M. Dreux

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.

Borate complexation of flavonoid-O-glycosides in capillary electrophoresis: I. Separation of flavonoid-7-O-glycosides differing in their flavonoid aglycone

Capillary electrophoresis was found to give significantly greater efficiency, selectivity and speed compared with high-performance liquid chromatography for the separation of mixtures of flavonoid-O-glycosides. The migration behaviour of selected flavonoid-O-glycosides and their flavonoid aglycones in free solution capillary electrophoresis and micellar electrokinetic capillary chromatography (MECC) was investigated. A mixture of flavonoid-7-O-glycosides and their flavonoid aglycones was resolved by MECC. A 70 cm × 50 μm I.D. fused-silica capillary column and a 50 mM sodium dodecyl sulphate-20 mM Tris-CHl running buffer (pH 7.1) were used for these electrophoretic separations. Under these neutral conditions, the retention mechanism is based on hydrophobic interactions betweeen flavonoids and the hydrophobic core of the micelles. Free zone capillary electrophoresis of borate complexes was more appropriate for the separation of a mixture of flavonoid-O-glycosides. In that case, the migration behaviour of these selected flavonoid-O-glycosides was explained by ionization of some hydroxyl groups and mainly by borate complexation of the sugar moiety. The identity of each flavonoid-O-glycoside was determined by using a fast-scanning multiple-wavelength UV detector and by recording its on-column UV spectrum in the range of 190–360 nm.

Borate complexation of flavonoid-O-glycosides in capillary electrophoresis. II. Separation of flavonoid-3-O-glycosides differing in their sugar moiety.

Capillary electrophoresis was found to give significantly higher efficiency, selectivity and speed than high-performance liquid chromatography for the separation of a mixture of flavonoid-3-O-glycosides differing in their sugar moiety. Boric acid running buffer (0.2 M, pH 10.5) was used for this electrophoretic separation. The migration order in free solution capillary electrophoresis (CE) and the selectivity of these flavonoid-3-O-glycosides can be mainly explained by in situ borate complexation of both the sugar moiety and the cis-1,2-hydroxyl groups on the flavonoid skeleton and, to a lesser extent, by the ionization of hydroxyl groups on the flavonoid skeleton due to alkaline pH conditions. The correlation of the electrophoretic mobilities with the configuration and conformation of the compounds is discussed.

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.

Separation of inorganic anions by ion-pair, reverse phase liquid chromatography monitored by indirect photometry

SummaryA new method for the analysis of inorganic anions is presented. It uses the most conventional liquid chromatographic system (reverse-phase chromatography) and the most common detector (ultraviolet). The nature of the ammonium ions used and their counter ions lead to different selectivities. The use of the indirect photometry allows detection of all anions.

Comparison of ethoxylated alcohols and polyethylene glycols by high-performance liquid chromatography and supercritical fluid chromatography using evaporative light-scattering detection☆

Abstract Ethoxylated alcohols are surfactants whose oligomer distributions are conventionally determined by high-performance liquid chromatography. However, this method cannot resolve individual oligomers with a high degree of ethoxylation. Further, normal- and reversed-phase liquid chromatography cannot give the fingerprints for both the ethoxylated alcohols and polyethylene glycols, the synthetic residues without surfactant properties. Supercritical fluid chromatography coupled with evaporative light-scattering detection was developed for the analysis of these mixtures which do not contain chromophores.

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.