Capillary isotachophoresis from the student point of view – images and the reality

J. Sep. Sci. 2006, 29, 2705 –2715 J. Petr et al. 2705

Jan PetrV�tezslav MaierJana Hor�kov�Juraj �evc�kZdenek Str�nsky

Department of AnalyticalChemistry, Palacky University,Tr�da Svobody 8, Olomouc, CzechRepublic

Review Article

Capillary isotachophoresis from the student point ofview – images and the reality*

A review of some fundamental aspects of ITP from the student point of view, imagi-nations of some basic facts and laws, use of ITP, and the recent trends are presented.The results of theoretical computations of ITP separation processes are added forcomparison of imaginations with the exact mathematical description.

Keywords: Boundary / Discontinuous electrolyte systems / Isotachophoresis / Kohlrausch regulat-ing function / Simulation /

Received: June 28, 2006; revised: July 28, 2006; accepted: August 4, 2006

DOI 10.1002/jssc.200600249

1 IntroductionCapillary ITP belongs to the group of electromigrationseparation techniques which have been playing a veryimportant role in solving various scientific problems, e. g.in genomics, proteomics, or metabolomics. ITP repre-sents a very impressive and powerful technique, whichhas formed principles for the development of other tech-niques, mainly CZE.

The 65th birthday of Professor Dr. Petr Bocek, one of thebards in the field of ITP (and one of the leading personsin following CZE and stacking techniques as well), hasled us to review some fundamental aspects of ITP forcomparison of students' images with the reality andrecent trends.

2 Theory

2.1 Discontinuous electrolyte systems

Discontinuous system in CE could be defined as a systemin which electric field strength is not the same in thewhole capillary [1]. The vector of electric field strength isaffected by concentrations and mobilities of electrolytecomponents [2] or by an additional electric field [3]. It isnecessary to understand that preseparation discontinu-ities (static) which are formed, e. g., by loading different

electrolytes into the capillary, are rearranged during themigration process and they cause dynamic changeswhich strongly influence the system behavior and sep-aration mechanism [2]. Every static discontinuity is chan-ged into the dynamic one after applying an electric fieldbecause of the movement of electrolytes’ charged species[4].

The simplest formation of the static discontinuity is pre-sent when a sample dissolved in diluted electrolyte isintroduced [5]. It causes a failure of the homogenous elec-tric field and forms a specific system behavior – forma-tion of system zones [4, 6–12]. This disturbance can alsobe applied to the stacking of analytes due to the differ-ences in analytes velocity in the sample zone and in theelectrolyte zone [13–16]. The use of partial filling tech-niques (PFT) presents another approach to discontinuouselectrolyte systems, where the heterogeneous electricfield is formed because of loading additives' plugs intocontinuous electrolyte [17, 18]. An interesting way of PFTis asymmetrical capillary filling published by Tesarov� etal. [19]. The idea of using different types of discontinuoussystems for influencing the separation and for analytestacking was introduced by the group of Bocek in the1980s [20–29]. Nowadays, a junction of different electro-lytes is used in many methods, e. g. dynamic pH junctionpreconcentration methods [30–34], moving chemicalreaction boundary techniques [35, 36], but the main well-known technique is the ITP where the junction of electro-lytes with different mobilities is present [37–39].

2.2 ITP separation

In ITP, a sample is introduced between two working elec-trolytes; the leading electrolyte (LE) is chosen to have thehighest mobility in the system whereas the second elec-trolyte, the terminating one (TE), is chosen to have the

Correspondence: Dr. Jan Petr, Department of Analytical Chem-istry, Palacky University, Tr�da Svobody 8, CZ-771 46 Olomouc,Czech Republic.E-mail: [email protected]: +420-585-634-433.

Abbreviations: KRF, Kohlrausch regulating function; LE, lead-ing electrolyte; TE, terminating electrolyte

*This paper is dedicated to the 65th birthday of Professor PetrBocek

i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

2706 J. Petr et al. J. Sep. Sci. 2006, 29, 2705 –2715

lowest mobility in the system. The discontinuity in theelectric field strength is purposely formed. For the expla-nation of the separation run we should propose a modelexample of separation of two ions (Fig. 1). EOF isneglected. For two sample ions A and B we can write the

relation: lLE A lA A lB A lTE. The zones start moving afterthe application of constant electric current. The sameelectric field strength in the sample zone causes differentspeed of ions A and B due to their mobilities. Ions Amigrate faster than ions B (Figs. 1b and c). After separa-tion, the steady state is formed and all zones migratewith the same speed (“iso” = same, “tacho” = speed)which is given by the speed of the leading coion (Fig. 1d)[37]. The same situation is shown on mathematicallysimulated concentration profiles during the ITP (Fig. 2): amixture of anions is loaded between LE and TE (Fig. 2a);after applying current, the zones are rearranged untilsharp boundaries are formed (Fig. 2d).

Electric field strength E is given by the equation:

E ¼ UL¼ I

Sj¼ j

j¼ j

FXn

i¼1

cizili

ð1Þ

where U is voltage, L is conductor (capillary) length, I iselectric current, S is conductor (capillary) area, j is cur-rent density, and j is specific conductivity given by theproduct of Faraday constant and the sum of product ofconcentration ci, charge zi, and mobility li of ith species.

The use of constant electric current in ITP (while in CZEnot) is visible from this equation. If we used constant vol-tage, spreading of the electric field strength would be thesame along the whole capillary. When we use constant


Figure 1. The run of ITP with presentation of electric fieldstrength E in particular coordinates x.

Figure 2. Mathematical simulations of ITP run in Simul 5.0. (a) t = 0 s, (b) t = 1.6 s, (c) t = 2.4 s, and (d) t = 4.0 s. LE: chloride(pK = –2.0, l = –79.1610 – 9 m2V – 1s – 1), TE: propionic acid (pK = 4.87, l = –37.1610 – 9 m2V – 1s – 1), counterion: b-alanine(pK = 3.43, l = 38.5610 – 9 m2V – 1s – 1), samples: A: acetate (pK = 4.76, l = –42.4610 – 9 m2V – 1s – 1), B: glycolate (pK = 3.89, l =–42.4610 – 9 m2V – 1s – 1), C: formiate (pK = 3.75, l = –56.6610 – 9 m2V – 1s – 1).

J. Sep. Sci. 2006, 29, 2705 –2715 ITP – images and reality 2707

electric current (current density), electric field strengthis indirectly proportional to the conductivity of eachzone and it is dependent on the zones' compositions:

j ¼ ELEjLE ¼ ETEjTE ¼ EAjA ¼ EBjB ¼ . . . ¼ Eiji ð2ÞThanks to the constant current density, discrete andsharp zones are created.

The same velocity in the steady state can be explained bya macroscopic image which starts with the fact that theleading ions have the highest mobility in the system.Ions of the sample and TE must migrate with the samevelocity as the leading ions because if it was not true, thedisruption of electric circuit would occur (an empty gapbetween electrolytes would be formed). Because of theconstant zone velocity in the steady state, we can derive asimple equation describing the relation between zones'mobilities:

v ¼ lLEELE ¼ lTEETE ¼ lAEA ¼ lBEB ¼ . . . ¼ liEi ð3ÞFrom this equation it is possible to explain a very impor-tant effect in ITP, the selfsharpening effect. The electro-lytes’ system is chosen to fill the mobility relation lLE A lA

A lB A lTE. From Eq. (3) proportional differences in electricfield strength in the zones are visible (ELE a EA a EB a ETE).When an ion leaves its zone due to diffusion, it getsslower (or faster) due to the higher (or lower) electricfield strength in the adjoining zone [37, 38]. Equations(2) and (3) are also applied in the basic calculations forthe determination of mobilities and dissociation con-stants by ITP [40–51].

2.3 Boundaries

The junction of two electrolytes forms two types ofboundaries: moving boundary and nonmoving boundary(Fig. 3). The movement of a boundary can be character-ized by a parameter W which is the electrolyte volume Vrelated to the capillary length L which the boundarypasses due to the charge of 1 C:

W2!1 ¼VQ¼ SL

Q¼ SL

It¼ S

Iv2!1 ð4Þ

where Q is the electric charge, S the capillary area, I theelectric current, t the time of moving, and v2!1 is thespeed of moving boundary.

The formation of a new zone “2” can be described by theions' migration into the zone nin and out of the zone nout.The number of ions (for ions A) can be derived from sim-ple equations:

nin ¼ VcA;2 ¼ SLcA;2 ¼ StvA;2cA;2 ¼ StI

Sj2lA;2cA;2

¼ tI

j2lA;2cA;2ð5Þ

where cA,2 is the concentration of ions A in zone 2, vA,2 isthe speed of ions A in zone 2, j2 is the conductivity ofzone 2 and lA,2 is the mobility of ions A in zone 2.

The molar balance between “in” and “out” migration ofA ions gives the moving boundary equation:

cA;2lA;2

j2�

cA;1lA;1

j1¼ W2!1 cA;2 � cA;1Þ

�ð6Þ

In Fig. 3, zone “1” does not contain ions B (cB,1 = 0), there-fore the migration of boundary is determined by themobility of ions which migrate out of the zone:

lB;2

j2¼ W2!1 ¼

SI

v2!1 ð7Þ

This equation clearly illustrates that the velocity ofboundary movement can be derived from the mobility ofthe component which migrates out of the zone.

In the place where the moving boundary was before theapplication of the potential, the nonmoving boundaryremains (boundary 3/2, Fig. 3). The parameter W for thisboundary is zero (L = 0); therefore with prerequisition ofconstant mobilities we can derive from Eq. (4) the equa-tion for nonmoving boundary:

cA;2

j2¼ cA;3

j3ð8Þ

It shows that the ratio of A ions concentrations in frontof the boundary and behind the boundary is dependenton the ratio of conductivities of the adjoining zones [37].

2.4 Dynamics of ITP, Kohlrausch regulatingfunction

In general, the description of dynamics of moving bound-aries and ITP is difficult. It starts with the mass transport(mass flux J), which can be formulated as the number oftransported species ni during a certain time t with unitedcolumn area S:

Ji ¼ni

tS¼ nixi

tSxi¼ nixi

Vt¼ civi ð9Þ


Figure 3. Formation of boundaries and their movement.


where xi is the length of capillary which ith substancepasses, V is the appropriate volume, ci is concentration ofith electrolyte component, and vi is the speed of the ithsubstance.

The mass flux (or mass current density) in electromigra-tion techniques is given by the sum of the flux due to thediffusion and the flux due to the electromigration. Theflux due to the diffusion (thanks to the chemical poten-tial gradient) is equal to –DiCci, which can be rewrittenfor simplification by using just x-axis (gradient along thecapillary):

Ji;dif ¼ �Didci

dxð10Þ

where Di is diffusion coefficient and dci=dx is the concen-tration gradient.

The electromigration contribution is the sum of the ionsmigration, the electroosmotic migration, and the hydro-dynamic flow of electrolyte:

Ji;mig ¼ ci vi þ vEOF þ vhf Þð ð11Þ

After neglecting the hydrodynamic flow, Eq. (11) can berewritten with respect to the formed electric potentialgradient @b=@x (only for x-axis), or for simplification justby using the electric field strength in a particular coordi-nate [37, 38, 52, 53] to:

Ji;mig ¼ sgnðziÞcili@b

@xþ civEOF ¼ sgnðziÞciliEþ civEOF ð12Þ

Concentration changes of the electrolyte componentsare given by motion of the mass and by mass changesdue to chemical reactions Ri. They are described by conti-

nuity equations, which are the basic ones in the theoryof electrophoresis; for x-axis only:

@ci

@t¼ � @

@xJi þ Ri ð13Þ

The general solution of Eq. (13) does not exist [52]. Sol-ving is possible after neglecting chemical reactions(Ri = 0); because in ITP the EOF is fully suppressed, it ispossible to postulate vEOF = 0. Then from Eqs. (9)– (13) thetransport equations can be derived:


Figure 4. Scheme of the KRF.

Figure 5. Model of moving boundary with the KRF. (a)t = 0 s, (b) t = 2.0 s, and (c) t = 12.0 s. Modeled by Simul 4.0program; A – : benzoate (pK = 4.20, l = –33.6610 – 9 m2V – 1

s – 1), B – : acetate (pK = 4.76, l = –42.4610 – 9 m2V – 1s – 1), R+:histidinium (pK = 6.04, l = 29.6610 – 9 m2V – 1s – 1); currentdensity 300 A/m2.


@ci

@t¼ Di

@2ci

@x2� j

@

@xcili

j

� �ð14Þ

These equations are solvable by numerical methods or bylinearization [37, 52]. Numerical methods, introduced inthe early 1980s [54–56], are used by various simulationprograms [57–60]; linearization was examined forunderstanding of the separations and for describing thesystem zones phenomena [61–64].

One of the solutions of transport equations for uni-univa-lent electrolytes is the well-known Kohlrausch regulat-ing function (KRF or x-function) [37, 38]. For understand-ing this function we can establish an image (Fig. 4): thesample is introduced between the LE and TE; EOF isneglected. In the definite capillary place with volume V,there is a certain number of species ni which are able tocarry the charge:

ni ¼ ciVi ¼ ciSxi ð15ÞFor preservation of the number of species, which cancarry the charge during the migration (constant currentdensity) adaptation of ions’ concentration must occur(Figs. 4b and c). The adaptation is dependent on the spe-cies ability to carry the charge that is given by the ratio oftheir concentration and mobility. This is described bythe x-function (the KRF), which is defined as a functiondependent on a position in the capillary:

xðxÞ ¼Xn

i¼1

cizi

lið16Þ

This can be described simply: in the capillary place x0,where was a certain zone, is due to the electromigrationa new zone. The composition of this novel zone is notrandom but it is defined by x-function that the value ofx-function in the point x0 is constant for the whole anal-ysis. If differences in electrolyte composition are formedbefore the separation (also in x(x)), the next electromigra-tion leads to the adjusting of concentration of migratingspecies with holding the x(x) values. A simulated modelexample of concentration profiles on the formed bound-ary with the calculation of the x-function values areshown in Fig. 5. The adjustment of A – concentration dueto proportional changes, thanks to migration of B – zone,is clearly shown (Figs. 5b, c) [37, 38, 52].

3 Practical aspects

3.1 Method development

The development of every setup in electromigration tech-niques is based on the same principles – to know themost information about a sample and a matrix [1, 2, 65].It is necessary to optimize the ionization of analytes, eva-luation of using additives etc. [1, 2, 37, 38, 66]. Very impor-tant and not trivial is the optimization of buffering sys-tem. This is because freely migrating H3O+ and OH – ionscan cause the canceling of ITP due to their highest mobi-lities in the system (llim

H3Oþ = 362.5610 – 9 m2V – 1s – 1;llim

H3Oþ = –205.5610 – 9 m2V – 1s – 1 [67]). This must be solvedjust by the counterions. With good buffering system, it ispossible that H3O+ or OH – have apparently the lowest


Table 1. Recommended electrolyte systems

Counterion pH TE

Analysis of anionsLE: HCl + counterion + EOF suppressing additive (e. g. polyvinylalcohol (PVA), hydroxymethylcellulose (HMC))

b-alanine 3.1–4.1 Caproic acid, propionic acide-amino-n-caproic acid (EACA) 4.1–5.1 Glutamic acid, propionic acid, MESCreatinin 4.5–5.5 MES, NaHCO3

Histidine 5.5–6.5 MES, HEPESImidazole 6.6–7.6 MES, VeronalTris 7.6–8.6 Glycine + Ba(OH)2, b-alanine + Ba(OH)2Ammediol 8.3–9.3 b-alanine + Ba(OH)2, GABA + Ba(OH)2Ethanolamine 9.0–10.0 EACA + Ba(OH)2

Analysis of cationsLE: KOH + counterion

Acetic acid 4.2–5.2 Acetic acid, GABAPropionic acid 4.4–5.4 Acetic acid, creatinineMES 5.7–6.7 Histidine, imidazoleVeronal 6.9–7.9 Imidazole, triethylendiamineAsparagine 8.3–9.3 Triethanolamine, trisGlycine 9.1–10.1 Triethanolamine, NH3

b-Alanine 9.8–10.8 NH3, ethanolamine


mobility and they can play the role of the terminator[26–29, 37, 68]. The behavior of water protolytic ions asthe terminating ions (correct terminator) and the distri-bution diagrams of ITP zones existence are still consid-ered to be fundamental information in the choice of ITPstacking process [26–29, 69, 70]. The use of Hirokawa’stable is still important in the case of method develop-ment; the table gives isotachophoretic indices: simulatedratios of potential gradients of the sample zone to that ofthe leading zone, the time-based zone lengths, the effec-tive charges, and the pH of the sample zones, in the pHrange 3–10, which can be used for the assessment ofseparability, quantitation, etc. [71]. Bocek et al. [37] withrespect to the previous articles presented a set of recom-mended electrolyte systems for the first analysis by ITP(see Table 1). The presence of a high mobility and a highconcentration ion in the sample can be used to design anITP separation, too. In present days, a lot of publishedworks use sodium or chloride ions in biological samplesas the leading coions which lead to the great separationwithout background effects from high salt-matrices [72–75]. Also the influence of the presence of complexationagent in separation system was described in the early1980s [76–79] and it was used later by many authors forimproving separations [80–84].

3.2 ITP preconcentration

Adjustment of a sample concentration to the leadingconcentration can be used for the precontration of ana-lytes. At this time, three main approaches based on thesame principle are presented in the literature: simple ITP[85–87], coupled column techniques (ITP/CZE, ITP/ITP)[88–92], and transient ITP [93–96]. Coupled column tech-niques are used in normal capillary modes as well as chipmodes. The first column is for preconcentration (ITPmechanism) and the second one is for separation (CZE orITP mechanism) [92]. The modern miniaturized chip var-iant can use the coupling of more than two channels andcan be optimized for a unique problem [90]. TransientITP is a one-column alternative where the isotachophore-tic process takes a certain time and then the most com-mon zone electrophoresis is proceeded. This can be rea-lized in three ways: T-S-T, L-S-L, BGE-S-BGE (Fig. 6). The firstone (T-S-T) is characterized by using TE as the BGE, the sec-ond one (L-S-L) by using LE as the BGE, and the third (BGE-S-BGE) by using another BGE than LE and TE. TransientITP process runs until the fast hydrogen or hydroxideions move through the zones and stop the ITP (and startCZE).

3.3 Recent progress in ITP

The state of ITP in many fields was reviewed by manyauthors [97–118]; the last comprehensive review from


Table 2. Recent applications and methods

Inorganic analysis References

Alkaline metals [120 –125]Heavy metal cations [72, 126 –129]Transition metal cations [130, 131]Rare-earth cations [132 –136]As, Se, Pd speciation [137 –141]Halide anions [73, 142 –147]Iodine speciation [148 –153]Sulfite, sulfate [88, 92, 154]Phosphates [87, 155 –157]Nitrate and nitrite [158 –163]

Organic analysis

Phenolic compounds [91, 164 –168]Amino acids [112, 169 –173]Hydroxy acids [128, 174 –181]Food preservatives [182, 183]Acetaldehyde, acrolein [184]Ethylglucuronide [185]Porphyrines [186]CTAB [187]Vitamins [188]Panthothenic acid [189]Heparin oligosaccharides [190, 191]Natural neurotoxins [192, 193]Aromatic and aliphatic sulfonates [194, 195]Pharmaceuticals [85, 86, 196 –205]

Biochemical applications

Lipids [206 –208]Lipoproteins [209 –214]Proteins [89, 111, 215 –223]DNA fragments [224 –226]Cell lysate [227]Glycoforms of erythropoietin [228]Glutathione [229, 230]

Miscellaneous

Whole-capillary transversescanning detection

[231]

Coupling of ITP with NMR [190, 192, 196, 232–237]Coupling of ITP with TOF-MS [238]Improvements in chip design [239 –241]Calibration calculations [242]Pseudo-ITP [243 –245]Visualization of the stacking [246]Measuring transport numbers [247, 248]Choice of ITP spacers [249]Combination of ITP with pH junc-tion focusing

[250]

Interaction studies [233, 251]Stacking by electrokinetic injection [252, 253]Tandem ITP/CZE strategy [254]Effect of nonionic surfactant [255]Million-fold stacking by tITP [256]Simulations for dynamic studies [257]Migration across membranes [258]ITP in free-flow [259]tITP in nonaqueous CE [260]


Gebauer and Bocek [119] was published in 2002. Fromthis date, over a hundred novel applications and meth-ods were published; a short survey of these works is givenin Table 2.

4 Conclusions and perspectivesCapillary ITP is a very important analytical separationtechnique which gives us ideal outcomes with sharpzones. This advantage with possibility of column cou-pling, lab-on-chip technology, and transient methodol-ogy will lead to the restart of the plentiful use of ITP forsolving many analytical problems. The next decade willkeep on the concentration possibility of ITP; probably itwill be online combined with other concentration tech-niques (like it was published for dynamic pH junction[250]); the usefulness of ITP as a concentrator will bedemonstrated on coupling of CE techniques with massspectrometric detection [254]. The progress of miniaturi-zation will lead to the optimization and use of multidi-mensional separation plates – chips – where the ITP con-centration step will be included [101, 261]. With respectto the recent trends, we could summarize that ITP willalso be a necessary analytical technique in the comingdecades.

The financial support from the Research Project MSM6198959216is gratefully acknowledged. The authors also acknowledge IvanaPetrov� for the corrections of English grammar.

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Capillary isotachophoresis from the student point of view – images and the reality