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.     

Electrochromatographic characterization of etched chemically-modified capillaries with small synthetic peptides

In this investigation, various capillary electrochromatographic (CEC) experiments have been employed to characterize the properties of etched, chemically-modified surfaces of open tubular capillary columns with peptides as solute probes and under conditions of variable voltage, temperature and solvent composition. The separation performance of etched capillaries with either n-octadecyl or liquid crystal moieties derived from a cholesterol phase bonded to the surface were compared. With the liquid crystal bonded species, interesting and significantly different variations in retention behavior of peptides are obtained compared to those observed with the corresponding n-octadecyl modified surfaces by changes in temperature, solvent composition and field strength. These peptide separations illustrate the usefulness of this CEC approach for practical applications, where both the retention characteristics of the charged analytes as well as the selectivity differences due to the surface properties of the etched chemically-modified surfaces of open tubular capillary columns can be rationally modulated. As in HPLC, appropriate choice of CEC experimental variables, including the chemical properties of the immobilized ligand(s), represents a powerful tool for optimizing resolution.     

Impact of pore structural parameters on column performance and resolution of reversed-phase monolithic silica columns for peptides and proteins

In this work, monolithic silica columns with the C4, C8, and C18 chemistry and having various macropore diameters and two different mesopore diameters are studied to access the differences in the column efficiency under isocratic elution conditions and the resolution of selected peptide pairs under reversed-phase gradient elution conditions for the separation of peptides and proteins. The columns with the pore structural characteristics that provided the most efficient separations are then employed to optimize the conditions of a gradient separation of a model mixture of peptides and proteins based on surface chemistry, gradient time, volumetric flow rate, and acetonitrile concentration. Both the mesopore and macropore diameters of the monolithic column are decisive for the column efficiency. As the diameter of the through-pores decreases, the column efficiency increases. The large set of mesopores studied with a nominal diameter of approximately 25 nm provided the most efficient column performance. The efficiency of the monolithic silica columns increase with decreasing n-alkyl chain length in the sequence of C18相似文献   

In-line coupling of low-pressure short capillary electrochromatography columns to electrospray ionisation mass spectrometry

A new in-house designed and constructed injection valve for capillary electrochromatography (CEC) based on a rotating injection part with compartments for the eluent as well as for the sample has been coupled to a mass spectrometer via a sheath flow electrospray ionisation (ESI) interface, using short capillary columns of 15 cm length. The CEC columns were packed with 3 microm C(18) bonded silica particles, and a mixture of peptides was analysed using an ammonium acetate/acetonitrile eluent. A significant increase in the signal-to-noise ratio was obtained when the peptides were dissolved in water with the same content of organic modifier as in the eluent with an addition of 0.5% (v/v) acetic acid. When the CEC analysis was performed without any additional pressure, the separation current sometimes dropped tremendously due to bubble formation, caused by different permeability in the first and packed part of the column causing an extremely low electroosmotic flow. The separation current was restored to its original value by applying only 7 bar at the inlet of the CEC column, and the separation performance for the test peptides was recovered. A comparison of the CEC performance of peptides in pure CEC mode and in low-pressure CEC mode is reported.     

Liquid chromatography of benzene derivatives on a porous methacrylate copolymer containing epoxy groups

Summary The retention of benzene derivatives with nonpolar and polar substituents on a porous methacrylate copolymer containing epoxy groups using both nonpolar and polar eluents was investigated. When n-hexane is used as the eluent, the retention of n-alkylbenzenes and polymethylbenzenes is weaker than that of benzene. In the case of benzene derivatives containing polar functional groups their capacity ratios (k) on this porous polymer increases approximately linearly with the increase of the adsorbate molecules dipole moment. The retention characteristics of the methacrylate copolymer were compared with that of a styrene-divinylbenzene copolymer and silica gels with a hydroxylated surface and with a surface modified by chemically bonded alkylsilyl groups.     

Practical aspects of using methacrylate-ester-based monolithic columns in capillary electrochromatography

Methacrylate-ester-based monoliths containing quaternary ammonium groups were prepared in situ in capillary columns and in simultaneous experiments in vials, employing thermal initiation. The chromatographic properties of the monoliths were determined with capillary electrochromatography (CEC), and their morphology was studied with mercury-intrusion porosimetry on the bulk materials. Materials with different, well repeatable pore-size distributions could be prepared. A satisfactory column-to-column and run-to-run repeatability was obtained for the electro-osmotic mobility, the retention characteristics (k-values) and the efficiency on the columns prepared and tested in the CEC mode. A relatively high electro-osmotic flow was observed in the direction of the positive electrode. The electro-osmotic mobility was found to be influenced only marginally by mobile-phase parameters such as the pH, ionic strength, and acetonitrile content. The retention behavior of the monolithic columns was similar to that of columns packed with C18-modified silica particles. Columns could be prepared with optimum plate heights ranging from 6 microm for unretained compounds to 20 microm for well retained (k=2.5) polyaromatic hydrocarbons. However, for specific analytes a - still unexplained - lower chromatographic column efficiency was observed.     

Capillary electrochromatography/nanoelectrospray mass spectrometry for attomole characterization of peptides

The successful coupling of capillary electrochromatography (CEC) to an ion trap mass spectrometer via a nanoelectrospray interface (nESI) is described. Using a conductively coated tip butted to the end of a CEC column, it was possible to obtain a stable spray without any sheath liquid being employed. Selected small peptides were separated with CEC columns (100 microm i.d./25 cm long) packed with 3 microm Hypersil C8 or C18 bonded silica particles with an eluent composed of ammonium acetate/acetonitrile. Peptide mixtures of desmopressin, peptide A, oxytocin, carbetocin and [Met(5)]-enkephalin were detected in the mid-attomole range, which is the lowest amount analyzed using CEC combined with MS detection. It was also observed that sensitivity can be compromised at higher separation voltages. We demonstrate that CEC/nESI-MS, at the current stage of development, represents one of the most sensitive systems for peptide analysis.     

A glycopeptide antibiotic chiral stationary phase for the enantiomer resolution of hydroxy acid derivatives by capillary electrochromatography

Separation of hydroxy acid enantiomers was achieved by using capillary electrochromatography (CEC) employing a chiral stationary phase (CSP) based on MDL 63,246 (Hepta-Tyr), a macrocyclic antibiotic of the teicoplanin family. The chiral selector was chemically bonded to 5 num diol-modified silica particles and the CSP mixed with amino silica (3:1 w/w) was packed into a 75 num ID fused-silica capillary. The CEC experiments were carried out by using an aqueous reversed-phase mode for the enantiomeric resolution of hydroxy acid compounds. Good enantioresolution was achieved for mandelic acid (MA), m-hydroxymandelic acid (m-OH-MA), p-OH-MA, and 3-hydroxy-4-methoxymandelic acid (3-OH-4-MeO-MA). The CEC system was less enantioselective towards 2-phenyllactic acid (2-PhL) and 3-PhL while mandelic acid methyl ester (MA-Et-Est) enantiomers were not resolved. Several experimental parameters, such as organic solvent type and concentration, buffer pH, capillary temperature, on enantioresolution factor, retention time, and retention factor were studied.     

Fast methods for screening of trichothecenes in fungal cultures using gas chromatography-tandem mass spectrometry

The excess adsorption isotherms of acetonitrile, methanol and tetrahydrofuran from water on reversed-phase packings were studied, using 10 different columns packed with C1-C6, C8, C10, C12, and C18 monomeric phases, bonded on the same type of silica. The interpretation of isotherms on the basis of the theory of excess adsorption shows significant accumulation of the organic eluent component on the adsorbent surface on the top of "collapsed" bonded layer. The accumulated amount was shown to be practically independent of the length of alkyl chains bonded to the silica surface. A model that describes analyte retention on a reversed-phase column from a binary mobile phase is developed. The retention mechanism involves a combination of analyte distribution between the eluent and organic adsorbed layer, followed by analyte adsorption on the surface of the bonded phase. A general retention equation for the model is derived and methods for independent measurements of the involved parameters are suggested. The theory was tested by direct measurement of analyte retention from the eluents of varied composition and comparison of the values obtained with those theoretically calculated values. Experimental and theoretically calculated values are in good agreement.     

Chiral separation of amides of amino acid on a (S)‐N‐(3,5‐dinitrobenzoyl)leucine‐N‐phenyl‐N‐propylamide‐bonded silica using nonaqueous capillary electrochromatography

(S)‐N‐(3,5‐dinitrobenzoyl)leucine‐N‐phenyl‐N‐propylamine‐bonded silica was used as a chiral stationary phase for separation of a set of racemic π‐acidic and π‐basic α‐amino acid amides in electrolyteless ACN‐water eluents by CEC in the RP and polar organic (PO) modes. The effect of the amount of water in the ACN‐water eluent on chiral separation was examined. As water is added to ACN, retention was shortened but resolution and selectivity deteriorated severely. Retention, enantioselectivity, and resolution factors obtained in 100% ACN were compared with those in an n‐hexane‐isopropanol eluent with a small amount of water by normal phase (NP) CEC. Much shorter retention times with comparable enantioselectivities were observed with 100% ACN, demonstrating the advantage of separation on (S)‐N‐(DNB)leucine‐N‐phenyl‐N‐propylamine‐bonded silica in PO‐CEC over NP‐CEC.     

Effects of Blending Bare-Silica and Reversed-Phase on Retention of Neutral Compounds in Capillary Electrochromatography

Most commercially available instruments for capillary electrochromatography (CEC) have a fixed configuration and lack the flexibility to use shorter columns. Applying a blended stationary phase (a phase consisting of a given ratio of bare silica and reversed phase material) can simulate columns of different length in CEC. The goal of this work was to examine the effect of the degree of blending of reversed-phase columns (with bare silica) on the speed of the separation of neutral compounds in CEC. Optimum column packing mixture was determined from the variation of the solute retention factors as a function of the ratios of blending of reversed-phase and bare silica. By adjusting the column composition, solute retention factors and the analysis run time were halved when compared to a pure reversed-phase column of the same length. Stationary phase blending can be considered as an additional parameter to mobile phase variation, column temperature and applied electric field for the optimization of selectivity and analysis time. By adjusting the stationary phase composition, mobile phase composition, column temperature and applied electric field, the analysis run time of neutral components was decreased more than 75% when compared to a separation obtained on neat reversed-phase column of the same dimensions. The linear dependence of the retention factors as a function of the blend ratio (reversed phase/bare silica) offers a framework for designing a “blended” packed capillary column for CEC separations.     

Comparison of the efficiency of microparticulate and monolithic capillary columns

A comparison is made between the efficiency of microparticulate capillary columns and silica and polymer-based monolithic capillary columns in the pressure-driven (high-performance liquid chromatography) and electro-driven (capillary electrochromatography) modes. With packed capillary columns similar plate heights are possible as with conventional packed columns. However, a large variation is observed in the plate heights for individual columns. This can only be explained by differences in the quality of the packed bed. The minimum plate height obtained with silica monolithic capillary columns in the HPLC mode is approximately 10 microm, which is comparable to that of columns packed with 5-microm particles. The permeability of wide-pore silica monoliths was found to be much higher than that of comparable microparticulate columns, which leads to much lower pressure drops for the same eluent at the same linear mobile phase velocity. For polymer-based monolithic columns (acrylamide, styrene/divinyl benzene, methacrylate, acrylate) high efficiencies have been found in the CEC mode with minimum plate heights between 2 and 10 microm. However, in the HPLC mode minimum plate heights in the range of 10 to 25 microm have been reported.     

丙基脲硅胶毛细管整体柱的制备及性能研究

采用溶胶-凝胶技术在毛细管中原位合成硅胶整体柱,通过表面化学修饰技术制备了极性的丙基脲硅胶整体柱.对所制备的整体柱柱性能进行了评价,考察了极性物质在该整体柱上的保留行为,并对其可能的保留机理进行了探讨.研究表明,该柱在亲水作用电色谱模式下能有效分离苯酚类极性小分子化合物.     

Comparison of separation behavior of benzodiazepines in packed capillary electrochromatography and open-tubular capillary electrochromatography

Packed column capillary electrochromatography (CEC), open-tubular CEC and microcolum liquid chromatography (LC) using a cholesteryl silica bonded phase have been studied to compare the retention behavior for benzodiazepines. It has been found that packed column CEC gives better resolution, faster analysis time than microcolumn LC for benzodiazepines maintaining similar selectivity except for some solutes which are charged species under the separation conditions. However, open-tubular CEC gave different selectivities to a larger extent for charged benzodiazepines from that which should be produced by the chromatographic properties of the cholesteryl silica phase. Charged species migration times are mainly influenced by electrophoretic mobility rather than the chromatographic interactions.     

Capillary electrochromatography with novel stationary phases. IV. Retention behavior of glycosphingolipids on porous and non-porous octadecyl sulfonated silica

In this investigation, capillary electrochromatography (CEC) with a novel stationary phase proved useful for the separation of neutral and acidic glycosphingolipids (GSLs). Four different gangliosides, namely G(M1a), G(D1a), G(D1b) and G(T1b), served as the acidic GSLs model solutes. The following four GSLs: galactosylceramide (GalCer), lactosylceramide (LacCer), globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) served as the typical neutral GSLs. The stationary phase, octadecyl sulfonated silica (ODSS), consisted of octadecyl functions bonded to a negatively charged layer containing sulfonic acid groups. Porous and non-porous ODSS stationary phases were examined. The retention behavior of the acidic and neutral GSLs was examined over a wide range of elution conditions, including the nature of the electrolyte and organic modifier and the pH of the mobile phase. The porous ODSS stationary phase yielded the separation of the four different gangliosides using a hydro-organic eluent of moderate eluent strength whereas the non-porous ODSS stationary phase permitted the separation of the four neutral GSLs with a mobile phase of relatively high eluent strength.     

Capillary electrochromatography on silica columns: factors influencing performance

Much capillary electrochromatography (CEC) work is carried out on bonded silica packings which offer many advantages: the number of such packings which are available; the fact that the chemistry of bonding and the separation process are fairly well understood; and the possibility of the transfer to CEC of existing HPLC methods. Packing methods for the preparation of CEC columns have been investigated. The problems inherent in the use of burned-in frits remains an obstacle, but can be at least partially overcome by minimising the length and by silanisation. The influence of a variety of mobile phase variables on aspects of CEC is in agreement with theory for: ionic strength, organic content (including isoeluotropy), and pH. Temperature can be used as a variable to change column selectivity in CEC. The influence of pH on electroosmotic flow (EOF) by changing the degree of ionisation of residual silanol groups is similar for a wide range of neutral bonded groups, but is much less marked for bonded sulphonic acid groups. The EOF may be reversed for bonded groups containing nitrogen.     

Separation of carbohydrates by liquid chromatography on silica gel,adding adsorption modificators to the eluent

Summary For the separation of carbohydrates by liquid chromatography, utilizing the intermolecular interaction with amino groups on the surface of the adsorbent, two types of adsorbents were used: silica gel modified by adding to the eluent a diamine, and a chemically bonded phase prepared by the reaction of -aminopropyl triethoxy silane with silica gel. Mono- di- and trisaccharides could be separated on silica gel modified by the adsorption of piperazin and ethylene diamine from the eluent. The separation capacity of columns packed with silica gel with bonded NH₂ groups and with silica gel having diamines adsorbed on its surface is similar. The retention volumes of 15 carbohydrates were measured on columns containing silica gel with hydroxylated surface and on silica gel modified with piperazin from the eluent consisting of acetone-water. Comparison of acetone-water and acetonitrile-water as the eluent showed that the former is also suitable for the analysis of carbohydrates.     

Development of standard operation procedures for the manufacture of n-octadecyl bonded silicas as packing material in certified reference columns for reversed-phase liquid chromatography

The development of standard operation procedures for the manufacture of a n-octadecyl bonded spherical silica packing from partially condensed tetraethoxysilane as silica source is described. The synthesis comprises five intermediate products and six synthesis steps which were examined according to their reproducibility and robustness. The results led to the optimisation of the manufacturing process for a n-octadecyl bonded silica. Correlations were drawn between the dynamic viscosity of the poly(ethoxy)siloxane (PES), the synthesis parameters, the resulting pore structural properties and particle size distribution of the silicas. Validated procedures were developed to manufacture spherical porous ultra-pure silicas with a specific surface area of 350 m2 g(-1) +/- 5% R.S.D., a specific pore volume of 1.0 ml (-1) +/- 3.7% R.S.D., an average pore diameter of 12.0 nm +/- 0.5% R.S.D. and an average particle diameter of 5 microm. Results are presented on trial batches and the final master batch which were both used as packing materials in reversed-phase liquid chromatography (RP-LC) columns. The latter columns were certified and accepted as an HPLC column as reference material (BCR-722) by the European Commission, Institute for Reference Materials and Measurements (IRMM), Geel, Belgium.     

Succinyl methacrylate polymerized in porous-layered phases for open-tubular capillary electrochromatography: Comparison with silica hydride monolayered phases

A polymer phase, which was constructed with butyl methacrylate (BMA), an ionizable monomer (mono-(2-(methacryloyloxy)ethyl) succinate (MES)), and a crosslinking agent (ethylene dimethacrylate), was first formed in a porous-layered open-tubular (PLOT) capillary. The PLOT capillary was characterized with SEM and electrophoretic flow as the pH level, ionic strength and addition of organic modifiers in the running buffers changed. In addition, a bare capillary and a silica hydride based capillary (SiH-MES), which bore a monolayered MES phase on it, were used to compared with the BMA-MES capillary. Besides optimizing the capillary electrochromatographic (CEC) conditions for each group of analytes, which were a mixture of nucleosides and thymine, flavonoids, and phenolic acids,comparison of the separation selectivity among analytes between the BMA-MES and SiH-MES capillaries was done according to the velocity and retention factors obtained from the CEC data. Overall, the polymeric phase formed in the PLOT mode was capable of preventing blockage of the columns and was superior to the monolayered phase bonding with the same ionizable ligands for application in CEC as well as to the bare silica phase in CE.     

Immobilisation and evaluation of a vancomycin chiral stationary phase for capillary electrochromatography

The macrocyclic antibiotic, vancomycin, is covalently bonded to LiChrospher diol silica packed columns and evaluated in capillary electrochromatography (CEC) both in the reversed-phase and polar organic mode. Initially, capillaries were packed with 5 microm LiChrospher 100 A diol silica and evaluated in CEC with a reversed-phase biphenyl-pyrene achiral test resulting in resolution and efficiency values of ca. 2.5 and 100000 plates meter(-1), respectively. Repeatability for this test (resolution and efficiency) was also examined and found to be acceptable for both run-to-run (n=5, 0.74% and 1.5%) and column-to-column (n=5, 3.4% and 9.0%), respectively. Similar results were obtained when the 10 microm LiChrospher 1000 A diol silica was examined with the exception of efficiency, where a reduced plate height value of four times lower was obtained compared to the 100 A material. A simple three step in-situ vancomycin immobilisation procedure was subsequently carried out on these packed diol columns. Selectivity was obtained for thalidomide enantiomers on this vancomycin chiral stationary phase in reversed-phase CEC with resolution and efficiency values of ca. 2.5 and 80000 plates meter(-1), with acceptable repeatability (n=8) 0.9% and 3.0%, respectively. Selectivity was also obtained for thalidomide enantiomers on this phase in the polar organic mode with resolution and efficiency values of ca. 2.5 and 120000 plates meter(-1), with acceptable repeatability (n=7) 0.9% and 2.0%, respectively. It was possible to deduce from a chemometric design carried out for evaluating the mobile phase component effects that organic modifier ratio, MeOH/MeCN, played a significant role in controlling both resolution and efficiency. It was also possible to separate a number of basic analytes including four beta-adrenergic blocking agents in the polar organic mode albeit with lower resolution and efficiency values, ca. 1.5 and 45000 plates meter(-1), respectively.