Separation of selected peptides by capillary electroendoosmotic chromatography using 3 microns reversed-phase bonded silica and mixed-mode phases.

The retention behaviour and selectivity of selected basic, neutral and acidic peptides have been studied by capillary electroendoosmotic chromatography (CEC) with Hypersil C8, C18, Hypersil mixed-mode, and Spherisorb C18/SCX columns, 250 (335) mm x 100 microns, packed with 3 microns particles, and eluted with mobile phases composed of acetonitrile-triethylamine-phosphoric acid (TEAP) at pH 3.0 using a Hewlett-Packard Model HP3DCE capillary electrophoresis system. The selected peptides were desmopressin (D), two analogues (A and B) of desmopressin, oxytocin (O) and carbetocin (C). The peptides eluted either before or after the electroendoosmotic flow (EOF) marker, depending on the concentration of acetonitrile used and the buffer ionic strength. The retention and selectivity of these peptides under CEC conditions were compared to their behaviour in free zone capillary electrophoresis (CZE), where the separation mode was based on the electrophoretic migration of the analytes due to their charge and Stokes radius properties. In addition, their retention behaviour in RP-HPLC was also examined. As a result, it can be concluded that the elution process of this group of synthetic peptides in CEC with a TEAP buffer at pH 3.0 is mediated by a combination of both electrophoretic migration processes and retention mechanisms involving hydrophobic as well as silanophilic interactions. This CEC method when operated with these 3 microns reversed-phase and mixed-mode sorbents with peptides is thus a hybrid of two well-known analytical methods, namely CZE and RP-HPLC. However, the retention behaviour and selectivity of the selected peptides differs significantly in the CEC mode compared to the RP-HPLC or CZE modes. Therefore this CEC method with these peptides represents an orthogonal analytical separation procedure that is complimentary to both of these alternative techniques.     

Life-time studies with capillary electrochromatography columns operated under different conditions

A test system has been established to permit the monitoring of the life-time performance of several reversed- phase capillary electrochromatography (CEC) columns. The retention factors, k(cec), peak symmetry coefficients, lambda(sym), and column efficiencies, N, of three neutral n-alkylbenzene analytes, namely ethyl-, n-butyl- and n-pentylbenzenes, were determined for Hypersil 3 microm n-octylsilica and n-octadecylsilica packed into CEC capillary columns of 100 microm I.D., with a packed length of 250 mm, and a total length of 335 mm. The performances of these CEC capillary columns were examined for a variety of eluents with pH values ranging between pH 2.0 - 8.0, similar to those employed to study the retention behaviour of peptides that we have previously reported. The relative standard deviation (RSD) of the retention factors (k(cec) values) of these n-alkylbenzenes, acquired with an eluent of (25 mM Tris-HCl, pH 8.0,)-acetonitrile (1:4, v/v), when the CEC capillary columns were used for the first time (virgin values), were 4% (based on data acquired with 4 CEC capillary columns) for the n-octyl bonded silica capillary columns, and 6% (based on 8 columns) for n-octadecyl bonded silica capillary columns. The RSD values of the k(cec) values of the n-alkylbenzenes for one set of replicates (n=6) with one CEC capillary column was < 0.5%. The theoretical plate numbers, N, for the virgin CEC capillary columns were ca. 60,000, whilst the observed N values for all new CEC capillary columns were > or = 40,000 for n-octyl bonded silica capillary columns and > or = 50,000 for n-octadecyl bonded silica capillary columns. The peak symmetry coefficients, lambda(sym), of the n-alkylbenzenes for virgin CEC capillary columns and for CEC capillary columns used for more than 1,000 injections were always in the range 0.95-1.05. The experimental results clearly document that the life-time performance of the CEC capillary columns depends on the eluent composition, as well as the nature of the analytes to which the CEC capillary columns are exposed.     

Theoretical study of the separation mechanism of ionizable compounds in capillary electrochromatography

The migration mechanism of ionizable compounds in capillary electrochromatography (CEC) is more complicated than in high performance liquid chromatography (HPLC) due to the involvement of electrophoresis and the second chemical equilibrium. The separation mechanism of ionizable compounds in CEC has been studied theoretically. The electrochromatographic capacity factors of ions (k~*) in CEC and in the pressurized CEC are derived by phenomenologicai approach. The influence of pH, voltage, pressure on k~* is discussed. In addition, the k~* of weak acid and weak base are derived based on acid-base equilibrium and the influence of pH on k~* is studied theoretically.     

Evaluation of polymeric methacrylate-based monoliths in capillary electrochromatography for their potential to separate pharmaceutical compounds

Polymeric methacrylate-based monoliths are evaluated in capillary electrochromatography (CEC) and pressurized capillary electrochromatography (p-CEC) for their potential in pharmaceutical analysis. Using a given polymerization mixture as a basis for the monolith synthesis, different mobile phase pH at constant organic modifier concentrations are tested in both CEC and p-CEC. The test set consists of basic, acidic, amphoteric, and neutral compounds, which are mainly pharmaceuticals. Because of the mainly hydrophobic character of the stationary phase, the interactions are largest when the compounds appear in an uncharged state, but some ion-exchange phenomena with negatively charged compounds can also be observed. In CEC, acidic substances are most retained at low pH. For amphoteric and neutral compounds, no preference regarding analyzing pH can be derived from these experiments. For basics, a high pH is chosen, but a reduced solvent strength is needed to enhance the retention of these compounds. The retention mechanism in p-CEC can also be assigned to both hydrophobic and ionic interactions. For acidic, amphoteric, and neutral compounds, acceptable retention is seen. For the basic compounds, the retention with a mobile phase containing 50% organic modifier is low, as in CEC. However, when the organic modifier content in the mobile phase is decreased, retention increases and the selectivity of the stationary phase is more pronounced. This mode of operation presents a possibility for separating some test mixtures, thus some potential for pharmaceutical analysis is seen. More efforts are needed to obtain higher efficiencies and better peak shapes, which might be solved by a further optimization of both the stationary phase synthesis and the mobile phase composition.     

Collective excitations in an ionic liquid

Collective dynamics in a representative model of ionic liquids, namely, 1-butyl-3-methylimidazolium chloride, have been revealed by molecular dynamics simulation. Dispersion of energy excitation, omega versus k, of longitudinal acoustic (LA) and transverse acoustic (TA) modes was obtained in the wave vector range 0.17 < k < 1.40 Angstroms(-1), which encompasses the main peak of the static structure factor S(k). Linear dispersion of acoustic modes is observed up to k approximately 0.7 Angstroms(-1). Due to mixing between LA and TA modes, LA spectra display transverselike component, and vice versa. Due to anisotropy in short-time ionic dynamics, acoustic modes achieve distinct limiting omega values at high k when the cation displacement is projected either along the plane or perpendicular to the plane of the imidazolium ring. In charge current spectra, branch with negative dispersion of omega versus k is a signature of optic modes in the simulated ionic liquid. Conductivity kappa estimated by using ionic diffusion coefficients in the Nernst-Einstein equation is higher than the actual kappa calculated by integrating the charge current correlation function. From TA spectra, a wave vector dependent viscosity eta(k) has been evaluated, whose low-k limit gives eta in reasonable agreement with experimental data.     

Theory for migration of ions in capillary electrochromatography

The fundamental migration theories for chromatography and electrophoresis are both based on a solution of the mass balance equation. The corresponding analysis for an electrochromatographic system has previously been published and is analysed in more detail in this paper. It is shown that the resulting equation, Eq. (8) in this paper, is in agreement with both electrophoretic and chromatographic theories and that when these migration modes are mixed a complicated migration behaviour emerge. These complications arise, if the comparison is done with electrophoretic theory, because the presence of the stationary phase creates a number of new restrictions on the system (electroneutrality on the stationary phase and simultaneous equilibrium for all components between the eluent and stationary phase). From a mathematical point of view, these restrictions make it difficult for the system to satisfy the coherence condition and this in turn may lead to an anomalous behaviour. To minimise the possibility for a complicated behaviour it is advisable to avoid too much mixing of the two migration mechanisms and/or to match the mobilities of the ionic components in the eluent phase with the mobility of the analyte ion.     

Comparison of CZE, open-tubular CEC and non-aqueous CE coupled to electrospray MS for impurity profiling of drugs

Open-tubular CEC and non-aqueous CE (NACE) methods were developed for the analysis of six pharmaceutical compounds and their respective process-related impurities, comprising 22 analytes in total with a range of functional groups and lipophilicities. These methods were assessed for orthogonality of analyte separation with respect to existing CZE-ESI-MS and HPLC-ESI-MS methods, in order to complement a generic analytical strategy for impurity profiling of pharmaceutical compounds. Open-tubular CEC, using etched and chemically modified capillaries, induced weak reversed-phase-type interactions between some of the analytes and the bonded phases (0.811相似文献   

对-叔丁基杯[8]芳烃键合硅胶制备及其毛细管电色谱性能研究

以γ-(环氧丙氧)丙基键合硅胶为前体,于硅胶表面键合环氧基,在催化剂存在下以杯芳烃钠盐开环制备杯芳烃键合硅胶固定相.该方法反应条件温和,适用性强.将这个新方法首次用于制备对-叔丁基杯[8]芳烃电色谱键合固定相(C8BS),采用加压电色谱初步评价其电色谱性能.研究结果表明,C8BS电渗流(Electrosmoticflow,EOF)较小,但通过控制键合反应及使用压力辅助电色谱可部分弥补上述不足.该固定相的EOF受流动相pH影响小(pH=3-8),同时大环配体屏蔽效应能有效地克服硅羟基引起的碱性化合物拖尾现象,这对电色谱分离具有重要意义.通过分步封尾研究EOF的来源发现,杯芳烃酚羟基对EOF有弱的贡献,这与报道的杯芳烃涂层具有径向电渗流调控能力相一致.     

Expressions for the C-term in the presence of pore flow

In the course of our work on capillary electrochromatography (CEC) we, as others, have found strong evidence that flow in pores of particles can be significant. Its magnitude relative to the interstitial flow is characterized by the flow reduction factor, omega. Indirect evidence for pore flow was obtained much earlier by others, when it was noted that plate height, especially the C-term part, was significantly smaller in electrically driven (ED) than in pressure drive (PD) systems. This was interpreted as enhanced mass transfer, for which the intra-particle flow was held responsible. More direct evidence was produced by us when the size-exclusion (SEC) behaviour of polymers was studied in ED systems. It was found that the effect of exclusion on migration velocity could vanish entirely, and large and small molecules were co-eluted. This can only be explained if omega approaches 1; flow within the pores being as large as the interstitial flow. Indeed, consideration of double layer overlap indicated that omega-values close to 1 can often be expected in CEC. These large values omega inspired us to reconsider the effect of pore flow on the mass transfer term. We have arrived at the conclusion that enhanced mass transfer cannot explain in itself the extremely small values for the reduced plate height, h, (<1) observed especially for weakly retained solutes. In fact, when the pore flow is equal in magnitude to the interstitial flow, an unretained solute moves as fast within the particle as in the interstices; there is no non-equilibrium generated and a mass transfer term in h is not expected. For the migration of the solute the system is essentially uniform. Thus, apart from the mass transfer enhancement, another factor plays a role in the decrease of the h-values. We have attempted to derive a suitable expression for this effect. Some results are presented here. In one approach the situation is compared to that of an open tubular column with moving pseudo-stationary phase on the wall, an experiment that has actually been carried out by Krejci et al., or with micellar electrokinetic chromatography. In that case the plate height is easily derived. The result says that the plate height is proportional to the square of velocity difference between the two zones. However, the analogy is not perfect, and another approach suggests a direct proportionality rather than a square law one. Finally, a more refined treatment could be made only for a slab, not for a sphere. Extrapolation of this result to a sphere is put forward as a tentative expression for this effect.     

The analysis of pharmaceutical bases on a silica stationary phase by capillary electrochromatography using aqueous mobile phases

Summary The capillary electrochromatographic (CEC) separation of a range of pharmaceutical bases was investigated on a commercially available silica stationary phase using aqueous mobile phases. The effects of mobile phase composition, buffer pH, applied voltage, and buffer anion on the retention behaviour of these bases were studied. Promising chromatography was obtained at pH 7.8 but was later found to be irreproducible. However, successful and reproducible chromatography of the bases was achieved at pH 2.3. We have previously demonstrated that the addition of mobile phase additives such as TEA-phosphate at low pH values has resulted in excellent CEC analysis of bases on reversed-phase packing materials. The same approach was applied to the analysis of bases on the silica phase in order to improve peak shape. Excellent chromatography was obtained for the analysis of strong pharmaceutical bases such as benzylamine, nortriptyline and diphenhydramine. The experimental investigations have shown that the CEC separation of a range of pharmaceutical bases can routinely be achieved with excellent peak shapes and peak efficiencies as high as 320,000 plates m⁻¹.     

Differences between retentions of various classes of aromatic hydrocarbons in reversed-phase high-performance liquid chromatography. Implications of using retention data for characterizing hydrophobicity

Capacity factors of a series of alkylbenzenes (C1-C10), 12 chlorobenzenes, 9 chlorotoluenes, 17 chloronaphthalenes and 65 chlorobiphenyls have been measured on an octadecylsilica column. Aqueous methanol of four different compositions (80-95% methanol) was used as eluent. Logarithms of capacity factors of all eluites are linearly related to the amount of organic modifier in the eluent. In addition, linear relationships between the solvent strength and the logarithms of capacity factors extrapolated to zero methanol have been revealed. The proportionality factors are dependent on the structures of the eluites. Thermodynamic consideration of the retention processes shows that, within each type of eluite, enthalpy-entropy compensation is found. The compensation temperatures are not significantly different for the various types of eluite. Furthermore, it is shown that the compensation temperatures increase with increasing water content of the eluent. Since the intercepts of the delta G0-delta H0 plots are not equal for the various types of eluite, it was concluded that the distribution processes causing retention of benzene, naphthalene and biphenyl are different. When only the free energies of retention (i.e. the capacity factors) of different types of eluite are compared, no accurate information on the hydrophobicity of the eluites can be obtained if aqueous methanol is used as eluent. Therefore the possibilities for relating or predicting other physico-chemical parameters of the test compounds, such as octan-1-ol-water partition coefficients with isocratic retention data, will be limited.     

Capillary electrochromatography with zwitterionic stationary phase on the lysine-bonded poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic capillary column

A polymer-based neutral monolithic capillary column was prepared by radical polymerization of glycidyl methacrylate and ethylene dimethacrylate in a 100 mum id fused-silica capillary, and the prepared monolithic column was subsequently modified based on a ring opening reaction of epoxide groups with 1 M lysine in solution (pH 8.0) at 75 degrees C for 10 h to produce a lysine chemically bonded stationary phases in capillary column. The ring opening reaction conditions were optimized so that the column could generate substantial EOF. Due to the zwitterionic functional groups of the lysine covalently bonded on the polymer monolithic rod, the prepared column can generate cathodic and anodic EOF by varying the pH values of running buffer during CEC separation. EOF reached the maximum of -2.0 x 10(-8) m2v(-1)s(-1) and 2.6 x 10(-8) m2v(-1)s(-1) with pH of the running buffer of 2.25 and 10, respectively. As a consequence, neutral compounds, ionic solutes such as phenols, aromatic acids, anilines, and basic pharmaceuticals were all successfully separated on the column by CEC. Hydrophobic interaction is responsible for separation of neutral analytes. In addition, the electrostatic and hydrophobic interaction and the electrophoretic migration play a significant role in separation of the ionic or ionizable analytes.     

Charge effects during surface analysis of poorly conducting inorganic materials

Summary The properties of surfaces and interfaces often determine the behaviour of materials in a given application: typical examples are all kinds of corrosion, segregation, sensor activity, wear and friction, adhesion of coatings, joining processes, sintering, etc. It is the aim of this paper to provide materials scientists with a better understanding of the limits of applying modern surface analysis methods (SIMS, AES, etc.) to materials with low room temperature electrical conductivity like most ceramics and glasses. One feature common to all methods deserves special attention: this is the influence of surface and near surface charges on the distribution of mobile ionic species in the near surface region of the sample to be analyzed. The origin of these charges and their order of magnitude as a function of the experimental parameters can be deduced from charge balance considerations: the main result is that self-compensating methods should be most favorable.     

On the bonding in doubly charged diatomics

Summary The potential energy of interacting atomic ions A⁺+B⁺ often shows a shallow local minimum separated by a broad potential barrier from the dissociation products at much lower energy. Early interpretations of dication potential shapes were based on the similarity of the electronic structure between isoelectronic neutral and ionic species and led to a picture of a chemical bond superimposed on a repulsive Coulomb potential. More recently, barriers in dication potentials have commonly been interpreted as avoided curve crossings involving covalent and ionic structures. In this paper, we demonstrate that the former model is the appropriate one except in cases with very small asymptotic ionic/covalent energy splittings. By deriving dication wavefunctions from their neutral isoelectronic counterparts, we obtain upper bound dication potential curves which show all the characteristic features. By further modeling induction effects, we arrive at an almost quantitative fit of accurateab initio dication potentials. The chemical bond plus electrostatic repulsion interpretation of dication interactions also explains why the accurate calculation of potential curves appears to be much more demanding for dications than for isoelectronic neutrals.Dedicated in the honor of Prof. Klaus Ruedenberg     

Separation and determination of biphenyl nitrile compounds by microemulsion electrokinetic chromatography with mixed surfactants

A mixture of six biphenyl nitrile compounds and three related substances with high hydrophobicity and similar structures was successfully separated by microemulsion electrokinetic chromatography (MEEKC) within 30 min. The microemulsion system contained 100 mM sodium dodecyl sulfate (SDS), 80 mM sodium cholate (SC), 0.81% v/v heptane, 7.5% v/v n-butanol, 10% v/v acetonitrile, and 10 mM borate. The addition of SC, organic modifiers, sample preparation, and temperature all showed remarkable effects on the separation. The capacity factor (k) was calculated by using dodecyl benzene as the marker for microemulsion, and the calculated partition coefficient log P(o/w) of the solutes was in the range of 3.35-7.38. The log k values matched well with the log P(o/w) with a correlation coefficient of 0.96. In addition, the linear correlation coefficients of each compound between peak area and concentration were from 0.996 to 0.998 with the repeatability RSD value < 1.2% for migration time and < 4.8% for peak area, and the highest theoretic plate number was > 586000. MEEKC was compared with micellar electrokinetic chromatography (MEKC) indicating that the former method is more suitable for this separation and can be used for the quality control of biphenyl nitrile compounds in the synthesis of liquid crystals.     

Modeling of retention behavior in capillary electrochromatography from chromatographic and electrophoretic data

A phenomenological approach was presented to describe the retention behaviors of solutes in capillary electrochromatography (CEC). Equations for calculation of the retention time and actual chromatographic retention factor for ionic solutes, weak monoprotic acid and weak monoprotic base were derived, which can be described by two general expressions regardless the charge status of the solute. The general expressions enable calculation of the retention time and retention factor in CEC from chromatographic and electrophoretic data, which were experimentally verified with a variety of compounds and a variety of CEC modes. Based on this approach, the chromatographic retention and the electrophoretic migration in the CEC systems studied were found to be two independent processes. The validity of this approach was also discussed.     

Fast Separation of Nucleosides by Capillary Electrochromatography on Non‐Endcapped Phenyl‐Bonded Silica Phase Using Short‐End Injection Method

The influence of several experimental parameters (pH, ionic strength, organic modifier content of hydro‐organic buffer) upon EOF, migration time, and retention factor has been studied in CEC with a phenyl‐bonded silica column on a model mixture of five nucleosides. This paper illustrates the current interest in CEC as a method of resolving complex mixtures of neutral and ionic solutes and demonstrates the potential of the short‐end injection method as a means of reducing analysis time.     

Capillary Electrochromatography of Pyrimidine Derivatives Using UV and Mass Spectrometric Detection

 The separation of pyrimidine derivatives by capillary electrochromatography (CEC) using either UV or mass spectrometric detection is described. For UV detection an aqueous phosphate carrier electrolyte containing acetonitrile is employed. The results are compared to the analysis of the same compounds by micellar electrokinetic chromatography in terms of selectivity, migration times, linearity, and detection limits. For the combination of CEC and mass spectrometry (MS) an inexpensive way to couple commercially available instruments is presented; the interface consists of an electrically grounded stainless steel connector (containing a stainless steel frit) serving as the electrode and coupling the CEC capillary with a fused silica transfer capillary to the MS instrument. Alternatively, a PEEK adapter combining the CEC capillary and a grounded stainless steel transfer capillary serving as the electrode is employed. To avoid the formation of hydrogen gas at the coupling piece or the transfer capillary, p-benzoquinone is added to the carrier electrolyte consisting of aqueous ammonium acetate and acetonitrile.     

Influence of temperature on the behaviour of small linear peptides in capillary electrochromatography

The influence of temperature, T, on the retention times, peak widths, peak symmetry coefficients and theoretical plate numbers of two small linear peptides, [Met5]enkephalin and [Leu5]enkephalin, has been studied with capillary electrochromatography (CEC) capillary columns of 100 microm I.D. and 250 mm packed length with a total length of 335 mm, containing 3 microm Hypersil n-octadecyl bonded silica. With increasing column temperature from 15 to 60 degrees C, the electroosmotic flow (EOF) and the column efficiencies increased, whereas the retention coefficients (Kcec) of both peptides decreased. A linear relationship was found between the EOF value and the square root of the temperature over this temperature range, with a linear regression correlation of 0.998. Non linear Van 't Hoff plots (In Kcec versus 1/T) were observed for these peptides between 15 and 60 degrees C, suggesting that a phase-transition occurred with the n-octadecyl chains bonded on the silica surface, affecting the CEC retention behaviour of these peptides. In CEC systems, the Kcec values of peptides incorporate contributions from both electrophoretic migration and chromatographic retention. Positive and negative Kcec values can, in principle, thus arise with these charged analytes. However, the Kcec values of the enkephalin peptides under all temperature conditions studied were positive with an eluent composed of water-50 mM NH4OAc/AcOH, pH 5.2-acetonitrile (5:2:3, v/v) and therefore the chromatographic component dominates the retention process with these small peptides under these conditions.     

Mass analysis in islands of stability with linear quadrupoles with added octopole fields

Mass analysis with linear quadrupole mass filters is possible by forming "islands" in the stability diagram with auxiliary quadrupole excitation. In this work, computer simulations are used to calculate stability boundaries, island positions, and peak shapes and ion transmission for mass analysis with linear quadrupole mass filters that have added octopole fields of about 2 to 4%. Rod sets with exact geometries that have quadrupole and octopole fields only in the potential, and round rod sets, with multipoles up to N = 10 (the twenty pole term) included in the calculations, show the same stability boundaries, island positions, and peak shapes. With the DC voltage applied to the rods so that the Mathieu parameter a < 0, conventional mass analysis is possible without the use of an island. With the DC polarity reversed so that a > 0, the resolution and transmission are poor preventing conventional mass analysis. In principle, mass analysis in an island is possible with operation at either of two tips. Provided the correct island tip is chosen for mass analysis, peak shapes comparable to those with a > 0 and no excitation are possible, both with a > 0 and with a < 0. In the latter case, the use of an island of stability allows mass analysis when the added octopole otherwise prevents conventional mass analysis.