Preconcentration of selenium compounds on a porous graphitic carbon column in view of HPLC-ICP-AES speciation analysis

The retention of organic selenium compounds on a porous graphitic carbon stationary phase was investigated. Different acids were studied as mobile phases to elute selenocystamine, selenoethionine, selenomethionine and selenocystine. Detection was achieved using inductively coupled plasma-atomic emission spectrometry to provide selenium-specific and sensitive detection. The separation of the four species was carried out using methanoic acid. An important on-column preconcentration was obtained when solutes were injected in nitric acid or trifluoroacetic acid (TFA) media. The large injection volume employed (2,500 µL) allowed us to reach low relative detection limits (2–6 µg/L). The method, employing TFA as injection solvent and methanoic acid as the eluent was found to be robust with respect to different matrices spiked with selenocompounds.     

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.     

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.     

Functionalization/passivation of porous graphitic carbon with di-tert-amylperoxide

Porous graphitic carbon (PGC) particles were functionalized/passivated in situ in packed beds at elevated temperature with neat di-tert-amylperoxide (DTAP) in a column oven. The performance of these particles for high performance liquid chromatography (HPLC) was assayed before and after this chemistry with the following analytes: benzene, toluene, ethyl benzene, n-propyl benzene, n-butyl benzene, p-xylene, phenol, 4-methylphenol, phenetole, 3,5-xylenol, and anisole. After the first functionalization/passivation, the retention factors, k, of these compounds decreased by about 5% and the number of theoretical plates (N) increased by ca. 15%. These values of k then remained roughly constant after a second functionalization/passivation but a further increase in N was noticed. In addition, after each of the reactions, the peak asymmetries decreased by ca. 15%, for a total of ca. 30%. The columns were then subjected twice to methanol at 100°C for 5h at 1 mL/min. After these stability tests, the values of k remained roughly constant, the number of plates increased, which is favorable, and the asymmetries rose and then declined, where they remained below the initial values for the unfunctionalized columns. Functionalized and unfunctionalized particles were characterized by scanning electron microscopy and BET measurements, which showed no difference between the functionalized and unfunctionalized materials, and X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry (ToF-SIMS), where ToF-SIMS suggested some chemical differences between the functionalized and unfunctionalized materials. In particular ToF-SIMS suggested that the expected five-carbon fragments from DTAP exist at higher concentrations on DTAP-functionalized PGC. First principle calculations on model graphitic surfaces suggest that the first addition of a DTAP radical to the surface proceeds in an approximately isothermal or slightly favorable fashion, but that subsequent DTAP additions are then increasingly thermodynamically favorable. Thus, this analysis suggests that the direct functionalization/passivation of PGC with DTAP is plausible. Chemometric analyses of the chromatographic and ToF-SIMS data are also presented.     

A selective method for the determination of benzo[a]pyrene in soil using porous graphitic carbon liquid chromatography columns

A back-flushing procedure using porous graphitic carbon (PGC) HPLC columns has been used successfully for the cleanup of soil samples for the determination of benzo[a]pyrene in ppb levels by an ODS-fluorescence HPLC column. The procedure was tested on nine random soil samples taken from an industrial area of the Kingdom of Bahrain. The mean percent recovery from the PGC column was 96% and the average coefficient of variation for the whole method was 5.2%.     

Porous graphitic carbon sorbents in biomedical and environmental applications

This review is written as a privilege of the work of Professor Mietek Jaroniec on surface phenomena, adsorption, chromatographic separations, chemistry of conventional and ordered nanoporous materials. The problems of the porous graphitic carbon (PGC) application in analytical field are presented. Special attention is paid on possibilities of use PGC as a specific sorbent and packing material for selective isolation and analytes enrichment from complex matrices by means of liquid-solid extraction techniques (SPE, SPME, MSPD) and as particular stationary phase in analytical chromatographic columns. Surface and adsorption properties as well as a unique mechanism of retention on porous graphitized carbon sorbents are described. As supplement the examples of application in biomedical and environmental area are added.     

Ion-pair chromatography on a porous graphitic carbon stationary phase for the analysis of twenty underivatized protein amino acids

The analysis of twenty underivatized protein amino acids has been achieved on porous graphitic carbon packing material (Hypercarb). Five perfluoroalkyl carboxylic acids (trifluoroacetic, heptafluorobutyric, nonafluoropentanoic, tridecafluoroheptanoic and pentadecafluorooctanoic acid) have been studied as ion-pairing reagent. Several parameters (equilibration time, quantities adsorbed onto the chromatographic support, concentration and nature of the ion-pairing reagent, as well as temperature effect) have been studied leading to the complete separation of these compounds in gradient elution mode. Evaporative light scattering detector has allowed the detection of these non UV–visible absorbent molecules. The chromatographic methodology developed can also be easily coupled with pneumatically assisted electrospray mass spectrometry.     

Temperature effect on HPLC retention of PCBs on porous graphitic carbon

Summary The effect of column temperature on solute retention was investigated for 10 PCBs with different degrees of planarity (degree of ortho position substitution). The variation of retention values (lnk) with temperature was measured using two solvents, n-hexane and dichloromethane, as mobile phase: retention decreases as temperature increases. The relationship between lnk values and temperature (1/T) exhibits a good linearity. The ΔH ⁰ values calculated are in the −2 to −5 kcal mole^{− 1} range: they are very similar for compounds with the same degree of ortho-substitution and are significantly higher for non-ortho substituted congeners. High temperature significantly improves separation efficiency (plate number significantly increases): this effect is the dominant factor controlling chromatographic resolution, which improves with increasing temperature. Operating at 40 °C, an efficient separation of planar PCBs was obtained with low solvent consumption.     

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.     

HPLC analysis of PCBs on porous graphitic carbon: Retention behavior and gradient elution

Summary The fractionation of PCB congeners into classes according to their planarity (i.e. amount of ortho substitution) by HPLC on porous graphitic carbon (PGC) as stationary phase has been investigated as a preliminary step before GC analysis, indispensable for a complete separation of PCB congeners. A systematic study of retention behavior on PGC eluted with different n-hexane-dichloromethane mixtures made it possible to design a linear binary gradient which separated PCB congeners in a reasonable time and with good performance. Relationships were obtained between retention behavior and the molecular structure of the PCB congeners. The beneficial effects of elevated temperature on separation efficiency were also investigated. The analysis conditions selected, i.e. continuous gradient separation at 40°C, were successfully used for fractionation of technical PCB formulations, e.g. Aroclor 1242.     

Retention studies of sulphated glycosaminoglycan disaccharides on porous graphitic carbon capillary columns

Summary The retention behaviour of highly polar and charged disaccharide isomers has been studied on porous graphitic carbon columns and experimental parameters were varied over wide a range, including concentration and type of mobile phase constituents as well as temperature. The hydrophilic and anionic sugar analytes were highly retained on this stationary phase compared to the octadecyl-derivatized silica packings more commonly used. E.g., an increase in retention with polarity of a solute and with temperature was observed. By isotherm measurements and nonlinear fitting of Langmuirian expressions to the experimental data the graphite surface appeared homogeneous with only one kind of active adsorption site for these kinds of compound which was furthermore supported by the linear Van't Hoff plots obtained by varying the temperature. The gain in free energy was found to be entropically driven after determination of the ΔH° and ΔS° values. However, enthalpy-entropy compensation behavior was not met.     

Chiral ligand-exchange chromatography of amino acids using porous graphitic carbon coated with a dinaphthyl derivative of neamine

In this paper, we describe the preparation and the evaluation of a porous graphitic carbon (PGC) column coated with a new dinaphthyl derivative of neamine for chiral ligand-exchange (LE) chromatography. It was shown that the graphitic surface/dinaphthyl anchor system efficiently (1.15 μmol/m²) and stably (three months of intensive use) adsorbs the neamine template onto the chromatographic support. The resulting coated PGC stationary phase showed appreciable LE-based enantioselective properties towards several native amino acids. Chromatographic separation of methionine enantiomers using a dinaphtyl neamine-based ligand-exchange chiral stationary phase     

Preliminary studies of supercritical-fluid chromatography on porous graphitic carbon with methylated cyclodextrin as chiral selector

Summary Dimethylated-β-cyclodextrins dynamically adsorbed on porous graphitic carbon have been used as chiral selectors in chiral supercritical-(or subcritical-) fluid chromatography. The kinetics of adsorption and desorption were studied with CO₂-methanol+dimethylated-β-cyclodextrins and CO₂-methanol as mobile phases. The system was proved to be stable and reproducible and to afford rapid enantiomer separations especially when performed with 95:5 CO₂-methanol+dimethylated-β-cyclodextrin as mobile phase. The versatility of the chiral system enabled the use of a variety of chiral selectors. It was found that enantiomer separation can vary largely as a function of the composition of commercial dimethylated-β-cyclodextrin mixture.     

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.     

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.     

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.     

Effects of the redox state of porous graphitic carbon on the retention of oligosaccharides

Retention of hydrophilic compounds on porous graphitic carbon (PGC) is afforded by polar interactions with induced dipoles within this polarizable stationary phase. These interactions depend on the redox state of PGC, which can be influenced by application of an electrical field or by chemical means. We explored the impact of oxidizing and reducing agents on the retention of fluorescence labeled neutral oligosaccharides. Malto-oligosaccharides were employed as simple model system. Subsequently, the effects on the retention of glycans typical for immunoglobulin G (IgG) antibodies were investigated. Chemical oxidation of the PGC surface increased the retention of all analytes tested. Selectivities were significantly altered by the redox treatment, emphasizing the need for controlling the redox state of PGC to achieve reproducible conditions. Furthermore a column pre-conditioning protocol is presented, which allowed for reproducible chromatography of neutral IgG glycans.     

Interference of the electrospray voltage on chromatographic separations using porous graphitic carbon columns

The electrospray ionization (ESI) voltage is shown to interfere with liquid chromatographic separations performed with packed porous graphitic carbon (PGC) capillary columns. This interference is ascribed to the presence of an electric field over the conductive column in the absence of an earth point between the column and the ESI emitter. The current evolved alters the chromatographic behavior of the catecholamine metabolite 3-O-methyl-DOPA significantly, as both peak splitting and a dramatic decrease in the retention time were observed. Furthermore, the response from the mass spectrometer was decreased by 33% at the same time. A related compound, tyrosine, exhibited decreased retention times but no peak splitting, whereas no shifts in the retention times (or peak splitting) were seen for the less retained dopamine and noradrenaline. When the current through the PGC column was eliminated by the use of an earth point between the column and the ESI emitter, the chromatographic behavior of the column was found to return slowly to normal after hours of equilibration with 60 : 40 (v/v) methanol-ammonium formate buffer of pH 2.9. The behavior of the PGC column with and without the earth point was found to be highly reproducible during a period of 1 month. We propose that the effect of the ESI voltage on the chromatographic behavior of the PGC column is due to associated redox reactions affecting both the PGC particles and the analytes. It is concluded that (for analytical reasons), care should be taken to ensure that no current is flowing through the chromatographic system when interfacing PGC columns, and conducting parts in general, to ESI mass spectrometry.     

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.