equal to
the mobile phase hold up volumne.

Sanket Patel
Quality Assurance
[email protected]