Quantitative Analysts of 5-Hydroxytryptamtne in Biological Material by High Perfornance Liquid Chromatography and Electrochmical Detection

Abstract

A quantitative method for the analysis of 5-hydroxytryptamine in biological material is described. The method is based on high performance liquid chromatography (HPLC) with electrochemical detection. A simple purification on a weakly acidic ion exchange resin prior to the analysis gives quite clean samples and permits concentration of diluted samples. The chromatographic separation is performed on a reverse phase column with organic modifier added to an aqueous eluent. With this analytical system 25 pg of 5-hydroxytryptamine can be detected.     

A Simple Liquid Chromatographic Method for Analysis and Isolation of the Unsaturated Components of Anacardic Acid

Abstract

Good analytical and preparative separation of the saturated, monoene, diene and triene components of anacardic acid have been achieved by reverse phase HPLC on a C₁₈ column by isocratic elution with methanol-aqueous acetic acid.     

A review of: “HPLC of Biological Compounds-A Laboratory Guide,by William S. Hancock & James T. Sparrow, (Volume 26 of the Chromatographic Science Series,Jack Cazes,Editor), Marcel Dekker,Inc., New York,NY, 1984, 361 pp., £39.75 (US)”.

Abstract

The analytical and preparative separation of the saturated, monoene, diene and triene constituents of cardanol and cardol have been achieved by reverse phase HPLC on a C₁₈ column using gradient elution.     

Controlling morphological and electro-optical properties via the phase separation in polymer/liquid-crystal composite materials

ABSTRACT

The polymer/liquid-crystal composite materials have been extensively studied for their potential applications. Various optical devices based on this composite material have been proposed and realised. The device performance is highly dependent on the phase separation of this composite material. Here, we investigate the photopolymerisation-induced phase separation in this composite material. Depending on the mass ratios between the polymer and the liquid crystal, the phase separation can be well controlled and subsequently affect the morphological and electro-optical properties. At a fixed ratio, we can realise either phase-separated composite films or conventional polymer-dispersed liquid crystal films with completely different optical properties. By carefully controlling the exposure conditions, the morphologies and electro-optical properties have been studied and optimised in details. With in-depth studies and optimisation, the photopolymerisation-induced phase separation technique could be utilised to realise many different optical functions based on the polymer/liquid-crystal composite materials.     

Use of Centrifugal Partition Chromatography and Proteins in The Preparative Separation of Amino Acid Enantiomers

Abstract

The growth of analytical methodologies for the separation of enantiomers has been impressive. Attention is now turning to the large scale separation of enantiomers. Often scaling-up sensitive analytical separations is ineffective and inefficient. Centrifugal partition chromatography (CPC) may be a viable alternative for the preparative separation of racemic mixtures in some cases. The use of proteins as chiral selectors in CPC is examined. Attention was focused on proteins that previously were used as bonded phases in analytical LC columns. The enzymatic properties of α-chymotrypsin allowed it to be used as a bioreactor in conjunction with CPC. When proteins are used as components of the stationary or mobile phase there can be problems with denaturation. However, when used in external incubation processes or as column bioreactors coupled with CPC, effective gram-scale separations can be performed. Tryptophan methyl ester was used as a model compound to evaluate this approach.     

Comparison of Column Packings for Isocratic High-Performance Liquid Chromatographic Analysis of Carotenoids Using Non-Aqueous Reversed-Phase

Abstract

Ten different columns are compared for the isocratic non-aqueous reversed-phase separation of carotenoids, using solvent mixtures of ethyl acetate-acetonitrile both with and without 0.1% n-decanol as modifier. Conditions were established for separation of a mixture of alfalfa carotenoids containing mainly neoxanthin, violaxanthin, lutein and β-carotene. The best material for use for rapid isocratic separation of all major components was a high carbon loading non-endcapped material with ODS functionality, although one endcapped C8 material gave similar results. The use of n-decanol as mobile phase modifier is imperative to rapidly condition new columns to give optimum peak shape and definition and system linearity.     

Preparation and Evaluation of P-tert-Butyl-calix[8]arene-bonded Silica Stationary Phase for High Performance Liquid Chromatography

ABSTRACT

A new p-fert-butyl-calix[8]arene-bonded silica gel stationary phase was synthesized through heterogeneous functionalisation of suspended porous silica. A characterization of its structure was carried out by using elemental analysis, FTIR and ¹³C solid state NMR spectroscopy. Chromatographic performance of the new packing material was investigated by employing polycyclic aromatic hydrocarbons (PAHs) as probes and using methanol-water as mobile phase. The investigations show that the new stationary phase behaves as a reversed phase stationary phase. The liquid chromatographic separation of PAHs solutes on the new bonded phase was compared with that on a p-tert-butyl-calix[4]arene-bonded silica stationary phase. The new p-tert-butyl-calix[8]arene-bonded phase exhibited higher retention and better separation selectivity, although the carbon content and coverage of the new packing material was lower than that of the p-tert-butyl-calix[4]arene bonded silica stationary phase. A possible retention mechanism for the new packing material was also proposed.     

Evaluation of Analytical Countercurrent Chromatographs: High-Speed Countercurrent Chromatograph-4000 Vs. Analytical Toroidal Coil Centrifuge

Abstract

Performance of two countercurrent chromatographic models, high speed countercurrent chromatograph (HSCCC-4000) and analytical toroidal coil centrifuge (TCC), is evaluated in terms of theoretical plate number, resolution factor and separation time to assess their analytical capability. A series of experiments was conducted to investigate the effects of internal diameter and length of the coiled column, and flow rate of the mobile phase on the separation of indole auxins in two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water at different volume ratios. The three components of indole auxins were completely resolved in 16 min with a HSCCC system equipped with a multilayer coil of a 0.55 mm I.D. PTFE tube with theoretical plates ranging from 1290 to 829. Similar separation was achieved in 24 min with a TCC systems equipped with a 0.3 mm I.D. PTFE tube with theoretical plates ranging from 1811 to 969. It is concluded that both systems have comparable analytical capability at the present stage of development.     

Chromatographic Isolation of Presumptive Peptide Flavor Principles from Red Meat

Abstract

Top round, bovine semimembranosus and adductor muscle was selected as a model for isolation of presumptive, low molecular mass (M_r) flavor peptides. The isolation and purification of these peptides (<5,000 M_r) from ‘cooked’ and ‘cooked-stored-recooked’ meat was developed by combining various chromatographic techniques. Peptide samples were initially made by preparing acetic acid extracts of meat followed by the removal of organic soluble lipids and carbohydrates by phase partition extraction. The lipid-free extracted material was subsequently subjected to size exclusion chromatography using Sephadex G-25 resulting in two major polypeptide groups with M_r of 1500 to 3000. This material was now available for further purification by both semipreparative and analytical reverse phase (RP) - high performance liquid chromatography (HPLC) for separation of hydrophilic and hydrophobic peptides. Separation of the peptides into these two groups is particularly important since the perception of sweet taste is usually associated with hydrophilic peptides while bitter (and often sour) taste is associated with hydrophobic peptides. Semipreparative RP-HPLC of peptides from the low M_r material revealed highly significant differences in the hydropilic and hydrophobic peptide composition of ‘cooked’ versus ‘cooked-stored-recooked’ samples i.e., the former appeared to have equal amounts of the two classes of peptides while the latter appeared to contain predominantly hydrophobic peptides. Peptides prepared semipreparatively were readily available for further examination by analytical RP-HPLC and analyzed by diode array detection. The latter method revealed major differences in the hydrophobic peptide components found in the two meat groups.     

Quantitative Analysis of Terbutaline (Bricanyl®) in Human Plasma with Liquid Chromatography and Electrochemical Detection Using On-Line Enrichment

Abstract

A liquid chromatographic method with electrochemical detection (LC-EC) has been developed for the quantitative analysis of terbuta-line in the range 5–50 pmole ml^?1 of human plasma. Terbutaline is isolated from 2 ml of plasma on an ion-exchange column and the eluate is concentrated on a hydrophobic precolumn on-line in the chromatographic system. The precolumn is then back-flushed for further separation onto a hydrophobic analytical column. The mobile phase is a methanol-aqueous buffer to which sodium perchlorate is added to achieve resolution from interfering peaks. A glassy carbon electrode is used for detection. Comparison has been made with gas chromatography-mass spectrometry (GC-MS) to examine the accuracy of the method.     

A Novel Approach to Reversed-Phase Preparative High-Performance Liquid Chromatography of Peptides

Abstract

In this study, we describe a novel approach to preparative liquid chromatography which takes advantage of the different relative hydrophobicities of components of a sample mixture, so that when a column is optimally loaded with an aqueous solution of the sample mixture, there is competition among the sample components for the adsorption sites on the hydrophobic stationary phase. The more hydrophobic components compete more successfully for these sites than more hydrophilic components, which are displaced and immediately eluted from the column. Thus, the major separation takes place in water. Subsequent treatment with an aqueous organic modifier is only required to wash retained components off the column and takes no part in the major separation process. This approach was applied to the preparative purification of mixtures of closely-related peptides, representing the crude peptide mixtures typically obtained from solid-phase peptide synthesis. The excellent separation profiles and high yields of pure peptide products on analytical columns reported in this study demonstrate that this methodology has great potential for preparative separation of a major component from hydrophilic and/or hydrophobic impurities.     

Liquid Chromatographic and Spectral Methods for the Differentiation of 3,4-Methylenedioxymethamphetamine (MDMA) from Regioisomeric Phenethylamines

Abstract

The analytical profiles are described for four amines, 3,4-methylenedioxymethamphetamine (MDMA) and three isomeric phen-ethylamines of MW = 193. These four amines all contain an identical 3,4-methylenedioxyphenyl moiety, thus the regioisomerism is within the carbon-carbon bond located alpha to the amine. Therefore these phenethylamines are regioisomeric within the imine fragment (m/z = 58) which is the base peak in the electron impact mass spectrum of MDMA. The ultraviolet absorption spectra for these compounds show the characteristic methylenedioxyphenyl group absorption bands in the 235 to 280 nm range. These amines may best be differentiated by chromatographic separation and are well resolved by liquid chromatographic techniques. The four regioisomeric amines were separated using an isocratic reversed-phase system consisting of a C₁₈ stationary phase and an acidic (pH) mobile phase. The elution order under these conditions appears to parallel the length of the carbon chain attached to the aromatic ring.     

Micromethod for Determination of Thiopental in Biological Fluids by High Performance Liquid Chromatography. Application to Therapeutic Follow-Up and Pharmacokinetic Study

Abstract

Thiopental is an ultra-short acting barbiturate, used either to induce anesthesia, or to manage intracranial hypertension associated with head injuries. A high performance liquid chromatography method vas proposed for the rapid and easy determination of thiopental in biological fluids. After precipitation of proteins with acetonitrile the chromatographic separation vas performed onto a reversed-phase column with an acetonitrile-perchlorate buffer mixture as mobile phase. Compounds were analysed at 300nm and the detection limit vas about 0.1 mg/1. The analytical procedure vas applied to the therapeutic follow-up in patients (8 childrens. 4 adults) treated with long-term infusion of thiopental after head injury, and to a pharmacokinetic study in patients receiving a single 1Y bolus of thiopental to induce anesthesia.     

Separation of Biological Pyridines by High Pressure Liquid Chromatography

Abstract

The separation of the six pyridine compounds which comprise the pyridine nucleotide cycle, nicotinamide adenine dinucleotide phosphate and para-aminobenzoic acid, a compound biologically related to these pyridines, can be achieved rapidly utilizing high pressure liquid chromatography. Optimum separation is accomplished using ion-ion pairing in reverse phase chromatography with a C₁₈ stationary phase and an aqueous mobile phase of 5mM pentanesulfonic acid and 25 mM KH₂PO₄. The effect of temperature on the separation is minimal. As little as 10 ng of these compounds is detected via absorption of ultraviolet light at a wavelength of 254 nm.     

RP-HPLC Separation of Highly Charged Quaternary Ammonium Salts

Abstract

The reverse phase HPLC separation of polycationic viologenes 1 – 4 and macrotricyclic quaternary ammonium salts 5 – 8 using ion pair conditions is described. Whereas the compounds 1 – 4 could be analysed on any of 4 stationary RP-18 phases tested, the cage compounds 5 – 8 were much more sensitive to the source of the matrix material. Optimal separation conditions for the latter up to eightfold positively charged macrocycles employ sodium perchlorate in acidic aqueous methanol using Nucleosil RP-18 or Lichrospher CH-18 columns.     

Toroidal Coil Countercurrent Chromatography: A Fast Simple Alternative to Countercurrent Distribution Using Aqueous Two Phase Partition: Principles,Theory, and Apparatus

Abstract

The principles, theoretical basis and equipment for continuous two phase toroidal coil chromatography are described. Rat liver homogenates were subjected to analytical subcellular fractionation by toroidal coil chromatography in a phase mixture of 3.3% (w/w) dextran T500, 5.4% (w/w) poly(ethylene glycol) 6000, 10 mM sodium phosphate-phosphoric acid buffer, pH 7.4, in 0.26 M sucrose containing 0.05 mM Na₂EDTA and 1 mM ethanol. The distribution of organelles, as reflected by their marker enzymes, was compared to that obtained by discrete counter-current partition in a 17 transfer apparatus. Toroidal coil chromatography showed enhanced resolution of certain organelles. In particular, almost complete separation of plasma membrane from endoplasmic reticulum was achieved and some resolution of plasma membrane from lysosomes was obtained. It is concluded that toroidal coil chromatography offers a potentially useful alternative approach to organelle separation techniques.     

An Overview of Analytical Chemistry in Australia

ABSTRACT

A brief overview of analytical chemistry research in Australia is presented and reference is made to the work of several research groups. Topics covered include the development of a longitudinally modulated cryogenic system for comprehensive gas chromatography, mixed-mode capillary electrochromatography for manipulation of separation selectivity of inorganic ions, new developments in chemical metrology, discontinuous flow analysis and its applications, the design and applications of an orthogonal acceleration time-of-flight mass spectrometer, and the use of chemiluminescence in a range of analytical techniques.     

Phase Systems and Post-Column Dithizone Reaction Detection for the Analysis of Organo-Mercurials by HPLC

Abstract

The suitability of HPLC (normal and reversed phase adsorption) with UV or post-column reaction detection for the analysis of organomercurials was investigated systematically. The separation of organomercurials is best carried out on a reversed phase system with a C-8 bonded phase material as the stationary phase and acetonitrile-aqueous sodium bromide mixtures as the mobile phase.

The precision and detection limit of the method and the efficiency of the extraction procedure were established. For the alkylmercury compounds the lowest limit of detection (80ppb) was obtained with the dithizone reaction detection and for the phenylmercury compounds with UV detection (60ppb). A chromatogram of a spiked fish (2ppmHg) and a river water sample (50ppbHg) is shown.     

Simplified Approach to HPLC Precolumn Fluorescent Labeling of Carbohydrates: N-(2-Pyridinyl)-glycosylamnes

Abstract

Reducing sugars were fluorescently labeled by condensation with 2-aminopyridine. The reaction was quantitative and the product was sufficiently stable so that a subsequent reduction step (as in reductive amination) proved unnecessary. The products have been characterized as N-(2-pyridinyl)-glycosylamines. Oligosaccharides labeled in this manner exhibit important analytical characteristics. They: a) are stable under both HPLC separation and prolonged storage; b) have improved chromatographic resolution; c) regenerate original material by weak acid hydrolysis.