1、Designation: E2865 12Standard 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, in the case of rev
2、ision, 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 containing biologica
3、l 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 does not purport to
4、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. Referenced Documents2.1 ASTM Standards:2E1470 Test
5、 Method for Characterization of Proteins byElectrophoretic MobilityE2456 Terminology Relating to Nanotechnology2.2 ISO Standards:3ISO 13099-1 Colloidal systems Methods for zeta-potential determination Part 1: Electroacoustic andelectrokinetic phenomenaISO 13099-2 Colloidal systems Methods for zeta-p
6、otential 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 Terms Specific to This Standard:3.3 Brownian motionis the random movement
7、of particlessuspended in a fluid caused by external bombardment bydispersant atoms or molecules.3.4 dielectric constantthe relative permittivity of a mate-rial for a frequency of zero is known as its dielectric constant(or static relative permittivity).3.4.1 DiscussionTechnically, it is the ratio of
8、 the amountof electrical energy stored in a material by an applied voltage,relative to that stored in a vacuum.3.5 electrophoretic mobilitythe motion of dispersed par-ticles relative to a fluid under the influence of an electrical field(usually considered to be uniform).3.6 isoelectric pointpoint of
9、 zero electrophoretic mobility.3.7 mobilitysee electrophoretic mobility.3.8 redox reactiona chemical reaction in which atomshave their oxidation number (oxidation state) changed.3.9 stability the tendency for a dispersion to remain in thesame form for an appropriate timescale (for example, theexperi
10、ment duration; on storage at 358K).3.9.1 Discussion In certain circumstances (for examplewater colloid flocculation) instability may be the desiredproperty.3.10 van der Waals forcesin broad terms the forcesbetween particles or molecules.3.10.1 DiscussionThese forces tend to be attractive innature (b
11、ecause such attractions lead to reduced energy in thesystem) unless specific steps are undertaken to prevent thisattraction.3.11 zeta potentialthe potential difference between thedispersion medium and the stationary layer of fluid attached tothe dispersed particle.3.12 zwitterionica molecule with a
12、positive and a nega-tive electrical charge.3.12.1 DiscussionAmino acids are the best known ex-amples of zwitterions.4. Summary of Practice4.1 IntroductionIt is not the intention of this guide tospend any significant time on the theory of zeta potential andthe routes by which a particle acquires char
13、ge within a system.Indeed it may be more appropriate to deal only with themovement or mobility of particles under an electrical fieldwhere conversion to zeta potential is not even attempted. The1This guide is under the jurisdiction of ASTM Committee E56 on Nanotech-nology and is the direct responsib
14、ility of Subcommittee E56.02 on Characterization:Physical, Chemical, and Toxicological Properties.Current edition approved Jan. 1, 2012. Published June 2012. DOI: 10.1520/E2865-12.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org
15、. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from International Organization for Standardization (ISO), 1, ch. dela Voie-Creuse, CP 56, CH-1211 Geneva 20, Switzerland, http:/www.iso.org.1Copyright ASTM International
16、, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.relevant text books (for example, see Hunter (1)4) should beconsulted along with the more academic ISO references(ISO 13099-1 and ISO 13099-2). The IUAPC report (2) is alsovery useful, albeit fairly theoretical, bu
17、t it does contain asection (4.1.2) entitled How and under which conditions theelectrophoretic mobility can be converted into z-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 so
18、le vendorsequipment, but this does not deal 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 whol
19、e so the environment that the particle resides 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 (C
20、a2+),iron (III) ions (Fe3+) or other impurities 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 unre
21、stricted mobility of aparticle in suspension. 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
22、 potential isattempted.4.3.3 Zeta potential 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 de
23、nsity of the particle with respectto the 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 mobil
24、ity is in effect deter-mined by the signal 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 r
25、easonably well understood in aqueous systems.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 di
26、ffuse layer will be difficult to identify 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 eff
27、ects such as electrode polarization (causing 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 (s
28、ingle atom or molecule?) of the double layer, 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).UE5z fka!6ph(1)where:UE= the e
29、lectrophoretic mobility (measured by instru-ment), = the dielectric constant of the dispersion medium,z = the (calculated) zeta potential,f(ka) = Henrys function (see below), andh = the viscosity of the medium (measured or as-sumed).4.3.5.1 It is important to specify the units of measurement asfailu
30、re 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 practical purposes (diffusiondistances of one metre are not routinely encountered
31、!) 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) recommended units for electropho-retic mobility are m2s-1V-1. This can be expresse
32、d as (ms-1)/4The boldface numbers in parentheses refer to a list of references at the end ofthis standard.FIG. 1 Equation (1)E2865 122(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 unit
33、s (for example, theoft-utilized mcm-1/Vs because 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 relativ
34、e permittivityof the material to vacuum whose relative permittivity is definedas 1.4.3.5.3 f(ka) is usually referred to as “Henrys function”where a is the radius of the particle. k is referred to as theDebye parameter and can be calculated from the electroniccharge, Boltzmanns and Avogadros constant
35、s, the absolutetemperature and the ionic strength. The charged region arounda particle falls to about 2 % of the surface charge at a distanceapproximately 3/k from the particle. For ionic strength around0.01 molL-1then 3/k is around 10 nm and for ionic strengtharound 10-5molL-1then 3/k is around 280
36、 nm (see Koutsoukoset al. (6). 1/k can be envisioned as the 9thickness9 of theelectrical double layer (the Debye length) and thus the units ofk are reciprocal length. Thus f(ka) is dimensionless andusually assigned the value 1.00 or 1.50. For particles in polarmedia the maximum value of f(ka) is tak
37、en to be 1.5(Smoluchowski approximation) and for particles in non-polarmedia the minimum value of f(ka) is 1 (Hckel approxima-tion). It is the former that we are considering in this text. Theliterature does indicate intermediate values for f(ka) but inmost biologically relevant media the value of 1.
38、5 is the mostappropriate.4.3.5.4 In terms of viscosity, h, the SI 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,
39、particularly inASTM standards, as centipoise (cP). Water at 293 K 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 *
40、 UE(2)where the value of K (collective proportionality constant) is 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 anelectri
41、c field, some degree of Brownian motion will also occurand may need to be considered. In biological media ofrelatively high ionic strength the Hckel model (f(ka)=1)forzeta potential calculation is inappropriate and the value off(ka) should be calculated from the measured size and theknown ionic stre
42、ngth (or measured conductivity) (see Fig. 2).4.3.6 Systems of positive charge 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
43、pH and may attract particles of oppositecharge 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 pH and additive concentration.4.3.7 It is of no value to state a zeta potenti
44、al 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.4.4 Biological Molecules and EntitiesAgain, a few obvi-ous points will need me
45、ntioning: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 field at the electrodewith the deposition of carbon (shown as electrode darke
46、ning)FIG. 2 Graphical Representation of the Henry Function and the ka Values for Four Example Particle Size and Ionic StrengthCombinationsE2865 123and gas evolution. This is a conventional redox reaction and isvirtually impossible to eliminate if organic materials interactwith or contact metal elect
47、rodesthe 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 the phenomenon. It may bevirtually impossible to
48、 eliminate such decomposition for somemolecules unless specific routes are takenfor example,isolation of the electrodes from the biological molecules witha porous membrane that allows ions but not larger molecules topass through. Measurements taken quickly and at lower volt-ages in combination with
49、a reduced electrode spacing (thusreducing the field) may also help in this regard but resolutionwill almost certainly be lost. Many hours are required in orderfor proteins to diffuse a few tens of millimeters; a distancebetween detection point and electrodes somewhat typical ofmany capillary based laser Doppler electrophoresis systems. Itis the slow timescales associated with the diffusion coupledwith measurement times of the order of minutes to tens ofminutes associated with laser Doppler electrophoresis that isthe
