ASTM E682-92(2000)
(Practice)Standard Practice for Liquid Chromatography Terms and Relationships
Standard Practice for Liquid Chromatography Terms and Relationships
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.
General Information
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Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation:E682–92 (Reapproved 2000)
Standard Practice for
Liquid Chromatography Terms and Relationships
This standard is issued under the fixed designation E 682; 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 (ε) indicates an editorial change since the last revision or reapproval.
1. Scope 3.1 Liquid Chromatography, abbreviated as LC, comprises
all chromatographic methods in which the mobile phase is
1.1 This practice deals primarily with the terms and rela-
liquid under the conditions of analysis. The stationary phase
tionships used in liquid column chromatography. However,
may be a solid or a liquid supported by or chemically bonded
most of the terms should also apply to other kinds of liquid
to a solid.
chromatography, notably planar chromatography such as paper
3.2 The stationary phase may be present on or as a plane
or thin-layer chromatography.
(Planar Chromatography), or contained in a cylindrical tube
NOTE 1—Although electrophoresis can also be considered a liquid
(Column Chromatography).
chromatographic technique, it and its associated terms have not been
3.3 Separation is achieved by differences in the distribution
included in this practice.
of the components of a sample between the mobile and
1.2 Since most of the basic terms and definitions also apply
stationary phases, causing them to move along the plane
to gas chromatography, this practice uses, whenever possible,
surface or through the column at different rates (differential
symbols identical to Practice E 355.
migration).
3.3.1 In Planar Chromatography, the differential migration
2. Referenced Documents
process will cause the sample components to separate as a
2.1 ASTM Standards:
series of spots behind the mobile phase front.
D 3016 Practice for Use of Liquid Exclusion Chromatogra-
3.3.2 In Column Chromatography, the differential migration
phy Terms and Relationships
process will cause the sample components to elute from the
E 355 Practice for Gas Chromatography Terms and Rela-
column at different times.
tionships
3.3.3 InDry-ColumnChromatography,mobilephaseflowis
E 1151 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
3.3.4 In Flash Chromatography, mobile phase flow is con-
term“ high-performance (or high-pressure) liquid chromatography,” ab-
tinuedafterthemobilephasehasreachedtheendofthecolumn
breviated as HPLC. This term was introduced to distinguish the present-
of dry medium until elution of the desired components is
day column chromatographic techniques employing high inlet pressures
achieved. Often low pressures, compatible with the materials
and columns containing small diameter packing from the classical
of construction of the column, are applied to the top of the
methods. The utilization of this term or any derivative term (for example,
HPLSC for high-performance liquid-solid chromatography) is not recom- column to speed up the elution.
mended.
3.4 The basic process of selective distribution during the
Similarly, the use of the term “high-performance thin-layer chromatog-
chromatographic process can vary depending on the type of
raphy,” abbreviated as HPTLC, describing newer variations of thin-layer
stationary phase and the nature of the mobile phase.
chromatography, is also not recommended.
3.4.1 In Liquid-Liquid Chromatography, abbreviated LLC,
the stationary phase is a liquid and the separation is based on
1 selective partitioning between the mobile and stationary liquid
This practice is under the jurisdiction of ASTM Committee E13 on Molecular
phases.
Spectroscopy and is the direct responsibility of Subcommittee E13.19 on Chroma-
tography.
3.4.2 In Liquid-Solid Chromatography, abbreviated as LSC,
Current edition approved Jan. 15, 1992. Published March 1992. Originally
the stationary phase is an interactive solid. Depending on the
published as E 682 – 79. Last previous edition E 682 – 79.
type of the solid, separation may be based on selective
Annual Book of ASTM Standards, Vol 08.02.
Annual Book of ASTM Standards, Vol 14.02. adsorption on an inorganic substrate such as silica gel, or an
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E682–92 (2000)
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
procedure. The changes are made to enhance separation or to
3.4.2.1 In this definition, Ion Exchange Chromatography is
considered to be a special case of LSC in which the interactive speed elution of sample components, or both. Such changes in
operating conditions may be continuous or step-wise.
solid has permanently bonded ionic sites and separation is due
to electrostatic interaction. 3.6 In the standard modes of liquid chromatography, the
stationary phase is more polar than the mobile phase. This is
3.4.2.2 In this definition, Ion Pair Chromatography is con-
referred to as Normal Phase Chromatography. The opposite
sidered to be a special case of LSC in which ionic counterions
case is also possible, in which the mobile phase is more polar
are added to the mobile phase to effect the separation of ionic
than the stationary phase. This version of the technique is
solutes. In this technique both electrostatic and adsorptive
called Reversed-Phase Chromatography.
forces are involved in the separation.
3.7 Planar Chromatography comprises two versions: paper
NOTE 3—Other terminology for this technique include, but are not
chromatography and thin-layer chromatography.
limited to, extraction chromatography, paired ion chromatography, soap
3.7.1 In Paper Chromatography, the process is carried out
chromatography, ion pair extraction chromatography, ion pair partition
onasheetorstripofpaper.SeparationisusuallybasedonLLC
chromatography, and ion interaction chromatography, but utilization of
inwhichwaterheldonthecellulosefibersactsasthestationary
these terms is not recommended.
phase. Separation based on LSC may also be utilized when the
3.4.2.3 In this definition, Affınity Chromatography is con-
paper is impregnated or loaded with an interactive solid.
sidered to be a special case of LSC in which special ligands are
3.7.2 In Thin-Layer Chromatography, the solid stationary
bonded to a stationary phase so that bio-specific interactions
phase is utilized in the form of a relatively thin layer on an
(for example, antibody/antigen, enzyme/substrate) may be
inactive plate or sheet.
invoked to effect the separation.
3.7.3 In any version of planar chromatography, the mobile
3.4.2.4 In this definition, Ion Chromatography is considered
phase may be applied in a number of ways. In normal usage,
to be a special application of LSC in which the ion exchange
Ascending, Descending, and Horizontal Development, the
mechanism is still effecting the separation. Special columns or
mobile phase movement depends upon capillary action. In
devices, after the separating column, may be needed to remove
Horizontal Development, the mobile phase may move pre-
higher concentrations of inorganic ions which might otherwise
dominantly linearly or radially. In Radial Development, the
interfere with the detectability using conductivity. See Practice
mobile phase is applied as a point source. Devices have been
E 1151 for further details of nomenclature for this technique.
employed which accelerate the mobile phase movement on
3.4.2.5 In this definition, Hydrophobic Interaction Chroma-
planar layers by pressure or centrifugal force.
tography, is considered to be a special application of LSC in
3.7.4 The Mobile Phase Front is the leading edge of mobile
which the separation is based upon interaction of the hydro-
phase as it traverses the planar media. In all forms of
phobic moieties of the solutes and the hydrophobic moieties of
development, including radial, the local tangent to the Mobile
the sites on a reversed phase packing. High to low salt
Phase Front is everywhere normal to the local direction of
gradients are used to effect this type of separation.
development.
3.4.3 In some cases, such as with bonded stationary phases,
3.7.5 Consecutive Developments of planar media may be
the exact nature of the separation process is not fully estab-
carried out after removal of the mobile phase from a previous
lished and it may be based on a combination of liquid-liquid
development. If the consecutive development is accomplished
and liquid-solid interactions.
in the same direction as previously, this is Multiple Develop-
3.4.4 In Steric Exclusion Chromatography, the stationary
ment. If a second development is accomplished at a right angle
phase is a noninteractive porous solid, usually silica or an
to the first development, this is Two-Dimensional Develop-
organicgel.Inthiscase,separationisaffectedbythesizeofthe
ment. Continuous development of planar media is possible by
sample molecules, where those which are small enough pen-
allowing evaporation of the mobile phase near the Mobile
etrate the porous matrix to varying extents and degrees while
Phase Front.
those that are largest are confined to the interstitial region of
3.7.6 Impregnation is the technique of applying a reagent
the particles. Thus, the larger molecules elute before the
to the planar media to effect an enhanced separation or
smaller molecules. See Practice D 3016 for further details of
detection. This impregnation is accomplished by dipping or
nomenclature for this technique.
spraying a reagent solution after the preparation of the me-
3.5 Inliquidchromatography,thecompositionofthemobile
dium, or by incorporating during the manufacturing process.
phase may be constant or changing during a chromatographic
4. Apparatus
separation.
3.5.1 The term Isocratic may be used when the composition
4.1 Pumps—The function of the pumps is to deliver the
of the mobile phase at the column inlet is kept constant during mobile phase at a controlled flow rate to the chromatographic
a chromatographic separation.
column.
3.5.2 The term Gradient is used to specify the technique 4.1.1 Syringe Pumps have a piston that advances at a
when a deliberate change in the mobile phase operating controlled rate within a smooth cylinder to displace the mobile
condition is made during the chromatographic procedure. The phase.
E682–92 (2000)
concentration column, but utilization of these terms is not recommended.
4.1.2 Reciprocating Pumps have a single or dual chamber
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 calibration marks define the sample volume. 4.4.1 Bulk Property Detectors measure the change in a
physical property of the mobile phase passing from the
4.3 Columns consist of tubes that contain the stationary
phase and through which the mobile phase flows. column. Thus a change in the refractive index, conductivity, or
dielectric constant of a mobile phase can indicate the presence
4.3.1 Separating Column is the column on which the sepa-
of eluting components.
ration of the solutes is accomplished.
4.4.2 Solute Property Detectors measure the physical or
4.3.2 Pre-column is a column that has been used classically
chemical characteristics of the component eluting from the
to precondition the mobile phase, placed between the pump
column. Thus, lig
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