ASTM E682-92(2011)
(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.
General Information
Relations
Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: E682 − 92 (Reapproved 2011)
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.
chromatography, abbreviated as HPTLC, describing newer variations of
1. Scope
thin-layer chromatography, is also not recommended.
1.1 This practice deals primarily with the terms and rela-
3.1 Liquid Chromatography, abbreviated as LC, comprises
tionships used in liquid column chromatography. However,
all chromatographic methods in which the mobile phase is
most of the terms should also apply to other kinds of liquid
liquid under the conditions of analysis. The stationary phase
chromatography, notably planar chromatography such as paper
may be a solid or a liquid supported by or chemically bonded
or thin-layer chromatography.
to a solid.
NOTE 1—Although electrophoresis can also be considered a liquid
chromatographic technique, it and its associated terms have not been 3.2 The stationary phase may be present on or as a plane
included in this practice.
(Planar Chromatography), or contained in a cylindrical tube
(Column Chromatography ).
1.2 Since most of the basic terms and definitions also apply
to gas chromatography, this practice uses, whenever possible,
3.3 Separation is achieved by differences in the distribution
symbols identical to Practice E355.
of the components of a sample between the mobile and
stationary phases, causing them to move along the plane
1.3 The values stated in SI units are to be regarded as
surface or through the column at different rates (differential
standard. No other units of measurement are included in this
migration).
standard.
3.3.1 In Planar Chromatography, the differential migration
2. Referenced Documents
process will cause the sample components to separate as a
series of spots behind the mobile phase front.
2.1 ASTM Standards:
D3016 Practice for Use of Liquid Exclusion Chromatogra-
3.3.2 InColumnChromatography, the differential migration
phy Terms and Relationships process will cause the sample components to elute from the
E355 Practice for Gas Chromatography Terms and Relation-
column at different times.
ships
3.3.3 InDry-ColumnChromatography,mobilephaseflowis
E1151 Practice for Ion Chromatography Terms and Rela-
stopped as soon as the mobile phase has reached the end of the
tionships
column of dry medium. This column can be glass or a rigid or
flexible solvent compatible plastic. Solute visualization and
3. Names of Techniques
recovery are from the extruded or sliced column packing.
NOTE 2—In the chromatographic literature one may often find the term
3.3.4 In Flash Chromatography, mobile phase flow is con-
high-performance (or high-pressure) liquid chromatography, abbreviated
tinuedafterthemobilephasehasreachedtheendofthecolumn
as HPLC.This term was introduced to distinguish the present-day column
chromatographic techniques employing high inlet pressures and columns
of dry medium until elution of the desired components is
containing small diameter packing from the classical methods. The
achieved. Often low pressures, compatible with the materials
utilization of this term or any derivative term (for example, HPLSC for
of construction of the column, are applied to the top of the
high-performance liquid-solid chromatography) is not recommended.
column to speed up the elution.
Similarly, the use of the term high-performance thin-layer
3.4 The basic process of selective distribution during the
1 chromatographic process can vary depending on the type of
This practice is under the jurisdiction of ASTM Committee E13 on Molecular
Spectroscopy and Separation Science and is the direct responsibility of Subcom-
stationary phase and the nature of the mobile phase.
mittee E13.19 on Separation Science.
3.4.1 In Liquid-Liquid Chromatography, abbreviated LLC,
Current edition approved Nov. 1, 2011. Published December 2011. Originally
the stationary phase is a liquid and the separation is based on
approved in 1979. Last previous edition approved in 2006 as E682 – 92 (2006).
DOI: 10.1520/E0682-92R11.
selective partitioning between the mobile and stationary liquid
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
phases.
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
3.4.2 In Liquid-Solid Chromatography, abbreviated as LSC,
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. the stationary phase is an interactive solid. Depending on the
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E682 − 92 (2011)
type of the solid, separation may be based on selective 3.5.1 The termIsocratic may be used when the composition
adsorption on an inorganic substrate such as silica gel, or an of the mobile phase at the column inlet is kept constant during
organic gel. In this definition, Ion-Exchange Chromatography a chromatographic separation.
is considered to be a special case of LSC in which the 3.5.2 The term Gradient is used to specify the technique
interactive solid has ionic sites and separation is due to ionic when a deliberate change in the mobile phase operating
interaction. condition is made during the chromatographic procedure. The
change is usually in mobile phase composition, flow rate, pH,
3.4.2.1 In this definition, Ion Exchange Chromatography is
considered to be a special case of LSC in which the interactive or temperature. The first-named change is called Gradient
Elution. Flow Programming is a technique where the mobile
solid has permanently bonded ionic sites and separation is due
to electrostatic interaction. phase linear velocity is changed during the chromatographic
procedure. The changes are made to enhance separation or to
3.4.2.2 In this definition, Ion Pair Chromatography is con-
sidered to be a special case of LSC in which ionic counterions speed elution of sample components, or both. Such changes in
operating conditions may be continuous or step-wise.
are added to the mobile phase to effect the separation of ionic
solutes. In this technique both electrostatic and adsorptive
3.6 In the standard modes of liquid chromatography, the
forces are involved in the separation.
stationary phase is more polar than the mobile phase. This is
referred to as Normal Phase Chromatography. The opposite
NOTE 3—Other terminology for this technique include, but are not
limited to, extraction chromatography, paired ion chromatography, soap case is also possible, in which the mobile phase is more polar
chromatography, ion pair extraction chromatography, ion pair partition
than the stationary phase. This version of the technique is
chromatography, and ion interaction chromatography, but utilization of
called Reversed-Phase Chromatography.
these terms is not recommended.
3.7 Planar Chromatography comprises two versions: paper
3.4.2.3 In this definition, Affınity Chromatography is con-
chromatography and thin-layer chromatography.
sidered to be a special case of LSC in which special ligands are
3.7.1 In Paper Chromatography, the process is carried out
bonded to a stationary phase so that bio-specific interactions
on a sheet or strip of paper. Separation is usually based on LLC
(for example, antibody/antigen, enzyme/substrate) may be
inwhichwaterheldonthecellulosefibersactsasthestationary
invoked to effect the separation.
phase. Separation based on LSC may also be utilized when the
3.4.2.4 In this definition,IonChromatography is considered
paper is impregnated or loaded with an interactive solid.
to be a special application of LSC in which the ion exchange
3.7.2 In Thin-Layer Chromatography, the solid stationary
mechanism is still effecting the separation. Special columns or
phase is utilized in the form of a relatively thin layer on an
devices, after the separating column, may be needed to remove
inactive plate or sheet.
higher concentrations of inorganic ions which might otherwise
3.7.3 In any version of planar chromatography, the mobile
interfere with the detectability using conductivity. See Practice
phase may be applied in a number of ways. In normal usage,
E1151 for further details of nomenclature for this technique.
Ascending, Descending, and Horizontal Development, the
3.4.2.5 In this definition, Hydrophobic Interaction
mobile phase movement depends upon capillary action. In
Chromatography, is considered to be a special application of
Horizontal Development, the mobile phase may move pre-
LSC in which the separation is based upon interaction of the
dominantly linearly or radially. In Radial Development, the
hydrophobic moieties of the solutes and the hydrophobic
mobile phase is applied as a point source. Devices have been
moieties of the sites on a reversed phase packing. High to low
employed which accelerate the mobile phase movement on
salt gradients are used to effect this type of separation.
planar layers by pressure or centrifugal force.
3.4.3 In some cases, such as with bonded stationary phases,
3.7.4 The Mobile Phase Front is the leading edge of mobile
the exact nature of the separation process is not fully estab-
phase as it traverses the planar media. In all forms of
lished and it may be based on a combination of liquid-liquid
development, including radial, the local tangent to the Mobile
and liquid-solid interactions.
Phase Front is everywhere normal to the local direction of
3.4.4 In Steric Exclusion Chromatography, the stationary
development.
phase is a noninteractive porous solid, usually silica or an
3.7.5 Consecutive Developments of planar media may be
organicgel.Inthiscase,separationisaffectedbythesizeofthe
carried out after removal of the mobile phase from a previous
sample molecules, where those which are small enough pen-
development. If the consecutive development is accomplished
etrate the porous matrix to varying extents and degrees while
in the same direction as previously, this is Multiple Develop-
those that are largest are confined to the interstitial region of
ment. If a second development is accomplished at a right angle
the particles. Thus, the larger molecules elute before the
to the first development, this is Two-Dimensional Develop-
smaller molecules. See Practice D3016 for further details of
ment. Continuous development of planar media is possible by
nomenclature for this technique.
allowing evaporation of the mobile phase near the Mobile
3.5 Inliquidchromatography,thecompositionofthemobile Phase Front.
phase may be constant or changing during a chromatographic 3.7.6 Impregnation is the technique of applying a reagent to
separation. the planar media to effect an enhanced separation or detection.
E682 − 92 (2011)
This impregnation is accomplished by dipping or spraying a unwanted sample components that otherwise might bind irre-
reagent solution after the preparation of the medium, or by versibly to the separating column. It has a volume of no more
incorporating during the manufacturing process. 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-
graphic processes.
4.1 Pumps—The function of the pumps is to deliver the
4.3.4 ConcentratorColumn is a small column placed in-line
mobile phase at a controlled flow rate to the chromatographic
at the loop injector for introducing a dilute sample which is
column.
collected into it before elution onto the separating column.
4.1.1 Syringe Pumps have a piston that advances at a
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
literature and with no clear distinction between them when the
4.2 Sample Inlet Systems represent the means for introduc-
actual column ID is examined. It is recommended that all
ing samples into the column.
descriptive terms regarding column ID be discontinued, that is,
4.2.1 Septum Injectors—Sample contained in a syringe is
packed column, 1000 µm ID×100 mm or open column, 250
introduced directly into the pressurized flowing mobile phase
µm ID×1 m.
by piercing an elastomeric barrier. The syringe is exposed to
4.3.6 Column Inlet is the end of a column where the mobile
pressure and defines the sample volume.
phase is introduced.
4.2.2 Septumless Injectors—Sample contained in a syringe
4.3.7 Column Outlet is the end of a column where the
is introduced into an ambient-pressure chamber, and the
mobile phase exits.
chamber is subsequently mechanically displaced into the
4.3.8 Frit is the porous element placed at the ends of a
pressurized flowing mobile phase. The syringe is not exposed
chromatography column, or in a special device for in-line
to pressure and defines the sample volume.
filtration to effect the removal of particulate material in the
4.2.3 Valve Injectors—Sample contained in a syringe (or
mobile phase or the sample solution.
contained in a sample vial) is injected into (or drawn into) an
ambient-pressure chamber which is subsequently displaced
4.4 Detectors are devices that respond to the presence of
into the pressurized flowing mobile phase. The displacement is
eluted solutes in the mobile phase emerging from the column.
by means of rotary or sliding motion. The chamber is a section
Ideally, the response should be proportional to the mass or
(loop) of tubing
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