Graphitic Carbon Stationary Phase Research Articles

Evaluation of the surface charge properties of porous graphitic carbon stationary phases treated with redox agents.

The effect of treatment of porous graphitic carbon (PGC) stationary phases with hydrogen peroxide and with sodium sulfite on the retention behavior of analyte compounds has been investigated using benzene, aromatic sulfonate ions, and benzyltrialkylammonium ions as model compounds. It is shown that the retention times of the cationic analytes are increased by treating the PGC column with the reducing agent, while decreased by treating it with the oxidizing agent. On the other hand, the retention times of the anionic analytes are decreased by treating the column with the reducing agent, while increased by treating it with the oxidizing agent. The effect of the redox treatment on the retention of benzene is negligibly small. The investigation of the ion-exchange property of the PGC packings have shown that PGC has anion-exchange property and the anion-exchange capacity is decreased by treating PGC with the reducing agent, whereas it is increased by treatment with the oxidizing agent. This means that the modification of the retention selectivity of the PGC stationary phases with redox treatment can be interpreted in terms of the change of the surface charge. The mechanism of chemical modification of the PGC stationary phase with redox treatment is discussed on the basis of the experimental results obtained on the ion-exchange capacity and the redox activity.

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 micro L) allowed us to reach low relative detection limits (2-6 micro g/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.

On-column derivatization using redox activity of porous graphitic carbon stationary phase: an approach to enhancement of separation selectivity of liquid chromatography.

A new on-column derivatization method based on the redox activity of porous graphitic carbon (PGC) packing materials was presented for enhancement of separation selectivity of HPLC. Two PGC packing materials were used as the solid redox agents as well as the stationary phase, and their redox activities were investigated using trans-1,2-diaminocyclohexanetetraacetate (DCTA) complexes of some metal ions as probe compounds. It was found that the redox property of PGC was modified by treating them with a solution containing a reducing agent, sodium sulfite or hydroxylammonium chloride. The original PGC packings oxidized Co(II)-DCTA to Co(III)-DCTA during elution, while the PGC treated with a reducing agent showed reduction activity converting Co(III)-DCTA to Co(II)-DCTA. These two cobalt complexes do not form their individual chromatographic zones but migrate as a single zone of their mixture on the PGC column contrary to the chromatographic behavior on a C18 bonded silica, on which Co(II)-DCTA and Co(III)-DCTA can be separated. Treatment of the PGC column with a reducing agent solution transforms the oxidative activity of the original PGC packing to a reductive one from the upper part of the column, so that the retention time of the cobalt complex can be controlled by changing the volume of the reducing agent solution to be used for treatment of the PGC column. The selective separation and determination of cobalt in a reference manganese nodule sample by the developed method was demonstrated.

Using principal component analysis for the study of the retention behaviour of phenol derivatives under reversed-phase conditions

The retention time of 11 ring-substituted phenol derivatives was measured on six different reversed-phase HPLC columns and the log k, theoretical plate number (N) and asymmetry factor (F) values were calculated for each solutes on each column. The similarities and dissimilarities among the columns and solutes were elucidated by principal component analysis followed with nonlinear mapping technique and cluster analysis. Calculations indicated that the retention characteristics of porous graphitized carbon stationary phase considerably deviate from those of octadecyl- and hexyl-coated silica, octadecyl-coated polystyrene-divinylbenzene polymer and polybutadiene-coated alumina. The differences among these columns were markedly smaller. The retention behaviour of aminophenols differed from those of other phenol derivatives proving the importance of molecular polarity in the retention. It was established that the mode of calculation slightly modifies the similarity and dissimilarity among the columns and solutes, therefore, the use of more than one calculation method is proposed.

Impurity profiling of non-nucleoside reverse transcriptase inhibitors by HPLC using a porous graphitic carbon stationary phase

Porous graphitic carbon (PGC) HPLC columns have been marketed for several years and have been shown to be superior to octadecyl silica columns for the separation of geometric isomers and closely related compounds. This makes them ideally suited to the determination of related substances and egradation products of drugs. In this study, PGC was used to improve the separation of related substances and degradation products of DPC 961, a new Non Nucleoside Reverse Transcriptase Inhibitor in development for the treatment of HIV infections. The porous graphitic carbon column dramatically improved the separation ofcis/trans isomeric related substances. In addition, the PGC column separated DPC 961 degradants which were not resolved from the main drug peak on a silica-based octadecyl column.

Role of the Na + ion on phenol derivatives/hydroxypropyl-β-cyclodextrin complex formation on porous graphitic carbon phase

The reversed-phase liquid chromatography retention of phenol derivatives was investigated over a concentration range of sodium chloride (0–10 −2 M) and hydroxypropyl-β-cyclodextrin (HP-β-CD) (0–35×10 −3 M) using a porous graphitic carbon (PGC) stationary phase and a methanol/water mixture (50:50 (v/v)) as the mobile phase. A theoretical treatment was developed to investigate the effect of the sodium chloride and hydroxypropyl-β-cyclodextrin on the equilibrium between the solutes with the PGC surface and the aqueous medium, respectively. The thermodynamic parameter variations were calculated using van’t Hoff plots. It was expected that the sodium ion acted on the solute–PGC association process by modifying the surface tension of both the bulk solvent and the PGC surface. The phenol derivative/HP-β-cyclodextrin complexation was shown to be entropically controlled for all the solutes except for the one which contained the –NO 2 group in its structure, i.e. the nitro phenol derivative. A comparison of the compensation temperature of the solute–PGC association process when sodium chloride and HP-β-CD concentration changed in the mobile phase led to the conclusion that these two modifiers acted via a variation in the hydrophobic effect.

High performance liquid chromatography of acarbose and its metabolite on porous graphitic carbon column

This paper describes the development of an analytical method for the determination of two pseudo-oligosaccharides, acarbose and its main metabolite. The analysis was carried out by liquid chromatography on a porous graphitic carbon stationary phase. The separation mechanism of these compounds is dominated by charge-induced interactions between the polar analyte and the polarizable surface of graphitic carbon. Several chromatographic parameters were investigated, including the nature and percentage of the organic solvent and acid modifier as well as column temperature. The best conditions were achieved with a mobile phase containing trifluoroacetic acid 0.1% and acetonitrile 8%. Under these conditions, the simultaneous resolution of acarbose and its metabolite as well as of their anomers was achieved, highlighting the potential of this stationary phase for challenging separations. Moreover, three detection methods are described and compared in terms of sensitivity. The evaporative light scattering detector was not sensitive enough for acarbose and metabolite determination in biological fluids. Mass spectrometry allowed a significant improvement in sensitivity but was not sufficient to permit the analysis of selected compounds in such complex matrices. However, this detector confirmed the presence of acarbose and its metabolite as well as the anomeric separation. Finally, fluorescence detection was found to be very sensitive after derivatization with 2-aminobenzamide and enabled the determination of selected compounds at a low concentration level.

Inorganic selenium speciation using HPLC-ICP-hexapole collision/reaction cell-MS

A chromatographic separation procedure based on the use of a porous graphitic carbon stationary phase and formic acid as eluent was employed to separate Se(IV) and Se(VI), inorganic selenium anions. Detection was performed by using an ICP-MS instrument equipped with a screened torch and a collision/reaction cell. The use of helium, dinitrogen oxide and hydrogen as collision gases was studied to improve selenium detection. The different configurations of the detection system were compared in terms of detection limits. Hydrogen gave the best results with detection limits of 12 and 24 ng L−1 for Se(IV) and Se(VI), respectively.

Chromatographic study of terpene-β-cyclodextrin complexes on porous graphitic carbon stationary phase

The effect of temperature on the retention of terpene derivatives on a porous graphitic carbon stationary phase has been investigated with methanol-water mobile phases containing β-cyclodextrin. Changes in Van't Hoff plots were evaluated in relation to the β-cyclodextrin concentration. Thermodynamic data were determined for both (i) solute transfer from the mobile phase to the PGC stationary phase and (ii) complexation of the solute with the β-CD. Enthalpyentropy compensation studies were performed for these two processes. Solute transfer was shown to be enthalpically driven and independent of terpene structure, β-cyclodextrin concentration, and mobile phase methanol content. The complexation reaction was shown to be entropically controlled and independent of terpene structure and mobile-phase composition. Comparison of the compensation temperatures of these two physicochemical processes led to the conclusion that each process had an similar influence on the global retention process.

Chemical Modification of Carbonaceous Stationary Phases by the Reduction of Diazonium Salts

This paper describes a new strategy for the creation of chemically modified carbonaceous stationary phases. The strategy exploits the electroreduction of arenediazonium salts as a means for functionalizing the surface of glassy carbon (GC) and porous graphitic carbon (PGC) stationary phases. The one-electron reduction of these salts forms an arene radical which then couples via a carbon-carbon linkage to the carbon framework at the surface of the stationary phase. Two arenediazonium-based modifiers were used in evaluating the potential utility of this strategy: 4-nitrobenzenediazonium tetrafluoroborate for the GC and PGC phases and 4-hexylbenzenediazonium tetrafluoroborate for only the PGC phases. Modifications were carried out by packing the phases into a column used for electrochemically modulated liquid chromatography. The effectiveness of the modifications was assessed by X-ray photoelectron spectroscopy and by comparing the liquid separation of a series of mixtures before and after coating deposition. For the nitrobenzyl-modified GC phase, the test mixture contained both anisole and fluoranthene. The performance of the nitrobenzyl- and hexylbenzyl-modified PGC stationary phases was characterized by the separations of substituted phenols (i.e., nitrophenol and resorcinol) and a few important pharmaceutical agents (i.e., hexobarbital, oxazepam, and nitrazepam). The potential utility of this modification procedure to form stationary phases that are stable upon extended exposure to aggressive mobile phases is discussed and briefly examined.

Chromatographic study of terpene derivatives on porous graphitic carbon stationary phase with β-cyclodextrin as mobile phase modifier

The stoichiometric coefficients and apparent formation constants ( K f) of α-terpineol, thymol, geraniol and linalool complexes with β-cyclodextrin (β-CD) were determined using HPLC with a porous graphitic carbon (PGC) chromatographic support. Measurements were performed with four different methanol–water mobile phases. All the terpene derivatives under study form 1:1 guest–CD complexes. Graphs of K f as a function of the mobile phase composition appeared different from those classically described for RP-C 18 and suggest that the PGC stationary phase could play an active role in the complexation process. Solute–CD inclusion and solute–stationary phase interactions may be involved in this specific behavior.

Ion Chromatography of Inorganic Anions on Graphitic Carbon as the Stationary Phase

Inorganic anions could be separated on porous graphitic carbon stationary phases in ion chromatography. Ion exchange between eluent anions and sample anions on the stationary phase was confirmed by the retention behavior and the possibility of indirect photometric detection. The elution order of anions was different from that observed for commercially available anion exchangers. Chloride, nitrate, and sulfate contained in tap water could be determined in 7 min.

SEPARATION OF UNDERIVATIZED SMALL PEPTIDES ON A POROUS GRAPHITIC CARBON COLUMN BY ION-PAIR CHROMATOGRAPHY AND EVAPORATIVE LIGHT SCATTERING DETECTION

Separations of small peptides (di-, tri- and tetrapeptides)using ion-pair chromatography and evaporative light scattering detection havebeen investigated with a porous graphitic carbon stationary phase, Hypercarb®.Five perfluorinated carboxylic acids (trifluoroacetic (TFA), pentafluoropropanoic(PFPA), heptafluorobutanoic (HFBA), nonafluoropentanoic (NFPA) and tridecafluoroheptanoic(TDFHA) acids) have been evaluated as ion-pairing agents. Influence of thepercentage of organic modifier, concentration, and chain length of the ion-pairingagent on the retention factors, k, has been examined. Equilibration timeswere also studied. Separations of dipeptides or tri- and tetrapeptides arepresented using HFBA and NFPA at 20 mM and TDFHA at 2.5 mM.

Relationships between Retention Factors and Analyte Hydrophobicity on Cyanopropyl and n-Octadecyl Bonded Silicas, Cross-Linked Polymers and Porous Graphitic Carbon Stationary Phases. Consequences for the Trace Analysis of Highly Polar Organic Compounds

LC retention data have been measured using various stationary phases with an emphasis on highly polar to moderately polar neutral organic compounds having octanol-water partition coefficients (Kow) in log units between 0 and 3. The relationships between the retention factor measured in water and the octanol-water partition coefficient are linear but with different slopes for octadecyl (C18) silicas, and two polystyrene divinylbenzene (PS-DVB) phases with low and high surface areas. These relationships confirm that highly cross-linked polymers can provide more than 1000-times higher retention values than C18 silicas for moderately polar analytes but close values for highly polar ones. They also explain why C18 silicas and polymers are equivalent for the separation of very polar analytes. In contrast, due to a different retention mechanism, no relation exists between the retention shown by porous graphitic carbons (PGC) and analyte hydrophobicity, but highly polar analytes are in general much more strongly retained than by any other sorbent. The potential of PGC for both the extraction and the separation of analytes is shown. Due to the difference in separation mechanism, PGC is the analytical phase that should be used for confirmation of the identity of analytes instead of a cyanopropylsilica column as recommended in some environmental procedures. Applications are presented for the trace-determination of triazines and polar degradation products in ground and surface water with detection limits below the 0.1 μg/L level.

Relationships between Retention Factors and Analyte Hydrophobicity on Cyanopropyl andn-Octadecyl Bonded Silicas, Cross-Linked Polymers and Porous Graphitic Carbon Stationary Phases. Consequences for the Trace Analysis of Highly Polar Organic Compounds

LC retention data have been measured using various stationary phases with an emphasis on highly polar to moderately polar neutral organic compounds having octanol-water partition coefficients (K ow ) in log units between 0 and 3. The relationships between the retention factor measured in water and the octanol-water partition coefficient are linear but with different slopes for octadecyl (C 18 ) silicas, and two polystyrene divinylbenzene (PS-DVB) phases with low and high surface areas. These relationships confirm that highly cross-linked polymers can provide more than 1000-times higher retention values than C 18 silicas for moderately polar analytes but close values for highly polar ones. They also explain why C 18 silicas and polymers are equivalent for the separation of very polar analytes. In contrast, due to a different retention mechanism, no relation exists between the retention shown by porous graphitic carbons (PGC) and analyte hydrophobicity, but highly polar analytes are in general much more strongly retained than by any other sorbent. The potential of PGC for both the extraction and the separation of analytes is shown. Due to the difference in separation mechanism, PGC is the analytical phase that should be used for confirmation of the identity of analytes instead of a cyanopropylsilica column as recommended in some environmental procedures. Applications are presented for the trace-determination of triazines and polar degradation products in ground and surface water with detection limits below the 0.1 μg/L level.

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.

Elution characteristics of natural cyclodextrins on porous graphitic carbon

The retention behavior of natural alpha-, beta-, and gamma-cyclodextrins on a porous graphitic carbon (PGC) stationary phase is investigated. Unusual retention properties for reversed-phase chromatographic conditions are observed with acetonitrile-methanol and water-methanol mixtures as mobile phases. It is assumed that the retention process is governed not only by the standard solvophobic effect but also by specific interactions described as "CD-PGC" effect. The retention factor versus the volumetric methanol fraction in the mobile phase show second-order curves expressing this double mechanism hypothesis. van't Hoff plots demonstrate the contribution of these two retention processes. The retention factor of each natural cyclodextrin is shown to depend on the mobile phase property to act as a proton acceptor, according to the solvent selectivity classification described by Snyder. The "CD-PGC" effect is interpreted as an equilibrium between different interactions: cyclodextrin-PGC stationary phase, London dispersion forces, and cyclodextrin-mobile phase hydrogen bonding. The balance of these interactions may monitor the orientation of the cyclodextrin molecule facing the carbon surface, which is therefore suspected to be the major parameter of this retention mechanism.

Use of principal component analysis for investigation of factors affecting retention behaviour of ceramides on porous graphitized carbon column

The potentiality of a porous graphitized carbon stationary phase was assessed for ceramide analysis in nonaqueous reversed phase chromatography with an evaporative light scattering detection. The influence of the nature of the mobile phase was first performed under isoeluotropic isocratic conditions. Eight mobile phase compositions in the presence or absence of triethylamine/formic acid were tested with a set of structurally relevant ceramides. Values obtained under these conditions for the peak heights, retention factors, plate numbers, asymmetry factors, relative retentions and Time-Corrected Calibrated Normalised Resolution Product were analysed by Principal Component Analysis. Principal Component Analysis was effective in attributing relationships between the chromatographic behaviour and analytical conditions. The relationship between the increase of the detector response and the presence of triethylamine/formic acid has been confirmed, and the main change of selectivity has been attributed to interactions provided by the weak solvent of the mobile phase. These conclusions have allowed the development of gradient conditions for the analysis of complex ceramide mixtures on porous graphitized carbon column.

Liquid chromatography analysis of phosphonic acids on porous graphitic carbon stationary phase with evaporative light-scattering and mass spectrometry detection

The analysis of several phosphonic acids has been investigated by liquid chromatography (LC), using porous graphitic carbon as stationary phase with mass spectrometry (MS) or evaporative light-scattering detection (ELSD). In both detection modes (MS and ELSD), the mobile phase must be volatile and, due to the porous graphitic carbon (PGC) properties, should promote electronic interactions. Among the various hydrogeno- and perfluorocarboxylic acids tested, trifluoroacetic acid (0.1%, v/v) was selected as electronic competitor for solute retention. The baseline resolution of a phosphonic acids mixture required a trifluoroacetic–acetonitrile gradient elution. This methodology was then applied to the identification of phosphonic acids in a spiked tap water sample. Quantitative analyses are successfully achieved with a good correlation coefficient.

Enantiomeric separations of benzodiazepines by electrochemically modulated liquid chromatography

Electrochemically modulated liquid chromatography (EMLC) has been applied to the enantiomeric separation of a group of benzodiazepines (i.e., oxazepam, lorazepam and temazepam) using a porous graphitic carbon (PGC) stationary phase and β-cyclodextrin (β-CD) as a chiral mobile phase additive. The basis of the separation derives from the reversible electrosorption of the additive onto PGC, resulting in a new approach for manipulating the chirality and selectivity of the stationary phase. Changes in the voltage applied ( E appl) to the stationary phase were found to alter the retention and the enantioselectivity of the separation, both of which reflect the dependence of the electrosorption of the additive on E appl. By exploiting these dependencies, mixtures of each of the three racemates could be resolved to differing extents while using the same isocratic elution condition. A general description of the retention process and the role of the apparent enantiomerization of oxazepam and lorazepam are briefly discussed.