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HPLC Terms

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The Terminology of Chromatography

 

I want to learn more about chromatography!

 

I want to learn more about reversed phase liquid chromatography!

 

Below you find a collection of the most important terms that are used in liquid 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. Tetraalkylammonium 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

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.

 

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