518 Electrophoresis 2003, 24, 518–535 Review Jozef L. Beckers1 Petr Boček2 1 Eindhoven University of Technology, Dempartment of Chemistry (SPO), Eindhoven, The Netherlands 2 Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Brno, Czech Republic The preparation of background electrolytes in capillary zone electrophoresis: Golden rules and pitfalls In this article the methodology of the design of suitable background electrolytes (BGEs) in capillary zone electrophoresis (CZE) is described. The principal aspects of the role of a BGE in CZE are discussed with respect to an appropiate migration behavior of analytes, including the transport of the electric current, the buffering of pH, the Joule heat, the electro-endosmotic flow (EOF) and the principal migration and detection modes. The impact of the composition of the BGE upon migration and detection is discussed. It is shown that the total concentration of the BGE is a principal factor and the adjustment of migrating analyte zones according to the Kohlrausch regulating function (KRF) is the principal effect in most of the sample stacking techniques. The number of co-ions and their properties are of key importance for peak shapes of the analyte peaks and for the existence of system zones. The detection of UV-transparent analytes may advanteously be done in the indirect UV mode, by using UV-absorbing co-ions, however, both peaks and dips may be expected in the UV trace in case of multiple co-ionic BGEs. Properties of BGEs can be predicted applying mathematical models and it is shown that with SystCharts, predictions can be given concerning the existence of system zones, detection modes and the peak shapes of analytes for a given BGE. Practical examples of methodological considerations are given in the design of suitable BGEs for four principal combinations of migration and detection modes. The properties of the BGEs selected are exemplified with experimental results. Golden rules are summarized for the preparation of suitable BGEs in CZE. Keywords: Background electrolyte / Capillary zone electrophoresis / Review Contents 1 2 2.1 2.2 3 3.1 3.2 3.3 3.4 3.5 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . Material and methods . . . . . . . . . . . . . . . . . . . Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The role of a BGE . . . . . . . . . . . . . . . . . . . . . . . Transport of the electric current . . . . . . . . . . . pH of the BGE . . . . . . . . . . . . . . . . . . . . . . . . . Joule heating . . . . . . . . . . . . . . . . . . . . . . . . . . The electroosmotic flow . . . . . . . . . . . . . . . . . Principal migration and detection modes . . . . 4 519 519 519 519 519 519 520 521 521 522 Correspondence: Prof. Petr Boček, Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Veveři 97, CZ-61142 Brno, Czech Republic E-mail: email@example.com Fax: +420-5-41212113 Abbreviations: KRF, Kohlrausch regulating function; SP, system peak; SZ, system zone; TBA, tetrabutylammonium; ZE, zone electrophoresis 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4.1 4.2 4.3 4.4 4.5 4.6 5 5.1 5.2 6 6.1 6.2 6.3 EL 5256 Impact of the composition of a BGE upon migration and detection . . . . . . . . . . . . . . . . . . The concentration of a BGE and stacking phenomena . . . . . . . . . . . . . . . . . . . . . . . . . . . Electrodispersion and peak shape . . . . . . . . . System zones in CZE . . . . . . . . . . . . . . . . . . . . The use of multiple co-ionic BGEs . . . . . . . . . The use of weak multivalent ionic species in BGEs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Peaks and dips . . . . . . . . . . . . . . . . . . . . . . . . . Prediction of properties of BGEs . . . . . . . . . . General considerations . . . . . . . . . . . . . . . . . . SystCharts . . . . . . . . . . . . . . . . . . . . . . . . . . . . Examples of BGEs . . . . . . . . . . . . . . . . . . . . . . BGE for analysis of cations with direct UV detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BGE for analysis of cations with indirect UV detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BGE for analysis of anions with direct UV detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523 523 524 525 526 526 526 527 527 528 530 530 531 532 0173-0835/03/0302–518 $17.501.50/0 Electrophoresis 2003, 24, 518–535 7 8 BGE for analysis of anions with indirect UV detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . 533 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534 1 Introduction If electrophoresis is applied in order to solve an analytical problem, important questions are “which electrophoretic technique should be used” and “which electrolyte system is needed”. Especially the latter question is still one of the key problems in the application of electrophoresis in analytical chemistry since its role is very complex. In this article we will discuss basic principles of the choice of background electrolytes (BGEs) in CZE. The BGE should primarily provide an appropiate migration of the analytes, i.e., a quantitative transport of their concrete zones along the detector in a reasonable time. Moreover, extraneous peak broadening and other migration interferences should not be present. When the primary requirements are fulfilled, and, a suitable BGE is designed which provides a suitable migration and detection behavior, the second question is how to reach the required selectivity in order to separate the analytes. The selectivity is not dealt with in this contribution since it is beyond the scope and size of this article and it may be found elsewhere . This article deals with the primary task and describes how to design a BGE for a good migration in CZE. At first, we describe the main purposes of the BGE, the requirements which should be met and the principal CZE modes which can be applied. After that, the main consequences of the composition of the BGE will be discussed from peak and dips in the indirect UV mode to the problems of system zones (SZs). Then, we give a short overview of mathematical models, which can be used to get insight in the properties of BGEs and the SystChart has been selected as an actual example. Finally, we describe the principles how to choose a BGE for an application and also some pitfalls are shown. All the above items are supplemented with theoretical and experimental results. For the experiments we used simple standard solutions to show clearly the actual individual effects and to give a telling description and explanation of them. and detection Ld of 50.0 cm. The wavelength of the UV detector was set at 214 nm. All experiments were carried out at an operating temperature of 257C and applying 10 kV, unless otherwise stated. Sample introduction was performed applying 5 s pressure injections at 3.46103 Pa (0.5 psi). Data analysis was performed using laboratorywritten data analysis program CAESAR. 2.2 Chemicals All chemicals were of analytical-reagent grade. Deionized water was used for the preparation of all buffer and sample solutions. 3 The role of a BGE The main purposes of a BGE are to provide the transport of electric current and the separation of the analytes. But if an electric current passes through a BGE, some additional phenomena occur, such as the electroosmotic flow (EOF) and the Joule heat, which play an important role in electrophoretic processes. In this section we will discuss the most important aspects of using a BGE. 3.1 Transport of the electric current In zone electrophoresis (ZE) the whole system is filled with a BGE, the purpose of which is to transport the electric current if a voltage is applied over the system and to provide an electric field strength E, according to modified Ohm’s law: Es = j 2.1 Instrumentation For all CZE experiments the P/ACE System 5000 HPCE (Beckman-Coulter, Fullerton, CA, USA) was used, applying a Beckman eCAP capillary tubing (75 mm ID) with a total length Lt of 57.0 cm and a distance between injection (1) where s refers to the specific conductivity (1/Om) and j to the current density (A/m2). In order that an electric current can pass through a solution if a voltage is applied over the system, the solution must contain charged particles and, i.e., that the BGE must consist of at least a cation and an anion. The specific conductivity s of a BGE, consisting of strong electrolytes, is given as: sF 2 Materials and methods 519 n X c i jz i mi j (2) i1 In this equation, F represents Faraday’s constant (96 490 C/mol) and ci (mol/m3), zi and mi (m2/Vs) refer to the concentrations, valences and mobilities of all ionic forms present in the BGE. Mobilities are defined as the migration velocity per unit of electric field strength and can be calculated from the ionic conductance or from electrophoretic data (see Section 3.4). In ZE the whole system CE and CEC 6.4 BGEs in CZE 520 J. L. Beckers and P. Boček Electrophoresis 2003, 24, 518–535 is filled with the same BGE, in order to keep parameters, such as the conductivity, the E, temperature and pH, as constant as possible. In that case any sample component S migrates through the BGE system with a constant electrophoretic velocity, vS, defined by: vs = meff,sE (3) where meff,S represents the effective mobility of a sample component related to a nonmoving solvent (see Section 3.4). The ionic species of the BGE with like charge as the sample components S are called co-ions, and the other ionic species are counterions. 3.2 pH of the BGE The regulation of the pH is an important purpose of the BGE in order to keep the migration velocity of weak electrolyte components and the velocity of the EOF constant. In that way a stable and reproducible migration behavior of the sample components can be obtained. The pH is of key importance for all electromigration phenomena in systems with weak electrolytes, i.e., weak bases, weak acids and ampholytes. The concepts of ionic mobility and effective mobility are used to describe the migration behavior, where the ionic mobility relates to the electromigration of a fully ionized substance and the effective mobility relates to the electromigration of a partially ionized substance. The effective mobility of a weak monovalent acid HA is given by : meff,A = aAmA (4) where aA represents the degree of dissociation of HA and mA is the ionic mobility of A2. The effective mobility of a weak acid base B is given by meff,B = (1 2 aA)mB (5) where aB represents the degree of dissociation of BH1 and mB is the ionic mobility of BH1. Obviously, for weak anionic and cationic ionic species the effective mobilities strongly depend on their pK values related to the pH of the BGE. As an example, in Fig. 1 the calculated relationships between effective mobilities and pH are given for (solid lines) a component BH1 (pK = 4.0, mB = 3061029 m2/Vs), a component HA (pK = 5.0, mA = 23061029 m2/Vs), and for an amphiprotic component (dashed line) containing both an acid group and a base group with ionic mobilities of 23061029 m2/Vs and 3061029 m2/Vs, respectively, and pK values of 3 and 10. It can be seen that the weak base (B), is practically fully protonated (BH1) at pH , pKB 2 2 and its effective mobility equals then the ionic mobility. For pH . pHB 1 2, the base (B) is practically neutral (nonprotonated) and its effective mobility is zero. A weak acid (HA) has an effec- Figure 1. Calculated relationships between effective mobility and pH for (solid lines) a component BH1 (pK = 4.0, mB = 3061029 m2/Vs), a component HA (pK = 5.0, mA = 23061029 m2/Vs) and for an amphiprotic component (dashed line) containing an acid group and a base group with ionic mobilities of 23061029 m2/Vs and 3061029 m2/Vs, respectively, and pK values of 3 and 10. tive mobility of zero for pH , pKA 2 2 and is practically fully ionized at a pH . pHA 1 2. Ampholytes behave like a weak base at low pH and like a weak acid at high pH. From the practical point of view, the larger the effective mobilities of substances, the faster their electrophoretic migration, and, in a suitable experimental arrangement, the shorter the time of analysis. However, it should be emphasized, that even substances with zero effective mobilities may move in the capillary due to the EOF, (see Section 3.4), and this EOF is also strongly dependent on the pH of the BGE used. Further in analogy with the curves in Fig. 1, it is obvious that bases with high pK values and acids with low pK values are practically fully ionized at moderate pH values and for these components the pH of the BGE is not so important for their migration behavior. Concerning the separation of substances, the pH of the BGE is of key importance, too, since the prerequisite for the separation of a couple of substances is that these substances differ sufficiently in their effective mobilities. This topic is not, however, dealt with here since its complexity is beyond the scope of this article, and, it can be found elsewhere . Concerning the pH of a BGE, one aspect should be mentioned here. The BGE must have some buffering capacity at the selected pH. It means that one of the ionic species of the BGE should have a buffering capacity, thus it must have a pK value near the desired pH. It is of no importance whether this is a co-ion or a counterion. It should be mentioned here that there are also procedures to reach fast migration, good separation and/or concentration by stacking where the BGE is not kept constant but is subjected to dynamic changes . Electrophoresis 2003, 24, 518–535 BGEs in CZE Here, usually a pH change, having the character of a step, gradient, or pulse is induced at one side of the capillary and this change migrates along the capillary due to the applied electric field strength [4–10]. Thus, it brings dynamic changes in the effective mobilities of sample components as well as of the BGE constituents as it meets them during its migration. In this way very selective separations and other migration effects may be reached. The formation of pH changes at one side of a capillary is easily done either by hydrodynamic modification of the composition of an electrolyte in an appropriate electrode chamber , or by a sophisticated automated electromigration three-pole-column system [5, 6]. In this system, the capillary is equipped with two electrode chambers at one end, with electrodes of the same polarity, and, by controlling the individual electric currents passing through these individual electrode chambers filled with mutually different electrolytes, one can control the fluxes of electromigrating ionic species (including H1 or OH2) passing through the capillary. It seems that these principles attracted recently new attention . 3.3 Joule heating If a voltage is applied across the capillary and an electric current passes through the capillary, the temperature of the solution in the capillary increases due to the dissipation of electric energy, i.e., production of the Joule heat. The Joule heat is conducted through the capillary wall into the thermostating medium (circulating air or liquid coolant) surrounding the capillary. It has already been shown that radial temperature profiles in the solution are negligible in comparison to the temperature drop across the capillary wall . Further it has been shown that the elevation of the mean temperature nT = T2To, where T is the mean temperature of the solution and To is the temperature of the coolant, is proportional to the Joule heat produced per unit length of the capillary used [13–15], nT = QEI (6) where I represents the electric current (A) and the quotient of proportionality Q depends first of all upon the coolant used (air or liquid) and for present commercial instruments with liquid cooling and the usual capillaries of 50 up to 100 mm ID and 360 mm OD, it amounts 1–2 Km/W. Obviously, the power used in the capillary should not exceed 0.5 W/m if the elevation of the temperature should be under 1 K . It is a general rule that the speed of the analysis is proportional to E (V/m), and, the Joule heat is proportional to EI (W/m). Hence, low-conductive BGEs, which bring low currents, are recommended. If 521 the specific conductance of the BGE is too high, the heat production will increase, and increasing temperature of the system brings extra peak broadening and a decrease in resolution. Recently, on-line monitoring of the temperature in CZE capillaries  reported on values for nT up to 20 K. 3.4 The electroosmotic flow If a BGE is in contact with the inner capillary wall, an electrical double layer is formed. If a voltage is applied across the separation capillary the BGE solution migrates with a velocity directly proportional to the electric field strength E, the so-called EOF, and analogous to the ionic mobility, the mobility of the EOF, mEOF, can be defined as: mEOF vEOF E (7) The mEOF mainly depends on pH, composition and concentration of the BGE and for silica capillaries the mEOF has a positive value, i.e., the EOF shows a cathodic migration. The consequence is that the apparent migration velocity of a species S, vapp, S, which is related to the capillary as a nonmoving frame, can be expressed as: vapp; S V Ld mapp; S E meff; S mEOF tS Lt (8) where Ld is the distance between injection and detection, Lt is the total length of the capillary, V is the voltage applied across the capillary and tS is the migration time of an ionic species S. From Eqs. (7) and (8) it can be concluded that the mEOF can be calculated from the migration time of the EOF by: mEOF Ld Lt VtEOF (9) and the effective mobility of an ionic species can be calculated from its migration time tS by: meff; S mapp; S mEOF Ld Lt VtS Ld Lt VtEOF (10) A benefit of the EOF in bare fused-silica capillaries is, that it causes movement of cations, neutral components, anions (if the absolute values of their anionic mobilities are smaller than that of the mEOF) and even a micellar phase into the same cathodic direction, and, thus, brings them to pass the detector. Obviously, for cathodic EOF, mEOF . 0, and the EOF accelerates cations. Anions with umeffu , mEOF are driven cathodic and may be detected in cathodic runs. Anions with umeffu . mEOF do not reach the detector at all. Because the mEOF affects the migration behavior of analytes, it is often important to control the EOF, i.e., we want to change or even to reverse the 522 J. L. Beckers and P. Boček mEOF. For that purpose surfactants can be added to the BGE. To show their effect on the EOF, the measured relationships between mEOF and the pH of BGEs are given in Fig. 2A, for (a) BGEs prepared from a solution of 0.01 M Tris adjusted to the desired pH by adding acetic acid (dashed line for the addition of formic acid) and (b) the same BGEs under the addition of 561024 M CTAB. For the determination of the mEOF, a 5 s pressure injection of a solution of mesithyloxide (30 mL/100 mL) was applied. It can be seen that the values of the mEOF vary between ca. 20–10061029 m2/Vs. The mEOF is strongly increasing at high pH owing to the twofold effect of increasing pH Electrophoresis 2003, 24, 518–535 and decreasing ionic strength. For BGEs at very high pH of 11–12, mEOF values up to 110–12061029 m2/Vs can be reached. For BGEs with the addition of CTAB, the mEOF varies between ca. 250 to 210061029 m2/Vs. In order to show the influence of the concentration of surfactants on the mEOF, we give in Fig. 2B the relationship between mEOF and the concentration of the surfactant CTAB in several BGEs at various pHs. Besides the concentration of the surfactant, also the composition of the BGE is important . It should be mentioned here that the reversal of the EOF by adding CTAB may often be accompanied by the existence of a micellar phase due to its low critical micellar concentration (cmc). 3.5 Principal migration and detection modes Figure 2. (A) Relationship between measured mobilities of the EOF and the pH of (a) diverse BGEs, (b) the same BGEs with the addition of 561024 M CTAB. (B) Measured relationships between mobilities of the EOF vs. the concentrations CTAB in BGEs consisting of (d) 0.01 M Tris adjusted to a pH of 4.5 by adding acetic acid; (s) 0.02 M MES adjusted to pH 6 by adding 4 M NaOH; (n) 0.01 M Tris adjusted to pH 7.5 by adding acetic acid; and (m) 0.005 M (NH4)2HPO4 adjusted to pH 9.0 by adding 1 M NaOH. Applied Voltage, 15 kV. There are two principal migration and two principal detection modes in ZE. Often this is not clearly indicated in published papers and, instead, practical names are used which may be misleading. In order to avoid misunderstandings, we will characterize the above modes. With respect to the polarity of a voltage across the capillary, two modes can be recognized. The cathodic mode: In this mode the anode is placed at the inlet and the cathode at the outlet. Because generally the mEOF is into the direction of the cathode, cations can be determined and even anions if the absolute values of their mobilities are smaller than that of the mEOF. This mode is often denoted by the terms: normal polarity, direct polarity or positive (1) voltage applied. The anodic mode: In this mode the cathode is placed at the inlet and the anode at the outlet. Because in fused-silica capillaries, the EOF mostly migrates into the direction of the cathode, only such anionic species can reach the detector, which have the absolute values of their mobilities higher than mEOF. In order to detect anions with a lower mobility, the EOF has to be suppressed or even reversed. This can be done by coating the inner surface of the capillary with an electroosmotically nonactive layer or by a dynamic coating, i.e., by adding surfactants to the BGE, such as FC 127 or CTAB. The anodic mode is, in laboratory practice, often called: reversed mode or negative-voltage mode. Sometimes, independently of the polarity used and types of analytes, the terms upstream and downstream are used and they denote the migration of analytes against or along the direction of the EOF. With respect to the detection we can distinguish between the direct and indirect mode. In the direct mode, the detector monitors directly the sample zones and the BGE serves as a blank. In the indirect mode, the detector monitors a suitable ionic component of the BGE and substitution of this component by ionic analytes. It should be mentioned that according to KRF, neutral components of the BGE are not replaced by ionic analytes. In case of Electrophoresis 2003, 24, 518–535 a UV detector, the indirect UV mode is applied for the detection of UV-transparent ions and a BGE is used with UV-absorbing properties. Generally, a co-ion is chosen, which is UV-absorbing . 4 Impact of the composition of a BGE upon migration and detection In Section 3 the main purposes of a BGE are discussed and the effect of the composition of the BGE on migration behavior is described. Sometimes, some other phenomena can occur and they can facilitate or disturb the separation process. In this section some phenomena are described, affecting the electrophoretic separation process. 4.1 The concentration of a BGE and stacking phenomena The concentration of the BGE can play an important part in the resolution of a separation in electrophoresis, dependent on several aspects. If a constant voltage is applied over the capillary, the electric current will increase for higher concentrations of the BGE and this can cause extra peak broadening due to a strong increase in the Joule heat. Further a higher concentration of the BGE gives a lower mobility of the EOF and by this, fast and medium fast anions cannot be determined in the cathodic mode. The mobilities of analytes are affected by the concentration of the BGE according to the Debye-HückelOnsager effect  and sometimes this can improve the separation, especially for multivalent analytes. Often there is a strong attraction force between positively charged analytes and the negatively charged wall of the silica capillary resulting in broad tailing sample zones. A high concentration of the BGE can decrease this adhesive behavior of the sample ions by a competitive adsorption of the co-ions of the BGE, thus increasing the resolution. The counterions of the BGE can show attraction forces with analytes and this complexation can be stronger at a high concentration of the BGE. There are, however, phenomena which are of paramount importance for migration behavior of analytes and are directly dependent on the concentration of the BGE. These phenomena are stemming from the existence of KRF. The KRF  (for recent paper on KRF see ) prescribes that the numerical value o (molVs/m5), for fully ionized monovalent ionic constituents, defined as: X ci (11) o jmi j i is locally invariant in time, i.e., once given for any position along the capillary, prior to applying the voltage, it does not change with time during the electrophoretic run. In BGEs in CZE 523 our case, the initial value of o is given by the BGE used to fill the capillary prior to the electrophoretic run. During the electrophoretic process, sample ions migrate into the separation capillary, substitute the co-ions of the BGE and their concentrations adjust to the o value of the BGE electrolyte. This process is called adjustment of the concentrations and belongs to the well-known characteristics of ITP [22–25]. In case that an analyte is introduced at low concentration as a broad sample pulse, the adjustment of its concentration to that of the original BGE manifests itself as the accumulation of this analyte into a narrow band. This process is called stacking . In many cases, the term stacking is supplemented with specific adjectives, e.g., field-enhanced sample stacking (FESS), field-amplified sample stacking (FASS) or large-volume sample stacking (LVSS) . However, the principle is always the same, the adjustment of concentrations and it is an intrinsic property of CE. Sometimes, one of the sample ionic components is present at a very high concentration, through which a complete separation is impossible. Higher concentrations of the BGE can give a better stacking process, resulting in a higher resolution. In order to elucidate this stacking mechanism, we give in Fig. 3 the simulated cationic concentration profiles at four different times, for electrophoresis of a sample of 0.001 M ephedrine. The model BGE consists of 0.01 M of Figure 3. Simulated cationic concentration profiles for an electrophoretic process of a sample of 0.001 M ephedrine (Eph, solid line) and 0.01 M of a model co-ion (dashed line). (a) Original concentration profile prior to the electrophoretic process. (b) After a short time the ephedrine zone is partially replaced. (c) Ephedrine is just totally stacked. (d) Ephedrine migrates in a zone electrophoretic way. After the ephedrine zone has passed, the BGE zone is restored. 524 J. L. Beckers and P. Boček Electrophoresis 2003, 24, 518–535 a co-ion and counterion with equal assumed mobilites of 2661029 m2/Vs. We applied all ionic mobilities at infinite dilution in the simulation. In Fig. 3a the initial concentration profiles are given. The original ephedrine sample solution is 0.001 M (solid line) and the concentration of the co-ion is 0.01 M (dashed line). After a short time (see Fig. 3b), the front of the sample cation passes through the original stationary boundary (between original sample zone and BGE), and, the sample is concentrated owing to the adaptation to the o value of the BGE. Simultaneously, at the rear boundary, the sample is partially replaced by the co-ion. Its concentration is adjusted to the value of o, valid in the original sample zone, according to KRF and is a little bit lower than that of ephedrine, because its mobility is lower than that of ephedrine (see Table 1). In Fig. 3c all amount of ephedrine is just stacked, and, although the destacking has already started, the concentrated ephedrine is practically 0.01 M in its zone according to the KRF. It should be stressed here that the sample is stacked only after it has passed its own original stationary front boundary, i.e., the sample stack is formed only at a site which was previously occupied by the BGE. In Fig. 3d we see that behind the migrating ephedrine zone, the concentration of the co-ion of the BGE has been restored at its original value of 0.01 M. There are four main consequences of KRF viz., (i) if a sample solution is introduced with an o value different from that of Table 1. List of cations and anions often used for the preparation of BGEs m6109 (m2/Vs) pK UV-absorbing Creatinine Ephedrine Histamine 37.2 28.3 65 32 4.828 10.1 9.95 6.0 1 1 1 1 Histidine Imidazole 29.6 50.4 6.04 7.0 1 1 Lithium Potassium Sodium Tris 40.1 76.2 51.9 29.5 14 14 14 8.08 2 2 2 2 Benzoic acid Phthalic acid 233.6 228.1 252.9 4.203 2.81 5.1 1 1 1 Phenyl acetic acid Sulfanilic acid Sulphamic acid 231.7 233.7 250.3 4.405 3.227 22 1 1 1 Acetic acid Formic acid Hydrochloric acid Phosphoric acid 242.4 256.6 279.1 234.6 261.4 271.5 4.756 3.752 22 2.16 7.21 12.67 2 2 2 2 2 the BGE this initial o value remains valid at the site of injection during the whole experiment; (ii) the site where the original sample was injected, shows always stationary boundaries; (iii) diluted sample zones will be concentrated and stacked at the beginning of this electromigration, i.e., when they cross their original front boundary; and (iv) when the co-ion of the BGE starts to penetrate into the sample zone, the destacking process starts, i.e., the sample zone is going to migrate in a zone electrophoretic way. Of course, when an EOF is present, the injection site migrates with the velocity of the EOF through the capillary and this is the reason why the EOF can be observed as a peak or as a dip in the baseline. If the initial o value of the sample is lower than that of the BGE a “waterdip” is visible, if this o value is higher a peak can be observed. Further it can be seen that the boundaries marginating the sample zones are moving and can be electrophoretically stabilised or not stabilized . The migrating ephedrine zone is a moving zone with a stabilized rear boundary whereas the front boundary has a nonstabilized fronting character, although the peak shape is practically symmetrical because of the small difference between the mobilities of ephedrine and co-ion. 4.2 Electrodispersion and peak shape Zone boundaries can be stabilized or nonstabilized  and generally this behavior is regulated by Ohm’s law. If an ionic species leaves its zone in the direction of its migration, and it reaches a site with higher electrical conductivity, i.e., a locally lower E, its velocity decreases and it will be overtaken by its own zone. In that case, we have a stabilized zone boundary. If it reaches a site with higher E, its velocity increases and a nonstabilized diffuse boundary is the result. In that case, the electrophoretic migration contributes to the broadening of the migrating zone and this is called electrodispersion. The electrodispersive effect is generally larger than peak broadening by diffusion. The degree of electrodispersion is related to the differences between the mobilities of sample ion and co-ion. For a large difference between these mobilities broad, asymmetric tri-angled peaks are obtained. As a rule of thumb, it can be stated that a peak is fronting, if the mobility of an analyte is higher than that of the co-ion, otherwise it is tailing. If the sample solution contains only a few closely spaced analytes of interest, it is possible to optimize the BGE by mobility matching. Here, a co-ion is chosen with a mobility close to that of the analytes of interest. In that case, the electrodispersion is minimum and the peaks are sharp and symmetric . The above rules are fully correct for strong electrolyte systems. For weak electrolyte systems the rules are for orientation only, since a more sophisticated approach is then needed, see Section 5. Electrophoresis 2003, 24, 518–535 4.3 System zones in CZE In CZE there are often zones, which migrate through an electrophoretic system and which do not contain any of the analytes and contain only ionic species of the BGE. These zones migrate with a specific mobility through the electrophoretic system and are often called “eigen zones”, “system zones” or “system peaks” [27– 32]. SZs play a very important role in the electrophoretic process, and, their behavior is frequently decisive for the failure or success of the analysis. The composition of these zones differ from the composition of the BGE with regard to the concentrations of the ionic species and the pH. One of the properties of SZs is that they interact with sample zones with a mobility close to that of the SZ and they deform the peak shape of the sample components and disturb the separation process by a strong peak broadening. Important questions are (i) when do SZs exist, (ii) what are their mobilities and (iii) what is their effect on the electrophoretic process. At this very moment we know that SZs are formed (1) in BGEs containing two co-ions, (2) in BGEs at very high or very low pH, whereby OH2 or H1 ions act as a second co-ion and (3) BGEs containing multivalent weak acids and bases at a pH round a pK value, in which two ionic forms of that component act as two co-ions. For the first case, when BGEs consisting of two co-ionic species are used, the mobilities of the SZs are lying in-between the mobilities of these two co-ions. The actual mobility of a SZ, mSZ, is closer to the mobility of the co-ion which is present in minority. For all other cases, no rules of thumb can be given, and the mSZ must be calculated with mathematical models or simulation programs (see Section 5). Generally, the BGEs in CZE 525 mSZ increases for lower pH and higher pH of the BGEs, respectively. BGEs containing weak multivalent cations and anions show a maximum mSZ around the pK values of the multivalent ionic species, where two ionic forms of that ionic species are present. In that case, both ionic forms of that multivalent ionic species seem to act like two co-ions. In order to give an impression of the mSZ, we give in Fig. 4A the calculated relationship (solid line) and measured values of the mSZ for several BGEs containing the two co-ionic species (m) potassium and histidine and (n) potassium and imidazole. The total concentration of the two co-ions was always 0.01 M and the composition was expressed as molar ratio (%) of potassium. The pH was adjusted to pH 5 by adding acetic acid in all cases. For a concentration of the coion potassium approaching zero, the mSZ is equal to the mobility of potassium, whereas for a potassium concentration approaching 100%, the mSZ is equal to the mobility of the minor component (imidazole or histidine). In Fig. 4B calculated values (solid lines) and measured values (m, n) are given for the mSZ of SZs present in electropherograms applying BGEs consisting of (a) 0.01 M histidine and (b) 0.006 M histidine adjusted to different pH by adding formic acid. They always show an increase of mSZ for low pH of the BGE. It is clear that SZs are only visible in electropherograms as system peaks (SPs)/dips if at least one of the ions of the BGE is UV-absorbing. But even if SZs are invisible, their effects on peak shapes of sample components are still visible in the electropherograms. Some examples are given in Section 6. Figure 4. (A) Calculated values (solid line) and measured values of the mSZ of system zones for different compositions of BGEs containing the two co-ionic species (m) potassium and histidine and (n) potassium and imidazole. The total concentration of the two co-ions was always 0.01 M and the composition is expressed as molar ratio (%) of potassium. The pH was adjusted to pH 5 by adding acetic acid. (B) Calculated values (solid lines) and measured values (m, n) for the mSZ of SZs in electropherograms applying BGEs consisting of (a) 0.01 M histidine and (b) 0.006 M histidine adjusted to different pH by adding formic acid. 526 J. L. Beckers and P. Boček 4.4 The use of multiple co-ionic BGEs It has already been mentioned that a strong peak broadening owing to electrodispersion is obtained if there is a big difference between the mobilities of sample components and co-ions of the BGE. Therefore, it might be concluded that the use of a multiple co-ionic BGE would be favorable for the determination of sample solutions containing several ionic species with a diversity of mobilities, because then there are multiple centers of symmetry [29, 33], in which the electrodispersion is minimum. Sorry to say, it is just only half of the truth. If a BGE contains n co-ions, n-1 SZs are present, which interact with sample zones having equal mobilities and deform their peak shapes. The mobility of SZs is already shown in Figs. 4A and B for BGEs containing two co-ions and for BGEs at extreme pHs. To demonstrate this phenomenon, we separated an equimolar sample mixture of 0.0005 M of K1, Na1, Li1 and Tris1 ions, applying a BGE consisting of (a) 0.01 M histidine and (b) a mixture of 0.005 M histidine and 0.005 M imidazole, both adjusted to pH 5 by adding acetic acid. The electropherograms for 5 s pressure injections are shown in Fig. 5. For the BGE containing a single co-ion histidine, the Tris1 zone is very sharp because its mobility equals that of histidine. All other zones are fronting and show an increased peak broadening owing to the larger differences between the mobilities of sample ions and histidine. Applying a BGE containing the two co-ions histidine and imidazole, both the Na1 zone and the Tris1 Electrophoresis 2003, 24, 518–535 zone are sharp, because their mobilities correspond with that of the co-ion imidazole and histidine, respectively. A small SZ is visible in the electropherogram 5b just behind the lithium dip. The calculated effective mobility of the SZ according to the SystChart of the BGE is ca. 3361029 m2/Vs (see Section 5 for the concept SystChart) and this value corresponds quite well with the value of ca. 3061029 m2/Vs obtained from Peakmaster . The SZ will be more pronounced if its mobility is closer to that of a sample peak and if only one of the co-ions is UV-absorbing. The presence of more counterions has no implications for the presence of SZs. 4.5 The use of weak multivalent ionic species in BGEs Although weak multivalent ionic species, present in a solution in different ionic forms in fast dynamic equilibrium with each other, generally migrate as a uniform single zone  with an average mobility defined by: meff n X i0 a i mi n X ci i0 ct mi (12) in which the subscript i refers to all ionic forms and ct to the total concentration of the ionic species, they sometimes seem to behave as a mixture of independent ionic species. In a BGE, prepared at a pH around a pK of a weak multivalent ionic species, two ionic forms of that ionic species are present simultaneously and SZs often exist [35–37]. The two ionic forms seem to act as two co-ions. By this effect, BGEs with multivalent ions are not useful for the complete mobility window of the analytes. In Section 6, we give examples of BGEs containing weak multivalent cationic and anionic species and present calculated mSZ values for these BGEs. In order to avoid the influence of SZs, it is advisable to use BGEs in its safe region. Unsafe regions can be defined as the mobility window of analytes with values of mSZ 6 10% . 4.6 Peaks and dips Figure 5. Separation of an equimolar sample mixture of 0.0005 M of K1, Na1, Li1 and Tris1 ions applying a BGE consisting of (a) 0.01 M histidine and (b) a mixture of 0.005 M histidine and 0.005 M imidazole, both adjusted to pH 5 by adding acetic acid. For further information see Section 4.4. Applying a UV detector, the detector signal depends on the change in concentration of a UV-absorbing component (chromophore). For UV-absorbing sample components using a UV-transparant BGE, all components are visible in electropherograms as peaks. For the detection of UV-transparant sample components, the indirect UV mode has to be applied. With indirect detection, a co-ion is chosen as chromophore. The sample components displace this chromophore present in the BGE and their zones are always visible as dips in the electropherogram. It should be mentioned, however, that a one-to-one displacement occurs only if the mobilities of co-ion and Electrophoresis 2003, 24, 518–535 BGEs in CZE 527 sample component are equal. In that case, the electrodispersion is minimum resulting in a minimum peak broadening and large plate numbers. Generally, the transfer ratio, indicating the number of moles of a component i of the BGE displaced by a mole of the sample component S, can be defined as : TRi cSi cBGE i cSS Dci cSS (13) where c refers to the total concentration of an ionic species, the subscripts refer to the sample component S and an ionic species i of the BGE and the superscripts refer to the sample zone S and the BGE, respectively. Large transfer ratios are to be preferred in order to get a large UV signal. These transfer ratios can be calculated with mathematical models or determined with simulation programs. The component i of the BGE can be either a co-ion or a counterion and the definition (13) can even be used for complicated BGEs containing several co-ions or counter ions. Calculations of transfer ratios show that the values can be either positive (increasing concentration of component i in the sample peak) or negative, for both co-ions and counterions and this can give a very complicated case for the UV signal, dependent on which ions are chromophores [39, 40]. Especially in case of two co-ions, remarkable TR values for the co-ions can be obtained, in which the concentration of one of the co-ions increases in the sample zone (positive TR) and the concentration of the other co-ions decreases (negative TR). This means that if the first co-ion is a chromophore a peak is visible in the electropherogram and if the second is a chromophore a dip will be obtained in the electropherogram. If more than one of the constituents of the BGE are chromophores the result is difficult to predict without knowledge of all physicochemical data and calculations. As an example of such a complicated situation, we give in Fig. 6 the electropherogram for the separation of a mixture of 0.0005 M Li1 and Na1 ions, applying a BGE consisting of a mixture of 0.005 M of the co-ions potassium and histidine adjusted to pH 3.43 by adding formic acid. In the electropherogram Na1 is a peak and Li1 is a dip and three SPs are visible, viz. the EOF dip SP1, a system peak SP2 due to the low pH of the BGE and an SP3 owing to the use of two co-ions. Because the mobilities of Na1 and SP3 are close together, the zones show an extra peak broadening. 5 Prediction of properties of BGEs 5.1 General considerations A particular BGE is characterized by its composition, i.e., by the selected co-ions and counterions and their concentrations. By this choice, all other parameters such Figure 6. Separation of a mixture of 0.0005 M Li1 and Na1 ions applying a BGE consisting of a mixture of 0.005 M of the co-ions potassium and histidine adjusted to pH 3.43 by adding formic acid. Measuring conditions: 3 s pressure injection; voltage, 5 kV; capillary length, 36.7 cm; distance between injection and detection, 30.0 cm. as the pH of the BGE, the ionic strength and the specific conductivity are fixed and can be calculated from the mobilities and pK values of all constituents. The mEOF can experimentally be determined applying such a BGE for a given capillary. If all foregoing characteristics of a BGE, as well as the mobilities and pK values of the analytes, are known and providing that we consider analytes having no specific mutual interactions and no interactions with the ionic species of the BGE, the migration behavior of analytes (apparent mobilities) and peak shapes can be predicted. Even the existence and mobilities of SZs can be predicted. Predictions can be made using (i) rules of thumb, (ii) mathematical models [40–45] and (iii) simulation programs [34, 46–48] based on mathematical models. A problem is often how to present the information obtained with mathematical models in such a way that the reader can use this information in a general way. In this section, we will present an example of a SystChart, in which all parameters calculated with a mathematical model [40, 41] are visualized in eight panels, and the calculated data are confirmed with a measured electropherogram. In all panels the relationship between a specific parameter versus the mobilities of the sample analytes are given for sample zones at a sample concentration of 561024 M. 528 J. L. Beckers and P. Boček 5.2 SystCharts As an example, we will discuss the SystChart of a BGE consisting of a mixture of 0.005 M potassium and 0.005 M histidine and 0.02 M acetic acid at a pH of ca. 4.7. This BGE is useful for both UV-transparent cations and UVabsorbing anions and in this system acetic acid acts as buffering ion. The SystChart is calculated for fully ionized analytes. In Table 1, all mobilities at infinite dilution and pK values are given for the ionic species used in the calculations. The setup of a SystChart  is always as follows. The panels (A)–(D) always describe the electrophoretic behavior of cationic analytes and the panels (E)–(H) that of anionic analytes for the specified BGE. In all relationships in the panels (A)–(D), discontinuities are present indicating the existence of an SZ, with a mobility (at infinite dilution) of ca. 4661029 m2/Vs. In all panels the dashed vertical lines refer to the mobilities of the coions, the dashed arrows indicate the mSZ and the dashed horizontal lines indicate the values of the given parameter in the BGE. The set of panels for this BGE is given in Fig. 7. In the panels (A) and (E) the pH in the sample zones are given, calculated for a sample zone with a cS of 561024 M. The calculated pH in the sample zones in this BGE do not differ so much from the pH of the BGE, except for sample cations with mobilities close to that of the SZ. In the panels (C) and (G) the ratios E1m1/E2m2 are given where the subscripts 1 and 2 refer to the parameters in the pure BGE and in a sample zone with a cS of 561024 M, respectively. The m refers to the values of the mobilities of a particular sample ionic species in the pure BGE zone and in the sample zone of 561024 M, respectively. This ratio can better be applied than often used ratios E1/E2 because for “weak” acids and bases, small shifts in pH can result in a large change in effective mobility and this effect often overrules the changes in E ratio. If the ratio E1m1/E2m2 is larger than unity the sample component in the BGE moves faster than that in the sample zone at a concentration of 561024 M and because the lowest concentration segment of the peak moves fastest, the peaks will be fronting. If the ratio is smaller than unity tailing peaks are the result. For fully ionized sample cations and anions, having mobilities mS = mco-ion, the ratio is unity. In the panels (B) and (F) the total concentrations are given of all cationic species of the BGE present in the sample peaks and in the panels (D) and (H) the total concentrations are given of all anionic species of the BGE present in the sample peaks. The panels (B) and (H) describe the concentrations of the co-ions and the panels (D) and (F) those of the counter ionic species. In the panels (B) and (H) a second horizontal dashed line indicates the total Electrophoresis 2003, 24, 518–535 concentration of the co-ions for a TR of unity, i.e., with a concentration value of 561024 M lower than that of the concentration in the BGE. The turn-over point for TR, i.e., the mobility of the sample ions at which the value of TR is unity, is generally not affected by pK values of the sample ions and equals the mobility of the co-ions. If ionic mobilities of sample components and co-ions are equal, there is a one-to-one displacement of sample and co-ions and in that case the concentration of the counterions equals that of the BGE. For this BGE, we see a selective displacement for cationic sample ions if the mobility of sample ions equals that of one of the co-ions. For sample cations with a mobility near the mobility of the SZ a strange effect is visible. One of the co-ions is displaced at a very large degree whereas the concentration of the other co-ion increases! If the latter co-ion is UV-absorbing, then this sample zone is visible as a peak in the electropherogram. We see in panel (B) that this is true for sample ions with a mobility higher than that of the SZ and lower than that of potassium. In panel (F) only a single relationship is given because the calculated concentration for the counterions histidine and potassium are nearly identical. To illustrate these theoretical predictions, the experimental electropherogram, for the separation of a mixture of 561024 M of Na1, Li1 and Tris1 ions applying this BGE, is given in Fig. 8A. Just according to Fig. 7B, the sodium zone is visible as a peak in the electropherogram and the components lithium and Tris are dips. The discontinuities in Fig. 7, indicate the existence of a SZ with a mobility of ca. 4661029 m2V21s21, and, really a system peak SP2 is visible just before the lithium dip in Fig. 8A. According to Fig. 7B clearly can be seen that the area of the lithium dip is enlarged compared with the area of the sodium peak and Tris dip. In accord with the theoretical prediction in Fig. 7C, the experiment in Fig. 8A shows that the sodium zone is visible as a tailing peak, lithium is a fronting dip and Tris is nearly symmetric and sharp. Further a system peak SP1 is visible corresponding to the low pH of the BGE with a very low mobility. Besides this presentation of calculated data a lot of other mathematical models and simulation programs can be used for the prediction of the suitability of a BGE. A sophisticated simulation program Peakmaster is recently published by Gaš et al.  and this simulation program can even be downloaded. In Fig. 8B the electropherogram is given, simulated with Peakmaster, for the same electrophoretic run as given in Fig. 8A. The simulated electropherogram resembles very well the measured electropherogram. Concerning the anions, the panels (E)–(H) predict that no SZs are present and this BGE is only suitable for UV-absorbing anions, because the changes in the UV signal owing to changes in the concentration of the Electrophoresis 2003, 24, 518–535 BGEs in CZE 529 Figure 7. SystChart for a BGE consisting of a mixture 0.005 M potassium, 0.005 M histidine and 0.02 M acetic acid at a pH of 4.7. For further information see Section 5.2. 530 J. L. Beckers and P. Boček Electrophoresis 2003, 24, 518–535 Figure 8. (A) Measured electropherogram for the separation of an equimolar mixture of 561024 M of Na1, Li1 and Tris1 ions applying a BGE consisting of 0.005 M K1 and histidine1 adjusted to a pH of 4.7 by adding acetic acid. (B) Simulation of the same separation. Description from left to right: sodium (peak), SZ 2 (vertical line representing discontinuity), lithium (dip), Tris (dip), SZ 1 (vertical line), and EOF (vertical line). UV-absorbing counterion histidine are too small. Examples of these separations are trivial and we do not include them here. 6 Examples of BGEs In this part we like to describe the general way of constructing typical BGEs for the principal modes of migration (cathodic and anodic) and of detection (direct and indirect), and, exemplify these considerations by using simple model samples. For the preparation of a BGE, we first have to know whether we want to work in the direct or indirect UV mode. If the analytes are UV-absorbing, we will work in the direct UV mode and in that case we can choose a UV-transparent cation and anion for the BGE. If the analytes are UV-transparent we will work in the indirect UV mode and in that case it is advisable to take a UV-absorbing co-ion. In order to obtain a separation, by controlling effective mobilities of analytes, the analytes must be (partially) charged, i.e., that for cations a pH will be chosen near or lower than its pK value and for anions near or higher than its pK value. One of the ionic species of the BGE should have a pK value near the desired pH in order to act as buffer. 6.1 BGE for analysis of cations with direct UV detection A UV-transparent cation, such as Tris or sodium should be chosen. Tris can be used as pH buffering co-ion between pH 7–9 because its pK value is 8, in combination Electrophoresis 2003, 24, 518–535 with all acids with a pK value lower than 8. For pH lower than ca. 7, the counterion must be buffering and its pK value defines the useful pH region. If sodium is used as co-ion, the counterions should always be buffering. All these BGEs show no SZ except at a very low pH. In order to obtain an optimum resolution, the effect of electrodispersion should be considered. A minimum peak broadening is obtained if the mobility of the co-ion matches those of the analytes. If two co-ions are used, there are invisible SZs present and they can disturb the separation and deform the peak shapes. To demonstrate this, we consider the separation of imidazole, histidine and creatinine. This mixture should easily be separated at a pH where the analytes are completely charged. In Fig. 9, the measured electropherograms are given for the separation of an equimolar mixture of 561024 M of imidazole, histidine and creatinine applying BGEs consisting of (a) 0.01 M potassium, (b) 0.01 M Tris and (c) 0.009 M potassium and 0.001 M Tris, all adjusted to pH 3.5 by adding formic acid. Applying a BGE containing the co-ion K1 (a), the analyte peaks are broad, especially histidine, because of the big difference between the mobilities of K1 and those of the analytes and all peaks are tailing because the co-ion has the highest mobility. Applying a BGE containing the co-ion Tris (b), the analyte peaks are narrower because the mobilities of analytes do not differ so much from that of Tris, especially that of histidine. Imidazole and creatinine are fronting, whereas Tris is practically symmetric. Figure 9. Measured electropherograms for the separation of an equimolar mixture of 0.0005 M of imidazole (Im), histidine (Hi) and creatinine (Cr) applying BGEs consisting of (a) 0.01 M potassium, (b) 0.01 M Tris and (c) 0.009 M potassium and 0.001 M Tris, all adjusted to pH 3.5 by adding formic acid. BGEs in CZE 531 Applying a BGE containing two co-ions potassium and Tris (c) an invisible SZ is present, with a mobility close to that of creatinine through which the peak shape of creatinine is totally disturbed. 6.2 BGE for analysis of cations with indirect UV detection For the separation of cations in the indirect UV mode, it is advisable to choose a UV-absorbing co-ion, such as histamin, histidine or imidazole. All these ions are weak and the buffering capacity of them can be used advantegeously. Especially histamin, with a pK of 9.95, is a UVabsorbing base that can be used at high pH. On the other hand, these co-ions may be combined with buffering anions, e.g., acetic acid and then the buffering capacity of the applied anionic species can be used at pH values near their pK values. BGEs consisting of histidine acetate and imidazole acetate give no SZs whereas the divalent histamine can give SZs as explained in Section 4.5. In order to demonstrate this behavior, we give in Fig. 10 the electropherograms for the separation of 5 s pressure injections of an equimolar mixture of 561024 M of (a, b) K1, Na1, Li1, Tris1, and tetrabutylammonium (TBA1) ions and (c, d) K1, Ba21, Na1, Li1, Tris1, and TBA1 ions applying BGEs consisting of (a, c) 0.01 M histidine and (b, d) 0.01 M imidazole, all adjusted to pH 5 by adding acetic acid. A good separation is easily obtained in Fig. 10 both applying (a) histidine and (d) imidazole acetate. If we add Figure 10. Separation of an equimolar mixture of 561024 M of (a, b) K1, Na1, Li1, Tris1, and TBA1 ions, and (c, d) K1, Ba21, Na1, Li1, Tris1, and TBA1 ions, applying BGEs consisting of (a, c) 0.01 M histidine and (b, d) 0.01 M imidazole, all adjusted to pH 5 by adding acetic acid. 532 J. L. Beckers and P. Boček Electrophoresis 2003, 24, 518–535 adjusted to pH 6.5 by adding acetic acid. At pH 6.5, histamine is for the larger part a monovalent cation and its competitive adsorption to the wall is reduced, through which the Ba21 dip is strongly tailing again. Further, the mobility of the SZ is much higher than it was at pH 5, and the SP is visible just before the lithium dip, see Fig. 11B (a). These electropherograms demonstrate that weak multivalent co-ions should be used for BGE preparation with reservation owing to the existence of SZs. For BGEs consisting of 0.01 M histamine adjusted to different pH by adding acetic acid we calculated the relationship between the mobility of the SZ and pH of the BGEs and the relationships are given in Fig. 12 using (o) the simulation program Peakmaster  and (d) SystCharts . Both calculations agree perfectly well, although the values obtained from SystCharts are higher because no corrections are made for relaxation and retardation effects according to Debye-Hückel-Onsager. Figure 11. (A) Separation of an equimolar mixture of 561024 M of (a) K1, Na1, Li1, Tris1, and TBA1 ions, and (b) K1, Ba21, Na1, Li1, Tris1 and TBA1 ions, applying a BGE consisting of 0.01 M histamine adjusted to pH 5.0 by adding acetic acid. (B) Separation of the same sample mixtures as used for (A), applying a BGE consisting of 0.01 M histamine adjusted to pH 6.5 by adding acetic acid. Ba21 to the sample we see that this divalent ion strongly adsorbs to the wall causing a fronting Ba21 dip with a very long tailing backside, and this indicates that a BGE cannot always be used for all samples. The same samples are analyzed with BGEs consisting of histamine acetate. In Fig. 11A the electropherograms for the separation of the same samples as used in Fig. 10 are given for the BGE consisting of 0.01 M histamine adjusted to pH 5 by adding acetic acid. The Ba21 dip is extremely sharp because its mobility equals that of the histamine21 ions and its adsorption to the negative wall charge is strongly suppressed probably by the competitive adsorption of the divalent co-ion. Remarkable is the presence of an SP in the electropherogram, although this SP does not disturb the separation because of its low mobility (see also Fig. 12). In Fig. 11B the electropherograms for the same separations are given applying a BGE of 0.01 M histamine Figure 12. Calculated mobilities of SZs in BGEs consisting of 0.01 M histamine adjusted to different pH by adding acetic acid, according to (s) Peakmaster and (d) SystCharts. For further information see Section 6.2. 6.3 BGE for analysis of anions with direct UV detection Actually, identical BGEs as used for the determination of cations in the direct UV mode can be applied. For example, a cation such as Tris or sodium should be selected in combination with a UV-transparent acid, in which one of the ionic species of the BGE should be buffering. Important is whether the separation can be carried out in the cathodic or anodic mode and this is determined by the mobility of the EOF. If the absolute value of the mo- Electrophoresis 2003, 24, 518–535 BGEs in CZE 533 bility of an anionic species is smaller than the mEOF, it can be analyzed in the cathodic mode otherwise it should be analyzed in the anodic mode. If the EOF is suppressed by the addition of an EOF-supressing agent, anions can always be analyzed in the anodic mode. It should be mentioned that the EOF modifier also can affect the migration behavior of the analytes  and that a second co-ion can be introduced causing SZs. Popular anionic species for BGEs are phosphate and borate. In case of multivalent phosphate buffers, invisible SZs are possible, disturbing the separation process. Peakmaster can be used for the calculation of the mobilities of SZs. 6.4 BGE for analysis of anions with indirect UV detection Here, a cation such as Tris or sodium in combination with a UV-absorbing acid as co-ion should be selected. In the case of Tris a pH between 7–9 is suitable otherwise the pK value of the acid determines the useful pH region. For a single monovalent acid no SZs can be expected. For multiprotic acids SZs are possible near the pK values where two charged particles of the acid exist. For BGEs consisting of 0.005 M phthalic acid adjusted to different pH by adding Tris, the relationship between the mobilities of the SZs versus the pH is given in Fig. 13, again using (o) the simulation program Peakmaster and (d) SystCharts. Although phthalate buffers are very popular for the determination of anionic species , the use of these buffers is limited owing to the presence of SZs, as can be seen in Figure 14. Electropherogram for the separation of an equimolar mixture of 0.002 M of tartrate (Ta), citrate (Ci), succinate (Su), and acetate (Ac) applying a BGE of 0.005 M phthalic acid and 0.0075 M Tris at pH 5.1. Fig. 13. In Fig. 14 the electropherogram is given for the separation of an equimolar mixture of 0.002 M of tartrate, citrate, succinate, and acetate applying a BGE of 0.005 M phthalic acid and 0.0075 M Tris at pH 5.1. The separation is carried out in the anodic mode and 561024 M CTAB is added to the BGE in order to reverse the EOF. This BGE is often presented as standard BGE for the determination of anions, but should not be used for anions with mobilities lower than that of acetate, owing to the presence of a SZ, visible in the electropherogram just behind acetate (at ca. 3.2 min.). The EOF is visible in Fig. 14 as a peak, because of the adaption of the concentration of the BGE to the high o value of the original sample solution according to KRF and a small SP is visible at ca. 2 min due to the addition of a second co-ion bromide of the CTAB. The latter SP can be avoided by adding cetyltrimethylammonium hydroxide (CTAOH) instead of CTAB. 7 Conclusions Figure 13. Calculated mobilities of SZs in BGEs consisting of 0.005 M phthalic acid adjusted to different pH by adding Tris, using (s) Peakmaster and (d) SystCharts. For further information see Section 6.4. The composition of a BGE, with respect to the number and sort of co-ions and counterions and their concentrations, is of key importance in CZE. The most important basic principles to design a suitable BGE, as well as, to avoid some related pitfalls, may be summarized as follows: (i) Suitable BGEs should contain enough ions to conduct the electric current. The concentration of ions should not be too high, however, in order to avoid excessive Joule heating. (ii) The voltage needed for a specific time of analysis can be calculated from the mobilities of the analytes. The corresponding current can be calculated from the electric conductivity of the BGE. 534 J. L. Beckers and P. Boček (iii) For a known applied voltage, the electric power can be calculated and, if needed, the concentration of the BGE or the diameter of the capillary can be decreased. (iv) At least one component of the BGE must have significant buffering capacity at the selected pH, i.e., the pH of the solution must be within the range pK 6 1 of the buffering component. (v) A co-ion of the BGE should be selected, in such a way that its mobility is close to those of sample ions; then the electrodispersion of sample peaks is minimum and peaks are practically symmetric and sharp. (vi) Samples with UV-transparent components may be detected by using the indirect detection mode with a BGE where a suitable UV-absorbing co-ion (preferably) or counterion is used. (vii) When indirect detection is applied and the BGE contains two co-ions, the analytes show always selective displacements of these co-ions, and, the detection record may show peaks or dips (positive or negative peaks) for the analyzed components. (viii) Generally, an EOF is present in fused-silica capillaries migrating to the cathode (cathodic EOF). Thus, cations and anions can be detected by using cathodic mode. Anions with mobilities (absolute value) lower than that of the EOF can be detected. Anions with higher mobilities should be analyzed in the anodic mode. (ix) The EOF can be suppressed (zero EOF) or even reversed (anodic EOF) by adding suitable surfactants, such as CTAB; however, the risk that it brings some more co-ions into the BGE should always be considered. (x) The use of multiple co-ions should be avoided (unless unavoidable for indirect detection) since it brings the formation of SZs. When two co-ions are used, an SZ with a mobility in-between the mobilities of these co-ions is present. (xi) The use of buffering multivalent weak co-ions should be avoided for the same reasons. (xii) BGEs at low and high pHs show the pronounced risk of disturbances due to the formation of SZs where H1 or OH2 act as the second co-ion. For BGEs consisting of, e.g., 1 M acetic acid, H1 ions are the sole cations. (xiii) Theoretical predictions, based on mathematical models and computer simulations, are very useful and enable one to examine the migration behavior of the BGE with a proposed composition as far as all the above aspects are concerned. 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