1、Designation: E2865 12 (Reapproved 2018)Standard Guide forMeasurement of Electrophoretic Mobility and Zeta Potentialof Nanosized Biological Materials1This standard is issued under the fixed designation E2865; the number immediately following the designation indicates the year oforiginal adoption or,
2、in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This guide deals with the measurement of mobility andzeta potential in systems co
3、ntaining biological material such asproteins, DNA, liposomes and other similar organic materialsthat possess particle sizes in the nanometer scale (100 nm).1.2 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.3 This standard do
4、es 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, health, and environmental practices and deter-mine the applicability of regulatory limitations prior to use.1.4 This internatio
5、nal standard was developed in accor-dance with internationally recognized principles on standard-ization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recom-mendations issued by the World Trade Organization TechnicalBarriers to Trade (TBT) Commit
6、tee.2. Referenced Documents2.1 ASTM Standards:2E1470 Test Method for Characterization of Proteins byElectrophoretic Mobility (Withdrawn 2014)3E2456 Terminology Relating to Nanotechnology2.2 ISO Standards:4ISO 13099-1 Colloidal Systems Methods for Zeta-Potential Determination Part 1: Electroacoustic
7、andElectrokinetic PhenomenaISO 13099-2 Colloidal Systems Methods for Zeta-Potential Determination Part 2: Optical MethodsISO 13321 Particle Size Analysis Photon CorrelationSpectroscopy3. Terminology3.1 DefinitionsDefinitions of nanotechnology terms canbe found in Terminology E2456.3.2 Definitions of
8、 Terms Specific to This Standard:3.2.1 Brownian motionis the random movement of par-ticles suspended in a fluid caused by external bombardment bydispersant atoms or molecules.3.2.2 dielectric constantthe relative permittivity of a ma-terial for a frequency of zero is known as its dielectric constant
9、(or static relative permittivity).3.2.2.1 DiscussionTechnically, it is the ratio of the amountof electrical energy stored in a material by an applied voltage,relative to that stored in a vacuum.3.2.3 electrophoretic mobilitythe motion of dispersed par-ticles relative to a fluid under the influence o
10、f an electrical field(usually considered to be uniform).3.2.4 isoelectric pointpoint of zero electrophoretic mobil-ity.3.2.5 mobilitysee electrophoretic mobility.3.2.6 redox reactiona chemical reaction in which atomshave their oxidation number (oxidation state) changed.3.2.7 stabilitythe tendency fo
11、r a dispersion to remain inthe same form for an appropriate timescale (for example, theexperiment duration; on storage at 358K).3.2.7.1 DiscussionIn certain circumstances (for examplewater colloid flocculation) instability may be the desiredproperty.3.2.8 van der Waals forcesin broad terms the force
12、sbetween particles or molecules.3.2.8.1 DiscussionThese forces tend to be attractive innature (because such attractions lead to reduced energy in thesystem) unless specific steps are undertaken to prevent thisattraction.3.2.9 zeta potentialthe potential difference between thedispersion medium and th
13、e stationary layer of fluid attached tothe dispersed particle.1This guide is under the jurisdiction of ASTM Committee E56 on Nanotech-nology and is the direct responsibility of Subcommittee E56.02 on Physical andChemical Characterization.Current edition approved Jan. 1, 2018. Published January 2018.
14、 Originallyapproved in 2012. Last previous edition approved in 2012 as E2865 12. DOI:10.1520/E2865-12R18.2For 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 standar
15、ds Document Summary page onthe ASTM website.3The last approved version of this historical standard is referenced onwww.astm.org.4Available from International Organization for Standardization (ISO), 1, ch. dela Voie-Creuse, CP 56, CH-1211 Geneva 20, Switzerland, http:/www.iso.org.Copyright ASTM Inter
16、national, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards,
17、Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.13.2.10 zwitterionica molecule with a positive and a nega-tive electrical charge.3.2.10.1 DiscussionAmino acids are the best known ex-amples of zwitterions.4. Summary of Practice4.1 Introduc
18、tionIt is not the intention of this guide tospend any significant time on the theory of zeta potential andthe routes by which a particle acquires charge within a system.Indeed it may be more appropriate to deal only with themovement or mobility of particles under an electrical fieldwhere conversion
19、to zeta potential is not even attempted. Therelevant text books (for example, see Hunter (1)5) should beconsulted along with the more academic ISO references (ISO13099-1 and ISO 13099-2). The IUAPC report (2) is also veryuseful, albeit fairly theoretical, but it does contain a section(4.1.2) entitle
20、d How and under which conditions the electro-phoretic mobility can be converted into -potential. TheCorbett and Jack paper (3) contains excellent practical advicefor measurement of protein mobility and is recommended.4.2 Test Method E1470 is based around a sole vendorsequipment, but this does not de
21、al with the basis of themeasurement or provide guidance in the practice of themeasurement. It is one intention of this guide to address thosedeficits.4.3 The following aspects need emphasis:4.3.1 Zeta potential is a function of the particulate system asa whole so the environment that the particle re
22、sides in (pH,concentration, ionic strength, polyvalent ions) will directlyinfluence the magnitude and, in certain circumstances, the signof the acquired charge. In particular, small quantities (parts permillion) of polyvalent ions (for example calcium ions (Ca2+),iron (III) ions (Fe3+) or other impu
23、rities can significantly affectthe magnitude of the zeta potential. It is obvious, but oftenignored, that there is no such concept of the zeta potential of apowder.4.3.2 The calculation of zeta potential from mobility mea-surement typically refers to the unrestricted mobility of aparticle in suspens
24、ion. In crowded environments (that is highconcentration) particle-particle interactions occur and themovement may be hindered. In this circumstance, although amovement can be detected and measured, it may provideinterpretation issues when a conversion to zeta potential isattempted.4.3.3 Zeta potenti
25、al tends only to be important in the sub-5m (and thus relevant to the sub-100 nm region considered inthis text) region where van der Waals attractive forces are of asimilar order of magnitude as inertial forces. Thus if sedimen-tation (function of size and density of the particle with respectto the
26、medium it resides) is occurring or has occurred, thesystem is clearly not ideal for a zeta potential or mobilitymeasurement. With significant settling the measurement ofmobility is obviously compromised. The lower limit formeasurement of electrophoretic mobility is in effect deter-mined by the signa
27、l to noise which is a complex function ofsize, concentration and relative refractive index of the particu-late system. An unambiguous statement of the lower size istherefore not possible.4.3.4 Zeta potential and its (assumed) relation to systemstability are reasonably well understood in aqueous syst
28、ems.The classic examples are indicated in Thomas Riddicks text(4). The obvious or stated link with formulation or productstability is not obvious for organic media where the counter-ions will be strongly bound to the particle surface and theposition of the diffuse layer will be difficult to identify
29、 in an(effectively) insulating external medium. Again, what is oftenforgotten, is that conductivity is required in the backgroundsolution (typically 0.001 molL-1sodium chloride (NaCl) isutilized) so that an electrical field can be correctly appliedwithout effects such as electrode polarization (caus
30、ing voltageirregularities) occurring. Mobility or zeta potential measure-ments should not be made in de-ionized water. In non-polardispersant liquids, conversion of observed mobility to zetapotential may need some understanding of the position andthickness (single atom or molecule?) of the double la
31、yer, butthis is not relevant to measurements in (aqueous) biologicalmedia.4.3.5 It is mobility (movement) that is usually measured andthe conversion to zeta potential relies on application of theHenry equation. (See also Fig. 1).UE5 f!6(1)5The boldface numbers in parentheses refer to a list of refer
32、ences at the end ofthis standard.FIG. 1 Equation (1)E2865 12 (2018)2where:UE= the electrophoretic mobility (measured byinstrument), = the dielectric constant of the dispersion medium, = the (calculated) zeta potential,f() = Henrys function (see below), and = the viscosity of the medium (measured or
33、assumed).4.3.5.1 It is important to specify the units of measurement asfailure to get these correct will lead incompatibility of units onthe right and left hand side of the above equation. The normalSI units (metre, kilogram, second) are not often utilized in thisarea as they are too large for pract
34、ical purposes (diffusiondistances of one metre are not routinely encountered!) seeadditional unit information in Ref. (5). We need to rememberthat the mobility and diffusion coefficient are a flux (and thusarea) per unit time. The mobility will be scaled by the field(volts/distance). Ref. (5) recomm
35、ended units for electropho-retic mobility are m2s-1V-1. This can be expressed as (ms-1)/(Vm-1) or a velocity per unit field. In practice, the electropho-retic mobility, UE, has more convenient units of m2/Vs Oftenmobilities are expressed in confused units (for example, theoft-utilized mcm-1/Vs becau
36、se this gives rise to mobilityvalues in the convenient 610 region). Mobilities expressedwith a negative sign imply a negative zeta potential.4.3.5.2 is the dielectric constant of the dispersion mediumdimensionless/no units as it is a ratio of the relative permittivityof the material to vacuum whose
37、relative permittivity is definedas 1.4.3.5.3 f() is usually referred to as “Henrys function”where is the radius of the particle. is referred to as theDebye parameter and can be calculated from the electroniccharge, Boltzmanns and Avogadros constants, the absolutetemperature and the ionic strength. T
38、he charged region arounda particle falls to about 2 % of the surface charge at a distanceapproximately 3/ from the particle. For ionic strength around0.01 molL-1then 3/ is around 10 nm and for ionic strengtharound 10-5molL-1then 3/ is around 280 nm (see Koutsoukoset al. (6). 1/ can be envisioned as
39、the “thickness“ of theelectrical double layer (the Debye length) and thus the units of are reciprocal length. Thus f() is dimensionless and usuallyassigned the value 1.00 or 1.50. For particles in polar media themaximum value of f() is taken to be 1.5 (Smoluchowskiapproximation) and for particles in
40、 non-polar media the mini-mum value of f() is 1 (Hckel approximation). It is theformer that we are considering in this text. The literature doesindicate intermediate values for f() but in most biologicallyrelevant media the value of 1.5 is the most appropriate.4.3.5.4 In terms of viscosity, , the SI
41、 physical unit ofdynamic viscosity is the pascal-second (Pas), (equivalent toNs/m2, or kg/(ms). Water at 293 K has a viscosity of0.001002 Pas. The cgs physical unit for dynamic viscosity isthe poise (P). It is more commonly expressed, particularly inASTM standards, as centipoise (cP). Water at 293 K
42、 has aviscosity of 1.0020 cP.NOTE 1At room temperature (assumed 298 K) in water, all of theexpressions are constants except for the (measured) mobility and theequation defers to:Zeta potential 5 K*electrophoretic mobility, UE; 12.85*UE(2)where the value of K (collective proportionality constant) is
43、12.85 ifthe zeta potential is to be stated in mV and this falls out naturally from theHenry equation if the deprecated mcm-1/Vs unit is used for electropho-retic mobility.4.3.5.5 As well as movement under the constraint of anelectric field, some degree of Brownian motion will also occurand may need
44、to be considered. In biological media ofrelatively high ionic strength the Hckel model (f()=1)forzeta potential calculation is inappropriate and the value of f()should be calculated from the measured size and the knownionic strength (or measured conductivity) (see Fig. 2).4.3.6 Systems of positive c
45、harge tend to provide moremeasurement difficulties from a practical perspective thanthose of inherent negative charge. This is because most organicmedia including plastic sample cells are inherently negativelycharged at neutral pH and may attract particles of oppositeFIG. 2 Graphical Representation
46、of the Henry Function and the a Values for Four Example Particle Size and Ionic Strength Combina-tionsE2865 12 (2018)3charge removing them from suspension and altering the wallpotential. It is useful to have some form of automation for pHadjustment for example a titrator. This eases the adjustmentof
47、 pH and additive concentration.4.3.7 It is of no value to state a zeta potential value withoutdescription of the manner in which it was measured togetherwith vital measurement parameters. Zeta potential without astated pH, ionic compostion, and electrolyte concentrationvalue is close to meaningless.
48、4.4 Biological Molecules and EntitiesAgain, a few obvi-ous points will need mentioning:4.4.1 Many materials such as proteins contain charges andmay be zwitterionic (contain both positive and negativecharges). These molecules can be quite labile and may absorband decompose readily under an electrical
49、 field at the electrodewith the deposition of carbon (shown as electrode darkening)and gas evolution. This is a conventional redox reaction and isvirtually impossible to eliminate if organic materials interactwith or contact metal electrodesthe electrical field over thelength of an adsorbed molecule is enormous in relation to thatbetween the electrodes themselves. Protocols need to be awareof this possibility and seek to minimize it after appropriateinvestigation of the magnitude of
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