Claire Elfakir

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.

Ion-pair reversed-phase liquid chromatography for determination of polar underivatized amino acids using perfluorinated carboxylic acids as ion pairing agent

Abstract An isocratic liquid chromatography method using volatile ion pairing agent and evaporative light scattering detection was developed for the analysis of 10 underivatized amino acids (Asp, Asn, Ser, Gly, Gln, Cys, Glu, Thr, Ala, Pro) which are not sufficiently retained and resolved in the LC system commonly used. Five perfluorinated carboxylic acids (trifluoroacetic, heptafluorobutyric, nonafluoropentanoic, tridecafluoroheptanoic and pentadecafluorooctanoic acid) have been evaluated as ion-pairing agents in various concentrations (0.25–1 mM) using three analytical reversed-phase columns (LiChrospher RP-Select B, Purospher RP-18e and Supelcosil ABZ+ Plus). The quantities adsorbed on the chromatographic supports and the equilibration times of each system are also studied. Excellent separation with satisfactory baseline resolution was achieved on the chromatographic system made up of a Purospher RP-18e and an aqueous mobile phase containing pentadecafluorooctanoic acid at 0.5 mM.

Parameter optimization for the analysis of underivatized protein amino acids by liquid chromatography and ionspray tandem mass spectrometry.

The analysis of the 20 underivatized protein amino acids by liquid chromatography ionspray tandem mass spectrometry is investigated in positive ion mode. First, by direct infusion, amino acid fragmentation was investigated based on the product-ion mass spectrum of the parent compound in three different collision energies (10, 20, 30 eV). Then, the relative abundance of fragment ions was studied as a function of the collision energy in order to select the product ion with the highest abundance and to obtain the maximum sensitivity for each amino acid, by using the optimum collision energy. Depending on the amino acid, the loss of H2O or NH3 or CH2O2 was selected as the product ion from the molecular ion [M+H]+ in selective reaction monitoring mode. 15 eV was chosen as a mean value of collision energy to obtain satisfactory sensitivity for the simultaneous determination of the 20 protein amino acids. In spite of the specificity of mass spectrometry, and in order to obtain maximum sensitivity, several pairs of amino acids had to be separated. The separation of these amino acids pairs was achieved in less than 20 min by using a porous graphitic carbon column and nonafluoropentanoic acid as ion-pairing reagent. Detection limits depending on the amino acid varied from 500 fmol to 40 pmol (using a 10 microl loop).

Determination of 20 underivatized proteinic amino acids by ion-pairing chromatography and pneumatically assisted electrospray mass spectrometry

A qualitative determination of 20 underivatized proteinic amino acids by LC-MS is reported. The need for chromatographic separation before mass spectrometry determination is demonstrated based on the study of several amino acid pairs which have some similar characteristics. Two suitable LC-MS systems are proposed for amino acid analysis. A preliminary optimization of these systems has been investigated using evaporative light scattering detection as these two detection modes have the same chromatographic requirements. The amino acid separation was achieved on a Purospher RP-18e or a Supelcosil ABZ+Plus column with tridecafluoroheptanoic acid or pentadecafluorooctanoic acid as volatile ion-pairing reagent in an acetonitrile-water mobile phase. In order to elute the most retained amino acids, an elution gradient based on simultaneously increasing the concentration of acetonitrile and decreasing the concentration of the ion-pairing reagent was used. The detection limits of the present work (without specialized optimization) varied from 0.5 to 1 mg 1(-1).

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.

Investigations on the chromatographic behaviour of zwitterionic stationary phases used in hydrophilic interaction chromatography

Two commercial stationary phases possessing a sulfobetaine zwitterionic bonded ligand (ZIC-HILIC and Nucleodur HILIC) were compared under hydrophilic interaction chromatographic (HILIC) conditions. First of all, the separation of 12 model compounds chosen among neurotransmitters and presenting a diversity of ionization states (anionic, cationic and zwitterionic) was studied under varied operating conditions. The effects of the percentage of acetonitrile, ammonium acetate concentration and temperature of the mobile phase were compared on the two columns. Secondly, a generally applicable retention model was established, based on chromatographic retention data (logk) acquired for 76 model compounds. The chosen compounds are small molecules presenting a wide diversity of molecular structures and are relevant to biomedical and pharmaceutical studies. To account for their retention behaviour, a modified version of the solvation parameter model was designed: two additional molecular descriptors were introduced, to account for ionic interactions with anionic and cationic species. The retention equations obtained allow a rationalization of the interactions contributing to retention and separation in the HILIC systems considered.

Approach to hydrophilic interaction chromatography column selection: application to neurotransmitters analysis.

This paper presents the comparison between 12 hydrophilic interaction liquid chromatography columns that are commercially available. The main factors influencing the retention and selectivity toward 12 neurotransmitters for these different HILIC systems have been studied. For additional information, the retention data have been analyzed statistically by factor analysis. Principal component analyses (PCA) were calculated to evidence different separation behaviour between the stationary phases, based on the retention data measured for three compound classes: anionic acidic compounds, cationic basic compounds and zwitterionic amino acids. Finally, a generic procedure is suggested for optimization of HILIC analyses, depending on the ionization state of the analytes.

Determination of inorganic anions on porous graphitic carbon using evaporative light scattering detection: Use of carboxylic acids as electronic competitors

Abstract Different volatile carboxylic acids have been investigated as electronic competitors to optimize the separation of 12 common anions (F − , H 2 PO 4 − , Cl − , IO 3 − , IO 4 − , Br − , BrO 3 − , SO 4 2− , ClO 3 − , NO 3 − , I − , ClO 4 − ) on a porous graphitic carbon (PGC) column with evaporative light scattering detection. The addition of pyridine in the aqueous acidic mobile phase improves the efficiency of the separation and the detector response. The retention of inorganic anions on non-modified PGC is dominated by electronic interactions based purely on donor–acceptor (charge transfer) interaction between the lone pair electrons of the solute and the π-electrons of the PGC.

Ion-pair reversed-phase liquid chromatography-electrospray mass spectrometry for the analysis of underivatized small peptides.

The single run analysis of 23 small peptides (principally glycyl and lysyl dipeptides) is performed by ion-pair reversed-phase liquid chromatography coupled with evaporative light scattering detection or electrospray (tandem) mass spectrometry. Several perfluorinated carboxylic acid homologues are evaluated with an octadecyl silica stationary phase (Supelcosil ABZ+ Plus). Among the perfluorocarboxylic acids tested the nonafluoropentanoic acid and the tridecafluoroheptanoic acid gave the best results. Special attention was paid to the separation of isomer/isobar dipeptides (e.g., Gly-Ile, Gly-Leu, Leu-Gly, as well as Gly-Gln, Gly-Lys, etc.) which is usually necessary in spite of the high specificity of mass spectrometry. Before LC-MS analysis, ion-spray fragmentation as well as optimization of MS parameters of the analysed peptides was investigated. The optimum collision energy of glysyl peptides, Ala-Gln, Asp-Asp and Asp-Asp-Asp (13-18 eV) was different from that of the lysyl peptides, Tyr-Glu and oxidised glutathione (25-32 eV). Limits of detection varied from 0.1 to 1.2 mg l(-1) for simple MS and 0.05 to 25 mg l(-1) for tandem MS.

Fast separation of triterpenoids by supercritical fluid chromatography/evaporative light scattering detector

The screening of plant material, the chemical composition, the abundance and the biological activity of triterpenoids are of a major economical importance. The classical analytical methods, such as TLC, GC, and HPLC are either little resolutive, or require derivatization steps, or fail in sensitivity. The supercritical fluid chromatography/evaporative light scattering detector (SFC/ELSD) coupling provides high resolution, fast analysis and higher responses for the analysis of triterpenoids. After the initial screening of seven stationary phases to select the well suited one, analytical conditions (modifier percentage, from 10 to 3%; backpressure (from 12 to 18 MPa) and temperature (from 15 to 25 °C) were studied to improve the separation, and ELSD detection of a standard mixture composed of 8 triterpenoids (oleanolic acid, erythrodiol, β-amyrin, ursolic acid, uvaol, betulinic acid, betulin, lupeol). Applied to apple pomace extracts, this method allows the separation of about 15 triterpenoid compounds, in less than 20 min, with isocratic conditions. Moreover, the ELSD response is dramatically higher than the one provided by UV detection, and avoids derivatization steps. An attempt to identify some compounds was done by collecting chromatographic peaks and further analyzing them with mass spectrometry. Complete identification or molecular formula could be proposed for 11 compounds. However, due to the presence of position and orientation isomers the absolute identification remains difficult, despite some retention rules deduced from the standard analysis.