Electrochromatography with a 2.7 mm inner diameter monolithic column

Monolithic columns of 2.7 mm I.D. have been prepared and used in electrochromatography (EC) separation. Although capillary electrochromatography (CEC) has higher separation efficiency, it displays some shortcomings, such as limited sample loadability and restricted concentration detectability etc. In this paper, we investigate the feasibility of EC separation with millimeter diameter monolithic columns. By using a designed preparation method of monolithic column packed with about 150 microm quartz sand, the effect of Joule heating can be reduced, and the processes of frit making and column packing can be avoided. The concentration detectability of the EC is improved comparing with that of CEC. Moreover, the separation efficiency of 52,000 plates/m was achieved with a 70 mm length and 2.7 mm I.D. monolithic column.     

The impact of column inner diameter on chromatographic performance in temperature gradient liquid chromatography

The impact of column inner diameter on chromatographic performance in temperature gradient liquid chromatography has been investigated in the present study. Columns with inner diameters of 0.32, 0.53, 3.2 and 4.6 mm were compared with respect to retention and efficiency characteristics using temperature gradients from 30 to 90 degrees C with temperature ramps of 1, 5, 10 and 20 degrees C min(-1). The columns were all of 15 cm length and were packed with 3 microm Hypersil ODS particles. Alkylbenzenes served as model compounds, and the mobile phase consisted of acetonitrile-water (50:50, v/v). The study revealed that the column ID is not a critical limiting factor when performing temperature programming in LC, at least for columns narrower than 4.6 mm inner diameter in the temperature interval 30-90 degrees C. The retention times for all components on all columns were highly comparable, with similar peak profiles without any signs of peak splitting. The use of mobile phase pre-heating when using the larger bore columns was avoided by starting the temperature gradients close to ambient. However, the relative apparent efficiency was inversely proportional to column inner diameter, making the capillary columns generally more functional towards temperature gradients than the larger bore columns with respect to chromatographic efficiency. In addition, the capillary columns possessed higher robustness towards temperature programming than the conventional columns.     

Size-exclusion separation of proteins using a biocompatible polymeric monolithic capillary column with mesoporosity

Biocompatible poly(ethylene glycol methyl ether acrylate-co-polyethylene glycol diacrylate) monoliths were prepared for size exclusion chromatography (SEC) of proteins in the capillary format using Brij 58P in a mixture of hexanes and dodecanol as porogens. The monolithic columns provided size separation of four proteins in 20 mM sodium phosphate buffer (pH 7.0) containing 0.15 M NaCl, and there was a linear relationship between the retention times and the logarithmic values of the molecular weights. Compared to SEC monoliths previously synthesized using a triblock copolymer of polyethylene oxide and polypropylene oxide, an increase in mesoporosity was confirmed by inverse size exclusion chromatography. As a result, improved protein separation in the high molecular weight range and reduced column back-pressure were observed.     

Surfactant-bound monolithic columns for separation of proteins in capillary high performance liquid chromatography

A surfactant-bound monolithic stationary phase based on the co-polymerization of 11-acrylamino-undecanoic acid (AAUA) is designed for capillary high performance liquid chromatography (HPLC). Using D-optimal design, the effect of the polymerization mixture (concentrations of monomer, crosslinker and porogens) on the chromatographic performance (resolution and analysis time) of the AAUA–EDMA monolithic column was evaluated. The polymerization mixture was optimized using three proteins as model test solutes. The D-optimal design indicates a strong dependence of chromatographic parameters on the concentration of porogens (1,4-butanediol and water) in the polymerization mixture. Optimized solutions for fast separation and high resolution separation, respectively, were obtained using the proposed multivariate optimization. Differences less than 6.8% between the predicted and the experimental values in terms of resolution and retention time indeed confirmed that the proposed approach is practical. Using the optimized column, fast separation of proteins could be obtained in 2.5 min, and a tryptic digest of myoglobin was successfully separated on the high resolution column. The physical properties (i.e., morphology, porosity and permeability) of the optimized monolithic column were thoroughly investigated. It appears that this surfactant-bound monolith may have a great potential as a new generation of capillary HPLC stationary phase.     

N-methylimidazolium-functionalized monolithic silica column for mixed-mode chromatography

The development of mixed-mode stationary phase to achieve multiple separation capabilities in one column is very important for high performance liquid chromatography. In this paper, a new specific stationary phase based on grafting N-methylimidazolium to a monolithic silica column was successfully prepared for performing capillary liquid chromatography. The characteristics of the column were evaluated by the separation of different types of compounds including inorganic anions, aromatic acids, nucleotides, polycyclic aromatic hydrocarbons, alkylbenzenes, and phenols. The mechanisms for the separation of these compounds were investigated and appeared to involve the mixed interactions including anion-exchange, hydrophilic, π-π, dipole-dipole, and hydrophobic interactions.     

Effect of decreasing column inner diameter and use of off-line two-dimensional chromatography on metabolite detection in complex mixtures

Capillary liquid chromatography coupled with electrospray ionization to a quadrupole ion trap mass spectrometer was explored as a method for the analysis of polar anionic compounds in complex metabolome mixtures. A ternary mobile phase gradient, consisting of aqueous acidic, aqueous neutral and organic phases in combination with an aqueous compatible reversed-phase stationary phase allowed metabolites with a wide range of polarities to be resolved and detected. Detection limits in the full scan mode for glycolysis and tricarboxylic acid cycle intermediates were from 0.9 to 36fmol. Using this system, 111+/-9 (n=3) metabolites were detected in Escherichia coli lysate samples. Reducing column I.D. from 50 to 25microm increased the number of metabolites detected to 156+/-17 (n=3). The improvement in number of metabolites detected was attributed to an increase in separation efficiency, an increase in sensitivity, and a decrease in adduct formation. Implementation of a second separation mode, strong anion exchange, to fractionate the sample prior to capillary RPLC increased the number of metabolites detected to 244+/-21 (n=3). This improvement was attributed to the increased peak capacity which decreased co-elution of molecules enabling more sensitive detection by mass spectrometry. This system was also applied to islets of Langerhans where more significant improvements in metabolite detection were observed. In islets, 391+/-33 small molecules were detected using the two-dimensional separation. The results demonstrate that column miniaturization and use of two-dimensional separations can yield a significant improvement in the coverage of the metabolome.     

Optimization of St John's Wort flavonoid separation by reversed phase liquid chromatography on a silica-based monolithic column

Summary An optimization procedure for the separation of flavonoids from St John's Wort by reversed-phase high-performance liquid chromatography on monolithic stationary phase is described. Three-component mobile phase systems are studied using experimental design methodology. The starting experimental domain is a triangle, each vertex of which is a pure component. From preliminary isocratic experiments, truncations in the domain are performed leading to a quadrilateral shape working domain with no symmetry. To cope with both separation and analysis time, desirability functions are used. Optimal conditions are finally given by binary systems and the four flavonoids are separated in less than seven minutes.     

Fabrication of a poly(styrene-octadecene-divinylbenzene) monolithic column and its comparison with a poly(styrene-divinylbenzene) monolithic column for the separation of proteins

In this paper, a poly(styrene-octadecene-divinylbenzene) (PS-OD-DVB) monolithic column was prepared in one step by introducing a C18 carbon chain as monomer. N,N-Dimethylformamide and decanol served as porogens to make a homogeneous polymerization mixture in a fused silica capillary (320 microm inner diameter). Its physical and chromatographic properties were compared with those of poly(styrene-divinylbenzene) (PS-DVB) monolithic column, which was also fabricated by in-situ polymerization in a fused silica capillary with the same inner diameter. Six standard proteins were used to evaluate the columns and their potential application for the separation of human hemoglobin was also discussed. It was shown that the PS-OD-DVB and PS-DVB monoliths appeared to have similar efficiency for rapid separation of six proteins within 3.5 min. The PS-OD-DVB monolith was found to have higher loading capacity and higher resolution for the separation of alpha and beta chains of hemoglobin because of the introduction of C18 carbon chains, and shows great potential for the separation of bio-macromolecules.     

Comprehensive two-dimensional high performance liquid chromatography system with immobilized liposome chromatography column and monolithic column for separation of the traditional Chinese medicine Schisandra chinensis

A comprehensive two-dimensional (2D) separation is one that employs two separation dimensions (columns) and draws on all of the available resolving power from each of the dimensions of separate the components in a sample. In this study, a comprehensive 2D chromatography approach was developed for the separation and identification of membrane permeable compounds in a famous traditional Chinese medicine of Schisandra chinensis. The first dimensional column was the immobilized liposome chromatography (ILC) column, which mimics the biological membranes and can be used to study drug-membrane interactions in liquid chromatography. Using an automatic ten-port switching valve equipped with two sample loops, the section of the first-dimension was introduced in the second-dimension consist of a silica monolithic column. More than 40 components in Schisandra chinensis were resolved by using the developed separation system and among them 14 compounds were identified interacting with the ILC column based on their retention action, UV and mass data. With this comprehensive 2D-HPLC system, the three-dimensional chromatographic fingerprints of Schisandra chinensis were preliminarily established and processed by using principal component analysis and hierarchical clustering analysis. The obtained information can distinguish the unacceptable samples of the quality control. The result demonstrated that the 2D biochromatography system has been demonstrated to have more advantages of finding strong binding bioactive components, providing an enhanced peak capacity, good sensitivity and powerful resolution biological fingerprinting analysis of complex TCMs, which was a useful means to control the quality of and to clarify the membrane permeability of the compounds in Schisandra chinensis.     

Preparation of a monolithic column for weak cation exchange chromatography and its application in the separation of biopolymers

A procedure for the preparation of a monolithic column for weak cation exchange chromatography was presented. The structure of the monolithic column was evaluated by mercury intrusion. The hydrodynamic and chromatographic properties of the monolithic column--such as back pressures at different flow rates, effects of pH on protein retention, dynamic loading capacity, recovery, and stability--were determined under conditions typical for ion-exchange chromatography. The prepared monolithic column might be used in a relatively broad pH range from 4.0 to 12.0 and exhibited an excellent separation to five proteins at the flow rates of both 1.0 and 8.0 mL/min, respectively. In addition, the prepared column was first used in the purification and simultaneous renaturation of recombinant human interferon gamma (rhIFN-gamma) in the extract solution with 7.0 mol/L guanidine hydrochloride. The purity and specific bioactivity of the purified rhIFN-gamma in only one chromatographic step were obtained to be 93% and 7.8 x 10(7) IU/mg, respectively.     

Effect of column diameter on efficiency in capillary supercritical fluid chromatography

Fused silica capillary columns with internal diameters from 100 to 25 μm were coated with SE-54 and evaluated under supercritical fluid chromatographic conditions using carbon dioxide as mobile phase. Experimental results compared well with theoretical predictions. At ten times the optimum mobile phase velocity and for a capacity factor, k of 3, efficiencies of 2300 to 5600 plates m^?1 were obtained for column diameters of 100 to 25 μm, respectively.     

A novel monolithic column for capillary electrochromatographic separation of oligopeptides

A monolithic column was prepared using l-phenylalanine as template and a covalent approach through the formation of Schiff base with o-phthalaldehyde (OPA). OPA, allylmercaptan, l-phenylalanine, and triethylamine were stirred at first, then methacrylic acid, 2-vinylpyridine, ethyleneglycol dimethacrylate, α,α-azobisisobutyronitrile, and 1-propanol were added to the reaction mixture. The resulting material was introduced into a capillary column. Following thermal polymerization, the template was then extracted with a mixture of HCl and methanol. The column was employed for the capillary electrochromatographic separation of oligopeptides. A capillary column of 75 (50) cm × 75 μm ID with a mobile phase of phosphate buffer (pH 7.0, 40 mM)/methanol (5%, v/v), an applied voltage of +15 kV, and detection at 214 nm, could baseline separate angiotensin I, angiotensin II, [Sar¹, Thr⁸] angiotensin, oxytocin, vasopressin, tocinoic acid, β-casomorphin bovine, β-casomorphin human, and FMRF amide within 20 min. The separation behavior of the templated polymer was also compared with that of the non-templated polymer. As a result, it can be concluded that the electrochromatographic separation of this set of peptides was mediated by a combination of electrophoretic migration and chromatographic retention involving hydrophobic, hydrogen bonding, electrostatic as well as the Schiff base formation with OPA in the cavity of the templated polymer.     

Polydimethylsiloxane-functionalized monolithic silica column for reversed-phase capillary liquid chromatography

A polydimethylsiloxane (PDMS)-modified monolithic silica column was prepared for performing reversed-phase capillary liquid chromatography. The prepared PDMS column has a permeability of 6.4×10(-14) m(2) with a plate height <9.2 μm. Alkylbenzenes and polycyclic aromatic hydrocarbons (PAHs) were well separated with the PDMS stationary phase, which exhibited similar selectivity and separation mechanism to that of octadecyl stationary phase. The hydrophobic interactions between the analytes and the PDMS stationary phase mainly play the roles for the separation of alkylbenzenes and PAHs. The characteristics of the PDMS column for the separation of alkylbenzenes and PAHs demonstrated that it would be a promising alternative to the octadecyl column.     

Novel zwitterionic polyphosphorylcholine monolithic column for hydrophilic interaction chromatography

A novel porous zwitterionic monolith was prepared by thermal co-polymerisation of 2-methacryloyloxyethyl phosphorylcholine (MPC) and ethylene glycol dimethacrylate (EDMA) within 100 μm I.D. capillaries. Mercury intrusion porosimetry, scanning electron microscopy (SEM), micro-HPLC (μ-HPLC), elemental analysis and ζ-potential analysis were used to evaluate the monolithic structure. No evidence of swelling or shrinking of the monolith in different polarity solvents was observed. A typical hydrophilic liquid chromatography (HILIC) mechanism was observed at high organic solvent content (acetonitrile >60%). The phosphorylcholine (PC) functionality has both a positively charged quaternary ammonium and a negatively charged phosphate group. For charged analytes, a weak electrostatic interaction was also observed by studying the influence of mobile phase pH and salt concentration on their retentions on the poly(MPC-co-EDMA) monolithic column. The optimised poly(MPC-co-EDMA) monolith showed very good selectivities for a range of polar test analytes, especially small peptides. This might be ascribed to the good biocompatibility of PC functionality. At low organic solvent content, baseline separation was also observed for a test mixture of seven alkylphenones by a reversed-phase separation mechanism.     

Influence of the diameter of monolithic capillary columns on their gas chromatography characteristics

Divinylbenzene polymer monolithic capillary columns were prepared on the basis of capillaries 0.01 to 0.53 mm in diameter. Separation properties of the columns were investigated with the use of a test mixture of light hydrocarbons. The permeability and C parameter in the Van Deemter equation were determined for all the columns. For the most part, the columns had similar characteristics: permeability was in the range (2.2 ± 0.2) × 10^?9 cm², with parameter C in the range (0.7 ± 0.2) × 10^?3 s (with n-butane as a sorbate). It was thus established that capillary diameter has only a slight effect on the efficiency of monolithic capillary columns (unlike packed capillary columns and microcolumns, whose properties, according to the literature data, depend strongly on the column diameter). The difference in properties between the narrowest monolithic column (capillary diameter 0.01 mm) and the others is explained by column overloading.     

Size separation of colloidally dispersed nanoparticles using a monolithic capillary column

We developed a method to separate colloidally dispersed nanoparticles on monolithic capillary columns. Silica nanoparticles were eluted according to their sizes, and the plots of the logarithm of the size of nanoparticles against their elution volume showed good linearity (r=0.992) over wide range of sizes. Because of the high porosity of the monolithic column (porosity; 88%), the column's length could be increased without clogging of the dispersed samples and the pressure in a long column (500 mm × 0.2 mm i.d.) was low, with a value of 5.8 MPa at a flow rate of 1 μL/min. We demonstrate that this method using monolithic capillary columns could be used as a powerful tool for size separation of nanometer-size materials, which will open a new pathway to quality control of nanomaterials in nanotechnology applications.     

Low-pressure gradient micro-ion chromatography with ultra-short monolithic anion exchange column

A 1.0 x 0.4 cm silica based monolithic anion exchanger has been produced and evaluated for use within a miniature gradient suppressed ion chromatography system based upon low-pressure micro-scale peristaltic pumps.