ASTM_E_1470_-_92_1998.pdf

Designation:E 1470 92(Reapproved 1998)Standard Test Method forCharacterization of Proteins by Electrophoretic Mobility1This standard is issued under the fixed designation E 1470;the number immediately following the designation indicates the year oforiginal adoption or,in the case of revision,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.1.Scope1.1 This test method describes a procedure for determiningthe electrophoretic mobility of proteins of molecular weightgreater than 10 000 Daltons.1.2 This test method uses automatic Electrophoretic LightScattering(ELS)principles to determine the electrophoreticmobility.1.3 The instrument2simultaneously measures the Dopplershifts of scattered light at four different angles to determine theelectrophoretic mobility distribution of protein particles.Themobility is expressed as m-cm/V-s(micron-centimeter/volt-second).1.4 This standard does not purport to address all of thesafety concerns,if any,associated with its use.It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2.Summary of Test Method2.1 A carefully dispersed,dilute suspension of the proteinparticles is loaded into the sample cell and is positioned in thepath of collimated laser light.The laser light directed ontoparticles moving at constant velocity under an applied electri-cal field.The laser light is scattered from moving particles,producing a Doppler shift proportional to the particles veloc-ity.2.2 The instrument response is essentially to a sinusoidal“beat”signal produced at the detector by mixing the scatteredlight and a reference(unscattered)beam.The frequency of the“beat”signal is equal to the difference Doppler shift andtherefore,to particle speed and direction.3.Significance and Use3.1 The prime purpose of this test method is to provide dataexpressed as either electrophoretic mobility or zeta potentialdistribution of protein particles.3.2 Both sellers and purchasers of protein particles will findthis test method useful to determine either mobility or zetapotential distributions for protein specifications,manufacturingcontrol,and development and research.4.Apparatus4.1 The apparatus for analysis consists essentially of a laserlight source,sample cell for introducing the sample,powersupply source,four 256 channel spectrum analyzers,micropro-cessors,and computer assembly.4.2 Sample chamber assembly,holds approximately 1 mLof sample and is composed of three basic parts.The two sidepieces are made of solid silver and contain hemisphericalcavities.Between the two side pieces is a fused silica glassinsert,running through it is a rectangular channel(3 mm wideby 1 mm high).The channel connects the two cavities.Fluidfills both cavities and the channel.Electrophoretic LightScattering measurements are made on particles in the channel.4.2.1 30 mL Plastic Accuvetts,(disposable)for preparing thesample.4.2.2 Membrane Filtering Device,0.2 m filters or finer.4.2.3 5 mL Sterile Plastic Syringe.4.2.4 8 Gage Blunt Tipped Hypodermic Needle.4.2.5 pH Meter.4.2.6 Standard Buffer Solution.5.Reagents and Materials5.1 Purity of ReagentsReagent grade chemicals shall beused in all tests.Unless otherwise indicated,it is intended thatall reagents shall conform to the specifications of the Commit-tee on Analytical Reagents of the American Chemical Societywhere specifications are available.3Other grades may be used,provided it is first ascertained that the reagent is of sufficientlyhigh purity to permit its use without lessening the accuracy ofthe determination.5.2 Suspending MediaThe sample media could be anystandard buffer solution(conductivity 2 s to 200 millisiemen).The media shall be filtered through 0.2 m or finer membranefilter.Select filter that is chemically compatible with the diluentused and with no extractables or surfactants present.Thesurfactants or extractables can influence the particles surfacechemistry.5.3 Rinse WaterDeionized or distilled water twice filtered1This test method is under the jurisdiction of ASTM Committee E-48 onBiotechnology and is the direct responsibility of Subcommittee E48.03 on UnitProcesses and Their Control.Current edition approved March 15,1992.Published May 1992.2The Coultert Delsa 440 instrument from Coulter Corporation has been foundsatisfactory.This instrument is available from Coulter Corporation,601 W.CoulterWay,Hialeah,FL 33010.3“Reagent Chemicals,American Chemical Society Specifications,”Am.Chemi-cal Soc.,Washington,DC.For suggestions on the testing of reagents not listed bythe American Chemical Society,see“Analar Standards for Laboratory U.K.Chemicals,”BDH Ltd.,Poole,Dorset,and the“United States Pharmacopeia.”1AMERICAN SOCIETY FOR TESTING AND MATERIALS100 Barr Harbor Dr.,West Conshohocken,PA 19428Reprinted from the Annual Book of A