1、Designation: E682 92 (Reapproved 2011)Standard Practice forLiquid Chromatography Terms and Relationships1This standard is issued under the fixed designation E682; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last rev
2、ision. A number in parentheses indicates the year of last reapproval. Asuperscript 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.1. Scope1.1 This practice deals primarily with the terms
3、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 technique
4、, 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 E355.1.3 The values stated in SI units are to be regarded asstandard. No other u
5、nits of measurement are included in thisstandard.2. Referenced Documents2.1 ASTM Standards:2D3016 Practice for Use of Liquid Exclusion Chromatogra-phy Terms and RelationshipsE355 Practice for Gas Chromatography Terms and Rela-tionshipsE1151 Practice for Ion Chromatography Terms and Rela-tionships3.
6、Names of TechniquesNOTE 2In the chromatographic literature one may often find the termhigh-performance (or high-pressure) liquid chromatography, abbreviatedas HPLC. This term was introduced to distinguish the present-day columnchromatographic techniques employing high inlet pressures and columnscont
7、aining small diameter packing from the classical methods. Theutilization of this term or any derivative term (for example, HPLSC forhigh-performance liquid-solid chromatography) is not recommended.Similarly, the use of the term high-performance thin-layer chromatog-raphy, abbreviated as HPTLC, descr
8、ibing newer variations of thin-layerchromatography, is also not recommended.3.1 Liquid Chromatography, abbreviated 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 chemicall
9、y bondedto a solid.3.2 The stationary phase may be present on or as a plane(Planar Chromatography), or contained in 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 th
10、em to move along the planesurface or through the column at different rates (differentialmigration).3.3.1 In Planar 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 diff
11、erential migrationprocess will cause the sample components to elute from thecolumn at different times.3.3.3 In Dry-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 com
12、patible plastic. Solute visualization andrecovery are from the extruded or sliced column packing.3.3.4 In Flash Chromatography, 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 pressure
13、s, compatible with the materialsof construction of the column, are applied to the top of thecolumn to speed up the 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 L
14、iquid-Liquid Chromatography, abbreviated LLC,the stationary phase is a liquid and the separation is based onselective partitioning between the mobile and stationary liquidphases.1This practice is under the jurisdiction of ASTM Committee E13 on MolecularSpectroscopy and Separation Science and is the
15、direct responsibility of Subcom-mittee E13.19 on Separation Science.Current edition approved Nov. 1, 2011. Published December 2011. Originallyapproved in 1979. Last previous edition approved in 2006 as E682 92 (2006).DOI: 10.1520/E0682-92R11.2For referenced ASTM standards, visit the ASTM website, ww
16、w.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.4.
17、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 anorganic gel. In this definition, Ion-Exchange Chromatographyis consi
18、dered 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 definition, Ion Exchange Chromatography isconsidered to be a special case of LSC in which the interactivesolid has permanently bonded ionic sites and separation i
19、s 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 ionic counterionsare added to the mobile phase to effect the separation of ionicsolutes. In this technique both electrostatic and adsorptiveforces are involved in
20、 the separation.NOTE 3Other terminology for this technique include, but are notlimited to, extraction chromatography, paired ion chromatography, soapchromatography, ion pair extraction chromatography, ion pair partitionchromatography, and ion interaction chromatography, but utilization ofthese terms
21、 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 ligands 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
22、 In this definition, Ion Chromatography is consideredto be a special application of LSC in which the ion exchangemechanism is still effecting the separation. Special columns ordevices, after the separating column, may be needed to removehigher concentrations of inorganic ions which might otherwisein
23、terfere with the detectability using conductivity. See PracticeE1151 for further details of nomenclature for this technique.3.4.2.5 In this definition, Hydrophobic Interaction Chroma-tography, is considered to be a special application of LSC inwhich the separation is based upon interaction of the hy
24、dro-phobic moieties of the solutes and the hydrophobic moieties ofthe sites on a reversed phase packing. High to low saltgradients are used 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-lishe
25、d and it may be based on a combination of liquid-liquidand liquid-solid interactions.3.4.4 In Steric Exclusion Chromatography, the stationaryphase is a noninteractive porous solid, usually silica or anorganic gel. In this case, separation is affected by the size of thesample molecules, where those w
26、hich are small enough pen-etrate the porous matrix to varying extents and degrees whilethose that are largest are confined to the interstitial region ofthe particles. Thus, the larger molecules elute before thesmaller molecules. See Practice D3016 for further details ofnomenclature for this techniqu
27、e.3.5 In liquid chromatography, the composition of the mobilephase may be constant or changing during a chromatographicseparation.3.5.1 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 Gradie
28、nt is used to specify the techniquewhen a deliberate change in the mobile phase operatingcondition is made during the chromatographic procedure. Thechange is usually in mobile phase composition, flow rate, pH,or temperature. The first-named change is called GradientElution. Flow Programming is a tec
29、hnique where the mobilephase linear velocity is changed during the chromatographicprocedure. The changes are made to enhance separation 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 chromatogra
30、phy, thestationary phase is more polar than the mobile phase. This isreferred to as Normal Phase Chromatography. The oppositecase is also possible, in which the mobile phase is more polarthan the stationary phase. This version of the technique iscalled Reversed-Phase Chromatography.3.7 Planar Chroma
31、tography comprises two versions: paperchromatography and thin-layer chromatography.3.7.1 In Paper Chromatography, the process is carried 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
32、 may also be utilized when thepaper is impregnated or loaded with an interactive solid.3.7.2 In Thin-Layer Chromatography, the solid stationaryphase 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 a
33、pplied in a number of ways. In normal usage,Ascending, Descending, and Horizontal Development, themobile phase movement depends upon capillary action. InHorizontal Development, the mobile phase may move pre-dominantly linearly or radially. In Radial Development, themobile phase is applied as a point
34、 source. Devices have beenemployed which accelerate the mobile phase movement onplanar layers by pressure or centrifugal force.3.7.4 The 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 Mobi
35、lePhase Front is everywhere normal to the local direction ofdevelopment.3.7.5 Consecutive Developments of planar media may becarried out after removal of the mobile phase from a previousdevelopment. If the consecutive development is accomplishedin the same direction as previously, this is Multiple D
36、evelop-ment. If a second development is accomplished at a right angleto the first development, this is Two-Dimensional Develop-ment. Continuous development of planar media is possible byallowing evaporation of the mobile phase near the MobilePhase Front.3.7.6 Impregnation is the technique of applyin
37、g a reagentto the planar media to effect an enhanced separation ordetection. This impregnation is accomplished by dipping orspraying a reagent solution after the preparation of the me-dium, or by incorporating during the manufacturing process.E682 92 (2011)24. Apparatus4.1 PumpsThe function of the p
38、umps is to deliver themobile phase at a controlled flow rate to the chromatographiccolumn.4.1.1 Syringe Pumps have a piston that advances at acontrolled rate within a smooth cylinder to displace the mobilephase.4.1.2 Reciprocating Pumps have a single or dual chamberfrom which mobile phase is displac
39、ed by reciprocating pis-ton(s) or diaphragm(s). The chamber volume is relatively smallcompared to the volume of the column.4.1.3 Pneumatic 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
40、 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 Septum InjectorsSample contained in a syringe isintroduced directly into the pressurized flowing mobile phaseby piercing an elastomeric barrier. The syringe is exposed to
41、pressure and defines the sample volume.4.2.2 Septumless InjectorsSample contained in a syringeis introduced into an ambient-pressure chamber, and thechamber is subsequently mechanically displaced into thepressurized flowing mobile phase. The syringe is not exposedto pressure and defines the sample v
42、olume.4.2.3 Valve InjectorsSample contained in a syringe (orcontained in a sample vial) is injected into (or drawn into) anambient-pressure chamber which is subsequently displacedinto the pressurized flowing mobile phase. The displacement isby means of rotary or sliding motion. The chamber is a sect
43、ion(loop) of tubing or an internal chamber. The chamber can becompletely filled, in which case the chamber volume definesthe sample volume, 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
44、 and through which the mobile phase flows.4.3.1 Separating Column is the column on which the sepa-ration of the solutes is accomplished.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 wit
45、h liquid-liquidseparations involving coated stationary phases, such a columncontained an excess of the coating phase to presaturate themobile 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
46、 based columnpacking materials. It is filled with large particle silica which isslowly dissolved by polar, ionic mobile phases. By so 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 b
47、etweenthe injector and the separating column. The purpose of thiscolumn is to be the final filter for the sample, adsorbingunwanted sample components that otherwise might bind irre-versibly to the separating column. It has a volume of no morethan120 the volume of the separating column. It may be fil
48、ledwith any material which will effectively remove the unwantedcomponents without interfering with subsequent chromato-graphic processes.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 s
49、eparating column.NOTE 4Other terminology for this technique include, but are notlimited to, trace enrichment column, collector column, 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, an
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