electrophoresis separation technique

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1958 S. P. Radko and A. Chrambach Electrophoresis 2002, 23, 19571972

about 10 nm (which is above the average size of most

globular proteins) to about 10 mm in diameter and we will

not consider the case of long chain-like macromolecules.

For the sake of simplicity, we will refer to those species as

particles or microparticles.

Since the overwhelming majority of surfaces possess anelectric charge in electrolyte solutions, electrophoretic

methods have long been among those employed to sepa-

rate or to analyze microparticles both in science and in

industry. Zone electrophoresis and isoelectric focusing in

media stabilized by a density gradient, rotation, or agar-

ose gels of low concentration have been employed to

separate microparticles with the purpose of profiling,

purification, or measuring their pI (e.g., [39]). Free-flow

electrophoresis was mostly used for preparative separa-

tion of particles on the basis of electrophoretic mobility

differences and isolation of the species of interest [10,

11]. Microelectrophoresis [12, 13] and Doppler laser velo-cimetry (DLV) [13] were applied for measuring electro-

phoretic mobilities of particles.

CE appears to be well suited for both mobility and p I

measurements, for profiling microparticle mixtures, and

quantification of microparticles. Even micropreparative

isolations of microparticles can be achieved, using the

standard CE instrumentation [14]. The technique allows

for on-line detection producing quantitative information

in the form of peak area or height, single sample analysis

in a serial fashion, and the possibility to carry out separa-

tion in the presence of flow (generated by electroosmotic

current). These features, which render CE automated andeasy to perform can be thought to make it a method of

choice for many applications where electrophoretic char-

acterization or separation of microparticles is needed.

A concern may arise as to mobility measurements in cap-

illary zone electrophoresis (CZE) since the relatively large

surface-to-volume ratio of thin capillaries, coupled with

the relatively large surfaces of microparticles, can result

in an interaction between particles and capillary walls

that affects particle mobility. Nonetheless, a consistency

of mobility values derived by CZE and DLV both for model

latex microspheres (at least, those possesing a net nega-

tive charge) [2] and for biological cells [15] was found in

general.

Capillary zone electrophoresis (CZE) is the simplest of

CE modes and straightforward to perform. In regard to

analytical separation and characterization of microparti-

cles, CZE has been the CE mode most widely employed.

The aim of the present paper is to review applications of

CZE to separation and characterization of microparticles

such as synthetic latex nano- and microspheres, organic

and inorganic colloids, lipoprotein particles, viruses, lipo-

somes, biological membrane vesicles, and biological

cells. Distinction will be made between largely artificial

particles with smooth surfaces and biological particles

with preponderantly hairy surfaces. Some aspects of

microparticle electrophoretic behavior such as electro-

phoresis of non-spherical particles, size-dependent elec-

trophoretic migration, peak broadening, migration of par-

ticle conglomerates, and particle electrophoretic migra-

tion in solutions of neutral polymers will be shortly

discussed.

2 Physical mechanisms underlying theelectrophoretic migration of particles

An exhaustive account of both the surface electrostatic

theories and the electrokinetic theory as well as refer-

ences to original publications can be found elsewhere

(e.g., [1619]). Here we are presenting a very schematic

description of the main ideas and results, relevant to the

subject of the review.

In an electrolyte solution, a charged particle is known to

be surrounded by the ionic atmosphere which is a diffu-

sive part of the electric double layer (EDL) formed due

to the separation of charge at the interface of the particle

surface-electrolyte solution. The diffusive part of the

EDL where counterions accumulate while coions are

depleted is characterized by its thickness, k1 (the Debye

length), which is an explicit function of the ionic strength,

I (k , I1/2 ), of the solution. When the distortion of that

atmosphere by the imposed electric field and by the

Brownian motion of the particle is neglected, the particles

translational motion results from the balance of three

forces [18, 19]. The driving force is exerted on the particle

by the external electric field (due to the particles electro-

kinetic charge) and is balanced by the Stokes viscous

drag and by an additional hydrodynamic force the

electrophoretic retardation force. This third force arises

from the electrophoretic motion of counterions in the

diffusive part of the EDL, in a direction opposite to that of

the particle motion. The ratio of the retardation force to

the viscous drag is of the order of kR [19]. The driving

force for the small (relative to k1 ) particles is thus

balanced mainly by the ordinary hydrodynamic resis-

tance (the Stokes drag). For the large ones, it is balancedby the electrophoretic retardation. In the latter case, fluid

is at rest outside of the EDL, and all drag is due to viscous

tension on the particle surface caused by electroosmotic

slipping.

The mobility, m, of a rigid nonconducting spherical particle

undergoing electrophoretic migration in a medium of vis-

cosity Z has been derived by Henry [20] for an arbitrary

value of kR as

m = (2ez/3Z) f(kR ) (1)


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Electrophoresis 2002, 23, 19571972 CZE of microparticles 1959

where e is the dielectric permittivity of the medium and

z the particles zeta potential. The zeta potential is an

electrostatic potential at the so-called shear surface

an imaginary surface in the proximity of the solid surface,

forming a sheath enveloping the particle. Thus, the parti-

cle migrates as a kinetic unit, accompanied by a certain

quantity of the surrounding liquid. Within the frame of

the classical electrokinetic theory (e.g., [17]), z-potential

(and therefore particle mobility) monotonically decreases

with increasing I due to a progressive screening of the sur-

face charge by counterions. The function f(kR ) ranges

from 1 at kR,,1 (the Hckel limit) to 1.5 at kR..1 (the

Smoluchowski limit). In fact, the increase occurs within

the kR-region of approximately 1 to 100 (f = 1.027 and

1.460 at kR = 1 and 100, respectively) [20]. The underlying

assumptions in deriving Eq. (1) were that the medium is

structureless and characterized by uniform e and Z, and

that values of z are smaller than 25 mV (at 257C).

One important feature of the ionic atmosphere, neglected

in the derivation of Eq. (1) is its distortion (polarization).

Once the particle moves, the ionic atmosphere lags

behind the particle, imposing an additional drag on parti-

cle motion, known as the relaxation effect. While affect-

ing the particle migration negligibly at small z-potentials

(z# 25 mV), this effect makes Eq. (1) invalid at moderate

or high z. The relaxation effect gives rise to a much stron-

ger dependence of m on kR [21] than that according to

Eq. (1). The other consequence of the relaxation effect is

that, at kR. 3, the electrophoretic mobility undergoes a

maximum as a function of z at a given kR (ibid.). There-fore, the particle mobility can in some cases become inde-

pendent of, or even start to decrease with, an increasing

z-potential. In practice, particles of higher surface charge

density can migrate at a particular kR slower than those

of lower charge density and the derivation of z-potential

from the mobility data may become ambiguous.

The z-potential (or the electrokinetic charge) that governs

the particles electrokinetics is known to depend on the

surface electrostatic potential, cs (or the particle surface

charge). The electrostatic potential near the charged sur-

face (and, thus, cs as its part) is basically calculated

according to the Gouy-Chapman theory of a diffuse dou-ble layer [17]. In the more elaborate theory, the so-called

Gouy-Chapman-Stern model, the ion adsorption onto the

surface is taken into account based on the Langmuir

adsorption isotherm (ibid. ). However, the relation of the

z-potential to the surface potential is not straightforward:

electrokinetically only the hydrodynamically mobile part

of the double layer is seen. A significant part of the

countercharge is thought to be embedded in a hydro-

dynamically stagnant layer (a layer between the shear

and particle surfaces), resulting in a sometimes substan-

tial discrepancy between the electrokinetic and the sur-

face charge (e.g., [22]). Though the stagnant layer is often

assumed to coincide with the Stern layer (a region en-

compassing ions and molecules adsorbed at the particle

surface), not all ions in the stagnant layer are necessarily

bound to the surface and thus electrophoretically

immobile. Their motility may be taken into account by

ion transport processes of one or another sort in the stag-

nant (or the Stern) layer [17, 23, 24] but such anomalous

surface conductance further complicates the interpreta-

tion of electrophoretic mobility in terms of particle surface

properties. Moreover, the coion adsorption may occur in

the Stern layer (e.g., [24] and references therein). Both the

anomalous surface conductance and the co-ion adsorp-

tion if operative result in the appearance of a maximum on

the curve ofm vs. I, a deviation from the behaviorexpected

according to the classical electrokinetic theory [17].

The other important implication in the derivation of Eq. (1)is the ideality of the particle surface. One may often

neglect irregularities on the particle surface if their char-

acteristic dimensions are much smaller than the particle

size and the Stokes drag is the dominant retarding force.

Yet, if the electrophoretic retardation is the dominant

force, it is necessary to compare the characteristic size

of these irregularities with the thickness of the EDL ( e.g.,

k1 = 3 nm in a 10 mM 1:1 electrolyte solution upon com-

plete dissociation [17]). An example of a particle with a

rough (or hairy) surface may be that with a layer of

neutral polymers adsorbed on, or grafted to, its surface

(e.g., [2527]). If the thickness of such hairy layer iscomparable with or exceeds k1, the drag on the particle

due to the electrophoretic retardation will increase. The

medium then is no more characterized by a uniform Z

and the bulk viscosity in Eq. (1) must be substituted by

some effective viscosity. However, the calculation of this

effective viscosity involves a number of parameters

characterizing the hairy layer [28], which are often not

experimentally accessible. Formally, the roughness of

the particle surface may be accounted for by assuming a

thicker stagnant layer that results in the outward shift of

the shear surface and a consequent reduction of z-poten-

tial [29]. In that case, a possibility of anomalous surface

conductance [22, 23] should be taken into account.

The hairy layer can also be composed of polyelectro-

lytes. The typical examples are biological cells and mem-

brane vesicles, or colloids with adsorbed, grafted, or

naturaly occuring layers of charged polymers on their sur-

faces. Such particles may be described as having a rela-

tively hard core covered with a polyelectrolyte layer

permeable to both solvent (water) molecules and small

electrolyte ions and are sometimes called soft particles

[30]. Their important feature is that the surface charges


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cannot be considered as being located at a thin interface

but must be viewed rather as being smeared out over a

substantial region adjacent to the particle core. The fric-

tion imposed by polyelectrolyte chains on the electro-

osmotic flow in this region also has to be taken into

account. A theoretical description of soft particle electro-

kinetics was pioneered by Jones [31] and Levine et al. [32]

They have considered the case of a thin EDL (compared

to the particle size) and low z-potential (25 mV) [31, 32].

Snabre et al. [33] have analyzed the case of an arbitrary z

(to model electrokinetic behavior of erythrocytes under

conditions of low ionic strength). In most reports on the

electrokinetics of soft particles, a numerical computer-

based modeling of particle electrophoretic behavior is

employed (e.g., [3235]). An analytical expression, though

of a quite complicated form, for the electrophoretic mobil-

ity of an arbitrary soft particle has also been obtained [36].

It should be noted that theoretical descriptions of soft

particle electrokinetics do not necessarily employ theconcept of a z-potential (e.g., [30, 36]).

3 CE of various microparticles

3.1 Polystyrene latex size standards

Polystyrene latex microspheres are widely used as model

systems for colloids. The advantage of polystyrene latices

(PSL) is that their sizes and geometrical shape are well

defined. Nowadays, the microspheres are commercially

available in a broad variety of sizes, each usually withina narrow size distribution. They possess a charge arising

from reaction with polymerization initiators or from

charged groups introduced upon polymerization (e.g.,

sulfate and carboxyl groups, respectively, for negatively

charged PSL). The surface electrical properties of PSL

are, however, still poorly understood. The reason is

thought to be a high degree of hydrophobicity of PSL

(charged groups cover as a rule only a few percent of

particle surface), which strongly influences adsorption

onto the particle surface. Moreover, most PSL possess a

charged polymer (hairy) layer on their surfaces (e.g.,

[26, 37, 38]).

A mixture of PSL of different sizes was the first particu-

late system used to demonstrate the utility of CZE for an

analytical separation of microparticles. Six latex micro-

spheres ranging in diameter from 39 to 683 nm were

electrophoresed in a bare fused-silica capillary of 50 mm

ID in buffers of low ionic strength [39]. The conclusion was

that the observed separation of PSL is a result of their

differential retardation due to particle-capillary wall inter-

actions since the observed mobilities did not correlate

well with the surface (titrated) charge or the charge-to-

mass ratio (ibid.). Ballou and co-workers [40, 41], investi-

gating the dependence on CZE operational parameters of

selectivity and efficiency of separation for microspheres

of known surface charge density (particles ranging from

30 nm to 1.16 mm in diameter, 75 mm ID capillaries) drew

the opposite conclusion, viz. that the separations were

electrophoretic in nature. However, it is thought that

neither a qualitative agreement between the electro-

phoretic mobility and the titrated surface charge nor the

absence of one allows for a firm conclusion regarding the

nature of PSL separation in CZE, since first, the electro-

phoretic migration is governed by the electrokinetic

charge and, second, the surface of polystyrene micro-

spheres is not smooth but has a rather complex structure.

Later on, the notion of the electrophoretic nature of PSL

separation in CZE was supported by observing a reason-

able agreement between electrophoretic mobilities of

negatively charged polystyrene microspheres, measured

by CZE and DLV [42]. It should be noted, however, that,when measured for the same microparticle concentration

and in the same electrophoretic buffer, absolute values

of mobility, derived by CZE were found to be about 10%

higher than those derived by DLV for all PSL (100300 nm

in diameter) studied. A similar discrepancy was observed

for mobilities of PSL particles (3001100 nm in diameter),

measured in the presence of a 100 mM sodium lauryl

sulfate [43]. Moreover, when the latex diameter was de-

creased to 100 nm, the absolute values of mobility, pro-

vided by CZE were about twice of those by DLV (ibid.).

The cause of such size-dependent discrepancy is

unclear but presumably arises from the inherent technical

differences between CZE and DLV. The authors [43] none-

theless speculate that the more pronounced polarization

of the EDL due to external electric fields, which are much

stronger in CZE than in DLV, may give rise to the observed

mobility increase. However, such polarization will result in

the relaxation effect that is known to impose an additional

drag and to consequently reduce particle mobility [18,

21]. The mobilities of positively charged PSL in CZE were

also found to differ substantially from those derived by

DLV, probably in view of the strong interaction of these

microspheres with negatively charged silica (10 mM phos-

phate buffer, pH 7.4) in bare 50 mm ID capillaries [42].

PSL of up to 1.1 mm in diameter produced well-defined

peaks evenin 25mm ID bare capillaries (though in thepres-

ence of detergents) [43]. Polystyrene microspheres of

up to 10 mm in diameter were demonstrated to electro-

phoretically migrate in 150 mm ID capillaries coated inter-

nally with linear polyacrylamide [44]. The UV detection

appears to be well suited to monitor particle migration,

presumably due to the scattering of UV light by the parti-

cles. Sample concentrations of 0.01 0.1% of solid latex

(30 nm to 1.16 mm diameter microspheres) allow for a


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detection with confidence at 254 nm in 75 mm ID capil-

laries [40]. Optical spectra of PSL suspensions were

shown to have a maximum in the short UV range for

50- to 200 nm diameter particles and a broad maximum

in the long UV range for 400- to 600 nm diameter parti-

cles [45]. Thus, the detection sensitivity for a particle of

a given size depends on wavelength. This fact was used

for on-line identification of peaks corresponding to PSL

of different sizes by monitoring separation at two wave-

lengths [43].

The fast, well-resolved analytical separations by CZE of

mixtures of both chemically different latices [40, 46] and

those with similar chemical composition but differing in

size were demonstrated [39, 43, 45, 47, 48]. It should be

noted that PSL of similar chemical composition exhibited

in general a size-dependent electrophoretic migration in

CZE. This phenomenon as well as a possible underlying

mechanism will be discussed in Section 4.2. The feasibil-ity to study physical interactions between different parti-

cles by CZE was shown by using acrylic and urethane

latices as model particles [46]. The interacting particles

were observed in form of a new peak appearing on the

electropherogram (ibid.).

3.2 Organic and inorganic colloidal particles

Surface electrical properties play a crucial role in colloid

stability and in such processes as filtration and electro-

filtration of colloidal particles, reversible osmosis, electro-

phoretic coating, etc. [49]. The electrophoretic mobility isan important experimental characteristic commonly used

to evaluate the electrical properties of a particle surface.

Besides, a compositional heterogeneity of colloidal sys-

tems is often of interest in research and industry. CZE

was utilized to characterize colloids by measuring their

electrophoretic mobilities [47, 5056] and by analytically

separating their mixtures [5458], for both organic [51,

56] and inorganic [47, 50, 5255, 57, 58] colloidal parti-

cles. As in the case of PSL, the UV detection presumably

based on light scattering by particles was used.

DLV is a technique commonly employed for measuring

colloid electrophoretic mobilities [49]. It appears of inter-est to compare the mobility data provided by both

methods. The electrophoretic mobilities of nanoparticles

of thorium phosphate (30 nm mean diameter) were meas-

ured by CZE and DLV [47]. Though the values obtained

were practically identical, the measurements were in fact

carried out in solutions differing about 2-fold in ionic

strength and thus no firm conclusion in this particular

case can be drawn regarding the consistency of mobility

values provided by the two methods. When the m values

obtained for iron oxide particles (70 nm diameter) by CZE

and DLV in the identical buffer were compared, the CZE

values were found to exceed those derived by DLV by

3050% (depending on the pH of 10 mM phosphate

buffer) [53]. The cause of this discrepancy is unclear.

One may speculate that particle interactions with the cap-

illary walls (CZE was carried out in a bare capillary) result

in some retention of iron oxides and thus in an apparent

increase of mobility. However, the peak shape is rather

symmetrical, thus not supporting such speculation (ibid.).

Perhaps, thermal or concentration effects are responsible

for the discrepancy. It is thought that special attention

must be paid to CZE operational parameters if a precise

value of m is of interest. A linear relation was reported [56]

between mobility of organic colloids (28350 nm in dia-

meter) in CZE and their values of z-potential derived by

DLV (the Zetamaster apparatus) in the identical buffer.

Unfortunately, theequation isnotprovided in [56], bywhich

the apparatus software calculated the reported values of

z. Hence, no conclusion on a degree of consistency ofmobilities measured by CZE and DLV may be drawn.

Sub-mm-sized polymeric particles of different composi-

tion, possessing carboxylate and borate moieties on their

surface were subjected to CZE in buffers supplemented

with salts of di- and trivalent metals [51]. Their mobitilies

were shown to decrease in the presence of the salts, as

should be expected assuming strong binding of the multi-

charged counterions onto the particle surface. CZE was

successfully used to study effects of pH, electrolyte con-

centration, and anion types on electrophoretic mobility

of metal oxide particles [5355]. It was shown [54] that,contrary to the classical electrophoretic theory, the mag-

nitude of m generally increased with increasing I for each

oxide studied. This finding was interpreted in terms of co-

ions (anion) binding into the Stern layer. The interpretation

agrees with a significant effect of anion type (phosphate,

carbonate, and borate anions) on the mobility of oxides

and, hence, on the selectivity of separation (ibid. ). CZE

carried out in indifferent electrolyte solutions (such as

those of sodium nitrate in these electrolyte systems,

there is no specific adsorption of ions to the surface of

the oxides) was utilized to measure isoelectric points of

metal oxides (ranging from 0.01 to 1 mm in diameter) and

to study effects of pH on the surface charge of the oxides[55]. Nearly baseline-resolved separations by CZE under

optimized conditions for various two- and three-compo-

nent mixtures of oxide particles were reported [57]. The

choice of the anion type and the proper ionic strength of

electrophoretic buffer were decisive (ibid. ). The electro-

phoretic migration in a size-dependent manner was

revealed in the CZE of colloidal gold nanoparticles [52]

and silica sols [58]. In the latter, the separation of fine silica

particles of different sizes was demonstrated. This parti-

cular kind of separation will be discussed in Section 4.2.


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3.3 Lipoprotein particles

Lipoprotein particles are involved in transport of water-

insoluble lipids in plasma ([59] and references therein).

These transport vehicles consist of an outer layer com-

posed of polar lipids and certain apolipoproteins and

are filled inside with neutral lipids. The lipoprotein parti-cles are heterogeneous in their lipid and apolipoprotein

composition and operationally devided into four major

fractions in accordance with their buoyant densities:

high (HDL, 813 nm diameter), low (LDL, 1825 nm dia-

meter), intermediate (IDL, 2535 nm diameter), and very

low (VLDL, 3080 nm diameter) density lipoproteins

(ibid.).

CZE was successfully applied to monitor Cu21catalyzed

lipid peroxidation in isolated LDL particles as well as

their modification by treatment with malondialdehyde

[60]. In both cases, particle surface is expected to be-come more negative. Indeed, the substantial increase

in particle mobility was observed. The use of 40 mM

methylglucamine-Tricine buffer (pH 9.0) was crucial for

obtaining a high separation efficiency, presumably due

to suppressing the interaction of LDL particles with

silica in bare capillaries (ibid.). CZE was also tested as

a method for profiling plasma lipoproteins, based on the

differences in surface properties of lipoprotein particles.

When subjected to CZE in a 50 mM Na-borate buffer

(pH 9.1), both intact HDL and LDL particles (detected

by UV absorbance) exhibited a single, though relatively

broad peak but showed no differences in mobility [61].

In the presence of 0.5 mM SDS in the buffer that did

not result in any appreciable lipoprotein delipidation,

HDL and LDL particles were found to substantially differ

in mobility [62]. However, under these conditions, VLDL

particles had practically the same mobility as LDL

(ibid. ). Perhaps, by varying composition, pH, and ionic

strength of buffers, one might reveal conditions where

all peaks would be near-baseline-resolved but the

authors do not report whether they explored such

option [61, 62]. It should be noted that capillary iso-

tachophoresis was demonstrated to be much more

successful than CZE in profiling plasma lipoproteins

[59, 6365].

3.4 Viruses

Electrophoresis of a tobacco mosaic virus in a thin capil-

lary (100 mm ID, 20 mM Tris-HCl buffer, pH 7.5, on-line

detection at 260 nm), demonstrated by Hjertn et al. in

1987 [66], was the first example of the application of CZE

to viruses. The migrating sample zone produced a rela-

tively narrow, well-defined peak. Later on, Grossman and

Soane [67] have used the tobacco mosaic virus as a

model particle to investigate the effect of orientation on

the electrophoretic mobility in CZE of rod-shaped poly-

ions (this effect will be discussed in Section 4.1). How-

ever, until recently, no attempts to utilize CZE for charac-

terizing viruses have been made.

Over the last few years, a series of papers dealing with

CZE of human rhinovirus (HRV) has been published by

Okun et al. [6870]. HRV, the main causative agent of

common cold infections, is an icosahedral particle of

approximately 30 nm in diameter. It consists of a RNA ge-

nome and a shell composed of capsid proteins (e.g., [71]).

When a HRV preparation was subjected to CZE in 50 mm

ID capillaries, the electropherograms showed one major

peak upon detection at both 205 and 254 nm. This peak

was unambiguously identified as originating from the

native virus (i) by heat denaturation of native virus sam-

ples, (ii) by enzymatic treatment of native and heat-dena-turated virus, (iii) by depletion of virus in the sample by a

specific monoclonal antibody [68]. The successful sep-

aration and identification of native virus and subviral B-

particles were also reported [69]. The latter are the end

product of structural rearrangements that HRVs undergo

during infection. In vitro, these rearrangements can be

induced by exposure to elevated temperature or to low

pH (ibid.). The electrophoretic buffer (100 mM borate buf-

fer, pH 8.3) was supplemented with a ternary mixture

of detergents at low concentrations tolerated by both

viruses and subviral particles. The detergents were found

to be important for preventing virus particle aggregationand adsorption to the capillary wall [69]. The utility of

CZE to differentiate between some HRV serotypes even

in crude preparations, based on differences in their

electrophoretic mobility in the borate buffer supplemen-

ted with 10 mM SDS, was demonstrated in [70]. In that

study, an infectivity assay was carried out on fractions

collected at the capillary outlet, thus allowing for the bio-

specific identification of CZE peaks.

A specific CZE-based approach, affinity capillary electro-

phoresis, was applied by the same group [7274] to study

binding of monoclonal antibodies and lipoprotein recep-

tor fragments to HRVs. The behavior typical for a ligandbinding was observed, viz., shifts in the virus mobility in

response to ligand (antibody or receptor fragment) con-

centrations, accompanied by a peak broadening at low

ratios of antibody (or receptor fragment) to HRV. The

quantitative analysis of this behavior has allowed one to

estimate the binding stoichiometry [7274] and the disso-

ciation constant [74] of the complexes. It appears that

CZE may be a usefull method both for a quality control

and quantification of virus production and for studying

ligand binding to virus particles.


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3.5 Liposomes and biological membrane

vesicles

Liposomes are quasispherical vesicles composed of

either one phospholipid bilayer (unilamellar vesicles) or

multiple concentric bilayers (multilamellar vesicles, MLV),

encapsulating aqueous space or spaces, respectively.They were increasingly being used in commercial pro-

ducts as well as in basic research (as a model for cellular

membrane behavior) and in studies on drug delivery (e.g.,

[75]). The electric properties of the liposome surface and

their characterization by different methods including elec-

trophoresis have been attracting a steady interest for dec-

ades (e.g., [76, 77] and references therein).

One advantage of liposomes is the relative ease with

which their surface can be manipulated by a choice of

lipid composition or by chemical modifications (e.g.,

[77]). The liposome size can also be controlled to some

extent. The average diameter commonly lies within the

sub-mm- and mm-size range, depending on the method

of preparation. By employing ultrasonication, vesicles of

2050 nm in diameter can be produced (small unilamellar

vesicles, SUV). Liposomes can be sized by extrusion, that

is, by passing them through membrane filters of defined

pore size. As a result, vesicles with a mean diameter lying

within the range of 100300 nm, though of a rather broad

size distribution, can be obtained [78]. Additionally, values

of the electrostatic potential on and near the surface of

the lipid bilayer were shown to be in good agreement

with those predicted by the relatively simple Gouy-Chap-

man-Stern model [76, 77, 79].

The CZE of liposomes were first reported by Tsukagoshi

et al. [80] and by Roberts et al. [81]. Liposomes consisting

of phosphatidylcholine (PC) or of its mixture with either

phosphatidylethanolamine (PE) or cholesterol (CH) were

subjected to CZE in bare 50 mm ID capillaries, using

10 mM carbonate buffer (pH 9.0) as an electrolyte solu-

tion [80, 82]. While SUV exhibited relatively narrow peaks,

MLV of the same composition exhibited broad ones and

mobilities the average absolute values of which appear to

lie well above that of SUV. This observation agrees with

the size-dependent electromigration of microparticles

(Section 4.2). It should be pointed out that PC is a zwitter-ionic lipid. Nonetheless, liposomes composed of PC can

have a non-zero z-potential and, thus, a non-zero m. The

cause of that fact is thought to be either adsorption of

electrolyte ions onto the liposome surface or the non-

Coulombic electrostatic potential due to a strong hydra-

tion of the lipid bilayer, or both [76, 77]. No strong inter-

actions of liposomes with capillary silica were observed

[80, 82]. By contrast, liposomes composed of a mixture

of PC, CH, and two charged constituents such as phos-

phatidic acid (negative charge) and a cationic membrane

dye exhitited a strong binding to silica when subjected

to CZE in 75 mm ID bare capillaries, using 9.5 mM phos-

phate buffer (pH 7.4) [81]. The mean diameter of the lipo-

somes, controlled by extrusion, was 350 nm. A capillary

conditioning by means of three preliminary injections of

the liposomes was found necessary to obtain reproduci-

ble peaks. This is the only report where such pronounced

liposome binding was seen. The liposomes had a nega-

tive electrophoretic mobility, likely due to uneven incor-

poration of the acid and the dye: a slight precipitation of

the dye was observed during liposome preparation (ibid.).

It is not clear why liposomes with a net negative charge

would interact so strongly with negatively charged silica.

Interestingly, dimyristoyl-PC, which was the major consti-

tuent (about 70%) of these liposomes, has a gel-to-fluid

transition temperature of 23.97C (e.g., [83]). This tempera-

ture may be close to that in the capillary during electro-

phoresis. In multicomponent bilayers, a lateral phase

separation is known to occur at the transition temperature[83]. One may speculate that the strong adsorption of the

liposomes, reported in [81] may be due to a nonuniform

redistribution of charged constituents upon a phase

separation.

Separation by CZE of liposomes similar in lipid composi-

tion but differing in size was demonstrated [84] (details

in Section 4.2). The utility of CZE to analyze SUV with

compositional heterogeneity (liposomes prepared by dis-

persing mixed lipid powders in aqueous solutions rather

than from lipids mixed in an organic solvent and dried

prior to the dispersing and, therefore, presumably homo-geneous in composition) was also shown [85]. CZE

appears to present a robust method for evaluating lipo-

some preparations in respect to heterogeneity in both

composition and size [8486]. Interestingly, extruded lipo-

somes prepared from PC, phosphatidylglycerol (PG),

and CH, mixed in an organic phase were found to exhibit

a sharp peak either adjacent to, or superimposed on,

the broad original liposome peak when subjected to CZE

in a 25 mM (or below this concentration) Tris-HCl buffer

(pH 8.0) [86]. No such peak was observed for the same

preparations in 50 mM buffer. The height and area of the

sharp peak were reported to depend on average lipo-

some size and surface charge density at a given bufferconcentration and the peak was shown to disappear if

the concentration of the sample buffer was elevated in

relation to the background electrolyte (ibid. ). The cause

of this phenomenon is questionable but an original lipo-

some compositional heterogeneity seems unlikely (unless

one is hypothetically induced by the electric field in the

capillary). Thus, in spite of the obvious utility of CZE for

studying liposome heterogeneity, caution in the inter-

pretation of experimental data should be exercised and

CZE conditions must be well examined.


8/16


CZE was employed in investigating the binding of amphi-

philic polymers to cationic and anionic liposomes [87] and

to evaluate relative variations in surface charge of lipo-

somes of different lipid compositions in various buffer

systems [88]. It was also suggested that CZE might be a

convenient method to assess the rigidity of a lipid bilayer

[89], assuming a reduced frictional hindrance experi-

enced by the elongated liposomes compared to that by

the spherical ones. Liposomes are known to undergo the

elongation and the subsequent orientation in electric

fields as shown by electric field-induced transient bi-

refringence (e.g., [90] and references therein), though

the extent of the deviation from the spherical shape

has not been estimated. In [89], a jump in liposome

mobility was indeed found at the temperature of the gel-

to-fluid (rigid-to-soft bilayer) transition. The softening

of membranes by incorporating CH or by using lipids with

shorter acryl tails did give rise to an increase of absolute

mobility (ibid. ). However, the interpretation of suchchanges in mobility is not straightforward. The increase

(about 50100%) in values of absolute mobility of MLV

upon the bilayer phase transition, measured by micro-

electrophoresis [91] was previously accounted for by a

change in surface charge density since a lipid head occu-

pies different areas in gelled or fluid states of the bilayer

[76, 77, 91]. The extruded liposomes are also assumed to

possess a substantial degree of elongation as a result of

the extrusion procedure ([92] and references therein).

Thus, one may not rule out that the observed differences

in mobility of the softened liposomes [89] are caused by

a shape variance of liposomes of different composition

due to extrusion (see also Section 4.2).

An original approach to measuring electrophoretic mobil-

ities of individual liposomes, using a home-made CE

setup has recently been published [93]. A diluted suspen-

sion of MLV (0.31.8 mm in size) was subjected to CE in

10 mM HEPES buffer (pH 7.5) supplemented with 250 mM

sucrose, in 50 mm ID capillaries, internally coated with a

neutral polymer to suppress the electroosmotic flow

(EOF). Individual liposomes labeled with fluorescein

encapsulated upon preparation were detected online by

means of a postcolumn laser-induced fluorescence (LIF)

detector. This approach allows one to similtaneouslydetermine mobility and apparent size (entrapped volume,

based on a signal intensity) of an individual liposome.

Interestingly, the MLV prepared by simply dispersing lipid

films (formed by drying of lipid mixtures in an organic

phase) in an aqueous phase exhibited apparently a sub-

stantial compositional heterogeneity as revealed by the

mobility distribution of MLV in a similar size range [93].

Aside from liposomes, this CE-LIF based approach was

applied to study mobility distributions of mitochondria

isolated from different cells by density gradient centrifu-

gation [94]. A mitochondrion-selective dye, 10-nonyl acri-

dine orange, was used for fluorescent labeling (ibid.). Use

of a continuous electrokinetic injection (50 mm ID capil-

laries, with an internal polymer coating to suppress EOF)

and a specific fluorescent dye, MitoTracker Green, which

accumulates in the mitochondrial inner membrane and

covalently binds to proteins, were shown to allow for

counting the organelles and for measuring protein abun-

dance in individual mitochondria [95]. CZE, using a com-

mercial instrumentation, was also employed in studying

microsomesmembrane vesicles formed from intracellu-

lar membranes upon mechanical disruption of the cell in

aqueous solutions [96]. Microsomes (100250 nm in size)

formed by membranes of the rough endoplasmic reticu-

lum were isolated from a homogenate of rat liver and sub-

jected to CZE in Tris-borate buffer (pH 8.3). While original

microsomes exhibited one major peak (detection at

280 nm), those incubated under conditions promoting

the delayed vesicle fusion were found to exhibit a quitecomplicated pattern (ibid.).

Several approaches to on-line detection of liposomes

were employed in CZE: the direct UV absorbance [85,

88, 89], the absorbance in the visual range when the ap-

propriate dye was incorporated into the membranes [81],

the laser-induced fluorescence with a fluorescent dye

either incorporated into the membrane [84], or entrapped

in the internal volume of the liposomes [84, 86, 93], and

the postcolumn chemiluminescence [80, 82, 97]. In the

latter case, eosin-containing liposomes were allowed to

migrate by EOF and mixed at the capillary tip with appro-

priate reagents to disrupt the vesicles and to induce thechemiluminescent signal. It should be noted that, if spe-

cies encapsulated into the liposome interior are detect-

able online by some of the detection techniques, two

peaks are typically recorded: one is due to liposomes

and another due to unencapsulated (free) species. The

change in the ratio of peak areas with time may easily

and rapidly provide useful information as to the perme-

ability of liposome membranes to these species as sug-

gested in [97].

3.6 Biological cells

The interest in analytical cell electrophoresis is based

on the expectation that electrostatic properties of the

cellular surface, manifest in cell electrophoretic mobility,

are related to cellular states of function and differentia-

tion, to the interaction with exogenic factors, or to cell

phenotypic identity. In many cases, this expectation is

fulfilled (e.g., [98, 99] and references therein). Micro-

electrophoresis and DLV have been the basic methods

employed for cell electrophoretic measurements for

decades (ibid. ). During the past decade, the interest in


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CZE as a new electrophoretic technique for analytical

cell separations and analyses has been continually

increasing.

Several years after the work of Hjertn et al. [66], showing

the feasibility of zone electrophoresis of bacterial cells in

thin fused-silica capillaries, Ebersole and McCormick

[100] have reported a CZE separation of a ternary mixture

of bacteria. CZE was performed in 100 mm ID bare capil-

laries in 4.5 mM TBE (Tris-borate-EDTA, pH 8.3) buffer,

employing UV (190 or 200 nm) detection. Four discrete

electrophoretic peaks were observed. Two of those peaks

were related to individual coccal species of a particular

bacterium and their chain assemblages, respectively.

The purity of fractions corresponding to near-baseline

separated peaks (the electroeluted fractions were manu-

ally collected at the tip of capillary) was found to be at

least 98% [100]. Most bacteria were shown to survive

the electrophoretic process and could be recovered in aviable form (ibid.). Later on, the separation of three bacte-

rial populations by CZE, using 250 mm ID, bare capillaries

of 1 to 3 m length and UV detection at 208 nm has been

demonstrated [101]. The peaks were broad compared to

those for most molecular species. This was attributed

to the inherent electrophoretic heterogeneity of bacterial

populations. As a result, a baseline-separation of different

bacterial populations can be realized only if there is a sub-

stantial difference in their mean mobilities. As a com-

promise between reproducibility, cell stability, and peak

width and height, the most appropriate conditions for

CZE of bacteria were a low ionic strength of electro-phoretic buffer (I = 0.0010.002), basic pH (710), and a

moderate field strength (slightly above 100 V/cm) [101].

A good agreement between bacterial electrophoretic

mobilities measured by CZE and microelectrophoresis

was demonstrated [15]. The lower detection limit was

found to be 108 cells/mL (or 2500 cells for the injected

volume of 25 nL) in 75 mm ID capillaries with detection at

214 nm. Interestingly, when electrophoresed in bare capil-

laries in a 10 mM MOPS buffer (pH 7.0), two out of three

bacterial populations studied exhibited two peaks. No

bimodal mobility distributions were revealed by DLV for

the same cell populations [15]. The authors proposed

that the cause of the observed discrepancy was thehigher resolving power of the capillary electrophoresis

(ibid. ). The bimodal mobility distribution was interpreted

in terms of interpopulational differences in the charge

density of the bacterial surface [102]. These differences

were postulated to be also responsible for the chromato-

graphically observed decrease in the affinity of the bac-

teria for glass beads with distance traveled through a

column (ibid.). The electrophoretic mobilities of a number

of bacterial cells, derived by CZE [103] were also used

to evaluate bacterial surface properties, based on the

concept of soft particles [30, 36, 104]. Mobilities were

measured using 50 mm ID bare capillaries and a 10 mM

phosphate buffer (pH 7.0 or 7.8), the ionic strength of

which was adjusted with NaCl to vary from 0.02 to 0.23.

The charge density per volume and a frictional character-

istic of the glycocalyx region were estimated by the best-

fitting of theoretical dependencies of m on I to the experi-

mental points. It should be noted that the mobilities were

reported to agree well with those measured by DLV for

the same set of bacteria [103, 104].

It appears that CZE is well suited for measuring electro-

phoretic mobilities of various bacteria. However, due to a

rather complex structure of their surfaces [71, 98], the

interpretation of the mobility in terms of surface electro-

statics is model-dependent. The mobility data for bacte-

rial cells, derived either by CZE or other techniques, may

be converted to z-potential values [71], using the Helm-

holtz-Smoluchowski equation (Eq. 1 with f(kR ) = 3/2)since bacterial size is relatively large and kR..1 for all

practical cases. It was realized early on that such conver-

sion is merely formal and, consequently in most cases of

electrophoresis of biological cells, results are expressed

in terms of electrophoretic mobilities, the measured

values [98, 99]. The more elaborate theories consider dif-

ferent dynamic processes, which may affect electro-

phoretic mobility and, hence, the derivation of z-values

from mobility data (e. g., the ion transfer within the stag-

nant layer, which is assumed to coincide with bacterial

walls, treated as a surface conductance [105]). Other

approaches do not employ the concept of z-potential at

all [103, 104].

The improved separation of a mixture of two bacterial

strains by CZE in a polymer solution (1% dextran, 10 kDa

molecular mass), compared to the separation in buffer

alone (10 mM phosphate buffer, pH 7.0) was demon-

strated [106]. When cells under the peaks were collected

at the tip of the capillary, the purity of the cell population

for each peak was found to be .98% and about 90% for

CZE in the buffered dextran solution and in buffer alone,

respectively. It was also shown that peak areas were well

correlated with the injected amount of cells, thus, allowing

for an easy quantification of mixed bacterial cultures by

CZE (ibid.).

A series of reports on CZE of bacterial and yeast cells,

carried out in the presence of low concentrations

(0.01250.025%) of a neutral polymer, polyethylene oxide

(PEO, molecular mass of 100 or 600 kDa), has recently

been published by Armstrong and co-authors [107111].

The striking feature of the reported separations was an

extremely high efficiency (of the order of 106 theoretical

plates/m), apparently due to the presence of a small

amount of PEO. The separations were performed in


10/16


100 mm ID uncoated capillaries, mostly in TBE buffer

(pH 8.3) diluted to a concentration of 0.5 mM. Either UV

[107110] or LIF [110, 111] detection was employed.

When electrophoresed bacteria were detected by ab-

sorbance at 214 nm, the peak area was found to correlate

with cell concentration in the sample, thus allowing for

on-line quantitation of bacteria in the sample [110]. The

feasibility of rapid identification and quantitation by CZE

of pathogenic bacteria in human urine or bacterial ingre-

dients in consumer products was demonstrated [108,

110]. It was shown that, using specific fluorescent dyes,

cell viability in single or mixed bacterial populations can

be determined on-line during a CZE run [110, 111]. The

CZE in the presence of PEO was also applied to separate

microbial aggregates [109] (Section 4.4).

Surprisingly, there were only two attempts to utilize CZE

for analysis of cells other than microbes. In an early

attempt, red blood cells (RBCs) were electrophoresedusing fluorinated ethylene-propylene copolymer (FEP)

tubing as a capillary [112]. A number of various electrolyte

solutions supplemented with glucose to maintain iso-

tonicity and with hydroxypropylmethylcellulose (0.1%)

to suppress EOF was tested as electrophoresis buffers.

Sample concentration was of the order of 105 cells/mL

and a well-defined peak was observed upon UV detection

at 206 nm when 0.45 mm ID FEP tubing was used. Yet, no

peak was observed in tubing of ID # 0.3 mm under the

same conditions [112]. Recently, human RBCs have

been separated by CZE at the single cell level, employing

a direct microscopic detection [113]. A single injection

of a very diluted blood was shown to produce tens of

peaks, all of them of almost equal height. Each peak was

attributed to a single RBC. This appoach allows one to

obtain a distribution of electrophoretic mobility in the

RBC population.

4 Special cases of microparticleelectrophoretic behavior

4.1 CZE of nonspherical particles: the effect

of the electric field-induced orientation

Compared to the electrophoresis of spherical particles,

which was well addressed both theoretically and experi-

mentally, there are not many systematic studies on

electrophoresis of ellipsoidal or rod-like particles (see,

e.g., [114] for relevant references). In the Smoluchowski

limit (kb..1, where b is the smallest characteristic

dimension of a nonspherical particle), the electrical driv-

ing force is entirely balanced by the electrophoretic retar-

dation and the electrophoretic mobility does not depend

on particle shape and orientation (e.g., [17]). Since the

ionic strength of electrolyte solutions employed in CZE

often lies within 0.0010.01 (k1 ranges approximately

from 10 to 3 nm, respectively), the condition of kb..k1

is not necessarily fulfilled for nonspherical microparticles,

and their mobilities, in principle, may be shape- and orien-

tation-dependent.

The effect of orientation of the rod-like particle on its

electrophoretic mobility was studied by the CZE of

tobacco mosaic virus (TMV) as a model particle [67].

TMV may be considered as a rigid rod of 15 nm in dia-

meter and approximately 340 nm in length. When sub-

jected to CZE in 50 mm ID capillaries, using 2 mM borate

(pH 8.4) buffer as an electrolyte solution, TMV was shown

to exhibit an about 8% increase in absolute mobility

upon increase of electric field strength (E ) from 100 to

400 Vcm1. No appreciable change in mobility was

observed for a latex microsphere (364 nm in diameter)

under identical conditions. The monotonic mobility in-crease with Ewas accounted for by a progressive aligning

of TMV particles in the direction of the electric field. It is

noteworthy that the calculation of the degree of orienta-

tion was carried out assuming the Stokes drag as the

only retarding force [67]. Yet, k1 in 2 mM borate used is of

the order of 10 nm. Comparing that to the characteristic

dimensions of TMV, it is clear that the electrophoretic

retardation force cannot be ignored for TMV particles

under the given conditions. Irrespective of the real inter-

play of retarding forces, the effect of orientation of even

such elongated particle as that of TMV on its electro-

phoretic mobility appears to be rather slight, at least in

the range of E commonly used in CZE. It is not as yetknown at the present time whether the selectivity of

separation in CZE of nonspherical rigid particles can be

regulated or improved to any practical degree by varying

the electric field strength.

4.2 Size-dependent electrophoretic migration

of microparticles

The size-dependent separation of microparticles was

first demonstrated by Hannig et al. [115]. Using an ana-

lytical free-flow electrophoresis apparatus, four PSL

(0.232.0 mm in diameter) species have been separated

according to size with a near-baseline resolution upon

decreasing the concentration of electrophoretic buffer

(Tris-borate, pH 8.6) from 0.3 M to 0.3 mM. The observed

separation was accounted for by the relaxation effect,

which can strongly enhance size-dependent mobility of

microparticles at low electrolyte concentration [115].

When microparticles of a similar or identical chemical

composition but differing in size (PSL [39, 45, 47, 49],

silica [56] and gold [51] nanoparticles, or liposomes [84,


11/16


85]) were subjected to CZE, they also exhibited a size-

dependent migration and particles in a mixture were in

most cases separated according to their size. Except for

the gold nanoparticles, all microparticles studied in CZE

exhibited a similar pattern: absolute mobility either did

not change or increased with increase in particle size.

The relaxation effect as the likely physical mechanism

underlying this phenonenon was suggested early on [58].

Recently, two studies aimed at pinning down the operat-

ing mechanism have been published [48, 84]. A set of

four latex microspheres of 138 to 381 nm in diameter

was electrophoresed in a 100 mm ID capillary coated

with linear polyacrylamide to suppress EOF, using a TBE

buffer (pH 8.3) of various dilutions (I ranged from 0.0003 to

0.005) [48]. A size-dependent migration was indeed found

to be an explicit function of kR. The selectivity of particle

separation exhibited a maximum as a function of ionic

strength. The relative increase in particle mobility was

about 1.5 over the kR-range of 7 to 87, compared to thatof 1.2, expected from Eq. (1) (ibid.). In a recent publication

of Vanifatova et al. [45], the increase in absolute mobility

of PSL particles was reported to be 1.7-fold over the

kR-range of 12100, while a 1.16-fold increase should

be expected according to the Henry formula within this

kR-range (Eq. 1 and [17]). Estimating the lower limit for

the z-potential as about 100 mV (based on mobility data

of [45, 48]) one should anticipate the relaxation effect to

appreciably contribute to the electrophoretic migration of

the particles. However, one complication with PSL is that

they appear to possess a hairy layer on the surface that

can to a large extent affect their electrokinetic behavior as

well. It is thought that the expansion of this layer upon

decreasing ionic strength of the electrophoretic buffer

has been responsible for the decrease in absolute mobil-

ity of the PSL microspheres with decreasing I [48].

Later on, extruded liposomes with defined mean size

were employed as model particles to elucidate the

mechanism of size-dependent migration [84]. The advan-

tages of liposomes for that purpose are that their surface

is smooth and that, changing the ratio of zwitterionic to

charged lipids, one may easily modify the surface charge

density for liposomes of similar size. Liposome prepara-

tions ranging in mean diameter from 120 to 500 nm and inPG/PC ratio from 1.6 to 0.14, were studied by CZE, using

100 mm ID capillaries coated with linear polyacrylamide

and Tris-HCl buffers (pH 8.0) of various dilution (I = 0.001

to 0.027). The electrophoretic behavior was found to be

qualitatively consistent with that expected if the relaxation

effect was the dominant operating mechanism giving rise

to a strong size-dependent migration of liposomes (e.g.,

the absolute mobility of highly charged liposomes in-

creased twice over a kR-range of approximately 725)

[84]. The separations by CZE of mixtures of liposome pre-

parations identical in composition but differing in mean

size were also demonstrated (ibid. ). It should be pointed

out that liposomes might undergo an elongation in the

electric fields employed in CZE [90], and that the extruded

liposomes are assumed to be intrinsically ellipsoidal [92].

To what extent, if at all, the deviation of liposomes from

sphericity can contribute to their electrophoretic be-

havior including the size-dependent separation is unclear.

Gold nanoparticles of 5 to 15 nm in diameter, subjected

to CZE in electrolyte solutions of low ionic strength (I =

0.00030.006, kR ranges from 0.3 to 3.7) demonstrated

a quite complicated pattern of size-dependent migration

[52]. At higher ionic strengths used (0.003 and 0.006), the

smaller particles migrated faster. Yet, the migration order

changes to the opposite at I = 0.0003 and 0.0006. For the

particle of a given size, mobility was found to decrease

with decreasing ionic strength. This is a clear indication

of ion binding onto the particle surface. The authors [52]have interpreted their findings by viewing the nano-

spheres as conducting, though at the same time realizing

that the gold particles with adsorbed ions are widely con-

sidered as nonconducting particles. They also assumed

that the particles possess an equal z-potential. However,

if a uniform surface charge density is assumed (which

is reasonable since the binding constant would hardly

depend on particle size) rather than a uniform z-potential,

the observed electrokinetic behavior may be accounted

for without invoking any particle conductance. In the

case of a small z-potential (# 25 mV), that is easily

demonstrated as follows: The surface density of the elec-

trokinetic charge, se, and z-potential are known to relateas (e.g., Eq. 2.3.37 of [17]):

z = Rse/e (11kR ) (2)

Equation (1) may be rewritten:

m = (2se/3Z) [f(kR)R/(11kR ) ] (3)

Note that for large kR values, z-potential (and therefore

particle mobility for species possessing a similar or iden-

tical surface charge density) becomes independent of

particle size. This is not true for small kR values where

mobility will be increasing with particle size. For instance,

upon changing kR from 0.3 to 0.8 (I = 0.0003), the ex-pected increase in absolute mobility is 1.9-fold, thus com-

pletely accounting for the 1.6-fold increase, which was

experimentally observed [52]. When a higher particle

charge and consequently z-potential is built up by the ion

binding at elevated ionic strengths, the relaxation effect

may come into play, overpowering the increases of m

with particle size. Note that within the particular kR-range

under study (1.3 to 3.7 at I = 0.006 [52]), the relaxation

of the ionic atmosphere is shown to decrease particle

mobility in absolute values with increasing kR [18, 21].


12/16


4.3 Source of peak width in CZE of

microparticles

The efficiency of microparticle separation in CZE has

been an object of study from early on. For latex micro-

spheres, the observed peaks were broad compared to

those for molecular species and, even under optimizedoperational conditions, the separation efficiency was

rather low (the number of theoretical plates was found to

vary within several hundreds to few thousands) [40, 41].

The intrinsic electrophoretic heterogeneity of particle

populations was suggested as a major source of peak

width in the CZE of microparticles [41], probably caused

by heterogeneity of their z-potential [55]. As was pointed

out by Petersen and Ballou [41], the substantial loss in

efficiency due to electrophoretic heterogeneity eases

restrictions on other sources of peak broadening, thus

allowing one to employ capillaries of larger innner dia-

meter, larger injection and detection volumes, and highervoltage.

At relatively high values of ionic strength (large kR ), the

electrophoretic heterogeneity is likely to arise from a

diversity of some characteristics of the particle surface

such as surface charge density and/or charge distribu-

tion, surface irregularities, etc. At lower ionic strengths

and/or high values of z-potential, the electrophoretic

mobility can become size-dependent and a contribution

of particle polydispersity (defined here as a heterogeneity

in size) to peak width may be expected. Indeed, an

increase of peak width for PSL microspheres was ob-

served upon decrease of ionic strength of the electropho-retic buffer [48]. However, it is not clear to what extent this

increase is caused by size heterogeneity of PSL and to

what by an outward expansion of the hairy layer, which

can introduce a new variance in the mobility distribution.

For liposomes of 100300 nm in mean diameter, the poly-

dispersity (expressed by a ratio of the standard deviation

of size distribution to the mean diameter for a given lipo-

some preparation) was found to correlate with the peak

width in CZE carried out in a buffer of low ionic strength

(25 mM Tris-HCl, pH 8.0) and at moderate E (200 Vcm1

or less) [86]. No correlation was found in 50 mM Tris-HCl

buffer. Thus, the liposome polydispersity appears to be

a dominant source of peak broadening at low ionic

strengths (ibid.).

In CZE of bacterial and yeast cells, observed peaks were

basically broad [101, 103, 106], except for a case of

electrophoresis in PEO solutions of low concentrations

[107111] that will be discussed in Section 4.5. The

peak broadening was suggested to result from an

electrophoretic heterogeneity of microorganisms, which

was attributed to intrapopulational variations in surface

properties. Interestingly, in a representative case of a

bacterium subjected to CZE in 1 mM, 2 mM, and 6 mM

TBE buffer, the peak width was found to increase (about

3-fold) with increasing buffer concentration [101]. This

peak broadening was assumed to manifest a progressive

destruction of cells upon increase of the ionic strength

(ibid.).

4.4 Spike-like peaks in CZE of microparticles

and particle aggregation

Particle aggregation in a suspension is well known to exist

and can effect the particle electrophoretic analysis. More-

over, in addition to pre-existing aggregates, a particle

aggregation can be brought about or speeded up by an

external electric field (e.g., [116] and references therein).

Both the pre-existing and the electrically driven aggrega-

tion are definitely not desirable in applications where

either separation of particles or their characterization ispursued. On the other hand, the detection of particle

aggregates by means of an electrophoretic technique

may be a useful approach if particle interactions are the

subject of study.

In the CZE of microparticles, multiple narrow peaks

(spikes), irreproducible in both height and migration times,

were often observed. Each spike was attributed to the

passing by a detector position of a large conglomerate

(or large aggregate) of particles. Spikes appeared on

electropherograms of particles of different types: inor-

ganic colloids [55, 57], liposomes [81], and microorgan-isms [103, 106]. For some sub-mm- and mm-sized oxide

particles, the aggregation may be promoted by decreas-

ing the buffer pH from 10 to 4 [55]. The aggregation

resulted in a decrease in area of the main smooth peak

on electropherograms and in an appearance of spikes

(ibid. ). The large aggregates observed as spikes were

found to comigrate with the main (smooth) peak

assumed to consist of single particles and small aggre-

gates [57]. Liposome aggregation (and probably partial

fusion) induced by incubation in an acidic buffer (pH,5)

also greatly increased the number of spikes observed on

electropherograms [81]. A noticible flocculation of lipo-

somes upon a 30 min incubation in such buffer was ob-served visually, accompanied by an increase in the lipo-

some mean diameter from 350 nm to 2.5 mm as meas-

ured by dynamic light scattering. Most spikes migrated

behind the main liposome peak in bare capillaries on

EOF and thus liposome aggregates appeared to possess

absolute mobilities higher than those of single liposomes

(ibid.). A similar behavior was found in CZE of microorgan-

isms: large cellular aggregates assumed to associate

with spikes exhibited in general higher absolute mobilities

compared to those of single cells migrating as a broad


13/16


peak [103, 106]. The longer the duration of sonication

of cell suspensions prior to CZE analysis was, the fewer

spike-like peaks appeared [106].

When microbial cells in the form of pre-existing multiple

aggregates were subjected to CZE in 0.5 mM TBE buffer

supplemented with 0.012% PEO (600 kDa molecularmass), several narrow spike-like peaks were observed

[109]. It should be noted that under these conditions,

even single cells migrate as a narrow zone, giving rise to

a spike-like peak [107111]. In a specially designed

experiment, the injections of pre-existing aggregates of

yeast cells, with a counted number of cells in the aggre-

gate, were carried out under microscopic control [109]. A

reproducible relationship was found between the mobility

and the aggregation number (the cluster size) under con-

ditions of the study: the absolute mobility linearly

increased with lnN, where N is the number of cells in the

aggregate (ibid.).

It is not clear why large aggregates would exhibit mobility

higher in absolute value than that of its constituent parti-

cles. In the case of cells, these are large at the scale of

kR, even in electrolyte solutions of low concentrations

used in CZE. Besides, the binary aggregates are known

to migrate with a velocity intermediate between the velo-

cities of particles they are composed of ([116] and refer-

ences therein). Except for the specific case of CZE of cell

aggregates in a dilute PEO solution, the migration time

of large aggregates was irreproducible. Roberts et al.

[81] suggested that the aggregates are formed during a

CZE run. Nonetheless, this still would not explain thehigher mobilities. Perhaps, larger aggregates are more

susceptible to interactions with capillary walls than small

ones. If so, each aggregate can randomly be retarded in

the capillary which, in the capillaries with EOF, would give

rise to apparent mobilities higher in absolute values than

those of single particles and small aggregates.

4.5 CZE of microparticles in solutions of neutralpolymers

The behavior of polymers in solution at a solid-liquid inter-

face was an area of large interest in colloidal science fordecades since polymers are known to both stabilize and

destabilize colloidal dispersions (e.g., [117, 118]). Electro-

phoresis, which to a large extent is a surface-related

phenomenon can be used for studying polymer behavior

at the interface (e.g., [119, 120]). On the other hand, one

may speculate that the electrophoretic migration of a

particle could be modified by its interaction with polymers

in a polymer surface-specific interaction manner, thus

introducing a new dimension into the electrophoretic

separation and analysis of particles. For instance, a differ-

ence in mobility of RBCs from Alzheimer patients and

from normal individuals was found in a buffered dextran

solution but not in the buffer alone [121, 122].

In CZE, solutions of water-soluble neutral polymers were

employed in an attempt to bring about or to enhance the

particle separation in a size-dependent manner, analo-

gous to that in gel electrophoresis. Using polystyrene

size standards as model particles, the particle retardation

(expressed by the retardation coefficient, KR, the slope of

a linear dependence of logm vs. polymer concentration)

in solutions of linear polyacrylamide (PA) was found to

be a biphasic function of particle size range [96, 123]. Up

to approximately 30 nm in diameter, KR increased line-

arly with particle diameter and was field strength-inde-

pendent. Above that size, the retardation became field

strength-dependent and its dependence on size of sul-

fated PSL became complex [123]. Moreover, the relative

decrease in mobility was considerably less than thatexpected based on the solution bulk viscosity and inverse

relation between m and Z (Eq. 1) [124, 125]. This behavior

was hypothetically interpreted in terms of (i) a layer with a

depleted polymer concentration (the depletion layer), pro-

gressively forming at the particle surface-polymer solu-

tion interface and (ii) a shear-dependent deformation of

the polymer network by the migrating particle [123125].

For carboxyl-modified latex (CML)microspheres, a steady

decrease of KR was observed with increasing diameter

within the range of 100500 nm (ibid.). Interestingly, CML

particles possess an extensive hairy layer, according to

the manufacturer (Interfacial Dynamics, Portland, OR,USA; Product Guide 7, 1994, p. 8). The interrelation of

the hairy and depletion layers and their mutual effect

on particle electrophoretic mobility are challenging theo-

retical problems ([120] and references therein). In a prac-

tical respect, it was shown that the selectivity of separa-

tion for CML microspheres (100450 nm in diameter)

might be increased by the presence of PA in the buffer

[126]. For 110- and 280-nm diameter microspheres which

comigrate in the absence of polymer, a baseline separa-

tion was demonstrated in 0.7% solution of PA (7001000

kDa molecular mass). However, peak broadening was

generally observed with increasing polymer concentra-

tion [124, 126]. Consequently, a maximum in resolution(when such exists) would correspond to a particular poly-

mer concentration for a given pair of particles (ibid.).

The remarkable effect of PEO on electrophoretic behavior

of microbial cells in CZE was demonstrated by Armstrong

and co-authors [107111] and further exploited for highly

efficient separations. In the presence of 0.01250.025%

PEO in the electrophoretic buffer, cells were found to

migrate within narrow zones giving rise to spike-like

peaks. As a representative case, a complete separation


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by CZE of the mixture of four microorganisms was pre-

sented [107]. The resolution was rather insignificantly

affected by the relative cell retardation induced by the

polymer (change in separation selectivity), compared to

that by peak sharpening. The mechanism of this phenom-

enon is unclear. PEO is known to induce aggregation of

various biological species but not at such low concen-

trations. Nevertheless, one may speculate that cells of

different type would partition upon aggregation induced

by PEO (and electric field?), forming specific cell type-

dependent clusters migrating at some intermediate velo-

city and thus reducing the intrapopulational diversity. The

other possibility is that binding of PEO to cells can some-

how eliminate the intrapopulational diversity in cell sur-

face properties, making the apparent z-potential uni-

form. Earlier, about 10-fold reduction in peak width was

reported for PSL microspheres electrophoresed in dilute

solutions of PA (0.20.25%, 18 000 kDa molecular mass)

[124]. The cause of this reduction was also in question.Irrespectively of the mechanisms responsible for the

zone sharpening in dilute polymer solutions, one may

conclude that the use of polymers for improving particle

separations has a potential that is not as yet explored to

any substantial degree at the present time.

5 Concluding remarks

CZE appears to be an effective and experimentally simple

way to analytically separate and characterize particles.

A great variety of on- and postcolumn approaches toparticle detection feasible in CZE is thought to make this

technique exceptionally suited for electrophoretic analy-

sis of particles in many applications. CZE has been

shown to be applicable to particulate species within a

wide size range, up to about 10 mm in diameter. The

migration of particles in CZE is electrophoretic in nature

though in particular cases a substantial contribution of

particle-capillary wall interactions to the apparent mo-

bility cannot be ruled out. Separations of particles are

mostly based on differences in their electrophoretic mo-

bilities. The effectiveness of those separations depends

on particular conditions of buffer, ionic strength, the pre-

sence of detergents or specific polymeric media. In mostapplications, the optimal conditions at this time must still

be found empirically. Except for the case of (quasi)spheri-

cal particles with smooth surface, the electrokinetic

theories can appear to provide only a very general

guidance in predicting and optimizing those conditions.

CZE is well suited for measuring particle mobility but

special attention must be paid to operational parameters

if a precise value of mobility is of interest. As in general, an

interpretation of particle mobility in CZE is model-de-

pendent to the degree that assumptions are made regard-

ing the surface, relative size, and geometry of the particle.

A particular difficulty in this respect arises from the fact

that real particle surfaces in most cases are not smooth

as assumed in the more simplistic models but rather

hairy and heterogeneous to unknown degrees and in

unknown ways.

Received March 1, 2002

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