Eluotropic strength in non-aqueous liquid chromatography with porous graphitic carbon

Protein adsorption can be either endothermic or exothermic depending upon the protein, the sorbent and process conditions. In the case of protein adsorption onto ion-exchange surfaces exothermic adsorption heats are usually characterized as representing the electrostatic interaction between two oppositely charged surfaces. Endothermic adsorption heats are typically characterized as representing protein reconfiguration and/or repulsive interactions between adsorbed molecules. In certain segments of the literature surface dehydration and solution non-idealities have been suggested as possible sources of endothermic heats of adsorption. Each of these phenomena was investigated during studies concerning the adsorption of bovine serum albumin and ovalbumin onto an anion-exchange sorbent. The results demonstrated that electrostatic repulsive interactions between adsorbed molecules appears to be a larger contributor to endothermic heats of adsorption than surface dehydration or solution non-idealities. The presence of mobile phase cations can reduce the magnitude of endothermic adsorption heats by screening repulsive interactions between adsorbed molecules. Although water release was not found to be a major contributor to endothermic adsorption heats, it is likely to be a contributor to the entropic driving force associated with the adsorption of bovine serum albumin.     

Rapid analysis of cyanuric acid in swimming pool waters by high performance liquid chromatography using porous graphitic carbon

Summary An approach is presented for reducing analysis times of cyanuric acid in swimming pool waters by high performance liquid chromatography (HPLC). The HPLC method exploits the unique selectivity of porous graphitic carbon (PGC) to fully resolve cyanuric acid from other pool interferences within 10 min. By carefully timing the injections, multiple injections can be made before the end of the initial chromatographic run, more than doubling sample throughout. The method utilizes 95% of a 50 mM phosphate buffer solution (pH 9.1) and 5% methanol (v/v) with UV detection at 213 nm. This approach yielded run times rivaling those of the fastest methods using silica columns, and with the benefits of increased sensitivity.     

Solvent effects on the retention of oligosaccharides in porous graphitic carbon liquid chromatography

Porous graphitic carbon (PGC) is known as well suited adsorbent for liquid chromatography of carbohydrates. In this work we report on systematic investigations of solvent effects on the retention mechanism of fluorescence labeled malto-oligosaccharides on PGC. The adsorption mechanism was found to depend on the type of organic modifier used in the mobile phase. Positive adsorption enthalpies and entropies, which have already been reported in the literature, were solely produced using acetonitrile. Both alternative solvents (tetrahydrofuran, 2-propanol) yielded in contrast negative enthalpies. As plausible retention mechanism for oligosaccharides on PGC applying acetonitrile as mobile phase component we propose the formation of a dense and highly ordered solvation layer of the PGC surface with the linear acetonitrile molecules. Adsorption of analyte molecules requires a displacement of numerous acetonitrile molecules, which explains the positive enthalpy and entropy values measured. The interplay of enthalpic and entropic contributions to the overall adsorption phenomena results in strongly temperature dependent chromatographic selectivity values.     

A microfabricated graphitic carbon column for high performance liquid chromatography

We report the first development of a novel, planar, microfluidic, graphitic carbon separations column utilizing an array of graphitic micropillars of diamond cross-section as the chromatographic stationary phase. 795 nm femtosecond laser ablation was employed to subtractively machine fluidic architectures and a micropillared array in a planar, graphitic substrate as a monolithic structure. A sample injector was integrated on-chip, together with fluid-flow distribution architectures to minimize band-broadening and ensure sample equi-distribution across the micro-pillared column width. The separations chip was interfaced directly to the ESI probe of a Thermofisher Surveyor mass spectrometer, enabling the detection of test-mixture analytes following their differential retention on the micro-pillared graphitic column, thus demonstrating the exciting potential of this novel separations format. Importantly, unlike porous, graphitic microspheres, the temperature and pressure resilience of the microfluidic device potentially enables use in subcritical H(2)O chromatography.     

High temperature to increase throughput in liquid chromatography and liquid chromatography-mass spectrometry with a porous graphitic carbon stationary phase

High separation temperatures in liquid chromatography and liquid chromatography-mass spectrometry with a porous graphitic carbon column were investigated. Separation temperature was varied up to 200 degrees C, and the effect on retention, analysis time, and sensitivity was measured. Analysis times were reduced more than six-fold, whilst baseline resolution was maintained. The impact of the separation temperature on signal-to-noise ratio with atmospheric pressure chemical ionisation or electrospray mass spectrometric detection was also investigated. The potential of using superheated water for the analysis of some very polar compounds is illustrated. Monitoring of column stability detected no loss of performance, due to the highly stable nature of the 100% carbon stationary phase.     

Retention characteristics of porous graphitic carbon in reversed-phase liquid chromatography with methanol-water mobile phases

The solvation parameter model is used to study the retention mechanism of neutral organic compounds on porous graphitic carbon with methanol-water mobile phases containing from 0-100% (v/v) methanol. The dominant contribution to retention is the cavity formation-dispersion interaction term, composed of favorable interactions in the mobile phase (hydrophobic effect) and additional contributions from adsorption on the graphite surface. Electron lone pair and dipole-type interactions in the adsorbed state result in increased retention. Hydrogen-bonding interactions are more favorable in the mobile phase resulting in lower retention. The changes in the system constants of the solvation parameter model for cavity formation-dispersion interactions and hydrogen-bond interactions are linearly related to the volume fraction of water in the mobile phase. The system constants for electron lone pair interactions and dipole-type interactions are non-linear and go through a maximum and minimum value, respectively, at a specific mobile phase composition. The solvation parameter model poorly predicts the retention properties of angular molecules. This is probably due to the failure of the characteristic volume to correctly model the contact surface area for the interaction of angular molecules with the planar graphite surface. General factors affecting the quality of model fits for adsorbents are discussed.     

Rapid analysis of triterpenic acids by liquid chromatography using porous graphitic carbon and evaporative light scattering detection

An original system which uses Porous Graphitic Carbon as support and a mixture of organic solvents as mobile phase is proposed for the analysis of triterpenic acids by liquid chromatography. The separation of betulinic acid, ursolic acid, oleanolic acid, and 18alpha- and 18beta-glycyrrhetinic acids was carried out within a short time and monitored by evaporative light scattering detection as universal detection method. Molecular modelling studies show that the main contribution to the selectivity comes from the electrostatic interaction characterised by the dipole moment of the products.     

High performance liquid chromatography of disaccharides on a porous graphitic carbon column applying post-column derivatization with benzamidine

Summary A sensitive post-column fluoresence method based on the reaction of reducing sugars with a fluorigenic reagent (benzamidine) under alkaline conditions and elevated temperature is described. Optimum reaction conditions suitable for the detection of fluorescent reaction products have been investigated. A porous graphitized carbon column (shandon Hypercarb) was employed as the chromatographic solid phase and elution was isocratic by an aqueous acetonitrile mixture.A special problem in sugar analysis is the tendendy of anomer formation giving doublet peaks. This tendency varies largely among the different sugars, and systematic studies on conditions that eliminate this problem were performed. Efficient and selective separations of a series of disaccharides were obtained using high temperatures and a low concentration of acetonitrile in the mobile phase. The detection limits (S/N=3) with an injection volume of 20 L ranged from 10–60 picomoles. Linear calibration graphs were obtained in the 2–20 M concentration range (r ²>0.99). A relative standard deviation between 1–3% (n=6) demonstrated good precision of the developed system.Deceased July 21, 1996     

Liquid chromatography analysis of monosubstituted sulfobutyl ether-beta-cyclodextrin isomers on porous graphitic carbon

The retention behaviour of the three positional isomers of monosubstituted sulfobutyl ether-beta-cyclodextrin was investigated on a porous graphitic carbon (PGC) column. The influence of the mobile phase composition (nature and concentration of organic and electronic modifiers) was studied as well as the effect of column temperature. These hydrophilic and anionic analytes were highly retained on the PGC stationary phase compared to octadecyl bonded phases. The retention is mainly governed by a reversed-phase mechanism with electronic interaction playing a secondary role. An increase in solute retention and efficiency with temperature was observed. Successful isocratic separation with satisfactory baseline resolution of the three isomers of monosubstituted sulfobutyl ether-beta-cyclodextrin was achieved at 75 degrees C on a Hypercarb column by using ammonium acetate as electronic modifier in water-acetonitrile (83:17). The chromatographic methodology developed can be easily used for relative quantification of each isomer within a mixture and can be applied for semi-preparative purification of each one. The evaporative light scattering detector allows the detection of these non UV-visible absorbing molecules.     

Trace-level determination of polar phenolic compounds in aqueous samples by high-performance liquid chromatography and on-line preconcentration on porous graphitic carbon

The use of porous graphitic carbon (PGC) was investigated for the trace enrichment and the on-line liquid chromatographic separation of polar phenolic compounds (phenol, di- and trihydroxybenzenes, aminophenols, etc.) from aqueous samples. Comparison between retentions obtained with PGC and with the copolymer-based sorbent PRP-1 showed similar variations of the capacity factors with the mobile phase composition, but an inverse retention order. The capacity factor of a very polar analyte, such as 1,3,5-trihydroxybenzene (phloroglucinol), is 1000 in pure water, whereas this analyte is not retained by C₁₈-silica and is poorly retained by PRP-1 (k′ = 3 in water). A precolumn packed with PGC can be coupled to a PGC analytical column for simple separation in the reversed-phase mode. This methodology has been applied to the direct determination of pyrocatechol, resorcinol and phloroglucinol below the 0.1 μg/1 level in a 50-ml sample.     

Evaluation of porous graphitic carbon as stationary phase for the analysis of fatty acid methyl esters by liquid chromatography

Summary Polyunsaturated fatty acids have been analysed as methyl esters by liquid chromatography on porous graphitic carbon and the results compared with those obtained on octadecyl bonded phases. Chromatographic behaviour on octadecyl bonded phases arises principally as a result of hydrophobic interactions with the bonded phase. Because the retention of analytes is greater on porous graphitic carbon than on octadecyl phases, organic mobile phases are required. When the number of double bonds is low (ca 1–3), the behaviour of porous graphitic carbon is similar to that of octadecyl bonded phases, but when this number increases stronger interactions with the flat surface of the graphite appear, resulting in new selectivity. These two ‘reversed-phase’ systems are considered complementary for separation of different fatty acid methyl esters. An additional advantage of porous graphitic carbon is that it enables isolation of hexadecartrienoic and hexadecadienoic acids, which are not available commercially.     

Analysis of 'ARN' naphthenic acids by high temperature gas chromatography and high performance liquid chromatography

Examination by high temperature GC (HTGC) of the methyl esters of the so-called 'ARN' naphthenic acids from crude oils of North Sea UK, Norwegian Sea and West African oilfields revealed the distributions of resolved 4-8 ring C80 tetra acids and trace amounts of other acids. Whilst all three oils contained apparently the same major acids, the proportions of each differed, possibly reflecting the growth temperatures of the archaebacteria from which the acids are assumed to have originated. The structures of the 4, 5, 7 and 8 ring acids are tentatively assigned by comparison with the known 6 ring acid and related natural products and an HPLC method for the isolation of the individual acids is described. ESI-MS of individual acids isolated by preparative HPLC established the elution order of the 4-8 ring acids on the HPLC and HTGC systems and revealed the presence of previously unreported acids tentatively identified as C81 and C82 7 and 8 ring analogues.     

Effects of modifiers in subcritical fluid chromatography on retention with porous graphitic carbon

The effect of different modifiers in subcritical fluid chromatography (SubFC) on interactions between solute and porous graphitic carbon (PGC) and between solute and carbon dioxide-modifier mobile phases was studied by the use of linear solvation energy relationships (LSERs). This study was performed to allow efficient optimization of the composition of the carbon dioxide-modifier mobile phase in regard of the chemical nature of the solutes to be separated. With all modifiers tested (methanol, ethanol, n-propanol, isopropanol, acetonitrile, tetrahydrofuran and hexane), the solute/stationary phase interactions are greater than the solute/mobile phase ones. Dispersion interactions and charge transfer between electron donor solute and electron acceptor PGC mainly explain the retention on this surface, whatever the modifier. These interactions are quite constant over the range of modifier percentage studied (5-40%). For acidic compounds, the retention variation is mainly related to the change in the basic character of mobile and stationary phase due to the variation of modifier percentage. Changes in eluting strength are mostly related to adsorption of mobile phase onto the PGC with methanol and acetonitrile, and to the increase of dispersion interactions between the solute and the mobile phase for other modifiers. Relationships between varied selectivities and solvation parameter values have been studied and are discussed in this paper.     

Separation of phenylureas by capillary electrochromatography on porous graphitic carbon

Summary In this work the potential of porous graphitic carbon (PGC) as a new stationary phase in, capillary electrochromatography has been explored. Its behavior under the action of an applied potential is described for the separation of phenylureas. First, it was shown that porous graphitic carbon enables high efficiency in capillary electrochromatography over a wide range of mobile phase velocities. It was then demonstrated that this material might be responsible for degradation of the solutes at frit-PGC interfaces. Although electrolytic degradation reactions are suspected to occur on this type of conductive material, voltamperometric measurements furnished no clear evidence. A specific injection procedure is proposed for avoiding degradation of the solutes at the inlet interface before their chromatographic separation. Comparison of the retention behavior of phenylureas on PGC in liquid chromatography and in capillary electrochromatography show that the retention propertiets of PGC are altered by application of an electrical field, because this modifies the donor-acceptor interactions between the solutes and the stationary phase.     

Separation of L-DOPA and four metabolites in plasma using a porous graphitic carbon column in capillary liquid chromatography

Summary A sensitive HPLC method with marbofloxacin (MAR) as internal standard and fluorescence detection is described for the analysis of ofloxacin (OFL) enantiomers in plasma samples. Plasma samples were prepared by adding phosphate buffer (pH 7.4, 0.1m), then extracted with trichloromethane.S-OFL,R-OFL, and the internal standard were separated on a reversed-phase column with water-methanol, 85.5∶14.5, as mobile phase. The concentrations ofS-OFL andR-OFL eluting from the column (retention times 7.5 and 8.7 min, respectively) were monitored by fluorescence detection withλ _ex = 331 andλ _em = 488 nm. The detection and quantitation limits were 10 and 20 ng mL⁻¹, respectively, forS-OFL and 11 and 21 ng mL⁻¹ forR-OFL. Response was linearly related to concentration in the range 10 to 2500 ng mL⁻¹. Recovery was close to 93% for both compounds. The method was applied to determination of the enantiomers of OFL in plasma samples collected during pharmacokinetic studies.     

Retention characteristics of porous graphitic carbon in subcritical fluid chromatography with carbon dioxide-methanol mobile phases

Numerous relationships usually used in high-performance liquid chromatography (HPLC) for describing the retention on porous graphitic carbon (PGC) have been applied in subcritical fluid chromatography, with CO2-methanol mobile phases. As reported in HPLC, octanol-water partition coefficient failed to fit the retention, whereas satisfactory results were obtained with the sum of partial negative charges. A better fit was reached by using the solvation parameter model, allowing a better understanding of the interactions developed between the solute, the stationary and the mobile phases. Results show that the dominant contribution to retention was given by the polarizability (E) and the volume (V), while the hydrogen-bond basicity (B) was not selected in the retention model, whatever the methanol content. The increase in methanol percentage favours the retention decrease, mainly through the volume for hydrophobic compounds, and through the hydrogen-bond acidity for polar compounds.     

Porous graphitic carbon: A versatile stationary phase for liquid chromatography

Chromatographic stationary phases based on porous graphitic carbon were invented 30 years ago, while columns have been commercially available for 20 years. This special occasion deserved a complete review on this material. In this paper, we describe current knowledge on graphitic carbon stationary phases, based on over 400 fundamental studies and applications.     

Comparison of hydrophilic-interaction, reversed-phase and porous graphitic carbon chromatography for glycan analysis

Hydrophilic-interaction liquid chromatography (HILIC), reversed-phase chromatography (RPC) and porous graphitic carbon (PGC) chromatography are typically applied for liquid chromatographic separations of protein N-glycans. Hence the performances of these chromatography modes for the separation of fluorescently labeled standard glycan samples (monoclonal antibody, fetuin, ribonuclease-B) covering high-mannose and a broad range of complex type glycans were investigated. In RPC the retention of sialylated glycans was enhanced by adding an ion-pairing agent to the mobile phase, resulting in improved peak shapes for sialylated glycans compared to methods recently reported in literature. For ion pairing RPC (IP-RPC) and HILIC ultra-high performance stationary phases were utilized to maximize the peak capacity and thus the resolution. But due to the shallow gradient in RPC the peak capacity was lower than on PGC. Retention times in HILIC and IP-RPC could be correlated to the monosaccharide compositions of the glycans by multiple linear regression, whereas no adequate model was obtained for PGC chromatography, indicating the significance of the three-dimensional structure of the analytes for retention in this method. Generally low correlations were observed between the chromatography methods, indicating their orthogonality. The high selectivities, as well as the commercial availability of ultra-high performance stationary phases render HILIC the chromatography method of choice for the analysis of glycans. Even though for complete characterization of complex glycan samples a combination of chromatography methods may be necessary.     

Separation of free sterols by high temperature liquid chromatography

Increasing the column temperature accelerates markedly elution in HPLC. The separation of five free sterols was studied on three packing materials that can withstand high temperatures. These stationary phases included graphitic carbon, a polymeric C18 silica, and a zirconia-based adsorbent. Measurements of retention data were made at up to 150 degrees C with mobile phases of different compositions. Since the columns tested afford different retention mechanisms, a variety of elution patterns were observed, with some being more advantageous than others for certain sterol separations. Effects observed include some selectivity improvements and some elution order reversals. The separation of free sterols in selected fruit juices is also presented. Albeit at the expense of a longer analysis time, the graphitic carbon column produced the best separation of the sterols in this study.