Neutral Mobile Phase Research Articles

Conformational Arrangement of Dissolved Humic Substances. Influence of Solution Composition on Association of Humic Molecules

Both the primary chemical structure and the conformational structure of humic substances are still a matter of debate. A traditional assumption is that humic substances are large polymers and may present linear or coiled conformations according to solution properties. We studied the conformational changes of humic and fulvic acids of different chemical nature by high-pressure size-exclusion chromatography (HPSEC) after dissolution in mobile phases differing in composition but constant in ionic strength (I = 0.05). Modification of a neutral mobile phase (0.05 M NaNO3, pH 7) by addition of methanol (4.6 × 10-7 M, pH 6.97), hydrochloric acid (<2 × 10-6 M, pH 5.54), and acetic acid (4.6 × 10-7 M, pH 5.69) produced, in the order, a progressive decrease in molecular size. Size diminishing was shown by increasingly larger elution volumes at a refractive index detector and by concomitant reductions of peaks absorbance at a UV−vis detector. The decrease of molecular absorptivity (the phenomenon of hypochromism) proved that size reduction of dissolved humic substances was due more to disruption of an only apparent high-molecular-size arrangement into several smaller molecular associations than to coiling down of a macromolecular structure. The most significant conformational changes occurred in acidic mobile phases where hydrogen bondings formation was induced, suggesting that the large and easily disruptable humic conformation was held together predominantly by weak hydrophobic forces. The size of molecular association varied with humic samples indicating a close relation between humic chemical composition and stability of conformational structure. Our results show that humic substances in solution are loosely bound self-association of relatively small molecules, and intermolecular hydrophobic interactions are the predominant binding forces. The stability of such a conformation in solution is attributed to the entropy-driven tendency to exclude water molecules from humic association and thus decrease total molecular energy. This model of dissolved humic substances based on the reversible self-association of small molecules rather than on the macromolecular random coil represents a new understanding that should contribute to predict the environmental behavior of contaminants in association with natural organic matter.

Sparteine as mobile phase modifier in the chiral separation of hydrophobic basic drugs on an α 1-acid glycoprotein column

The effect of the chiral diamine sparteine as a cationic mobile phase modifier on the retention and enantioseparation of five hydrophobic basic drugs on an α 1-acid glycoprotein column was studied. Sparteine produced good results over the whole range of its concentration (1–15 m M) in the phosphate buffer mobile phase at pH 6.0 for the selected drugs (alprenolol, propranolol, promethazine, chlorpheniramine and disopyramide). When used as a sole modifier, sparteine greatly reduced retention without significant interference in the enantioseparation of the compounds. Sparteine was also applied in combination with 2.5% of the neutral modifier 2-propanol with good results as well. This study shows sparteine's potential both as a substitute for neutral mobile phase modifier and as an auxiliary in reducing amounts of neutral modifier.

Analysis of Benzoquinolines and Acridines in a Brazilian Diesel Oil by Particle Beam LC/MS and HPLC/UV

Abstract Nitrogen bases were isolated from a Brazilian diesel distillate by acid extraction. With minimum sample preparation and clean-up, nitrogen bases were analyzed by particle beam LC/MS and HPLC with photo diode-array detection using reversed phase chromatography. Benzoquinolines were identified as the major nitrogen containing compounds in this basic fraction. By using neutral mobile phases, benzoquinoline homologues were separated, enabling rapid class characterization as well as preparative HPLC isolation of individual benzoquinoline homologues. Acidified mobile phases, however, exhibited greater resolution for individual isomers. UV spectroscopy was used to differentiate various types of benzoquinolines (e.g. benzo[f]quinolines, benzo[h]quinolines, and acridines). Acridines can be easily distinguished from benzo[h]- or benzo[f]quinolines by its different UV absorption spectrum. Benzo[h]- and benzo[f]quinolines were differentiated by the differences in the first derivative of the absorbance (dA/dδ) vs. wavelength. To confirm the identification, nitrogen bases were also analyzed by conventional GC/MS methods.

Determination of erythromycin A by liquid chromatography and electrochemical detection, with application to salmon tissue

The chromatographic performance of erythromycin A (EA) is improved significantly over that achieved on polymeric columns by using a sterically shielded octyldiisopropylsilica (Zorbax Rx-C 8) column and a neutral mobile phase consisting of 5 m M aqueous sodium perchlorate—acetonitrile (50:50). This mobile phase facilitates electrochemical detection of EA at the 3-pmol level. Temperature control of both column and detection cell is important for minimizing detector noise and drift. A clean-up procedure, based on aminopropylsilica solid-phase extraction, allows the detection of EA in salmon flesh down to the 0.2-ppm level. Some of the metabolites of EA that retain the tertiary amine may also be detected by this method.

Fluorometric Determination of Indomethacin in Serum by High Performance Liquid Chromatography Using In-Line Oxidation with Hydrogen Peroxide

Abstract A fluorometric method has been developed for the determination of indomethacin In serum by high performance liquid chromatography. Indomethacin after deproteinization with acetonitriie was separated by reversed-phase chromatography with a neutral mobile phase (pH 7.0, 35% acetonitriie In phosphate buffer) containing hydrogen peroxide as a fluorogenic reagent, and was fluorometrically detected (Ex 358nm and Em 462nm) via in-line oxidation at high temperature (180 °C). The calibration curve was linear over the range of 2.5-15.0μg/ml by injecting a volume of 20μl of deproteinized serum. The detection limit (signal-to-noise ratio=3) for indomethacin in serum was 0.5μg/ml using a 20-μl aliquot of deproteinized serum.

Fluorometric Determination of Methotrexate in Serum by High-Performance Liquid Chromatography Using In-Line Oxidation with Hydrogen Peroxide

A fluorometric method has been developed for the determination of methotrexate in serum by high-performance liquid chromatography. Methotrexate after deproteinization with 2 M perchloric acid was separated by reversed-phase chromatography with a neutral mobile phase (pH 7.0, 8% acetonitrile in 25 mM phosphate buffer) containing 25 mM hydrogen peroxide as a fluorogenic reagent, and was fluorometrically detected (Ex 379 nm and Em 457 nm) via in-line oxidation at high temperature (160°C), The calibration curve could be made linear over the range 1 × 10-6 - 1 × 10-5 M by injecting a volume of 20 μl of deproteinized serum. The detection limit (signal-to-noise ratio=3) for methotrexate in serum was 2 × 10-8 M using a 50-μl aliquot of deproteinized serum. Comparison with the fluorescence polarization immunoassay gave a good correlation coefficient of 0.949.

Alkylamine/Phosphoric Acid as a Universal Buffer System for Basic and Acidic Mobile Phases in HPLC

Abstract This paper describes the production of a universal buffer system which can be used for acidic, neutral and basic mobile phases in HPLC. The mixture of alkylamine and phosphoric acid diluted with water gives the possibility to carry out separations with basic buffers on reversed phase columns with a silica gel support. The organic bases (n-butylamine/triethylamine) are less aggressive against the silica gel than mineral bases. The influence of different pH-values upon the quality of the separations is tested with some selected basic and acidic compounds. In addition to the tests with an ODS column we investigated the chromatographic behaviour (at different pH-values) of a new HPLC material (Polymer Gel) which is not based upon a silica gel support.

Automated determination of amoxycillin in biological fluids by column switching in ion-pair reversed-phase liquid chromatographic systems with post-column derivatization

Amoxycillin, a polar aminopenicillin, is rather unstable in biological fluids. Degradation can be prevented by fast sample pretreatment and storage at −70°C or below. After pH adjustment, it is stable in biological fluids for over 16 h. The samples were handled by automated chromatography overnight. The chromatographic system consisted of a small guard column, two analytical columns separated by a switching valve, a post-column reactor and a fluorescence detector. The chromatographic events and the calculation of results were handled by a computing integrator. The chromatography was based on ion-pairing principles. An efficient clean-up of the biological fluids was obtained by a heart cut from the first column, where the neutral mobile phase contained hexyl sulphate. In the second column the organic anion was exchanged for a large quaternary ammonium compound; amoxycillin was then retained as an ion pair. The composition of the mobile phases had to be designed carefully in order to avoid a disturbance of the chromatographic performance on the last column. An adequate selectivity and sensitivity was obtained by a post-column derivatization with fluorescamine. Detection limits were 10 and 25 ng/ml for plasma and urine,respectively, and the interassay precisions at low levels (350 and 2000 ng/ml for plasma and ca. 5% (R.S.D.).

Retention indices of some ergopeptines in reversed-phase high-performance liquid chromatography

The chromatographic behaviour of some naturally occurring ergot alkaloids of the peptide type (“ergopeptines”) and their isomers was examined by reversed-phase high-performance liquid chromatography. A satisfactory separation of eight natural ergot alkaloids and their isomers on LiChrosorb RP-18 is achieved with a neutral mobile phase of acetonitrile—water at pH 7 without addition of alkaline modifiers such as triethylamine or carbonate. For a better correlation of retention data with chemical structure, retention indices have been calculated using the homologous series of 2-ketoalkanes as reference compounds. On the octadecylsilica columns, the differences in retention indices (Δ I values) were nearly constant for particular compounds or molecular fragments, even when the absolute I values differed by 100 units or more.

Two-dimensional separations and behaviour of alkaloids of various polarities on unmodified silica gel in polar and very polar neutral or acidic mobile phases

New separation procedures for alkaloids of similar polarity and structure or of very different polarity and structure, based upon two-dimensional thin-layer chromatography on unmodified silica gel under mild conditions are described. Separation factors and separation mechanisms based on the structure of the bases and mobile phase composition are discussed for some examples of very efficient procedures.