Tyge Greibrokk

High-temperature liquid chromatography

The present state of the active use of elevated temperatures in liquid chromatography is reviewed, including the effects on retention, selectivity and efficiency. Separations in aqueous mobile phases as well as non-aqueous media are discussed, with particular emphasis on narrow-bore columns.

Temperature programming in liquid chromatography

The present state of temperature programming in liquid chromatography, including electrochromatography, is reviewed. A relatively limited number of papers making use of temperature programming as a technique in LC has been published so far, while the various effects of temperature are frequently discussed. As a part of the ongoing trend of miniaturization in chromatography, the active use of temperature as a variable in LC is expected to increase significantly. This paper includes an overview of the effects of temperature on column efficiency, retention, and selectivity, particularly in relation to narrow-bore columns, instrumental features, and some selected applications.

Determination of chlorinated phenols by high-performance liquid chromatography

The separation of mono-, di-, tri- and tetrachlorinated phenols and pentachlorophenol has been investigated by high-performance liquid chromatography using three different systems, adsorption chromatography on silica and reversedphase chromatography on a polar bonded phase (aminoalkyl) and a non-polar bonded phase (octadecyl). A satisfactory group separation as well as a satisfactory separation of isomers could not be obtained by adsorption chromatography on silica. A reasonable separation of isomers was obtained with amino columns, but the group separation was not satisfactory. The octadecyl columns were superior with reference both to separation of groups and of isomers within these groups as well as to stability and speed of analysis. A mixture of nineteen different phenols was resolved on a C18 column with a 30-min linear gradient of 56–80% methanol and 44-20% of 0.02 M KH2PO4 (pH 4.0).

Simultaneous determination of citalopram, fluoxetine, paroxetine and their metabolites in plasma by temperature-programmed packed capillary liquid chromatography with on-column focusing of large injection volumes.

A miniaturized temperature-programmed packed capillary liquid chromatographic method with on-column large volume injection and UV detection for the simultaneous determination of the three selective serotonin reuptake inhibitors citalopram, fluoxetine, paroxetine and their metabolites in plasma is presented. An established reversed-phase C8 solid-phase extraction method was employed, and the separation was carried out on a 3.5-microm Kromasil C18 0.32x300 mm column with temperature-programming from 35 (3 min) to 100 degrees C (10 min) at 1.3 degrees C/min. The mobile phase consisted of acetonitrile-45 mM ammonium formate (pH 4.00) (25:75, v/v). The non-eluting sample focusing solvent composition acetonitrile-45 mM ammonium formate (pH 4.00) (3:97, v/v) allowed injection of 10 microl or more of the plasma extracts. The method was validated for the concentration range 0.05-5.0 microM, and the calibration curves were linear with coefficients of correlation >0.993. The limits of quantification for the antidepressants and their metabolites ranged from 0.05 to 0.26 microM. The within and between assay precision of relative peak height were in the range 2-22 and 2-15% relative standard deviation, respectively. The within and between assay recoveries were in the 61-99 and 54-92% range for the antidepressants, respectively, and between 52-102 and 51-102% for the metabolites.

Reversed-phase chromatography of peptides

Summary The chromatographic behaviour of underivatized peptides on reversed-phase packings has been examined. To study the effects of different amino acids in peptides, simple model substances, such as amino acids and dipeptides, were chosen. The capacity factors of amino acids, l,l -dipeptides and diastereoisomeric dipeptides were measured, and side-effects such as loss by adsorption, double-peak formation and load-retention dependence were studied. Of the four different reversed phases examined, the ODS packings appeared to give both the best selectivity and the best efficiency in the separation of most peptides and diastereoisomers. A marked difference in the retention of basic functions was obtained on two ODS packings of different manufacture.

Recent advances in on-line multidimensional liquid chromatography

Multidimensional liquid chromatography (MD LC) has, due to enhanced peak capacity compared to one-dimensional LC, become an important analytical tool for separating components in complex samples, e.g. in proteomics. MD LC can be performed both on-line and off-line. Because of the advantages like possible automation and minimal sample loss, on-line MD LC has appeared to be very attractive also for high throughput analysis. This review includes only on-line coupled MD LC. Different aspects related to the compatibility of separation principles and column dimensions are highlighted and recent applications using on-line mainly two-dimensional (2D) LC have been included.

Determination of polyamines by pre-column derivatization with σ-phthalaldehyde and ethanethiol in combination with reversed-phase high-performance liquid chromatography

Abstract The polyamines spermine, spermidine, putrescine and cadaverine were treated with o -phthalaldehyde and ethanethiol. After reaction time of 90 sec., the stability of the derivatives was examined in the reaction medium, in an ethyl acetate extract and in different mobile phases used for high-performance liquid chromatography on reversed phase columns. The stability of the ethanethiol derivatives was improved compared to the 2-mercaptoethanol derivatives. Increasing concentrations of alcohol in the reaction medium increased the stability, but a significantly higher stability was obtained by including a simple and rapid extraction with ethyl acetate. With a mobile phase of N,N-dimethylcyclohexylamine (0.1 M ) and phosphoric acid (0.2 M ) in a 70–90% methanol gradient, decomposition on the column was minimized and a satisfactory resoluton was obtained. A detection limit of less than 0.5 pmol of each polyamine was obtained. by fluorescence detection.

Evaluation of amperometric detectors for high-performance liquid chromatography: analysis of benzodiazepines

Abstract The design of amperometric detectors for high-performance liquid chromatography is discussed. A simple flow cell with interchangeable working electrodes made from glassy carbon, carbon paste and mercury is described, and its performance is compared with commercially available cells, using both constant-potential and pulse-measuring techniques. The analysis of nitrazepam, diazepam and chlordiazepoxide was used as a model system; the detectors were used in the reduction mode and the mobile phase was methanol—water (60:40) containing 0.05 M ammonium acetate. The effects of various experimental parameters are reported. The detection limit was found to depend strongly on the reduction potential: at −0.93 V vs. Ag, ng of nitrazepam could be detected, whereas at −1.30 V the detection limit was 30 ng, owing to the high background current at this potential. A potential more negative than −1.1 V must be used for the detection of diazepam and chlordiazepoxide; at −1.30 V the detection limit was 300 ng for these compounds.

Group separation of oil residues by supercritical fluid chromatography

Abstract The group separation by supercritical fluid chromatography of high-boiling residues (b.p. higher than 350°C) or North Sea oil is described. The chromatographic separation used a three-column system (cyano and silica and silver-impregnated silica) with column-switching and back-flushing with supercritical carbon dioxide as the mobile phase. The flame ionization detector could be used directly by placing the restrictor in the jet. The quantitative aspects are discussed in relation to the results obtained by liquid chromatographic methods.

Improving the resolution of neuropeptides in rat brain with on-line HILIC-RP compared to on-line SCX-RP.

Our two already established on-line 2-D LC systems, a strong cation exchange-RP chromatography (SCX-RP) system and a hydrophilic interaction LC (HILIC)-RP 2-D LC system, were compared to explore which system is best suited for our further studies of differences in cerebral neuropeptide expression as a function of hypoxia-caused stress. The same mass spectrometer and database search parameters were applied in both systems. In total, 19 first dimension fractions were collected with the novel on-line HILIC-RP system, including a Hypercarb SPE column that was applied to trap the compounds not retained on a Kromasil C18 enrichment column. In contrast, six fractions were collected in the SCX-RP method, due to practical limitations of this traditional on-line 2-D LC system. With the on-line HILIC-RP system three times more peaks were detected. It was observed that most of the compounds eluted in the first two fractions in the SCX-RP method, while in the 2-D HILIC-RP method there seemed to be no correlation between peaks detected and fraction number. Thus, from this systematic study it seems that on-line HILIC-RP chromatography is the method of choice for comparative peptidomics of cerebral neuropeptides in future studies.