1、Designation: D 3016 97 (Reapproved 2003)Standard Practice forUse of Liquid Exclusion Chromatography Terms andRelationships1This standard is issued under the fixed designation D 3016; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision,
2、the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.INTRODUCTIONLiquid exclusion chromatography (LEC) began as “gel filtration chromatography” which isattributed to Pora
3、th and Flodin.2With the invention of new column packings by Moore3fororganic-phase work a new form of LEC developed which commonly became known as gel permeationchromatography or GPC. Liquid exclusion chromatography is a form of liquid chromatography (someother forms being partition, ion-exchange, a
4、nd adsorption) and as such is the preferred name for thetechnique; however, the reader must be aware that other names are common in the literature, the mostprevalent being those cited above. LEC differs from all other chromatographic techniques in that onlythe exclusion mechanism may be operative if
5、 meaningful data are to result. Most other chromato-graphic mechanisms operate in essentially the opposite way, that is, with small molecules exiting first.Any combination of mechanisms causes confusion and is misleading.Liquid exclusion chromatography as used for the analysis of polymers has grown
6、and matured sincethe first issuance of this practice in 1972. Therefore, some infrequently used or “outdated” terms havebeen deleted and some modern practices (or terms) have been included. Modern developments includethe use of constant-volume pumps, use of “microparticle” column packings and much s
7、mallercolumns, and automated data-handling procedures. In addition, SI units as recommended in ASTMStandard IEEE/ASTM SI 10 for Metric Practice are now used.1. Scope*1.1 This practice covers the definitions of terms and sym-bols most often used in liquid exclusion chromatography.Wherever possible, t
8、hese terms and symbols are consistentwith those used in other chromatographic techniques.4Asadditional terms and relationships are developed, they will beincorporated.NOTE 1There is no similar or equivalent ISO standard.2. Referenced Documents2.1 ASTM Standards:IEEE/ASTM SI 10 Standard for Use of th
9、e InternationalSystem of Units (SI): The Modern Metric System53. Terminology Apparatus Definitions3.1 absolute detectorsdevices that sense and measure theabsolute concentration or other physical property of solutecomponents contained in the eluate.3.2 by-pass or loop injectorthe injector most common
10、 inliquid exclusion chromatography and which utilizes a samplechamber that can be filled with sample while it is temporarilyexternal to the flowing liquid stream. It can be manipulated bymeans of a valving device to sweep the sample with eluent intothe column.3.3 collection devicesdevices used to co
11、llect discreteportions of an eluate according to a preset cycle (for example,times, volume, etc.).1This practice is under the jurisdiction ofASTM Committee D20 on Plastics andis the direct responsibility of Subcommittee D20.70 on Analytical Methods.Current edition approved March 10, 2003. Published
12、April 2003. Originallyapproved in 1972. Last previous edition approved in 1997 as D 3016 97.2Porath, J., and Flodin, P., Nature, NTRWA, Vol 183, 1959, p. 1657.3Moore, J. C., Journal of Polymer Science, JPYAA, Part A, Vol 2, 1964, p. 835.4Journal of Gas Chromatography, JCHSB, Vol 66, 1968.5For refere
13、nced 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 Document Summary page onthe ASTM website.1*A Summary of Changes section appears at the end of this standard.Copyr
14、ight ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.4 column end-fittingsdevices that prevent the columnpacking from passing through them but which are permeable tothe eluent (solvent or solution).3.5 columnstubes that contain the column pac
15、king.3.6 detectorsdevices that sense and measure the concen-tration or other physical property of solute components in thesolution (eluate) passing through.3.7 differential detectorsdevices that sense and measurethe difference in a physical or chemical property between asolution (solvent containing
16、solute components) and a refer-ence liquid (for example, solvent alone).3.8 direct injectora device for introducing a sample froma source external to the column directly onto the column (forexample, septum-syringe injector).3.9 pumpany device that causes mobile phase to flowthrough the columns.3.10
17、sample inlet systema means of introducing samplesonto the column.4. Reagent Definitions4.1 column packingthe stationary phase which consists ofmicroporous material and the stationary liquid phase containedin the pores.4.2 eluatethe liquid emerging from the column.4.3 eluentthe mobile phase or solven
18、t used to sweep orelute the sample (solute) components into, through, and out ofthe column. Its composition is the same as the stationary liquidphase.4.4 solutesdissolved substances that, in LEC, are causedto pass through the column and to influence the detectorresponse.5. Performance in Terms of Re
19、solution, ColumnEfficiency, and Precision5.1 Resolution is the resultant of two effects, the separatingpower of the column packing and the efficiency or peakbroadening. The separating power of the column packing isdependent on pore size and pore volume. Peak broadeningdepends on the nature of the co
20、lumn packings, on how well thecolumns are packed, and on instrumental components externalto the columns. The equations used in LEC (GPC) are similarto those used in other chromatographic techniques. Referencemay be made to any standard chromatography text. Resolutionfor any two samples is defined by
21、 the following equation:R1,25 2VR12 VR2!/W11 W2! (1)where subscripts 1 and 2 refer to samples 1 and 2. (See tablesfor symbols used in this equation.) For complete separation,R1,2must be 1.25 or greater.5.2 Column efficiency is a measure of peak spreading or therate of generation of variance with col
22、umn length. For amonodisperse material, efficiency is the number of theoreticalplates, N, for the entire system defined as follows: (See tablesfor symbols used in this equation.)N 5 16VR/W!2or s5N/VR(2)This expression includes all contributions to peak broaden-ing.5.3 Precision and accuracy are used
23、 according to theiraccepted definitions. Precision is inherent to the system. Bothprecision and accuracy are dependent on the method ofcalibration and treatment of the data as well as on the resolvingpower of the columns. The accuracy must be determined bycomparison with other methods. For example,
24、the molecular-weight distribution can be compared with that obtained fromequilibrium ultracentrifugation. More commonly the weight-and number-average molecular weights computed from theLEC (GPC) trace are compared with those measured by lightscattering, (both static and on-line), osmometry, and on-l
25、ineviscometry.6. Readout Definitions6.1 chromatograma plot of detector response againstvolume of eluate emerging from the system. An idealizedchromatogram obtained with a detector providing differentialresponse is shown in Fig. 1.6.2 The definitions that follow apply to chromatogramsobtained directl
26、y by means of differential detectors:6.2.1 baselinethe portion of the chromatogram recordingthe detector response when only eluent emerges from thecolumn.6.2.2 half width, GHthe retention volume interval of aline parallel to the peak base, bisecting the peak height, andterminating at the sides of th
27、e peak.6.2.3 peakthe portion of the chromatogram recording thedetector response while a single component or a singledistribution (two or more sample components that emergetogether as a single peak) is eluted from the column.6.2.4 peak area, BGDHCABthe area enclosed betweenthe peak and the peak base.
28、6.2.5 peak base, BCan interpolation of the baseline be-tween extremities of the peak.6.2.6 peak height, ADthe dimension from the peak maxi-mum to the base measured in the direction of detectorresponse.6.2.7 peak width, EFthe retention volume interval of thesegment of peak base intercepted by tangent
29、s to the inflectionpoints on either side of the peak.7. Volume Parameters7.1 Volume parameters expected to be involved in LEC arelisted in the glossary of terms found in Tables 1 and 2. TheseFIG. 1 Typical ChromatogramD 3016 97 (2003)2terms are intended for use where the primary mechanism ofseparati
30、on depends on the size of the solute molecules. Thehydrodynamic volume of polymers usually may be related toVRindependent of molecular weight and structure, for example,in describing the exclusion limit. Hydrodynamic volume isrelated to the product of intrinsic viscosity and molecularweight. For sma
31、ll molecules, molar volume is a generallyapplicable parameter for correlation with retention volume.Molar volumes can be readily calculated from experimentallymeasured densities or estimated at any temperature of interestby use of critical properties. Of course, determination of themolecular weight
32、distribution of a polymer by LEC requires aknown relationship between molecular weight of the polymerand retention volume.8. Presentation of Data8.1 Calibration procedures and data should be included inall LEC publications as follows:8.1.1 Number, length, inner diameter, exclusion limit, andtheoreti
33、cal plate numbers of the columns.8.1.2 Type of column packing, solvent, temperature, soluteconcentration, and flowrate.8.1.3 Type and average molecular weights of the calibrationpolymers, together with the limiting viscosity number (intrinsicviscosity) of these polymers in the above solvent at opera
34、tingtemperature. In case of small molecules, the molar volumes ofthe calibration samples should be given.8.1.4 Concentration of calibration solution and injectionvolume.8.1.5 Retention volume or time for each calibration sample.8.2 Sample procedures and data for unknown polymersamples should be incl
35、uded as in 8.1.1-8.1.5, except anyunknown data in 8.1.3.8.3 Calculation procedures should be described. If thecalculations are carried out by computer, the origin of theprogram should be reported. The same applies to any curveTABLE 1 Recommended List of Terms and Symbols for Use in LECPart IPART ILE
36、C PARAMETERSNo. Parameter SymbolUnitsADefinitionsSI Common1 Peak width W cm3ml The distance between the baseline intercepts of linesdrawn tangent to the points of inflection of the trace2 Peak width at half height W1/2cm3ml Peak width measured parallel to baseline at 50 % of itsheight3 Peak area A c
37、m2cm2The area enclosed between the peak and its base,ABGDHCA in Fig. 14 Retention volume VRcm3ml Volume of liquid that has passed through the systemfrom middle of sample injection period to peakmaximum5 Volume of mobile phase in interstitialvolume or void volumeVocm3ml Volume of mobile phase in the
38、interstices between thegel particles6 External volume Vextcm3ml Contributions to V by all system components external tocolumn7 Stationary liquid volume Vicm3ml Total carrier volume contained within the porous support8 Total liquid volume Vtcm3ml Vi+Vo+ Vext9 Hydrodynamic volume Vhcm3mol1ml/mol A pol
39、ymer molecular property proportional to.See Part II.10 Exclusion limit Vh,maxcm3mol1ml/mol Max Vhthat entered into pore11 Column plate count or number oftheoretical plates for systemN 16(R/W)2for Gaussian peak12 Effective plates N(eff) 16(VRVo)/W213 Reduced HhH/ Dw14 Height equivalent to theoretical
40、 platefor systemH mm mm L/N where L is the column length15 Resolution R1,22VR1 VR2/W1+ W216 Specific resolution RsR1,2/log10(M2/M1)17 Mobile phase velocity cms1cm/s F divided by internal cross-sectional area of the column18 Eluent flowrate F cm3s1ml/min Eluent volume flow at column temperature19 Dis
41、tribution coefficient K (VRVo)/Vt20 Solute capacity factor k8RVo)/Vo=KVt/Vo21 Diffusion coefficient of solute Dscm2s1cm2/s22 Molar volume Vmcm3mol1cm3/mol A basic molecular property23 Calibration curve Relationship between a molecular parameter (such as M,Vm,orVh) and VR24 Standard deviation for a G
42、aussiancurves cm3ml W/4=(12)W1/2 3 (2 3 ln2) 1/2=(N/VR)1/225 Pore diameter mm mm (determined by physical measurement)26 Time t smin27 Temperature T K C For fundamental calculations28 Column inlet pressure P Pa psi29 Pressure drop Dp Pa psi30 Weight w kg g31 Density r kgm3g/cm3AIt is recommended that
43、 reported values first be stated in the units as actually measured followed by SI units in parentheses if SI is not the unit of measurement.D 3016 97 (2003)3TABLE 2 Recommended List of Terms and Symbols for Use in LECPart IIPART IIPARTICLE AND MOLECULAR PARAMETERSNo. Parameter SymbolUnitsADefinition
44、sSI Common1 Molecular weight M gmol1g/mol The sums of all the atomic weights of the atoms in amolecule2 Weight differential distributionof molecular weightsfw(M) Plot of weight population density as a function of Mor the frequency distribution of M3 Number-average molecularweightMngmol1g/mol First m
45、oment or mean of the number distribution ofmolecular weights can be expressed in terms offw(M):*fw(M)dM/*(1/M)fw(M)dM4 Weight-average molecular weight Mwgmol1g/mol First moment or mean of the weight distribution ofmolecular weight:* Mfw(M)dM/* fw(M)dM5 Z-average molecular weight Mzgmol1g/mol First m
46、oment or mean of the Z-distribution ofmolecular weights which can be expressed in termsof fw(M):*M2fw(M)/* Mfw(M) dM6 Z + 1-average molelcular weight Mz+ 1 gmol1g/mol First moment or mean of the Z+1distribution ofmolecular weights which can be expressed in termsof fw(M):*M3fw(M)dM/* M2fw(M)dM7 Polyd
47、ispersity factor P or d Mw/Mn8 Intrinsic viscosity hm3kg1dl/g See ASTM Method D 2857, Test for Dilute SolutionViscosity of Polymers, Annual Book of ASTMStandards, Vol 08.02.9 Mark-Houwink intrinsic viscos-itymolecular weight relationh=KMawhere K and a are empirically determinedparameters for a given
48、 polymer solvent system at aspecific temperature10 Viscosity average molecularweightMvgmol1g/mol (h/K)1/a=* Mafw(M)dM/* fw(M)dM1/a11 Instrument symmetrical spread-ing parameterX1 Used to correct for symmetric band broadening. SeeMethod D 3536, Test for Molecular WeightAverages and Molecular Weight D
49、istribution ofPolystyrene by Liquid Exclusion Chromatography(Gel Permeation ChromatographyGPC), AnnualBook of ASTM Standards, Vol 08.03.1/2!FMnt!Mnu!1Mwu!Mwt!G12 Instrument skewing parameter X2 Used to correct for nonsymmetric band broadening.See Method D 3536.Mnt!/Mnu!#Mut!/Muu!# 2 1Mnt!/Mnu!#Mut!/Muu!# 1 113 Uncorrected molecular weightaveragesMn(u)Mn(w)gmol1g/mol Number and weight average molecular weightsuncorrected for ins
