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PubMed1988

Design and application of an expert system for mobile phase optimisation in reversed-phase liquid chromatography.

The selection of the optimum composition for the mobile phase in reversed-phase high-performance liquid chromatography (HPLC) is a complex task; conventional approaches require the expenditure of significant amounts of time by the analyst, particularly for complex mixtures of solutes of biological origin. Some of the existing strategies for the automated optimisation of mobile phase composition (e.g. Simplex), may fail if the elution order of the components changes; or they may require that standards be chromatographed in order to establish the retention behaviour of each component in a mixture (e.g. resolution mapping). These problems may be overcome if the retention behaviour of each individual solute can be established from the chromatogram of the mixture. In this regard, components can be tracked by exploiting the spectral information generated by a rapid scanning photodiode array detector. Unfortunately this information is often insufficiently detailed to allow an unambiguous model of retention behaviour to be constructed. The system developed by the Authors uses these spectral data as a basis for constructing one or more hypothetical retention models, each of which is refined or rejected as further information is obtained during the progress of the experiment. To improve the reliability of the retention models proposed by the system, the spectral data are utilised in a number of tests designed to assess the purity of each chromatographic peak. The information so generated may be used in conjunction with any previously acquired spectral data both to select an appropriate method for extracting spectra for each component from the matrix of (A, lambda, t) data and to establish reliability parameters for the resultant spectra. The development and philosophy of the expert system developed for eluent optimisation in reversed-phase HPLC is discussed.

Journal Article↗
PubMedDate not supplied

Solid-phase extraction and optimized separation of doxorubicin, epirubicin and their metabolites using reversed-phase high-performance liquid chromatography.

A reversed-phase isocratic high-performance liquid chromatographic method is described in which a formal structured procedure was applied to predict the mobile phase composition giving optimal baseline resolution of the clinically important anticancer agents doxorubicin and 4'-epidoxorubicin (epirubicin), their principal metabolites, and daunorubicin (internal standard). These formal statistical procedures included the simultaneous techniques of solvent selectivity triangle and factorial design for range-finding preliminary studies, followed by use of the modified simplex, a sequential procedure. These were used to select the parameters of organic modifier, buffer strength and pH necessary for use with a Spherisorb ODS 1 column, to achieve optimal separation of eight anthracycline solutes. Ultraviolet and fluorescence detection was used (lambda ex = 254 nm, lambda em = 560 nm), and the latter gave a low detection limit for doxorubicin in serum of 1 ng ml-1. The optimal mobile phase composition was determined to be acetonitrile-0.06 M Na2 HPO4 containing 0.05% (v/v) triethylamine adjusted to pH 4.6 with 0.03 M citric acid (35:65, v/v). A solid-phase extraction method was developed to enable the selective isolation of anthracyclines by adsorption onto C8 Bond-Elut cartridges, and is based on extraction of serum spiked with a mixture of the anthracycline solutes. The anthracyclines were eluted using acetonitrile-0.2 M Na2 HPO4 containing 0.05% (v/v) triethylamine adjusted to pH 3.6 with 0.1 M citric acid (67.5:32.5, v/v). Reproducible recoveries for doxorubicin (94 +/- 8%) and for epirubicin (96 +/- 8%) were obtained (n = 5). In particular, recoveries for the 7-deoxyaglycone metabolite (99%) were higher than other extraction methods cited.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗
PubMed1992

On-line phase-transfer catalysed dansylation of phenolic compounds followed by normal-phase liquid chromatography with fluorescence detection.

A study of the on-line phase-transfer catalysed dansylation of phenolic compounds is presented. The extraction-dansylation is performed in the extraction coil of a home-made flow-injection extraction unit. After phase separation, the organic phase is fed to a normal-phase liquid chromatographic system with fluorescence detection. Ethynyloestradiol, oestradiol and paracetamol are used as test compounds. The influence of temperature on the reaction is examined. Calibration graphs showed good linearity (r greater than 0.996) and limits of detection are satisfactory (8 x 10(-7) M for ethynyloestradiol, 2 x 10(-6) M for oestradiol and 5 x 10(-7) M for paracetamol). The method is not applicable for the assay of oxychinoline, phenylephrine and morphine.

Acetaminophen↗
PubMed2005

Centrographic analysis of 1-phase versus 2-phase treatment for Class II malocclusion.

INTRODUCTION: Cephalometric analyses have been used by orthodontists to track growth and monitor treatment effects. Most of these analyses have normative values to which patients are compared, but some "normal" patients vary quite a bit from the normative values. The centrographic analysis is a visual analysis with no angles to measure or normative values to compare. After a reference plane is developed, the relative position of variable landmarks can be seen. METHODS: We used the centroid centrographic analysis to study the effects of 1-phase and 2-phase orthodontic treatment. Phase 1 treatment consisted of bionator (n = 66), headgear/biteplane (n = 69), or observation (n = 65) until a Class I molar relationship was achieved or 2 years had elapsed. After 1 year, all subjects underwent full orthodontic treatment with fixed appliances. RESULTS: Centrographic analysis showed that early treatment has effects on the mandible. However, the differences were not apparent by the end of fixed appliance treatment. CONCLUSIONS: The skeletal effects of phase 1 treatment disappear by the end of fixed appliance treatment.

Activator Appliances↗
PubMed2004

Correlation between lifetime heterogeneity and kinetics heterogeneity during chlorophyll fluorescence induction in leaves: 1. Mono-frequency phase and modulation analysis reveals a conformational change of a PSII pigment complex during the IP thermal phase.

The relationship between the fluorescence lifetime (tau) and yield (Phi) obtained in phase and modulation fluorometry at 54 MHz during the chlorophyll fluorescence induction in dark-adapted leaves under low actinic light has been investigated. Three typical phases have been identified: (i) linear during the OI photochemical rise, (ii) convex curvature during the subsequent IP thermal rise, and (iii) linear during the PS slow decay. A similar relationship has been obtained in the fluorescence induction for the fluorescence yield measured at 685 nm plotted versus the fluorescence yield measured at 735 nm. A spectrally resolved analysis shows that the curvature of the tau-Phi relationship is not due to chlorophyll fluorescence reabsorption effects. Several other hypotheses are discussed and we conclude that the curvature of the tau-Phi relationship is due to a variable and transitory nonphotochemical quenching. We tentatively propose that this quenching results from a conformational change of a pigment-protein complex of Photosystem II core antenna during the IP phase and could explain both spectral and temporal transitory changes of the fluorescence. A variable blue shift of the 685 nm peak of the fluorescence spectrum during the IP phase has been observed, supporting this hypothesis.

Adaptation, Physiological↗
PubMed2004

Evaluation of an embedded polar C4 phase for hydrophobic protein analysis by reversed-phase liquid chromatography.

A C4 column (InertsilWP300 C4) was developed for a protein analysis in reversed-phase chromatography. Polar groups were embedded on this phase. And this column was evaluated with low concentration of trifluoroacetic acid (TFA) and formic acid. Test C4 phases that controlled carbon loading and commercially available columns were compared with this embedded C4 column. The test column with higher carbon loading showed good separations of proteins at low concentrations of TFA, although hydrophobic proteins were broadened. On the other hand, embedded polar C4 packings showed ideal retention behavior for proteins, which is independent of ion-pairing effects of additives in mobile phase. The reason was concluded to be the shielding effects of polar groups on the sorbents, which reduce silanophilic interactions with polar groups of proteins.

Chromatography, High Pressure Liquid↗
PubMed2004

Reversed-phase retention thermodynamics of pure-water mobile phases at ambient and elevated temperature.

The use of pure water at superheated temperatures, between 100 and 200 degrees C, as a mobile phase for reversed-phase separations is explored. The thermodynamics of the retention process at low temperature (15-55 degrees C) are compared to the thermodynamics at elevated temperature (125-175 degrees C). Significant differences in the enthalpy of the retention process are observed between the two temperature ranges. This is possibly due to changes in the hydrogen-bond network of the pure-water mobile phase, which would change the solvation, and therefore retention, of non-polar solutes. The change in thermodynamic values between the two temperature regions invalidates extrapolation of retention as a function of temperature between the two temperature regions for the prediction of room-temperature pure-water retention factors. The thermodynamic changes observed as the temperature is increased are similar to those seen when mobile phase composition is changed (by adding organic modifier) at constant temperature.

Hydrogen Bonding↗
PubMed2005

Study of overloading of basic drugs and peptides in reversed-phase high-performance liquid chromatography using pH adjustment of weak acid mobile phases suitable for mass spectrometry.

Serious losses in column efficiency for ionised basic drugs and peptides occur due to overloading of C18 phases when weak acid mobile phases of low ionic strength suitable for mass spectrometric detection are used. Measurable changes in retention time and efficiency can be observed even for levels around 0.01 microg of basic drugs on 0.46 cm I.D. columns; the overloading process is a continuum, rather than an event which takes place only once a certain threshold value has been reached. Overloading can be reduced by increasing the mobile phase pH, e.g to the pKa of the weak acid, which increases the ionic strength, allowing a greater degree of ion pairing and perhaps also physical screening of the adsorbed solute ions by buffer ions. Buffer capacity is also optimum at its pKa. Silanol ionisation and kinetic tailing effects were mostly absent at aqueous pH up to 4.75 on the hybrid inorganic-organic C18 phase employed in this study, except when analysing the most highly charged peptides. The exact cause of overloading of these ionic species is unclear.

Buffers↗
PubMed2005

Determination of lipophilicity by reversed-phase high-performance liquid chromatography. Influence of 1-octanol in the mobile phase.

Lipophilicity was evaluated using a novel RP-HPLC stationary phase (Discovery-RP-Amide-C16) with and without 1-octanol added to the mobile phase. A set of 46 drugs and flavonoids characterized by a broad structural diversity and a wide log Poct range (-0.69 to 5.70) was selected for this study. This set consists of neutral solutes and solutes with acidic or ampholytic functionalities which were maintained neutral at pH 2.5 or 4. In our conditions, the addition of 1-octanol in the mobile phase proved a key factor to derive a lipophilicity index log k(w) highly correlated with log Poct for all investigated solutes. 1-Octanol improved the correlation between log Poct and log k(w) mainly by influencing the retention behavior of the solutes with log Poct values below +3. This study brings additional evidence that under proper experimental conditions of stationary and mobile phases, RP-HPLC is a very useful method to obtain log Poct values.

1-Octanol↗
PubMed2005

Adsorbed solution model for prediction of normal-phase chromatography process with varying composition of the mobile phase.

The adsorbed solution model has been used to predict competitive adsorption equilibria of the solute and the active component of mobile phase in a normal-phase liquid chromatography system. The inputs to the calculations were the single adsorption isotherms accounting for energetic heterogeneity of the adsorbent surface and non-ideality of the mobile phase solution. The competitive adsorption model has been coupled with a model of the column dynamics and used for simulating of chromatography process at different mobile phase composition. The predictions have been verified by comparing the simulated and experimental chromatograms. The model allowed quantitative prediction of chromatography process on the basis of the pure-species adsorption isotherms.

Adsorption↗
PubMed2006

Effect of the mobile phase composition on the adsorption behavior of tryptophan in reversed-phase liquid chromatography.

Single-component adsorption isotherm data of l-tryptophan on a C18-bonded silica column were acquired by frontal analysis (FA), with aqueous mobile phases containing 2.5, 5, and 7.5% of acetonitrile (ACN) or 7, 10, 15, and 20% of methanol (MeOH). Most of these isotherms have two inflection points and three different parts. The low and the high concentration parts exhibit langmuirian behavior. The intermediate part exhibits anti-langmuirian behavior. The inflection points shift toward higher concentrations with increasing mobile phase concentration in ACN or MeOH, which causes the differences in the isotherm profiles. The nature of the organic modifier and its concentration affect only the isotherm profile and the numerical values of its parameters, not the nature of the best model, which is the bi-Moreau model in all cases. The isotherm profiles depend on the experimental conditions because they affect the intensity of the adsorbate-adsorbate interactions. Overloaded band profiles of tryptophan were recorded with the seven mobile phase compositions. They were used to determine the best values of the isotherm coefficients by the inverse method (IM) of chromatography. There is an excellent agreement between the values of these parameters obtained by FA and by IM. Increasing the concentration of either ACN or MeOH in the mobile phase causes a slight decrease in the saturation capacities of the low and the high energy sites, and in the adsorption constant of the low energy sites. The adsorption constant of the high energy sites increases with increasing concentration of either solvent or is little affected. The adsorbate-adsorbate interaction constants of both low and high energy sites increase for both solvents. Saturation capacities of the high energy sites are higher for ACN than for MeOH.

Acetonitriles↗
PubMed2004

Determination of avilamycin A and B in pig faeces by solid phase extraction and reverse-phase HPLC assay.

A HPLC method is described for the simultaneous determination of avilamycin A and B in pig faeces, following extraction using acetonitrile and normal-phase solid phase extraction. The HPLC stationary phase was Kromosil 5 micro C-18 with a mobile phase of 48% acetonitrile and 52% 0.01N ammonium acetate buffer, pumped at a flow rate of 1 ml/min. Detection was by UV absorbance at 295 nm and an injection volume of 50 microl was used. Recovery from faeces was >98% and intra-assay precision (CV) was <9.0% for both compounds. The lowest limit of quantification was 0.9 mg/kg (avilamycin A) and 0.2 mg/kg (avilamycin B) with an accuracy of <15% error. No interference was seen from endogenous materials in pig faeces and commonly used veterinary antibiotics.

Animals↗
PubMed2005

Effect of the temperature and mobile phase composition on the retention behavior of nitroanilines on ligand-exchange stationary phase.

This paper deals with the separation of isomers of nitroaniline by liquid chromatography using the ligand-exchange technique. The chromatographic separations were performed on the ligand-exchanger sporopollenin. The sporopollenin used as support of stationary phase was modified with carboxylated-ethylenediamine matrix and was loaded with cobalt(II) ions. Using the column packed with cobalt(II) loaded carboxylated diaminoethyl sporopollenin [Co(II)-CDAE-S], the retention behavior of 3- and 4-nitroanilines was investigated. The mobile phase used, was a mixture of 0.05 M NH(4)OH in ethanol-water. The resolution was strongly affected by the presence of ammonium hydroxide in the mobile phase and a concentration of 0.05 M was shown to be necessary for the separation of analytes. To study the effects of temperature on the resolution, column runs were also performed at various temperatures (15-60 degrees C). With increasing temperature, a decreased interaction between the solutes and the ligand-exchanger was observed. Consequently, the best results were obtained using a mixture of 0.05 M NH(4)OH in ethanol-water (10:90, v/v) as the mobile phase at a column temperature of 35 degrees C. Ligand-exchange chromatography on the Co(II)-CDAE-S could be a useful alternative method for the separation of nitroaniline.

Aniline Compounds↗
PubMed2006

Characterization and comparison of the chromatographic performance of different types of reversed-phase stationary phases.

The chromatographic performance of several base-deactivated stationary phases was evaluated with a specific chromatographic test. Seven basic test compounds, possessing different physico-chemical properties were injected on different supports with two mobile phases: one at pH 7.0 (acetonitrile-phosphate buffer, 40:60, v/v), and the other at pH 3.0 (acetonitrile-phosphate buffer, 15:85, v/v). Chromatographic parameters obtained under these conditions were treated by principal component analysis (PCA) to separate base deactivated supports according to their silanol activity (pH 7.0 mobile phase) and hydrophobic properties (pH 3.0 mobile phase). The information given by the specific test column evaluation was improved with complementary chemometric tools such as hierarchical cluster analysis. The same base deactivated supports were also tested following a general test procedure issued from the literature and obtained fundamental properties (in particular silanol activity and hydrophobicity) were compared with column evaluation obtained with the specific test: results were in good agreement, although the use of the specific test offered a better differentiation between numerous base-deactivated supports.

Chemical Phenomena↗
PubMed1996

Regulation of gene expression of various phase I and phase II drug-metabolizing enzymes by tamoxifen in rat liver.

The objective of the present investigation was to evaluate the effect of tamoxifen (TAM) on the gene expression of different phase I and phase II drug-metabolizing enzymes. Groups of male and female F344/NCr rats were administered either corn oil or TAM (2.8 to 45 mg/kg body wt x 14 days) dissolved in corn oil by gavage. An additional group of rats received a diet supplemented with phenobarbital (PB, 500 ppm). Northern blot analyses of total liver RNA were conducted using [32P]-labeled cDNA or oligonucleotide probes coding for different sulfotransferase (ST); UDP-glucuronosyltransferase (UGT), glutathione S-transferase (GST), epoxide hydrolase (EPH) or cytochrome P450 (CYP) mRNA transcripts. In male rats, TAM increased the levels of STel, STa and STpl mRNAs, whereas PB increased only the STel mRNA. In female rats, there was no expression of STel and STHA mRNA in either control or TAM-treated animals. TAM and PB increased UGTBe/p mRNAs in all rats, whereas UGTml mRNA was elevated only in PB-treated animals. EPH mRNA was elevated markedly in all rats treated with TAM and PB, whereas GSTya/ye mRNA was highly increased by PB, but only marginally increased by TAM. Finally, TAM increased CYP3A1 mRNA, and slightly increased CYP2B1 mRNA, whereas PB highly elevated mRNAs for both of these CYP genes. In conclusion, treatments of rats with TAM increased the mRNA levels of many phase I and phase II drug-metabolizing enzymes, and this pleiotypic response to TAM seems to be different from other prototype inducers such as PB or dioxin (TCDD).

Animals↗
PubMed2000

Determination of acetic acid in aqueous samples, by water-phase derivatisation, solid-phase microextraction and gas chromatography.

The direct derivatisation of acetic acid with n-hexyl chloroformate and with benzyl bromide in water was evaluated. With n-hexyl chloroformate, acetic acid did not give the n-hexyl acetate derivative, but the reaction of acetic acid with benzyl bromide in aqueous solution resulted in the formation of benzyl acetate. The derivatisation of acetic acid with benzyl bromide and the headspace solid-phase microextraction (SPME) of benzyl acetate were optimised. Under optimum conditions, the limit of detection for acetic acid was 260 nM, and the relative standard deviation of the overall procedure at 1.10(-4) M acetic acid was 15.6% (n = 10). A linear response was obtained in the 1 x 10(-4) to 5 x 10(-6) M concentration range (R2 = 0.993, n = 6). Although Carbowax-divinylbenzene (CW-DVB)-coated fibres exhibited a higher extraction capacity for benzyl acetate, polyacrylate (PA) was selected, because its mechanical stability was better than that of CW-DVB fibres. Moreover, the relative standard deviation of the SPME was better with PA (1.5%, n = 10 at 1 x 10(-5) M) than with CW-DVB-coated fibres (8.0%, n = 10 at 1 x 10(-5) M). Thus, a new analytical method for the quantitative determination of micromolar concentrations of acetic acid in the aqueous phase was developed. This method is based on water-phase derivatisation with benzyl bromide, headspace SPME with PA fibres and GC-FID. It was observed experimentally that benzyl alcohol formed by hydrolysis of the reagent affected the fibre-gas phase partitioning of benzyl acetate.

Acetic Acid↗
PubMed2000

Enantioselective reversed-phase and non-aqueous capillary electrochromatography using a teicoplanin chiral stationary phase.

Enantiomeric separation of chiral pharmaceuticals is carried out in aqueous and non-aqueous packed capillary electrochromatography (CEC) using a teicoplanin chiral stationary phase (CSP). Capillaries were slurry packed with 5 microm 100-A porous silica particles modified with teicoplanin and initially evaluated using a non-aqueous polar organic mode system suitability test for the separation of metoprolol enantiomers (Rs = 2.3 and 53000 plates m(-1)). A number of pharmaceutical drugs were subsequently screened with enantioselectivity obtained for 25 racemic solutes including examples of neutral, acidic and basic molecules such as coumachlor (Rs = 3.0 and 86000 plates m(-1)) and alprenolol (Rs = 3.3 and 135000 plates m(-1)) in reversed-phase and polar organic mode, respectively. A statistical experimental design was used to investigate the effects of non-aqueous polar organic mobile phase parameters on the CEC electroosmotic flow, resolution and peak efficiency for two model solutes. Results primarily indicated that higher efficiency and resolution values could be attained at higher methanol contents which is similar to findings obtained on this phase in liquid chromatography.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2000

Chromatographic study of terpene derivatives on porous graphitic carbon stationary phase with beta-cyclodextrin as mobile phase modifier.

The stoichiometric coefficients and apparent formation constants (Kf) of alpha-terpineol, thymol, geraniol and linalool complexes with beta-cyclodextrin (beta-CD) were determined using HPLC with a porous graphitic carbon (PGC) chromatographic support. Measurements were performed with four different methanol-water mobile phases. All the terpene derivatives under study form 1:1 guest-CD complexes. Graphs of Kf as a function of the mobile phase composition appeared different from those classically described for RP-C18 and suggest that the PGC stationary phase could play an active role in the complexation process. Solute-CD inclusion and solute-stationary phase interactions may be involved in this specific behavior.

Chromatography, High Pressure Liquid↗