ASTM E682-92(2019)
(Practice)Standard Practice for Liquid Chromatography Terms and Relationships
Standard Practice for Liquid Chromatography Terms and Relationships
ABSTRACT
This practice deals with the terms, parameters, symbols, units, and relationships used in liquid column chromatography. Most of the terms covered herein should also apply to other kinds of liquid chromatography, notably planar chromatography such as paper or thin-layer chromatography. These terms include the names of techniques, apparatuses and reagents, the parameters and symbols used for chromatogram data readout, and the retention parameters, symbols and units.
SCOPE
1.1 This practice deals primarily with the terms and relationships used in liquid column chromatography. However, most of the terms should also apply to other kinds of liquid chromatography, notably planar chromatography such as paper or thin-layer chromatography.
Note 1: Although electrophoresis can also be considered a liquid chromatographic technique, it and its associated terms have not been included in this practice.
1.2 Since most of the basic terms and definitions also apply to gas chromatography, this practice uses, whenever possible, symbols identical to Practice E355.
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
General Information
Relations
Standards Content (Sample)
This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E682 − 92 (Reapproved 2019)
Standard Practice for
Liquid Chromatography Terms and Relationships
This standard is issued under the fixed designation E682; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
This standard has been approved for use by agencies of the U.S. Department of Defense.
1. Scope 3. Names of Techniques
NOTE 2—In the chromatographic literature one may often find the term
1.1 This practice deals primarily with the terms and rela-
high-performance (or high-pressure) liquid chromatography, abbreviated
tionships used in liquid column chromatography. However,
as HPLC.This term was introduced to distinguish the present-day column
most of the terms should also apply to other kinds of liquid
chromatographic techniques employing high inlet pressures and columns
chromatography, notably planar chromatography such as paper containing small diameter packing from the classical methods. The
utilization of this term or any derivative term (for example, HPLSC for
or thin-layer chromatography.
high-performance liquid-solid chromatography) is not recommended.
NOTE 1—Although electrophoresis can also be considered a liquid
Similarly, the use of the term high-performance thin-layer
chromatographic technique, it and its associated terms have not been
chromatography, abbreviated as HPTLC, describing newer variations of
included in this practice.
thin-layer chromatography, is also not recommended.
1.2 Since most of the basic terms and definitions also apply
3.1 Liquid Chromatography, abbreviated as LC, comprises
to gas chromatography, this practice uses, whenever possible,
all chromatographic methods in which the mobile phase is
symbols identical to Practice E355.
liquid under the conditions of analysis. The stationary phase
may be a solid or a liquid supported by or chemically bonded
1.3 The values stated in SI units are to be regarded as
to a solid.
standard. No other units of measurement are included in this
standard.
3.2 The stationary phase may be present on or as a plane
1.4 This international standard was developed in accor- (Planar Chromatography), or contained in a cylindrical tube
dance with internationally recognized principles on standard-
(Column Chromatography).
ization established in the Decision on Principles for the
3.3 Separation is achieved by differences in the distribution
Development of International Standards, Guides and Recom-
of the components of a sample between the mobile and
mendations issued by the World Trade Organization Technical
stationary phases, causing them to move along the plane
Barriers to Trade (TBT) Committee.
surface or through the column at different rates (differential
migration).
2. Referenced Documents
3.3.1 In Planar Chromatography, the differential migration
2.1 ASTM Standards:
process will cause the sample components to separate as a
D3016 Practice for Use of Liquid Exclusion Chromatogra-
series of spots behind the mobile phase front.
phy Terms and Relationships
3.3.2 InColumnChromatography,thedifferentialmigration
E355 Practice for Gas Chromatography Terms and Relation-
process will cause the sample components to elute from the
ships
column at different times.
E1151 Practice for Ion Chromatography Terms and Rela-
3.3.3 InDry-ColumnChromatography,mobilephaseflowis
tionships
stopped as soon as the mobile phase has reached the end of the
column of dry medium. This column can be glass or a rigid or
flexible solvent compatible plastic. Solute visualization and
This practice is under the jurisdiction of ASTM Committee E13 on Molecular
Spectroscopy and Separation Science and is the direct responsibility of Subcom- recovery are from the extruded or sliced column packing.
mittee E13.19 on Separation Science.
3.3.4 In Flash Chromatography, mobile phase flow is con-
Current edition approved Sept. 1, 2019. Published September 2019. Originally
tinuedafterthemobilephasehasreachedtheendofthecolumn
approved in 1979. Last previous edition approved in 2011 as E682–92 (2011). DOI:
10.1520/E0682–92R19.
of dry medium until elution of the desired components is
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
achieved. Often low pressures, compatible with the materials
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
of construction of the column, are applied to the top of the
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. column to speed up the elution.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E682 − 92 (2019)
3.4 The basic process of selective distribution during the smaller molecules. See Practice D3016 for further details of
chromatographic process can vary depending on the type of nomenclature for this technique.
stationary phase and the nature of the mobile phase.
3.5 Inliquidchromatography,thecompositionofthemobile
3.4.1 In Liquid-Liquid Chromatography, abbreviated LLC,
phase may be constant or changing during a chromatographic
the stationary phase is a liquid and the separation is based on
separation.
selective partitioning between the mobile and stationary liquid
3.5.1 The termIsocratic may be used when the composition
phases.
of the mobile phase at the column inlet is kept constant during
3.4.2 InLiquid-SolidChromatography, abbreviated as LSC,
a chromatographic separation.
the stationary phase is an interactive solid. Depending on the
3.5.2 The term Gradient is used to specify the technique
type of the solid, separation may be based on selective
when a deliberate change in the mobile phase operating
adsorption on an inorganic substrate such as silica gel, or an
condition is made during the chromatographic procedure. The
organic gel. In this definition, Ion-Exchange Chromatography
change is usually in mobile phase composition, flow rate, pH,
is considered to be a special case of LSC in which the
or temperature. The first-named change is called Gradient
interactive solid has ionic sites and separation is due to ionic
Elution. Flow Programming is a technique where the mobile
interaction.
phase linear velocity is changed during the chromatographic
3.4.2.1 In this definition, Ion Exchange Chromatography is
procedure. The changes are made to enhance separation or to
considered to be a special case of LSC in which the interactive
speed elution of sample components, or both. Such changes in
solid has permanently bonded ionic sites and separation is due
operating conditions may be continuous or step-wise.
to electrostatic interaction.
3.6 In the standard modes of liquid chromatography, the
3.4.2.2 In this definition, Ion Pair Chromatography is con-
stationary phase is more polar than the mobile phase. This is
sidered to be a special case of LSC in which ionic counterions
referred to as Normal Phase Chromatography. The opposite
are added to the mobile phase to effect the separation of ionic
case is also possible, in which the mobile phase is more polar
solutes. In this technique both electrostatic and adsorptive
than the stationary phase. This version of the technique is
forces are involved in the separation.
called Reversed-Phase Chromatography.
NOTE 3—Other terminology for this technique include, but are not
3.7 Planar Chromatography comprises two versions: paper
limited to, extraction chromatography, paired ion chromatography, soap
chromatography, ion pair extraction chromatography, ion pair partition chromatography and thin-layer chromatography.
chromatography, and ion interaction chromatography, but utilization of
3.7.1 In Paper Chromatography, the process is carried out
these terms is not recommended.
on a sheet or strip of paper. Separation is usually based on LLC
3.4.2.3 In this definition, Affınity Chromatography is con- inwhichwaterheldonthecellulosefibersactsasthestationary
sidered to be a special case of LSC in which special ligands are phase. Separation based on LSC may also be utilized when the
bonded to a stationary phase so that bio-specific interactions paper is impregnated or loaded with an interactive solid.
(for example, antibody/antigen, enzyme/substrate) may be
3.7.2 In Thin-Layer Chromatography, the solid stationary
invoked to effect the separation.
phase is utilized in the form of a relatively thin layer on an
3.4.2.4 In this definition,IonChromatography is considered inactive plate or sheet.
to be a special application of LSC in which the ion exchange
3.7.3 In any version of planar chromatography, the mobile
mechanism is still effecting the separation. Special columns or
phase may be applied in a number of ways. In normal usage,
devices, after the separating column, may be needed to remove
Ascending, Descending, and Horizontal Development, the
higher concentrations of inorganic ions which might otherwise
mobile phase movement depends upon capillary action. In
interfere with the detectability using conductivity. See Practice
Horizontal Development, the mobile phase may move pre-
E1151 for further details of nomenclature for this technique.
dominantly linearly or radially. In Radial Development, the
3.4.2.5 In this definition, Hydrophobic Interaction mobile phase is applied as a point source. Devices have been
Chromatography, is considered to be a special application of employed which accelerate the mobile phase movement on
LSC in which the separation is based upon interaction of the planar layers by pressure or centrifugal force.
hydrophobic moieties of the solutes and the hydrophobic
3.7.4 The Mobile Phase Front is the leading edge of mobile
moieties of the sites on a reversed phase packing. High to low
phase as it traverses the planar media. In all forms of
salt gradients are used to effect this type of separation.
development, including radial, the local tangent to the Mobile
3.4.3 In some cases, such as with bonded stationary phases, Phase Front is everywhere normal to the local direction of
the exact nature of the separation process is not fully estab- development.
lished and it may be based on a combination of liquid-liquid
3.7.5 Consecutive Developments of planar media may be
and liquid-solid interactions.
carried out after removal of the mobile phase from a previous
3.4.4 In Steric Exclusion Chromatography, the stationary development. If the consecutive development is accomplished
phase is a noninteractive porous solid, usually silica or an in the same direction as previously, this is Multiple Develop-
organicgel.Inthiscase,separationisaffectedbythesizeofthe ment. If a second development is accomplished at a right angle
sample molecules, where those which are small enough pen- to the first development, this is Two-Dimensional Develop-
etrate the porous matrix to varying extents and degrees while ment. Continuous development of planar media is possible by
those that are largest are confined to the interstitial region of allowing evaporation of the mobile phase near the Mobile
the particles. Thus, the larger molecules elute before the Phase Front.
E682 − 92 (2019)
3.7.6 Impregnation is the technique of applying a reagent to 4.3.3 Guard Column is a protector column placed between
the planar media to effect an enhanced separation or detection. the injector and the separating column. The purpose of this
This impregnation is accomplished by dipping or spraying a column is to be the final filter for the sample, adsorbing
reagent solution after the preparation of the medium, or by unwanted sample components that otherwise might bind irre-
incorporating during the manufacturing process. versibly to the separating column. It has a volume of no more
than ⁄20 the volume of the separating column. It may be filled
with any material which will effectively remove the unwanted
4. Apparatus
components without interfering with subsequent chromato-
4.1 Pumps—The function of the pumps is to deliver the
graphic processes.
mobile phase at a controlled flow rate to the chromatographic
4.3.4 ConcentratorColumn is a small column placed in-line
column.
at the loop injector for introducing a dilute sample which is
4.1.1 Syringe Pumps have a piston that advances at a
collected into it before elution onto the separating column.
controlled rate within a smooth cylinder to displace the mobile
NOTE 4—Other terminology for this technique include, but are not
phase.
limited to, trace enrichment column, collector column, and sample
4.1.2 Reciprocating Pumps have a single or dual chamber
concentration column, but utilization of these terms is not recommended.
from which mobile phase is displaced by reciprocating pis-
4.3.5 Column sizes with various internal diameters (ID) and
ton(s)ordiaphragm(s).Thechambervolumeisrelativelysmall
lengths can be made. Larger columns present no problems
compared to the volume of the column.
concerning nomenclature, but columns with small internal
4.1.3 PneumaticPumps employ a gas to displace the mobile
diameters are now being used. As pointed out by Basey and
phase either directly or through a piston or collapsible con-
Oliver as many as nine terms (capillary, microcapillary,
tainer. The volume within these pumps may be large or small
narrow bore capillary, micro, microbore, ultramicro, narrow
as compared to the volume of the column.
bore, small bore, and small diameter) have been seen in the
4.2 Sample Inlet Systems represent the means for introduc-
literature and with no clear distinction between them when the
ing samples into the column.
actual column ID is examined. It is recommended that all
4.2.1 Septum Injectors—Sample contained in a syringe is
descriptive terms regarding column ID be discontinued, that is,
introduced directly into the pressurized flowing mobile phase
packed column, 1000 µm ID×100 mm or open column,
by piercing an elastomeric barrier. The syringe is exposed to
250µm ID×1 m.
pressure and defines the sample volume.
4.3.6 Column Inlet is the end of a column where the mobile
4.2.2 Septumless Injectors—Sample contained in a syringe
phase is introduced.
is introduced into an ambient-pressure chamber, and the
4.3.7 Column Outlet is the end of a column where the
chamber is subsequently mechanically displaced into the
mobile phase exits.
pressurized flowing mobile phase. The syringe is not exposed
4.3.8 Frit is the porous element placed at the ends of a
to pressure and defines the sample volume.
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