Early stages of fat crystallisation evaluated by low-field NMR and small-angle X-ray scattering

Low-field time-domain nuclear magnetic resonance (NMR; 20 MHz) is commonly used in the studies of fats in the form of solid fat content (SFC) measurements. However, it has the disadvantage of low sensitivity to small amounts of crystalline material (0.5%), thus often incorrectly determining crystallisation induction times. From spin–lattice relaxation rate measurements (R₁) during the isothermal crystallisation measurements of cocoa butter between 0.01 and 10 MHz using fast field cycling NMR, we learnt previously that the most sensitive frequency region is below 1 MHz. Thus, we focused on analysing our 10-kHz data in detail, by observing the time dependence of R₁ and comparing it with standard SFC_NMR and SFC determinations from small-angle X-ray scattering (SFC_SAXS). Although not reflecting directly the SFC, the R₁ at this low frequency is very sensitive to changes in molecular aggregation and hence potentially serving as an alternative for determination of crystallisation induction times. Alongside R₁, we also show that SFC_SAXS is more sensitive to early stages of crystallisation, that is, standard SFC_NMR determinations become more relevant when crystal growth starts to dominate the crystallisation process but fail to pick up earlier crystallisation steps. This paper thus demonstrates the potential of studying triacylglycerols at frequencies below 1 MHz for obtaining further understanding of the early crystallisation stages of fats and presents an alternative and complementary method to estimate SFC by SAXS.     

Rapid analysis of 14 ultraviolet absorbents in plastic food contact materials by supercritical fluid chromatography on Sub-2-micron particles

Abstract

A rapid and reliable method based on supercritical fluid chromatography (SFC) technology was established for the analysis of 14 ultraviolet (UV) absorbents in plastic food contact materials. The separation was optimized by employing different chromatographic conditions including stationary phases, organic modifiers, column temperatures and back pressures to achieve appropriate selectivity and resolution. The 14 UV absorbents were analyzed within 4.5?min on the sub-2-micron particles column (ACQUTY UPC² HSS C18?SB) via gradient elution with methanol as the organic modifier. The calibrations were carried out in the concentration range of 0.1–50.0?mg/L (except for UV-329 and UV-320 were 0.2–50.0?mg/L, UV-360 was 0.3–50.0?mg/L), and excellent linearity was observed with correlation coefficients (R²) greater than 0.99. The limits of quantification (LOQ) were in the range of 0.10–0.30?µg/g. The recoveries for accuracy were ranged from 90.4 to 114.1%, and the relative standard deviation (RSD) for repeatability was between 0.67 and 7.23%, respectively. The established method was successfully applied for the analysis of UV absorbents in five plastic food contact materials samples. The SFC method developed in this study can provide an alternative route for routine analysis of UV absorbents in the future.     

Spectrofluorimetric Determination of Antimony

Abstract

A spectrofluorimetric procedure for the determination of micromolar concentrations of antimony(III) was devised based on its reduction of cerium(IV) to produce fluorescent cerium(III). The method was optimized and the reaction was fast enough in hydrochloric acid media without the need for iodide or osmium(VIII) as catalysts. Linear calibration graphs were obtained in the range 1-10 10^?6M. The standard deviation for determining 5 × 10^?6M antimony(III)(10 times) was 1.43 × 10^?7M and the relative error was -3.4 %. The method was applied to the determination of antimony(III) in its mixture with antimony(V), total antimony was later determined after reduction with mercury metal in deoxygenated solutions. The affect several reducing agents on the determination of antimony-was also examined.     

Development of capillary supercritical fluid chromatography/Fourier transform infrared spectrometry

Summary The feasibility of a new technique, supercritical fluid chromatography/Fourier transform infrared spectrometry (SFC/FTIR), for separation and identification of nonvolatile and labile compounds in complex mixtures is discussed in this report. The design of a high pressure (flow cell) interface is described. The results of a continuing study of possible mobile phases for SFC/FTIR are presented. Finally, SFC/FTIR was applied to a standard test mixture. The current detection limits of one component of the mixture, 2,6-di-t-butylphenol, are presented.     

Chromatographic resolution of closely related species: Drug metabolites and analogs

In this study, we investigate the separation of a variety of mixtures of drugs, metabolites, and related analogs including representatives of the carbamazepine, methylated xanthine, steroid hormone, nicotine, and morphine families using several automated chromatographic method development screening systems including ultra high performance liquid chromatography, core–shell HPLC, achiral supercritical fluid chromatography (SFC), and chiral SFC. Of the 138 column and mobile phase combinations examined for each mixture, a few chromatographic conditions afford the best overall performance, with a single achiral SFC method (4.6 × 250 mm, 3.0 μm GreenSep Ethyl Pyridine, 25 mM isobutylamine in methanol/CO₂) affording good separation for all samples. Four of these mixtures were also resolved by achiral SFC on the Luna HILIC and chiral SFC Chiralpak IB columns using methanol or ethanol with 25 mM isobutylamine as polar modifiers. Modifications of standard chromatography screening conditions afforded fast separation methods (from 1 to 5 min) for baseline resolution of all components of each of these challenging sets of closely related compounds.     

Development of an Isocratic SFC Method for Four Centrally Active Muscle Relaxant Drugs

Abstract

A reproducible and selective supercritical fluid chromatography (SFC) method was developed for the estimation of four centrally active muscle relaxants, viz., chlorzoxazone, methocarbamot, tizanidine, and baclofen, using guaifenesin (expectorant, muscle relaxant) as the internal standard. The effect of temperature, pressure and the modifier concentration on retention of the five solutes has been explored and the parameters optimised. An arbitrary mixture of 5 components was base line resolved on a JASCO- RP- C₁₈ (150 × 4.6 mm) 5μ metaphase column with a tertiary mobile phase of 9.1 % modifier [methanol containing 2 % glacial acetic acid, v/v] in carbon dioxide at 1.5 ml/min, 50°C temperature, and 12.75 MPa outlet pressure. UV detection at 235 nm was employed. Without the acetic acid in the mobile phase it was not possible to elute tizanidine and baclofen. With the additive, all the analytes produced symmetrical peaks. Quantitative validation concentration ranges have been found, and the performance data evaluated.     

Total nitrogen in wastewater analysis: Comparison of Devarda’s alloy method and high temperature oxidation followed by chemiluminescence detection

Two methods for determining total nitrogen (TN) contents of complex wastewaters were compared: Devarda’s alloy method and a method involving high temperature oxidation (HTO) followed by chemiluminescence detection (CLD). In the latter multiple standard additions were used for quantification purposes since the CLD response was found to be dependent on the structure of the analysed nitrogen-containing compounds. For both methods, sample dilution steps were necessary to achieve acceptable accuracy. The accuracies for selected compounds used in tests were within the 65–105% range. In a long-term interlaboratory investigation the HTO-CLD method yielded significantly (p < 0.05) higher results than Devarda’s alloy method and greater precision (within-assay repeatability approximately 1% RSD, while the corresponding within-day repeatability for Devarda’s alloy method was approximately 7% RSD).     

The coupling of supercritical fluid chromatography and field ionization time-of-flight high-resolution mass spectrometry for rapid and quantitative analysis of petroleum middle distillates

We report the first coupling of supercritical fluid chromatography (SFC) with field ionization time-of-flight high-resolution mass spectrometry (FI-ToF HRMS), in parallel with ultraviolet (UV) detection and flame ionization detection (FID), for rapid and quantitative analysis of petroleum middle distillates. SFC separates petroleum middle distillates into saturates and 1- to 3-ring aromatics. FI generates molecular ions for hydrocarbon species eluted from the SFC. The high resolution and exact mass measurements by ToF mass spectrometry provide elemental compositions of the molecules in the petroleum product. The amounts of saturates and aromatic ring types were quantified using the parallel SFC-FID assisted by SFC-UV. With a proper carbon-number calibration, the detailed composition of the petroleum middle distillate was rapidly determined.     

Spectrofluorimetric determination of melamine formaldehyde in solid and liquid forms and in wastewater

A new, simple and sensitive spectrofluorimetric method for determination of trace amount of melamine formaldehyde (MF) was developed. In phosphate buffer solution of pH 7.4 MF can remarkably quench the luminescence intensity of toluylene red (NR) at λ_em = 590 nm due to formation of NR-MF ion associate complex. The luminescence intensity of NR-MF complex was in proportion to the concentration of MF and used as photo probe for its determination. The dynamic range for the determination of MF is 7.5–52.5 ppm with detection limit of 4.4 ppm. The method is relatively free from interferences from coexisting substances and used successfully for the determining of MF in powder and liquid forms and in wastewater produced from MF industries. The average recoveries and standard deviations of 98.3 ± 0.6, 98.1 ± 0.6 and 97.3 ± 0.5% were achieved for determination of MF in solid, liquid forms and wastewater, respectively     

Retention in SFC at high density

The elucidation of the detailed mechanism of retention in supercritical fluid chromatography (SFC) is a complex problem, especially for high-density supercritical fluid mobile phases. Martire and Boehm developed a theory of SFC retention based on a statistical thermodynamic treatment, and introduced a density-squared equation for retention in SFC: In k = a + b/T + cρ + dρ/T + eρ²/T. In this paper the systems n-alkane-polydimethylsiloxane-CO₂ and n-carboxylic acid methyl ester-polydimethylsiloxane-CO₂ have been treated using the method introduced by Martire and Boehm. In the ranges of reduced temperature from 1.079 to 1.293 and reduced SF mobile phase density from 0.705 to 1.632, two problems were discovered in the treatment of SFC retention behavior by the method of Martire and Boehm: (1) the experimental value of the site of CO₂ in the lattice field, r_a, of the two systems was ca 0.9, which is obviously different from the theoretical values of 3.6 or 5.4; and (2) the difference between experimental and calculated retention values increased with increasing SF mobile phase density. The minimum in SFC retention predicted by Martire and Boehm was not observed. On the basis of some assumptions and the experimental data, the following retention equation was derived: In k = a + b/T + cρ + dρ/T + eρ²/T + fρ³/T + gρ⁴/T. This equation was consistent with the experimental data and can conveniently be used to explain other SFC retention behavior.     

Demonstration of a linear composition gradient during water saturation of CO2 in supercritical fluid chromatography

Summary Water was added to CO₂ by saturation to increase the solvation power of the mobile phase in supercritical fluid chromatography. The saturation was performed at a temperature above the boiling point of water (100°C) to increase the amount of water which could be loaded homogeneously into the CO₂ (2.5–3.0 mol% water as compared to about 0.25 mol% water at 25°C). A linear composition of water was produced by altering the density of the CO₂ during saturation. Modifications to the injector and CO₂ transfer lines prevented phase separation as a result of the instrumentation used in capillary supercritical fluid chromatography (SFC). After fitting vapor-liquid equilibria data to pressure, density, and temperature conditions, approximately 2.5–3.0 mol% of water was introduced in a linear gradient at 110°C. The effect of water on SFC performance was evaluated with standard steroid compounds. This paper provides further evidence for the need to examine vapor-liquid equilibria data prior to SFC.     

Quantification of Retinoid Compounds by Supercritical Fluid Chromatography Coupled to Ultraviolet Diode Array Detection

Liposoluble vitamins are widely analyzed due to their significant antioxidant activity. Quantification by liquid or gas chromatography is often time consuming and requires sample treatments prior to the analysis. Supercritical fluid chromatography (SFC), especially with the developments of new commercial systems, is nowadays considered as a credible alternative to standard chromatography. It provides a reduced acquisition time as well as sensitivity similar to that of liquid chromatography. To illustrate the new capabilities of SFC, six organic compounds related to the vitamin A family, all-trans-retinal, all-trans-retinol, all-trans-retinoic acid, retinyl propionate, retinyl acetate, and all-trans-retinyl palmitate, were analyzed and quantified. The choice of the column chemistry, co-solvent, the linearity and reproducibility of the method, and the matrix effect are discussed in detail. Best separation was finally obtained using a diphenyl column, with an excellent linearity over three orders of magnitude and limits of quantification in the low picomole range. Finally, the method was used for the quantification of retinyl palmitate in a pharmaceutical product with minimal sample preparation.     

Indirect Determination of Lidocaine by Atomic Absorption Spectrophotometry

Abstract

A new procedure for determining lidocaine chlorhydrate is described. The method consists of extracting an ion pair composed of Lidocaine and the inorganic complex Co(SCN)₄ ^2- into an organic solvent (1, 2 dichloroethane), then measure the Co in the organic phase by A.A.S. at 240.7 mm. Optimal experimental conditions concerning pH, Co(SCN)₄ ^?2 concentration, shaking time, phase ratio, number of extractions and linear range for calibration are studied. The method has a standard deviation of 10^?2. Two pharmaceutical preparations containing lidocaine, have been analyzed with very good results.     

Simultaneous Determination of Procaine Penicillin G and Benzathine Penicillin G by Second Derivative Spectrophotometry

Abstract

A new method for determining the binary mixtures of procaine penicillin G and benzathine penicillin G using “zero-crossing” second derivative spectrophotometry is described. Calibration graphs were linear up to 8.80 10^?5 M for procaine penicillin G and 4.40 10^?5 M for benzathine penicillin G. The method was realized in ethanol/water medium (30 % v/v). A complete and exhaustive statistical analysis of the experimental data was realized to demonstrate the validity of method. The method was applied for determining procaine penicillin G and benzathine penicillin G synthetic mixtures with good results. The procedure does not require any separation step.     

Spirobifluorene-Based o-Carboranyl Compounds: Insights into the Rotational Effect of Carborane Cages on Photoluminescence

9,9′-Spirobifluorene-based closo-o-carboranyl ( SFC1 and SFC2 ) compounds and their nido-derivatives (nido- SFC1 and nido- SFC2 ) were prepared and characterized. The two closo-compounds displayed major absorption bands assignable to π–π* transitions involving the spirobifluorene group, as well as weak intramolecular charge-transfer (ICT) transitions between the o-carboranes and their spirobifluorene moieties. The nido-compounds exhibited slightly blueshifted absorption bands resulting from the absence of the ICT transitions corresponding to the o-carborane moieties due to the anionic character of the nido-o-carboranes. While SFC1 exhibited only high-energy emissions in THF at 298 K (only from locally excited (LE) states assignable to π–π* transitions on the spirobifluorene group), remarkable emissions in the low-energy region were observed in the rigid state such as in THF at 77 K and in the film state. SFC2 displayed intense emissions in the low-energy region in all states. The fact that neither of the nido-derivatives of SFC1 and SFC2 exhibited low-energy emissions and the TD-DFT calculation results of each closo-compound clearly verified that the low-energy emission was based on ICT-based radiative decay. The conformational barriers from each relative energy calculation upon changing the dihedral angles around the o-carborane cages for both compounds confirmed that the rotation of the o-carborane cages and terminal phenyl rings for SFC1 is freer than that for SFC2 .     

Determination of Oxygenates in Gasoline by GC×GC

Comprehensive two‐dimensional gas chromatography (GC×GC) has been applied to the quantitation of oxygenates in reformulated gasoline. Target oxygenates were C₁–C₄ alcohols, tert‐pentanol, methyl tert‐butyl ether (MTBE), diisopropyl ether (DIPE), ethyl tert‐butyl ether (ETBE), and tert‐amyl methyl ether (TAME). These were separated from the gasoline matrix using a volatility‐based selectivity in the first chromatographic dimension, followed by a mixed‐phase polarity/shape selectivity in the second dimension. The high resolving power of this stationary phase combination completely separated all oxygenates except DIPE, ETBE, and TAME, which exhibited coelution with other nonpolar gasoline components. Oxygenates quantitation was achieved with the use of an internal standard, an FID detector, and calibration curves. Quantitation results are in good agreement with ASTM and EPA standard methods. When coupled with our previous method for BTEX and aromatics, a single GC×GC method can now quantitate MTBE, alcohols, BTEX, and aromatics in a one‐hour analysis.     

Chlorine-Selective Detection in Supercritical Fluid Chromatography using a Coulson Electrolytic Conductivity Detector

Abstract

A Coulson electrolytic conductivity detector has been coupled successfully to a supercritical fluid chromatograph, providing chlorine-selective detection. This involves minimal modification of the detector and no modification of the SFC or its operation. The general operating parameters of the SFC/CECD system were established by direct injection of three probe compounds: chlordane, γ-lindane, and hexachlorobenzene. Next, the SFC separation of a test mixture containing phenol, γ-lindane, and several chlorinated phenols and related compounds was optimized using UV-Vis and FID detection. The mixture was then separated using the same chromatographic method but with the CECD in line with the UV-Vis detector. The response of the CECD was linear and selective for chlorinated compounds. Limits of detection for the test mixture by CECD ranged from 80 – 250 ng/μL, corresponding to approximately 14 – 44 ng of chlorine on column, and were dependent on chromatographic conditions, but independent of analyte structure.     

Development and validation of a HPLC method for simultaneous quantitation of gatifloxacin, sparfloxacin and moxifloxacin using levofloxacin as internal standard in human plasma: application to a clinical pharmacokinetic study

A highly selective, sensitive and accurate HPLC method has been developed and validated for the estimation of three fluoroquinolones (FQs) viz., gatifloxacin (GFC), sparfloxacin (SFC) and moxifloxacin (MFC) with 500 microL human plasma using levofloxacin (LFC) as an internal standard (IS). The sample preparation involved simple liquid-liquid extraction of GFC, SFC, MFC and IS from human plasma with ethyl acetate. The resolution of peaks was achieved with phosphate buffer (pH 2.5)-acetonitrile (80:20, v/v) at a flow rate of 1 mL/min on a Kromasil C(18) column. The total chromatographic run time was 18.0 min and the simultaneous elution of GFC, SFC, MFC and IS occurred at approximately 10.8, 12.8, 17.0 and 6.0 min, respectively. The method proved to be accurate and precise at linearity range of 100-10,000 ng/mL with a correlation coefficient (r) of > or =0.999. The limit of quantitation for each of the FQs studied was 100 ng/mL. The intra- and inter-day precision and accuracy values found to be within the assay variability limits as per the FDA guidelines. The developed assay method was applied to a pharmacokinetic study in human volunteers following oral administration of 400 mg GFC tablet.     

Reverse Phase HPLC Determination and Murine Pharmacokinetics of Arabinosyl-5-azacytosine

Abstract

A sensitive and specific reverse phase HPLC assay has been developed to measure the new antitumor agent arabinosyl-5-aza-cytosine (ara-AC) in biological fluids at concentrations as low as 50 ng/ml (0.2 μM). This assay also detects arabinosyl-N-formyl-guanylurea (AGU-CHO), the initial hydrolytic metabolite of ara-AC. 2′-Deoxy-5-azacytidine, an analogue with similar chemical stability, is used as an internal standard. Chromatographically interfering plasma ribosides are removed by solid phase extraction on a phenyl boronic acid cartridge. Separation of ara-AC, AGU-CHO and internal standard is then accomplished isocratically (1% CH₃CN in 10 mM pH 6.8 phosphate buffer) on fully carbon loaded and end-capped C₈ and C₁₈ columns connected in tandem. The compounds of interest are detected by UV absorption at 240 nm and total analysis time is 20 min. This assay has been used to determine bolus dose plasma kinetics in male BDF₁ mice given 200 mg/kg ara-AC as a tail vein injection. Plasma elimination of the ara-AC is triphasic with a terminal phase half-life of 52 min and the elimination of the AGU-CHO metabolite parallels that of the parent drug. Analysis of ara-AC in human plasma indicates that this method is suitable for determining drug disposition and pharmacokinetics in human subjects.     

Performance comparison between packed column supercritical fluid chromatography (SFC) and HPLC using cyclandelate as the model analyte

A reproducible and fast method has been developed for the assay of cyclandelate in bulk and drug forms using packed column supercritical fluid chromatography using dicyclohexyl phthalate (DCHP) as internal standard. The drug and the internal standard were resolved by elution with supercritical fluid carbon dioxide doped with 14.29% (v/v) methanol on an RP-C₁₈ column and detected spectrophotometrically at 228 nm. Chromatographic figures of merit using C₈, C₁₈, cyano and phenyl columns have been assessed. Parallel experiments have been performed by HPLC and the data have been compared. Supercritical fluid extraction using CO₂ modified with a small amount of methanol was found to give quantitative analytical recoveries of cyclandelate from a dosage form. SFC has been shown to be a viable, faster alternative technique to HPLC generating less disposable waste.