1、Designation: E 682 92 (Reapproved 2006)Standard Practice forLiquid Chromatography Terms and Relationships1This standard is issued under the fixed designation E 682; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last r
2、evision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.This standard has been approved for use by agencies of the Department of Defense.1. Scope1.1 This practice deals primarily with the ter
3、ms and rela-tionships used in liquid column chromatography. However,most of the terms should also apply to other kinds of liquidchromatography, notably planar chromatography such as paperor thin-layer chromatography.NOTE 1Although electrophoresis can also be considered a liquidchromatographic techni
4、que, it and its associated terms have not beenincluded in this practice.1.2 Since most of the basic terms and definitions also applyto gas chromatography, this practice uses, whenever possible,symbols identical to Practice E 355.2. Referenced Documents2.1 ASTM Standards:2D 3016 Practice for Use of L
5、iquid Exclusion Chromatogra-phy Terms and RelationshipsE 355 Practice for Gas Chromatography Terms and Rela-tionshipsE 1151 Practice for Ion Chromatography Terms and Rela-tionships3. Names of TechniquesNOTE 2In the chromatographic literature one may often find theterm“high-performance (or high-press
6、ure) liquid chromatography, abbre-viated as HPLC. This term was introduced to distinguish the present-daycolumn chromatographic techniques employing high inlet pressures andcolumns containing small diameter packing from the classical methods.The utilization of this term or any derivative term (for e
7、xample, HPLSCfor high-performance liquid-solid chromatography) is not recommended.Similarly, the use of the term high-performance thin-layer chromatog-raphy, abbreviated as HPTLC, describing newer variations of thin-layerchromatography, is also not recommended.3.1 Liquid Chromatography, abbreviated
8、as LC, comprisesall chromatographic methods in which the mobile phase isliquid under the conditions of analysis. The stationary phasemay be a solid or a liquid supported by or chemically bondedto a solid.3.2 The stationary phase may be present on or as a plane(Planar Chromatography), or contained in
9、 a cylindrical tube(Column Chromatography).3.3 Separation is achieved by differences in the distributionof the components of a sample between the mobile andstationary phases, causing them to move along the planesurface or through the column at different rates (differentialmigration).3.3.1 In Planar
10、Chromatography, the differential migrationprocess will cause the sample components to separate as aseries of spots behind the mobile phase front.3.3.2 In Column Chromatography, the differential migrationprocess will cause the sample components to elute from thecolumn at different times.3.3.3 In Dry-
11、Column Chromatography, mobile phase flow isstopped as soon as the mobile phase has reached the end of thecolumn of dry medium. This column can be glass or a rigid orflexible solvent compatible plastic. Solute visualization andrecovery are from the extruded or sliced column packing.3.3.4 In Flash Chr
12、omatography, mobile phase flow is con-tinued after the mobile phase has reached the end of the columnof dry medium until elution of the desired components isachieved. Often low pressures, compatible with the materialsof construction of the column, are applied to the top of thecolumn to speed up the
13、elution.3.4 The basic process of selective distribution during thechromatographic process can vary depending on the type ofstationary phase and the nature of the mobile phase.3.4.1 In Liquid-Liquid Chromatography, abbreviated LLC,the stationary phase is a liquid and the separation is based onselecti
14、ve partitioning between the mobile and stationary liquidphases.3.4.2 In Liquid-Solid Chromatography, abbreviated as LSC,the stationary phase is an interactive solid. Depending on thetype of the solid, separation may be based on selectiveadsorption on an inorganic substrate such as silica gel, or ano
15、rganic gel. In this definition, Ion-Exchange Chromatography1This practice is under the jurisdiction of ASTM Committee E13 on MolecularSpectroscopy and Separation Science and is the direct responsibility of Subcom-mittee E13.19 on Separation Science.Current edition approved Sept. 1, 2006. Published S
16、eptember 2006. Originallyapproved in 1979. Last previous edition approved in 2000 as E 682 92 (2000).2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards D
17、ocument Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.is considered to be a special case of LSC in which theinteractive solid has ionic sites and separation is due to ionicinteraction.3.4.2.1 In this
18、 definition, Ion Exchange Chromatography isconsidered to be a special case of LSC in which the interactivesolid has permanently bonded ionic sites and separation is dueto electrostatic interaction.3.4.2.2 In this definition, Ion Pair Chromatography is con-sidered to be a special case of LSC in which
19、 ionic counterionsare added to the mobile phase to effect the separation of ionicsolutes. In this technique both electrostatic and adsorptiveforces are involved in the separation.NOTE 3Other terminology for this technique include, but are notlimited to, extraction chromatography, paired ion chromato
20、graphy, soapchromatography, ion pair extraction chromatography, ion pair partitionchromatography, and ion interaction chromatography, but utilization ofthese terms is not recommended.3.4.2.3 In this definition, Affnity Chromatography is con-sidered to be a special case of LSC in which special ligand
21、s arebonded to a stationary phase so that bio-specific interactions(for example, antibody/antigen, enzyme/substrate) may beinvoked to effect the separation.3.4.2.4 In this definition, Ion Chromatography is consideredto be a special application of LSC in which the ion exchangemechanism is still effec
22、ting the separation. Special columns ordevices, after the separating column, may be needed to removehigher concentrations of inorganic ions which might otherwiseinterfere with the detectability using conductivity. See PracticeE 1151 for further details of nomenclature for this technique.3.4.2.5 In t
23、his definition, Hydrophobic Interaction Chroma-tography, is considered to be a special application of LSC inwhich the separation is based upon interaction of the hydro-phobic moieties of the solutes and the hydrophobic moieties ofthe sites on a reversed phase packing. High to low saltgradients are u
24、sed to effect this type of separation.3.4.3 In some cases, such as with bonded stationary phases,the exact nature of the separation process is not fully estab-lished and it may be based on a combination of liquid-liquidand liquid-solid interactions.3.4.4 In Steric Exclusion Chromatography, the stati
25、onaryphase is a noninteractive porous solid, usually silica or anorganic gel. In this case, separation is affected by the size of thesample molecules, where those which are small enough pen-etrate the porous matrix to varying extents and degrees whilethose that are largest are confined to the inters
26、titial region ofthe particles. Thus, the larger molecules elute before thesmaller molecules. See Practice D 3016 for further details ofnomenclature for this technique.3.5 In liquid chromatography, the composition of the mobilephase may be constant or changing during a chromatographicseparation.3.5.1
27、 The term Isocratic may be used when the compositionof the mobile phase at the column inlet is kept constant duringa chromatographic separation.3.5.2 The term Gradient is used to specify the techniquewhen a deliberate change in the mobile phase operatingcondition is made during the chromatographic p
28、rocedure. Thechange is usually in mobile phase composition, flow rate, pH,or temperature. The first-named change is called GradientElution. Flow Programming is a technique where the mobilephase linear velocity is changed during the chromatographicprocedure. The changes are made to enhance separation
29、 or tospeed elution of sample components, or both. Such changes inoperating conditions may be continuous or step-wise.3.6 In the standard modes of liquid chromatography, thestationary phase is more polar than the mobile phase. This isreferred to as Normal Phase Chromatography. The oppositecase is al
30、so possible, in which the mobile phase is more polarthan the stationary phase. This version of the technique iscalled Reversed-Phase Chromatography.3.7 Planar Chromatography comprises two versions: paperchromatography and thin-layer chromatography.3.7.1 In Paper Chromatography, the process is carrie
31、d outon a sheet or strip of paper. Separation is usually based on LLCin which water held on the cellulose fibers acts as the stationaryphase. Separation based on LSC may also be utilized when thepaper is impregnated or loaded with an interactive solid.3.7.2 In Thin-Layer Chromatography, the solid st
32、ationaryphase is utilized in the form of a relatively thin layer on aninactive plate or sheet.3.7.3 In any version of planar chromatography, the mobilephase may be applied in a number of ways. In normal usage,Ascending, Descending, and Horizontal Development, themobile phase movement depends upon ca
33、pillary action. InHorizontal Development, the mobile phase may move pre-dominantly linearly or radially. In Radial Development, themobile phase is applied as a point source. Devices have beenemployed which accelerate the mobile phase movement onplanar layers by pressure or centrifugal force.3.7.4 Th
34、e Mobile Phase Front is the leading edge of mobilephase as it traverses the planar media. In all forms ofdevelopment, including radial, the local tangent to the MobilePhase Front is everywhere normal to the local direction ofdevelopment.3.7.5 Consecutive Developments of planar media may becarried ou
35、t after removal of the mobile phase from a previousdevelopment. If the consecutive development is accomplishedin the same direction as previously, this is Multiple Develop-ment. If a second development is accomplished at a right angleto the first development, this is Two-Dimensional Develop-ment. Co
36、ntinuous development of planar media is possible byallowing evaporation of the mobile phase near the MobilePhase Front.3.7.6 Impregnation is the technique of applying a reagentto the planar media to effect an enhanced separation ordetection. This impregnation is accomplished by dipping orspraying a
37、reagent solution after the preparation of the me-dium, or by incorporating during the manufacturing process.4. Apparatus4.1 PumpsThe function of the pumps is to deliver themobile phase at a controlled flow rate to the chromatographiccolumn.4.1.1 Syringe Pumps have a piston that advances at acontroll
38、ed rate within a smooth cylinder to displace the mobilephase.E 682 92 (2006)24.1.2 Reciprocating Pumps have a single or dual chamberfrom which mobile phase is displaced by reciprocating pis-ton(s) or diaphragm(s). The chamber volume is relatively smallcompared to the volume of the column.4.1.3 Pneum
39、atic Pumps employ a gas to displace themobile phase either directly or through a piston or collapsiblecontainer. The volume within these pumps may be large orsmall as compared to the volume of the column.4.2 Sample Inlet Systems represent the means for introduc-ing samples into the column.4.2.1 Sept
40、um InjectorsSample contained in a syringe isintroduced directly into the pressurized flowing mobile phaseby piercing an elastomeric barrier. The syringe is exposed topressure and defines the sample volume.4.2.2 Septumless InjectorsSample contained in a syringeis introduced into an ambient-pressure c
41、hamber, and thechamber is subsequently mechanically displaced into thepressurized flowing mobile phase. The syringe is not exposedto pressure and defines the sample volume.4.2.3 Valve InjectorsSample contained in a syringe (orcontained in a sample vial) is injected into (or drawn into) anambient-pre
42、ssure chamber which is subsequently displacedinto the pressurized flowing mobile phase. The displacement isby means of rotary or sliding motion. The chamber is a section(loop) of tubing or an internal chamber. The chamber can becompletely filled, in which case the chamber volume definesthe sample vo
43、lume, or it can be partially filled, in which casethe syringe calibration marks define the sample volume.4.3 Columns consist of tubes that contain the stationaryphase and through which the mobile phase flows.4.3.1 Separating Column is the column on which the sepa-ration of the solutes is accomplishe
44、d.4.3.2 Pre-column is a column that has been used classicallyto precondition the mobile phase, placed between the pumpand the injector. In the instance of its use with liquid-liquidseparations involving coated stationary phases, such a columncontained an excess of the coating phase to presaturate th
45、emobile phase so it would not strip the same phase from thecoated stationary phase during the separation. Its predominateuse today is as a protector column for silica based columnpacking materials. It is filled with large particle silica which isslowly dissolved by polar, ionic mobile phases. By so
46、doing,the silicate saturated mobile phase cannot dissolve the silicabackbone of the analytical or preparative column.4.3.3 Guard Column is a protector column placed betweenthe injector and the separating column. The purpose of thiscolumn is to be the final filter for the sample, adsorbingunwanted sa
47、mple components that otherwise might bind irre-versibly to the separating column. It has a volume of no morethan 1/20 the volume of the separating column. It may be filledwith any material which will effectively remove the unwantedcomponents without interfering with subsequent chromato-graphic proce
48、sses.4.3.4 Concentrator Column is a small column placed in-line at the loop injector for introducing a dilute sample whichis collected into it before elution onto the separating column.NOTE 4Other terminology for this technique include, but are notlimited to, trace enrichment column, collector colum
49、n, and sampleconcentration column, but utilization of these terms is not recommended.4.3.5 Column sizes with various internal diameters (ID) andlengths can be made. Larger columns present no problemsconcerning nomenclature, but columns with small internaldiameters are now being used. As pointed out by Basey andOliver3as many as nine terms (capillary, microcapillary,narrow bore capillary, micro, microbore, ultramicro, narrowbore, small bore, and small diameter) have been seen in theliterature and with no clear distinction between them when theactual column ID is examined. It
