Separation of peptides by pressurized capillary electrochromatography

A pressurized electrochromatography (pCEC) instrument with gradient capability was used in this work for separation of peptides. Three separation modes, namely, pCEC, high-performance liquid chromatography and capillary electrophoresiscan be carried out with the instrument. In pCEC mode, the mobile phase is driven by both electroosmotic flow and pressurized flow, facilitating fine-tuning in selectivity of neutral and charged species. A continuous gradient elution can be carried out conveniently on this instrument, which demonstrates that it is more powerful than isocratic pCEC for separation of complicated samples. The effects of applied voltage, supplementary pressure and ion-pairing agents on separation of peptides in gradient pCEC were investigated. The effects of flow-rate of the pump and the volume of the mixer on resolution were also evaluated.     

Retention behaviour of peptides in capillary electrochromatography using an embedded ammonium in dodecacyl stationary phase

A highly water-soluble new cyclodextrin (CD) derivative 2-O-acetonyl-2-O-hydroxypropyl-beta-CD (2-AHP-beta-CD) was synthesized and tested as an effective chiral selector for the capillary zone electrophoretic resolution (Rs) of several basic and acidic analytes. The primary purpose of the research was to explore the capability of the 2-AHP-beta-CD as chiral selectors on comparison with the neutral CDs such as beta-CD, DM-beta-CD and HP-beta-CD. Substitution with 2-O-acetonyl-2-O-hydroxypropyl group at the secondary hydroxyl sites of the CD is aimed at influencing the magnitude and selectivity of analyte-CD interactions. The chiral resolution was strongly influenced by the concentration of the CDs and buffer pH. 2-AHP-beta-CD showed the best enantiomer resolution properties among the tested compounds, while the other CDs showed inferior or no performances at all.     

High temperature and temperature programming in capillary electrochromatography

In electrochromatography, solvent electrophoretic mobility and solute partitioning are temperature dependent processes. If temperature variations are controlled, solute selectivity and analysis times can be tailored. In this study the feasibility of temperature programming in capillary electrochromatography (CEC) was demonstrated using a reversed-phase CEC mode. The outcome of programmed separations was compared with isothermal, isocratic and isorheic (constant flow) separations. The combined effects of column temperature and mobile phase flow-rate changes during the separation run, resulted in up to a 50% reduction in the separation run time, without adversely affecting the quality of separation. For capillary electrochromatography, temperature programming may be a valuable alternative to solvent programming modes because of the great technical difficulties associated with carrying out solvent gradient elution.     

Rapid separation of peptides and proteins by isocratic capillary electrochromatography at elevated temperature

The use of capillary electrochromatography (CEC) for the separation by isocratic elution of synthetic peptides, proteins as well as the tryptic digest of cytochrome c has been demonstrated. The monolithic porous stationary phase was prepared from silanized fused-silica capillaries of 75 microm I.D. by in situ copolymerization of vinylbenzyl chloride and ethylene glycol dimethacrylate in the presence of propanol and formamide as the porogens. The chloromethyl groups at the surface of the porous monolith were reacted with N,N-dimethylbutylamine to form a positively charged chromatographic surface with fixed n-butyl chains. Results of studies on the influence of temperature and mobile phase composition on the retention and selectivity of separation by CEC demonstrated the feasibility of rapid polypeptide analysis and tryptic mapping at elevated temperature with high resolution and efficiency. Typically the chromatography of a tryptic digest of cytochrome c took about 5 min at 55 degrees C and 75 kV/m with hydro-organic mobile phases containing acetonitrile in 50 mM phosphate buffer, pH 2.5. For peptides and proteins plots of logarithmic k'cec against acetonitrile concentration were nonlinear, whereas Arrhenius plots for the mobilities were nearly linear. Comparison of the separation of such samples under conditions of CEC and capillary zone electrophoresis (CZE) indicates that the mechanism of separation in CEC is unique and leads to a chromatographic profile different from that obtained by CZE.     

Separation of peptides by strong cation-exchange capillary electrochromatography

Separation of small peptides on ion-exchange capillary electrochromatography (IE-CEC) with strong cation-exchange packing (SCX) as stationary phase was investigated. It was observed that the number of theoretical plates for small peptides varied from 240000 to 460000/m, and the relative standard deviation for t0 and the migration time of peptides were less than 0.57% and 0.27%, respectively for ten consecutive runs. Unusually high column efficiency has been explained by the capillary electrophoretic stacking and chromatofocusing phenomena during the injection and separation of positively charged peptides. The sample buffer concentration had a marked effect on the column efficiency and peak area of the retained peptides. The influences of the buffer concentration and pH value as well as the applied voltage on the separation were investigated. It has been shown that the electrostatic interaction between the positively charged peptides and the SCX stationary phase played a very important role in IE-CEC, which provided the different separation selectivity from those in the capillary electrophoresis and reversed-phase liquid chromatography. A fast separation of ten peptides in less than 3.5 min on IE-CEC by adoption of the highly applied voltage was demonstrated.     

Joule heating and determination of temperature in capillary electrophoresis and capillary electrochromatography columns

This article reviews the progress that has taken place in the past decade on the topic of estimation of Joule heating and temperature inside an open or packed capillary in electro-driven separation techniques of capillary electrophoresis (CE) and capillary electrochromatography (CEC), respectively. Developments in theoretical modeling of the heat transfer in the capillary systems have focused on attempts to apply the existing models on newer techniques such as CEC and chip-based CE. However, the advent of novel analytical tools such as pulsed magnetic field gradient nuclear magnetic resonance (NMR), NMR thermometry, and Raman spectroscopy, have led to a revolution in the area of experimental estimation of Joule heating and temperature inside the capillary via the various noninvasive techniques. This review attempts to capture the major findings that have been reported in the past decade.     

Modelling of migration behaviour of inorganic anions in ion-exchange capillary electrochromatography

A theoretical model to explain the observed mobility of inorganic anions in capillary electrochromatography (CEC) using ion-exchange (IE) stationary phases has been derived. The model divides contributions to the observed mobility of an analyte ion into capillary electrophoretic (CE) and IE components. The CE component includes the influence of varying the ionic strength of the background electrolyte on the electrophoretic mobility of the analyte, while the IE component accounts for the variation in retention of the analyte ion caused by changing the composition of the background electrolyte. The model was verified using a mixture of UV-absorbing inorganic ions in electrolytes of differing eluotropic strength in both packed and open-tubular CEC systems, with excellent agreement (r2 > 0.98) for both systems. Values of constants in the model equation determined by nonlinear regression were used to estimate the relative strengths of the interactions of different analytes with the stationary phase and these were found to agree well with elution orders observed in conventional IE chromatography.     

Chromatographic behaviour of peptides on a mixed-mode stationary phase with an embedded charged group by capillary electrochromatography and high-performance liquid chromatography

Retention behaviour of biological peptides was investigated on a stationary phase bearing an embedded quaternary ammonium group in a C21 alkyl chain by both high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). In HPLC experiments, variation of acetonitrile (ACN) content in the mobile phase showed that peptides are mainly separated by RP mechanism. The weak or negative retention factors observed as compared to C18 silica stationary phase suggested the involvement of an electrostatic repulsion phenomenon in acidic conditions. Comparison of HPLC and CEC studies indicated that (i) ion-exclusion phenomenon is more pronounced in HPLC and (ii) higher ACN percentage in mobile phase induce for some peptides an increase of retention in CEC, pointing out the existence of mechanisms of retention other than partitioning mainly involved in chromatographic process. This comparative study demonstrated the critical role of electric field on peptide retention in CEC and supports the solvatation model of hydrolytic pillow proposed by Szumski and Buszewski for CEC using mixed mode stationary phase in CEC.     

Theory of capillary electrochromatography

The present state of the theory of capillary electrochromatography (CEC) is reviewed. Emphasis is placed on electroosmosis and the electrical double layer, and the generally good understanding of the factors affecting the electroosmotic flow in CEC columns. The relation of CEC to other electrically driven separations are described, along with band broadening, and the influence of column temperature in CEC. The theoretical potential of CEC is assessed from the standpoint of current and future column technology, and likely future application areas are described.     

Instrumental developments in capillary electrochromatography

Summary An instrumental device for capillary electrochromatography (CEC) is described that permits conditioning of packed columns at pressures up to 100 bar, in or on-column fluorescence detection and electrokinetic sample injection. Capillaries with varying inner diameters (75, 100, 150 m) packed with octadecyl silica gel (3 m) are compared for efficiency and detection sensitivity. The influence of counter pressure on electroosmotic velocity and plate number is discussed.     

Applications of capillary electrochromatography

Applications performed by capillary electrochromatography (CEC) in all its modes, namely packed column CEC (packed-CEC), open tubular CEC (OT-CEC) and pressure-assisted CEC (pseudo-CEC), and published by June 1999 are reviewed. The review is divided into (i) separation of neutral, acidic and basic analytes with the main goal of evaluating column and system performance, (ii) separation according to field of application and/or chemical class, and (iii) separation of chiral analytes.     

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.     

Separation,detection and quantitation of peptides by liquid chromatography and capillary electrochromatography

This review discusses different liquid chromatographic and capillary electrochromatographic approaches to the separation and quantitation of peptides using silica-based and polymeric-based columns with emphasis on liquid chromatography. Mass spectrometry detection and quantitation of peptides using labeled and label-free procedures, will also be discussed, as well as the effect of amino acids’ properties on the solubility of peptides, an important parameter that influences the selection of the mobile phase. A discussion of different column packing materials, reversed-phase, cyclodextrins, macrocyclic antibiotics, porous graphitic carbon, mixed-phases, and normal-phase will be included, as well as a short discussion of multi-dimensional approaches for the separation of complex peptide mixtures.     

Preparation of capillary columns coated with linear polymer containing hydrophobic and charged groups for capillary electrochromatography

A linear polymer-coated capillary was prepared by in-capillary copolymerization of N-tert-butylacrylamide (TBAAm) with a charged monomer, 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), after the capillary pretreatment with a bifunctional reagent. The coated capillaries were applied in capillary electrochromatographic (CEC) separation of small neutral compounds. Hydrophobic groups in the linear polymer, which were immobilized onto the capillary surface, functioned as the stationary phase in reversed-phase CEC separation, and charged groups in the linear polymer generated electroosmotic flow (EOF) along the column. The coated capillaries were prepared by a simple procedure. Moreover, the reproducibility with respect to EOF rate and migration times of the solutes was excellent. The results for CEC separation of small molecules using the linear polymer-coated capillaries are presented.     

Recent progress in capillary electrochromatography

Capillary electrochromatography (CEC) continues to captivate many separation scientists. A remarkable activity is apparent from the numerous publications in the literature using CEC. A review of the most recent progress in CEC is presented herein, covering an extensive fraction of the literature on CEC published from the year 1997 until the beginning of 2000. Most of the recent developments have concentrated on column technology.     

Recent applications in capillary electrochromatography

The most recent and important applications in capillary electrochromatography (CEC) are summarized, covering literature published since May 2001. A selection of new developments in stationary phases for CEC is highlighted, and enantiomeric separations and chiral stationary phases are discussed. Also, CEC applications of biological molecules, pharmaceuticals, and applications in the field of industrial and environmental analysis are summarized. For this review three modes of CEC were taken into account, i.e., packed-column CEC, CEC using monolith technology, and open-tubular CEC.     

Recent applications in capillary electrochromatography

In this review research papers on the application of CEC are summarized that have been published between May 2003 and May 2005. First, a short overview is given of trends and developments in CEC that may increase the applicability of the separation technique. Next, application-oriented research using CEC is described in biochemical studies, including proteomics and genomics, in the analysis of food and natural products, and in pharmaceutical, industrial, and environmental analysis.