ASTM E682-92(2006)
(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, apparatus 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 E 355.
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.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 2006)
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 Department of Defense.
Similarly, the use of the term high-performance thin-layer chromatog-
1. Scope
raphy, abbreviated as HPTLC, describing newer variations of thin-layer
1.1 This practice deals primarily with the terms and rela-
chromatography, is also not recommended.
tionships used in liquid column chromatography. However,
3.1 Liquid Chromatography, abbreviated as LC, comprises
most of the terms should also apply to other kinds of liquid
all chromatographic methods in which the mobile phase is
chromatography, notably planar chromatography such as paper
liquid under the conditions of analysis. The stationary phase
or thin-layer chromatography.
may be a solid or a liquid supported by or chemically bonded
NOTE 1—Although electrophoresis can also be considered a liquid
to a solid.
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
1.2 Since most of the basic terms and definitions also apply
(Column Chromatography).
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
2. Referenced Documents
surface or through the column at different rates (differential
2.1 ASTM Standards:
migration).
D3016 Practice for Use of Liquid Exclusion Chromatogra-
3.3.1 In Planar Chromatography, the differential migration
phy Terms and Relationships
process will cause the sample components to separate as a
E355 Practice for Gas Chromatography Terms and Rela-
series of spots behind the mobile phase front.
tionships
3.3.2 In Column Chromatography, the differential migration
E1151 Practice for Ion Chromatography Terms and Rela-
process will cause the sample components to elute from the
tionships
column at different times.
3.3.3 InDry-ColumnChromatography,mobilephaseflowis
3. Names of Techniques
stopped as soon as the mobile phase has reached the end of the
NOTE 2—In the chromatographic literature one may often find the
column of dry medium. This column can be glass or a rigid or
term“high-performance (or high-pressure) liquid chromatography, abbre-
flexible solvent compatible plastic. Solute visualization and
viated as HPLC. This term was introduced to distinguish the present-day
recovery are from the extruded or sliced column packing.
column chromatographic techniques employing high inlet pressures and
3.3.4 In Flash Chromatography, mobile phase flow is con-
columns containing small diameter packing from the classical methods.
tinuedafterthemobilephasehasreachedtheendofthecolumn
The utilization of this term or any derivative term (for example, HPLSC
of dry medium until elution of the desired components is
for high-performance liquid-solid chromatography) is not recommended.
achieved. Often low pressures, compatible with the materials
of construction of the column, are applied to the top of the
This practice is under the jurisdiction of ASTM Committee E13 on Molecular
column to speed up the elution.
Spectroscopy and Separation Science and is the direct responsibility of Subcom-
3.4 The basic process of selective distribution during the
mittee E13.19 on Separation Science.
chromatographic process can vary depending on the type of
Current edition approved Sept. 1, 2006. Published September 2006. Originally
stationary phase and the nature of the mobile phase.
approved in 1979. Last previous edition approved in 2000 as E682 – 92 (2000).
DOI: 10.1520/E0682-92R06.
3.4.1 In Liquid-Liquid Chromatography, abbreviated LLC,
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
the stationary phase is a liquid and the separation is based on
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
selective partitioning between the mobile and stationary liquid
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. phases.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E682–92 (2006)
3.4.2 In Liquid-Solid Chromatography, abbreviated as LSC, 3.5.1 The term Isocratic may be used when the composition
the stationary phase is an interactive solid. Depending on the of the mobile phase at the column inlet is kept constant during
a chromatographic separation.
type of the solid, separation may be based on selective
adsorption on an inorganic substrate such as silica gel, or an
3.5.2 The term Gradient is used to specify the technique
organic gel. In this definition, Ion-Exchange Chromatography
when a deliberate change in the mobile phase operating
is considered to be a special case of LSC in which the
condition is made during the chromatographic procedure. The
interactive solid has ionic sites and separation is due to ionic
change is usually in mobile phase composition, flow rate, pH,
interaction. or temperature. The first-named change is called Gradient
Elution. Flow Programming is a technique where the mobile
3.4.2.1 In this definition, Ion Exchange Chromatography is
phase linear velocity is changed during the chromatographic
considered to be a special case of LSC in which the interactive
procedure. The changes are made to enhance separation or to
solid has permanently bonded ionic sites and separation is due
speed elution of sample components, or both. Such changes in
to electrostatic interaction.
operating conditions may be continuous or step-wise.
3.4.2.2 In this definition, Ion Pair Chromatography is con-
3.6 In the standard modes of liquid chromatography, the
sidered to be a special case of LSC in which ionic counterions
stationary phase is more polar than the mobile phase. This is
are added to the mobile phase to effect the separation of ionic
referred to as Normal Phase Chromatography. The opposite
solutes. In this technique both electrostatic and adsorptive
case is also possible, in which the mobile phase is more polar
forces are involved in the separation.
than the stationary phase. This version of the technique is
NOTE 3—Other terminology for this technique include, but are not
called Reversed-Phase Chromatography.
limited to, extraction chromatography, paired ion chromatography, soap
3.7 Planar Chromatography comprises two versions: paper
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.
onasheetorstripofpaper.SeparationisusuallybasedonLLC
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, Ion Chromatography 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 Chroma-
mobile phase is applied as a point source. Devices have been
tography, is considered to be a special application of LSC in
employed which accelerate the mobile phase movement on
which the separation is based upon interaction of the hydro-
planar layers by pressure or centrifugal force.
phobic moieties of the solutes and the hydrophobic moieties of
3.7.4 The Mobile Phase Front is the leading edge of mobile
the sites on a reversed phase packing. High to low salt
phase as it traverses the planar media. In all forms of
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.
smaller molecules. See Practice D3016 for further details of
3.7.6 Impregnation is the technique of applying a reagent
nomenclature for this technique.
to the planar media to effect an enhanced separation or
3.5 Inliquidchromatography,thecompositionofthemobile detection. This impregnation is accomplished by dipping or
phase may be constant or changing during a chromatographic
spraying a reagent solution after the preparation of the me-
separation. dium, or by incorporating during the manufacturing process.
E682–92 (2006)
4. Apparatus with any material which will effectively remove the unwanted
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 Concentrator Column is a small column placed in-
column.
line at the loop injector for introducing a dilute sample which
4.1.1 Syringe Pumps have a piston that advances at a
is 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 Pneumatic Pumps employ a gas to displace the
diameters are now being used. As pointed out by Basey and
mobile phase either directly or through a piston or collapsible
Oliver as many as nine terms (capillary, microcapillary,
container. The volume within these pumps may be large or
narrow bore capillary, micro, microbore, ultramicro, narrow
small 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 3 100 mm or open column, 250
by piercing an elastomeric barrier. The syringe is exposed to
µm ID 31m.
pressure and defines the sample volume.
4.3.6 ColumnInlet istheendofacolumnwherethemobile
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.
chromatography column, or in a special device for in-line
4.2.3 Valve Injectors—Sample contained in a syringe (or
filtration to effect the removal of particulate material in the
contained in a sample vial) is injected into (or drawn into) an
mobile phase or the sample solution.
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 or an internal chamber. The chamber can be
concentration of solute in the mobile phase. Detectors may be
completely filled, in which case the chamber volume defines
divided either according to the type of measurement or the
the sample volume, or it can be partially filled, in which case
principle of detection.
the syringe cali
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.