Contribution of Eigenmobility Shifts to the Separation of Peptides in

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Contribution of Eigenmobility Shifts to the Separation of Peptides in Capillary Electrophoresis with Aqueous−Acetonitrile Background Electrolytes Chadin Kulsing, Reinhard I. Boysen, and Milton T. W. Hearn* Australian Centre for Research on Separation Science (ACROSS), Centre for Green Chemistry, School of Chemistry, Monash University, Melbourne, Victoria 3800, Australia S Supporting Information *

ABSTRACT: In this investigation, the mobility of system eigenpeaks in capillary electrophoresis (CE) was experimentally found to decrease when the background electrolyte (BGE) contained higher percentages of acetonitrile. In order to explain this observation, the effects of changes in the pH and ionic strength of the BGE on the pKa and actual mobility of each constituent in the system were determined, and the results evaluated in terms of their theoretical basis. Utilizing the derived values of each of these parameters, the software Peakmaster was then applied to simulate the eigenpeak mobility. Although general trends for BGEs with different acetonitrile contents could be simulated, these simulations did not exactly match the experimental results. To account for this divergence between theory and experimental practice, the consequences of tube radial distribution of the organic solvent in an aqueous−organic system within the capillary and the effects of radial ion distribution leading to the electro-osmotic flow mobility (EOF) are proposed to be the cause of this deviation. Consequently, the Debye−Hü ckel approximation and Boltzmann distribution function were employed to calculate the amount of each constituent across the radius of the capillary. The inhomogeneous radial distributions of the constituents in the BGE and the organic solvent were simplified to a 1-dimensional problem based on a 4-constituent BGE approximation. A high level of correlation was then achieved between the experimental results and the corresponding CE separations simulated using Peakmaster. In addition, cancellation or suppression of the peak broadening was experimentally and theoretically demonstrated by taking advantage of the influence of a second independent system eigenpeak. The outcome from these studies was a new way to achieve sharpening of specific peaks in the CE separations of peptides.

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INTRODUCTION Capillary electrophoresis (CE) is a very useful analytical technique in separation science, often providing enhanced selectivity, high separation efficiencies,1−3 and fast analysis times for a wide variety of chemical and biological compounds.4−7 Changes in the pH or ionic strength of the background electrolyte (BGE) may however cause peak broadening of the analytes, superficially in a random fashion. This effect has been explained in terms of the concepts of electro-dispersion and system eigenpeak(s).8,9 An eigenpeak occurs when the BGE is perturbed by the presence of a sample zone in the capillary.10 When the eigenpeak migrates along the capillary at a position close to that of the analyte, the analyte peak shape can become broadened due to the so-called “bowtie” pattern of the local electric field strength within the sample zone.11 This behavior can be explained by the change in the velocity slope of the sample, as evidenced by the indirect absorption signal in the UV or mass spectrum or from the conductivity profile along the capillary.8,12 Current theories used to explain the migration behavior of low molecular mass polar analytes in CE employing BGEs containing many constituents are based on nonlinear relation© XXXX American Chemical Society

ships, which require numerical methods for the simulation of the corresponding electropherograms.11,13,14 Alternatively, these mathematical relationships can be transformed to a set of linear equations that account for perturbations in the concentration of each constituent from its initial value. These linear equations can be analytically solved using a matrix approach.8,12 On the basis of these latter approaches, Jaros et al.15 developed the software Peakmaster to simulate the effect of various parameters in CE, such as pH, conductance or ionic strength of the BGE, as well as to generate in silico electropherograms with direct and indirect UV profiles or conductivity profiles for CE systems with different BGEs, which may contain many types of ionic constituents,9 including micelles.16 Often organic solvents are used as components of BGEs to achieve selectivity advantages over their corresponding fully aqueous systems. Moreover, the solubility of some lipophilic molecules can be enhanced by the presence of organic Received: September 1, 2016 Accepted: November 7, 2016

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DOI: 10.1021/acs.analchem.6b03438 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry solvents,17 and ion pair formation and ionization properties can be fine-tuned.18 Včeláková et al.17 have illustrated that changes in the system eigenpeak mobilities of BGEs can result from changes in the pH, pKa, ionic strength and the limiting mobility of each constituent, when a pure solvent, e.g. methanol, is used and these results compared to those obtained with the corresponding fully aqueous system. These investigators also used Peakmaster to simulate the effects of changes in BGE composition with methanol-based solvent systems. Compared to studies on the eigenpeak behavior with such pure organic solvent systems, little attention has however been paid to the effects on the eigenpeak mobility of mixed aqueous−organic solvent systems. Acetonitrile has been frequently used in CE as a preferred organic solvent component of BGEs. Various effects on CE separations have been investigated, such as changes in resolution and selectivity,19,20 the impact on the EOF or pH of the BGE, as well as the pKa of the ions in the system,18,21−23 when the acetonitrile content in the BGE is changed. As acetonitrile is an aprotic solvent, it is more likely to form solvent clusters in water rather than form an extended hydrogen bonded network.24−28 This property results in acetonitrile/water mixtures behaving experimentally like two (or more) phases under certain conditions.29−31 Moreover, recent theoretical studies on the electric field dependence of phase equilibria of binary Stockmayer fluid mixtures have confirmed that two phase behavior can be induced upon exposure to sufficiently high electric field strengths, e.g., even up to 5 × 106 Vm−1, with the shapes of the vaporization and condensation curves changing, and the disappearance of the critical point and appearance of an azeotropic point.32 Under such conditions, the constituent molecules of a binary fluid mixture can separate into two partially miscible fluid phases, one of which is enriched with the more polar molecules. Recently, the two-phase behavior of acetonitrile−water mixtures in capillary liquid chromatography has also been reported and confirmed by fluorescence spectroscopy.33−35 Further, in CE, tube radial distribution of an organic solvent, as well as the radial distribution of ions near to the inner wall surface of a fused silica capillary, can be expected.36 Potentially, both of these effects can have a profound effect on eigenpeak behavior. The aim of this study was first to provide simulation-based explanations of the eigenpeak behavior of BGEs containing water−acetonitrile mixtures using Peakmaster and, second, to verify these predictions with experimental separations of peptides using fused silica capillaries with BGEs of different acetonitrile concentrations. These simulations initially took into account the effects of different changes in BGE properties, such as pH and ionic strength. Changes in the eigenpeak mobility were then further evaluated from theoretical and practical perspectives to test the hypothesis that radial distribution of the organic solvent and the BGE ions can also influence the migration behavior, and particularly the peak shape, of analytes within the capillary.

Q water (Millipore, Bedford, MA), followed by 0.1 M NaOH (for 10 min), and extensively with water again. Before use, the capillary was flushed with water for 5 min under 1 bar pressure, then with several capillary volumes of the background buffer electrolyte (sonicated by ultrasonication for 15 min). The composition of the background electrolyte system was 20 mM HCOONH4 at pH 2.98 or pH 6.80, containing various (0, 10, 20 and 25% v/v) percentages of acetonitrile, whereby HCOONH4 was titrated with formic acid until the desired pH was obtained. Acetonitrile (HPLC grade) was obtained from Biolab Scientific Pty Ltd. (Sydney, Australia). Ammonium formate and 100% acetic acid were purchased from BDH Chemicals Australia Pty. Ltd. (Kilsyth, Australia). NaOH and formic acid 99% (v/v) were obtained from AJAX Chemicals (Sydney, Australia). Five peptide standards (angiotensin II, Gly-Tyr, Val-Tyr-Val, leucine enkephalin, and methionine enkephalin) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). These peptides (0.1 mg mL−1) were dissolved in 20% (v/v) acetonitrile in 20 mM HCOONH4 at pH 2.98 or pH 6.80 before injection (the solution pH was measured before the addition of acetonitrile). The CE studies were carried out using an Agilent Technologies 3D G1600AX capillary electrophoresis system, operated at +20 kV, and equipped with a diode array detector. Pressure was externally applied via the pressure control system of the CE instrument. An Agilent Technologies 1100 series LC/MSD-SL ion trap mass spectrometer was connected to the capillary via an Agilent Technologies G1607A orthogonal electrospray interface (Agilent Technologies, Waldbronn, Germany). Electrical contact at the electrospray needle tip was established via a liquid sheath flow delivered by an Agilent Technologies 1100 series isocratic LC pump. The system was interfaced with Agilent Technologies ChemStation and MSD Trap Control software for instrument control and data collection. The theoretical basis and computational procedures employed for data analyses are described in the Supporting Information (SI).

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RESULTS AND DISCUSSION Upon addition of acetonitrile to a BGE, the activity coefficients of all constituents are changed.37 These changes result in a shift in both the pKa’s and the actual mobilities of all constituents, including the eigenpeak mobility and overall EOF. This shift in the eigenpeak mobility can be illustrated by the change in the position of the suppression zone relative to the analyte peak zones in the UV or mass electropherograms. Furthermore, the effect of the EOF36 on both the radial distribution of all components and the tube radial distribution of the aqueousacetonitrile system in the capillary33,35 also need to be considered, since these phenomena could also affect the shift in the eigenmobility. To evaluate these effects further, in this study, simulation procedures have been implemented and the findings compared to experimental observations (for further details about the methods employed refer to the theory and computational procedures section in the SI). Interestingly, as discussed below, segregation of the aquo-acetontrile mixtures into partially enriched binary phases appears to occur with all CE experiments undertaken in the current study with an electric field strength of +25 kV.m−1, which is well below the calculated levels of electric field strength with binary Stockmayer fluid mixtures.32 Eigenmobility and Analyte Peak Broadening with Aqueous Acetonitrile BGEs. In the absence of acetonitrile in the BGE at pH 2.98, the five peptides (their physicochemical

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MATERIALS AND METHODS Bare fused-silica capillaries, with an internal diameter of 50 μm (outer diameter 363 μm) and a total length of 80 cm, were purchased from Agilent Technologies (Waldbronn, Germany). A transparent UV detection window was made 26 cm from the inlet of the capillary by burning the outer polyimide-coating with a flame. The fused silica capillaries were washed with MilliB


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Analytical Chemistry

Figure 1. Electropherograms of five peptides (1−5, refer to the Table S1) dissolved in 20% (v/v) acetonitrile and 20 mM HCOONH4 at pH 2.98, using a CE capillary of 80 cm effective path length, and a buffer electrolyte containing 0%, 10%, 20%, and 25% (v/v) acetonitrile (A, B, C, and D, respectively) and 20 mM HCOONH4, pH 2.98; hydrodynamic injection 0.5 s, ramping of separation voltage in 0.3 min to 20 kV (∼13 μA), 5 μL/ min SL flow rate, dry gas 5 L/min and nebulizer gas pressure 27579 Pa (4 psi) and nebulizing temperature 200 °C. The migration positions of the eigenpeaks (or suppression zones) are highlighted by the dashed rectangles. The corresponding UV electropherograms at 214 nm from Peakmaster simulations for a CE capillary having 26 cm effective length are depicted in the panel to the right.

An explanation for the peak suppressions, such as those shown in Figure 1, is that they are caused by the system eigenpeak due to electro-dispersion.10,11,13,14,38 After a capillary has been equilibrated with a BGE, when a sample of different composition and concentration to that of the BGE is injected, perturbation of the BGE equilibrium occurs. This perturbation (eigenpeak) moves through the capillary and is influenced by both the magnitude of the electric field strength and the EOF. The number and behavior (namely eigenmobility, eigenpeak shape, and amplitude) of the generated eigenpeaks are governed by the constituents in the BGE.10 In the present study, the eigenmobility, which depends on the pKa, mobility, and concentration of HCOOH and the NH4+, H3O+, and OH− ions, can be categorized as a two-constituent system,10 if contributions from the H3O+ and OH− ions are excluded. Peak suppression for peptides 1, 2, and 5, respectively (Figure 1B− D), can be attributed to comigration of these peptides with an eigenpeak. Furthermore, at higher concentrations of acetonitrile, eigenmobility became slower, since the presence of acetonitrile in the BGE changed all governing parameters of the eigenpeak.

properties are listed in Table S1) were separated with good resolution and peak shape (Figure 1A). An important parameter to be considered in such CE-MS studies is the “target mass”, with relatively high intensities for the m/z signals achieved when the target masses are close to the actual masses of the analytes. In the present studies, the target mass of the MS detector was set in all experiments at m/z = 500. The peak intensity of peptide 3 with a m/z = 239.1 in the electropherogram (Figure 1A, left panel) was consequently lower relative to the intensities of the other peptides, due to its m/z value being significantly different to the target m/z of 500. The peak height of peptide 3 experimentally appeared to be much higher (at the same concentration as the other peptides) in the UV electropherogram at 214 nm (Figure 1A, right panel). Moreover, when the percentage of acetonitrile in the BGE was progressively increased from 10 to 20 and then to 25% (v/ v) (Figure 1B−D), besides a change in the overall migration times of the analytes, it was observed that different peaks were suppressed. For example, a 25% (v/v) acetonitrile concentration led to suppression of the peak for peptide 5 at longer migration times (Figure 1D). C


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Table 1. Effects of Adding Acetonitrile (ACN) into the BGE at pH 2.98 (Containing 0% ACN, NH4+ with the pKa of 9.25 and a Mobility of 76.2 × 10−9 m2v−1s−1, HCOOH with the pKa of 3.752 and a Mobility of 56.6 × 10−9 m2v−1s−1, Which Resulted in the Eigenmobility of 24.345 × 10−9 m2v−1s−1) and the Resulting Changes in Eigenmobility Values According to Peakmaster Calculations phenomena after adding acetonitrile into BGE at pH 2.98 (0% ACN) +

pKa of NH4 decreases by 1 unit pKa of HCOOH increases by 1 unit mobility of NH4+ is lower by 1 × 10−9 m2v−1s−1 mobility of NH4+ is higher by 1 × 10−9 m2v−1s−1 mobility of HCOO− is lower by 1 × 10−9 m2v−1s−1 (more negative) mobility of HCOO− is higher by 1 × 10−9 m2v−1s−1 (less negative) pH increases by 1 unit (by increasing pKa of HCOOH)

eigenmobility change (10−9 m2v−1s−1) 0 −21.176 −0.160 0.159 −0.095 0.097 −21.354

Figure 2. Simulated UV electropherograms of five peptides, 1−5, with pKa-values approximated to be the same as their pI values (refer to the Table S1 for additional information) and various mobility values (iteratively calculated by regression procedures to allow the simulation data to fit to the experimental data with a coefficient of determination, r2, of ≥0.90); the composition of the BGEs was the same as given in the Legend to Figure 1, with 0, 10, 20, and 25% (v/v) acetonitrile (A−D, respectively), assuming only the pKa shift values as calculated in Table S2 by using the limiting mobility of HCOO− and NH4+ ions fixed at 56.6 and 76.2 × 10−9 m2v−1s−1, respectively, with the simulated electropherograms obtained using the same simulation approach as that in D except the limiting mobility of NH4+ was set at 16.2 × 10−9 m2v−1s−1 (E). Plot of the calculated eigenpeak mobility (with the EOF subtracted, black curve) derived according to Peakmaster versus the effective mobility of NH4+ ions at 25% (v/v) acetonitrile is shown in (F). The solid circle is the point indicating the effective mobility of NH4+ ions (12.1 × 10−9 m2v−1s−1) required to have the calculated eigenmobility close to that found in the experiment at 25% (v/v) acetonitrile (3.2 × 10−9 m2v−1s−1) with the corresponding simulated electropherogram shown in E, and the solid square is the point resulting in the electropherogram shown in D.

pH, which resulted in a delay of the eigenmobility.10,12 Deviations between the experimental and calculated pH values arise from differences in the actual versus predicted activity coefficients after the addition of acetonitrile. Although an algorithmic correction can be included into the Peakmaster program,39 to accommodate these differences in activity coefficients, nevertheless significant variations in the Debye− Hückel parameters of different buffers, especially those containing organic solvents,17,40 will lead to additional variances. The Peakmaster algorithm does not incorporate these changes in Debye−Hückel parameters for organic solvents.17 In order to improve the simulation of the electropherograms shown in Figure 1, the derived pKa values (presented in Table

The identical behavior observed with both MS and UV detection (Figure 1, left and right panels, respectively) indicated that the peak broadening results were not caused by a malfunction of one or other of these detectors. On the basis of the calculations with Peakmaster, the possible effects of acetonitrile in the BGE on the eigenmobility are summarized in Table 1. From these data, it can be noted that when acetonitrile was added to the BGE, two phenomena in particular caused a significant decrease in the eigenmobility, namely an increase in the pKa of HCOOH and the decrease in the mobility of the NH4+ ions. The contributions of the change in pKa and mobility of each constituent to the eigenmobility shift were then resimulated by Peakmaster. The pKa shifts after adding more acetonitrile to the BGE (Table S2) led to an increasing D


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Figure 3. (A) Eigenmobility, with the EOF values subtracted (y-axis), calculated without the correction for mobility change and radial distribution (shown as a line), and the corresponding experimented data (shown as dots) at various percentages of acetonitrile (x-axis), size of error bars corresponding to the possible position of the eigenpeaks shown in Figure 1, (B) Calculated NH4+ ion concentration at the capillary inner wall surface (outer phase of the capillary content) due to the EOF contribution using different percentages of acetonitrile, with a NH4+ ion concentrations in the bulk buffer electrolyte of 20 mM, (C) Calculated NH4+ ion concentrations at the acetonitrile-rich phase associated with the capillary inner wall surface (at ARP, [NH4+]A) due to the phase separation contribution at different percentages of acetonitrile in the ARP keeping the NH4+ ion concentration in the water-rich phase of the lumen region (at WRP, [NH4+]A) = 20 mM, (D) simulation result of the effect of NH4+ ion concentrations at the capillary inner wall surface (with the NH4+ ion moving slowly with a limiting mobility of 30 × 10−9 m2v−1s−1) needed to fit experimental pH and eigenmobility at 0−25% (v/v) acetonitrile with the NH4+ ion concentrations in the bulk buffer electrolyte fixed at 20 mM in the lumen region of the capillary.

S2) were incorporated into the Peakmaster program, which then returned the calculated pH after corrections for the calculated change in the ionic strength. The comparison between the calculated and experimentally obtained pH values (see Table S4) indicated that a change from a fully aqueous to an aqueous−acetonitrile system results in a decrease in the respective BGE pH values. According to the trend shown in Table 1, this pH decrease causes the eigenpeak to advance more quickly, especially for BGEs where the pH values fell into the range from pH 5 to pH 9.12 However, simulations with the corrected pKa values alone (Figure 2) were still not sufficient to adequately generate the electropherograms shown in Figure 1 and resulted in incorrect calculations of the corresponding eigenmobility shift. These findings confirmed that mobility corrections for the BGE ions should thus be taken into account. By correcting for both the changes in the BGE pKa’s and ion mobilities, the simulation of the eigenmobility at, for example, 25% (v/v) acetonitrile then matched the experimental findings, compare Figure 1D (experiment) and Figure 2E (simulation). Interestingly, the value of the effective mobility of NH4+ ions with a fully aqueous BGE, when compared to that obtained with a BGE containing 25% (v/v) acetonitrile, was six times greater (reduced from 76.2 × 10−9 to 12.1 × 10−9 m2v−1s−1, as illustrated in Figure 2F). Consideration of the Impacts of Radial Distribution of Ions and Solvents in Capillaries. Since the inner wall surface of a fused silica capillary is negatively charged (at pH values >2), a higher NH4+ concentration is expected to occur there (i.e., the region proximal to the wall) than will occur in the lumen (i.e., in the center) of the capillary, due to participation of the double layer effect.36 However, the mobility of the NH4+

ions in the interfacial region of the double layer will be lower than that in the center of the capillary. This behavior arises because at the electro-stagnant zeroth plane layer of the capillary surface, characterized by a surface potential Ψ0, the mobility of the NH4+ ions becomes zero, since r = R, resulting in the Bessel function term becoming zero (refer to the eq S9). The diffuse layer, on the other hand, extends from the edge of the β-plane, which contains the electrokinetic slip plane of the double layer associated with the electrokinetic ζ-potential, into the lumen of the capillary (and to the bulk BGE). The NH4+ ions in this diffuse layer will be distributed according to their random thermal motion and the influence of electrical forces. Due to an overall decrease in the average value of the NH4+ ion mobility in these zones of the double layer, a slower eigenmobility will arise. The consequences of the dramatic reduction in the mobility of the NH4+ ions as the acetonitrile content of the BGE is increased can be considered to be one reason for the observed decreases in the eigenmobility (Table 1). Similarly, the H3O+ ion concentration within the outer regions of the electro-stagnant β-plane (Stern layer), when compared to the bulk BGE in the lumen of the capillary, will also become higher. Higher H3O+ ion concentrations will lead to higher pKa values of HCOOH near to the inner wall surface of the capillary. This property is the other main contributor to the decreased eigenmobility, as illustrated in Table 1. Higher pKa values of HCOOH will decrease the eigenmobility, and upon addition of more acetonitrile to the BGE, the readjusted pKa value is expected to lead to the BGE pH becoming higher (see also the Table S4). Experimentally, the influence of these double layer effects on the eigenmobility shift was particularly evident upon addition of E


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Figure 4. Extracted mass electropherograms at m/z = 524, 380.1, 239.1, 556.2, and 574.1 of peptides 1−5, respectively, dissolved in the solution conditions described in the legend of Figure 1, using 0%, 5%, 10%, 15%, and 20% (v/v) acetonitrile (A−E, respectively) with the capillary electrophoresis run with a buffer electrolyte of 20 mM HCOONH4, pH 6.80. The predicted positions of the sharpening zone are highlighted as dashed rectangles. Part F shows the simulated electropherograms corresponding to the experimental results shown in part A, and illustrates the impact of the sharpening zone generated between the region occupied by two eigenpeaks, with the parameters simulated with a 20 mM HCOONH4 buffer electrolyte of pH 6.8 with one basic analyte (1, pKa = 7.32) and four acidic analytes (their pKa values were chosen to be the same as the pI values of the peptides detailed in Table S1), with the limiting mobility of each sample computationally adjusted to allow the plot of intensity versus time to replicate the electropherogram shown in panel of part A. The vertical dotted box lines represent the eigenpeaks with the position of the EOF marker (one of the eigenpeaks) fixed at 7.6 min.

ARP, in the region proximal to the wall of the capillary in a manner analogous to that observed for capillary HPLC.33 For example, with a bulk BGE containing 25% (v/v) acetonitrile, due to the occurrence of phase separation, the WRP is expected to contain less than 25% (v/v) acetonitrile, while more than 25% (v/v) acetonitrile will be present in the ARP. In the extreme (hypothetical) case, the percentage of acetonitrile in the WRP and the ARP could become 0 and 100%, respectively. The composition of the ARP in the region proximal to the capillary inner wall surface will lead to an increase in the amount of NH4+ ions near to the inner surface and reduction in their mobility as well as increase the pKa value of HCOOH (refer also to Figure S1 and Table S3). Overall, the contributions from these two effects will also reduce the eigenmobility. By assigning a value for the WRP to be 0% acetonitrile and [NH4+]WRP to be 20 mM, the NH4+ ion concentrations in the ARP (denoted by [NH4+]ARP values) were calculated (as listed in the Table S3) and the results are plotted in Figure 3C. These calculations were performed based on the assumption that a Boltzmann distribution occurred (see also the eqs S16−S19). It can be concluded from this evaluation that the decrease in eigenmobility observed with 25% (v/v) acetonitrile in the bulk BGE (Figure 1A) was caused by the higher concentration of more slowly moving NH4+ ions near to the capillary surface as illustrated in Figure 3B,C. Moreover, as the percentage of acetonitrile in the ARP becomes higher than that present toward the center of the capillary with 25% (v/v) acetonitrile in

acetonitrile to the BGE, as shown in Figure 1D with a BGE containing 25% (v/v) acetonitrile, and more generally as the acetonitrile content in the BGE was progressively increased from 10 to 25% (v/v), Figure 3A. Moreover, this decrease in eigenmobility correlated with an exponential increase in the calculated NH4+ ion concentration, and resultant change in EOF as shown in Figure 3B for a BGE containing 25% (v/v) acetonitrile (data calculated using the eqs S8−S11). However, changes in the EOF alone may not be the sole phenomenon governing the magnitude of the eigenmobility, since the EOF for a BGE containing 0% acetonitrile was higher than that at 10% (v/v) acetonitrile (Figure 3A), yet a slower eigenmobility was observed at 0% acetonitrile. As a consequence (at least) two processes must be associated with the slower mobility of the NH4+ ions and the decrease in eigenmobility. This concept of a higher NH4+ ion concentration moving more slowly near to the inner wall surface of the capillary with BGEs containing higher percentages of acetonitrile is supported further when the effects in a capillary of tube radial distribution of an aqueous-acetonitrile system (solvent radial distribution) are taking into consideration.33−35 An acetonitrile−aqueous BGE moving along a capillary under the influence of an electric field can be treated as a two-phase system with a water-rich phase and an acetonitrile-rich phase within the capillary. If this occurs, for bulk BGEs containing less than 50% (v/v) acetonitrile, then the BGE is expected to exhibit properties consistent with a more water-rich phase, WRP, at the center (lumen) of the capillary and a more acetonitrile-rich phase, F


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acetonitrile was added to the BGEs, further decreases in the eigenpeak mobilities occurred (via the mechanisms discussed above), resulting in the position of the resharpening peak zone progressively moving toward higher migration times in the electropherograms. Consequently, the peak shapes of peptide 2, 3, 4, and 5 were progressively sharpened at higher acetonitrile concentrations (Figure 4B−E), while the position of peptide 1 was moved out of the resharpening zone with 25% (v/v) acetonitrile (Figure 4E). Effect of Pressure on the Sharpening Zone Position. When pressure (50 mbar) was applied to the CE capillary with a bulk BGE containing 7.5% (v/v) acetonitrile in the same direction as that of peptide migration (e.g., toward the detector), the peak height for peptide 2 was dramatically suppressed in these experiments (Figure 5). This behavior is

the bulk BGE, this behavior will further increase the pKa values of HCOOH near to the surface of the capillary compared to HCOOH molecules nearer to the center (lumen) of the capillary, leading to the slower eigenmobility, as shown in Figure 3A. By taking the effects of radial distribution on eigenmobility into account, the concentrations of NH4+ ions near to the inner surface of the capillary (c.f. the amount shown in Figure 3D) were used to simulate (with Peakmaster) the experimental eigenmobility for BGEs containing higher concentrations of acetonitrile as shown in Figure 3A. The increasing trends evident for the experimental results were in agreement with these simulations when the calculated profiles included the influence of the radial distribution on the EOF and phase separation (Figures 3B,C). In other words, the nonlinearity of the experimental results shown in Figure 3A can be satisfactorily accounted for when the effect of radial distribution of BGE ions in a capillary, as well as pKa, ionic strength, and mobility corrections, are included. For the purposes of the calculations and simulations performed in this investigation, the buffer system was viewed as two completely separated phases. However, it can be noted that if any electrolyte component exhibited a radial distribution across the capillary beyond the double-layer where the EOF arises, then this effect would inevitably lead to nonuniform mobility values and result in dispersion of all constituents. Simulation of this more complex behavior would of necessity require calculations to be based on more dynamic third (or higher) order models and expressions of such buffer systems. Effect of Two System Eigenpeaks: Cancelation of Peak Broadening. After an analyte peak zone has been broadened under the influence of an eigenpeak, the possibility exists for the peak broadening effect to be canceled, leading to resharpening of the peak. This resharpening will arise when another eigenpeak is appropriately introduced close to the broadened peak of the analyte. Thus, if a 20 mM HCOONH4 buffer at pH 6.80 was used as the BGE, for example, then such an effect could be realized with the peptide peaks resharpened (Figure 4). This effect was achieved by shifting the sharpening region in the electropherograms relative to the peptide peaks to the right (toward higher migration times) as higher percentages of acetonitrile were added, from the position of peptide 1 to between peptides 1 and 2, from the position of peptide 2 to between peptides 2 and 3, and so on (Figure 4A−E). The effect of the presence of two eigenpeaks (i.e., associated with the EOF and a second eigenpeak) is illustrated by the simulation shown in Figure 4F. Consistent with attainment of this peak sharpening (Figure 4) was the observation that all peptide peaks became broader when they migrated close to the EOF peak (the eigenpeak with zero mobility). At pH 6.80, the calculated charge values of all peptides were close to zero (refer to the Table S1). However, as apparent from the experimental results under these BGE pH conditions, the effects of the second eigenpeak (the “not EOF peak”) perturbed the electric field pattern generated by the EOF peak, canceling to some extent its effect on peak broadening. Thus, peak resharpening appears to be confined to a small zone, with only some broadened analyte peaks resharpened, i.e. those analytes with mobility values closer to the mobility of the second eigenpeak rather than the EOF peak. Hence, only the peak for peptide 1 with a suitable mobility at 0% ACN became sharpened in the resharpening zone (enclosed by the green rectangle in Figure 4A). As more

Figure 5. Overlay of the extracted mass electropherograms at m/z = 524, 380.1, and 556.2 of the peptides 1, 2, and 4 (as detailed in Table S1) dissolved in the solution conditions described in the legend to Figure 1, with the capillary electrophoresis run with a buffer electrolyte of 7.5% (v/v) acetonitrile 20 mM HCOONH4, pH 6.80, with the application of either no pressure or alternatively with 50 mbar additional pressure, with the positions of sharpening zones highlighted as dashed rectangles.

consistent with a relative shift of the sharpening zone position toward a lower migration time. Before application of pressure, the sharpening zone was located between peptide 1 and 2 (Figure 5, right panel) resulting in the resharpening of these two peaks. However, only a minor part of the sharpening zone was occupied by peptide 2. Therefore, a small shift in the zone by the application of pressure led to a lower migration time, and resulted in the electro-migration of peptide 2 falling outside this zone. Consequently, the peak shape of peptide 2 was not sharpened and remained a broad peak at 50 mbar (Figure 5, left panel). Interestingly, when applied in the same direction as peptide migration this effect of pressure appears to be analogous to the effect seen for a decrease in acetonitrile content from 20 to 10% (v/v). This behavior was evident from the appearance of a similar peak shape for peptide 2 under these two conditions (Figure S2A). Importantly, application of pressure in the reverse direction to the EOF gave rise to a similar peak shape for peptide 2 as seen when an increased percentage of acetonitrile was employed, e.g. from 20 to 25% (v/v), in the BGE (refer to the Figure S2C).

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CONCLUSIONS In this study, the effects of various acetonitrile concentrations in aqueous BGEs were studied with a main focus on their impact on the eigenmobility in the CE analysis of small peptides. The decrease in eigenmobility after adding acetonitrile into the 2constituent BGEs (HCOO− and NH4+ ions) at pH 2.98 was experimentally investigated to establish the impact of the position of the suppression zone relative to the position of the G



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sample peaks in the electropherograms. The results were also simulated using the program Peakmaster, which indicated that the decrease of eigenmobility was mainly due to an increase in the pKa of HCOOH and a decreased mobility of NH4+ ions, as a consequence of adding higher percentages of acetonitrile to the BGE. With a BGE containing 25% (v/v) of acetonitrile, there was a dramatic decrease in eigenmobility. This finding could not be explained in terms of a one-dimensional simulation, based solely on the shift in pKa or the activity coefficients of all constituents. By using the Debye−Hückel approximation and Boltzmann distribution, the concept of a radial distribution of NH4+ ions and H3O+ ions in the capillary, caused by the EOF and a phase separation of the acetonitrile− water system, can be proposed (refer also to the Figure S1). The conclusion that a concentration gradient of NH4+ ions in the BGE exist from the center of the capillary to near to the capillary inner wall surface was the key to fully explain the dramatic decreases in the eigenmobility at higher acetonitrile concentrations. This conclusion was also consistent with the requirement for an increasing concentration of NH4+ ions to exist near to the capillary inner wall surface with BGEs of higher percentages of acetonitrile in order to fit the experimental results with theory. These findings also illustrate the important role that radial ion distribution may play in the control of eigenmobility. This study has focused on the use of bare silica capillaries for the separation of peptides with BGEs compatible with MS detection. However, similar analytical methods, exploring the impact of eigenmobility, are equally relevant to other types of capillaries with modified inner wall surfaces, whereby their surface charges and zeta potentials can be chemically altered and the radial distribution of the ions in the BGE rationally varied. The investigations as described herein are thus expected to provide a new way to characterize the performance of modified open tubular capillaries for the separation of peptides or other classes of analytes, providing complementary information to that obtained from migration time and EOF measurements.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b03438. Theory and computational procedures, supplementary Tables S1−S4, and supplementary Figures S1 and S2 (PDF)

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AUTHOR INFORMATION

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*Phone: +61+3+99054547. E-mail: [email protected] (M.T.W.H.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was funded from an ARC Special Research Centre Grant (No: DP130104060) awarded to Professor Milton Hearn at the Centre for Green Chemistry of Monash University. H


Article

Analytical Chemistry (40) Porras, S. P.; Riekkola, M.-L.; Kenndler, E. Electrophoresis 2003, 24, 1485−1498.

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Contribution of Eigenmobility Shifts to the Separation of Peptides in

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