Separation and identification of chiralN-acylcalix[4]arene amino acid derivatives by use of reversed-phase HPLC

Summary This work focuses on the separation and identification of p-tert butyl calix[4]arene derivatives. An isocratic mixture of 65% (v/v) acetonitrile and 35% (v/v) water with 0.1% (v/v) phosphoric acid was used as the mobile phase. A Bio-Rad Bio-Sil ODS-5S (250 mm×4 mm) column was used as the stationary phase with UV detection of the analytes at 274 nm. The reversed-phase high performance liquid chromatographic method which was developed provided baseline separation of five p-tertbutyl calix[4]arene derivatives in twenty minutes.     

A Measurement Technique for Organic Nitrates and Halocarbons in Ambient Air

A cryogenic preconcentration/high-resolution gas chromatographic technique has been developed for the rapid, simultaneous quantitation of C₁–C₄ organic nitrates and halocarbons in ambient air. Whole-air samples are collected in Tedlar^TM bags by an evacuated-chamber method. Samples were stable in 0.010-cm-thick bags for 24 h if they were immediately stored in a freezer at −25°C. Analytes in a 50-cm³ air sample were efficiently preconcentrated on fused-silica beads at −180°C and thermally desorbed at 30°C. High-resolution gas chromatography with a cross-linked polydimethylsiloxane fused-silica capillary column and an electron-capture detector were used for separation and quantitation of the analytes. An analysis time of about 12 min was facilitated by sample cryofocusing at −180°C and oven temperature programming. Recoveries of the analytes by the evacuated-chamber method were better than 95%. The sensitivity of the technique for sample volumes of 50 cm³ is in the sub-parts-per-trillion by volume (ppt[v]) range for many of the analytes, with an average precision of about ±5% for analytes at levels of about 10 ppt(v).     

High-separation performance of chromatographic capillaries coated with MOF-5 by the controlled SBU approach

Recently developed MOF surface-coating techniques, the controlled SBU approach (CSA) for the generation of MOF-5, and the use of self-assembled monolayers have been combined to generate a wall-bonded, crosslinked stationary phase for gas chromatographic capillary columns displaying excellent performance in the separation of natural gas components. The chromatographic performance of this new type of column has been compared to the state-of-the-art solution for this separation problem, namely a coated silica column of the porous layer open tubular (PLOT) type. Chromatographic parameters such as separation, resolution, and tailing factors, as well as plate numbers and heights in the case of isothermal operation, have been determined. Kinetic and thermodynamic parameters characterizing the analyte-stationary phase interaction have been determined for various C1-C4 analytes.     

Phase-transfer catalytic determination of phenols as methylated derivatives by gas chromatography with flame ionization and mass-selective detection

A microemulsion electrokinetic chromatographic (MEEKC) method was developed for the separation of six catechins, specific marker phytochemicals of Cistus species. The MEEKC method involved the use of sodium dodecyl sulfate (SDS) as surfactant, heptane as organic solvent and butan-1-ol as co-solvent. In order to have a better stability of the studied catechins, the separation was performed under acidic conditions (pH 2.5 phosphate buffer). The effects of SDS concentration and of the amount of organic solvent and co-solvent on the analyte resolution were evaluated. The optimized conditions (heptane 1.36% (w/v), SDS 2.31% (w/v), butan-1-ol 9.72% (w/v) and 50 mM sodium phosphate buffer (pH 2.5) 86.61% (w/v)) allowed a useful and reproducible separation of the studied analytes to be achieved. These conditions provided a different separation profile compared to that obtained under conventional micellar electrokinetic chromatography (MECK) using SDS. The method was validated and applied to the determination of catechin and gallocatechin in lyophilized extracts of Cistus incanus and Cistus monspeliensis.     

Simultaneous determination of artificial sweeteners, preservatives, caffeine, theobromine and theophylline in food and pharmaceutical preparations by ion chromatography.

A novel ion chromatographic method was proposed for the simultaneous determination of artificial sweeteners (sodium saccharin, aspartame, acesulfame-K), preservatives (benzoic acid, sorbic acid), caffeine, theobromine and theophylline. The separation was performed on an anion-exchange analytical column operated at 40 degrees C within 45 min by an isocratic elution with 5 mM aqueous NaH2PO4 (pH 8.20) solution containing 4% (v/v) acetonitrile as eluent, and the determination by wavelength-switching ultraviolet absorbance detection. The detection limits (signal-to-noise ratio 3:1) for all analytes were below the sub-microg/ml level. Under the experimental conditions, several organic acids, including citric acid, malic acid, tartaric acid and ascorbic acid, did not interfere with the determination. The method has been successfully applied to the analysis of various food and pharmaceutical preparations, and the average recoveries for real samples ranged from 85 to 104%. The levels of all analytes determined by this method were in good agreement with those obtained by the high-performance liquid chromatographic procedure. The results also indicated that ion chromatography would be possibly a beneficial alternative to conventional high-performance liquid chromatography for the separation and determination of these compounds.     

Separation performance of graphene oxide as stationary phase for capillary gas chromatography

Graphene oxide(GO) has attracted extensive attention due to its unique properties and potential applications.Here,we report the investigation of GO nanosheets as a stationary phase for capillary gas chromatographic(GC) separations.The GO column,fabricated by a new one-step coating approach,showed average McReynolds constants of 308,suggesting the medium polar nature of the GC stationary phase.The GO stationary phase achieves good separation for analytes of different types with good peak shapes,especially for H-bonding analytes,such as alcohols and amines.The different retention behaviors of GO stationary phase from the conventional stationary phase may originate from its multiple interactions with analytes,involving H-bonding,dipole-dipole,π-π stacking and dispersive interactions.Moreover,GO column showed good separation reproducibility with relative standard deviation(RSD%) less than 0.24%(n = 5) on retention times of analytes.     

Liquid chromatographic resolution of racemic rasagiline and its analogues on a chiral stationary phase based on (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid

A liquid chromatographic chiral stationary phase based on (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid was applied to the resolution of 15 analytes, including racemic rasagiline, a chiral drug for the treatment of Parkinson's disease, and its analogues. The composition of mobile phase was optimized to be ethanol/acetonitrile/acetic acid/triethylamine (80:20:0.2:0.3, v/v/v/v) by evaluating the chromatographic results for the resolution of five selected analytes under various mobile phase conditions. Under the optimized mobile phase conditions, racemic rasagiline was resolved quite well with a separation factor of 1.48 and resolution of 2.71 and its 14 analogues were also resolved reasonably well with separation factors of 1.06–1.54 and resolutions of 0.54–2.11. Among 15 analytes, racemic rasagiline was resolved best except for just one analyte. The analyte structure–enantioselectivity relationship indicated that racemic rasagiline has the most appropriate structural characteristics for resolution on the chiral stationary phase.     

Evaluation of tetraglyme for the enrichment and analysis of volatile organic compounds in air

A recently developed method for the sampling and analysis of volatile organic compounds in air has been evaluated. The system is based on the enrichment of analytes in tetraethylene glycol dimethyl ether or tetraglyme, a water-soluble organic liquid. The subsequent analysis consists of dispersion of a sample aliquot in water followed by purge-and-trap and gas chromatographic separation. Physico-chemical data were investigated for 10 volatile organic compounds, providing information on the possibilities and limitations of the tetraglyme method. The target analytes included chlorinated alkanes and alkenes, and monocyclic aromatic hydrocarbons. Air/tetraglyme partition coefficients Kat were determined over an environmental relevant temperature range of 2-25 degrees C to evaluate sorption efficiencies and estimate breakthrough volumes at the sampling stage. At 2 degrees C breakthrough volumes (allowing 5% of breakthrough) ranged from 5.8 (1,1-dichloroethane) to 312 l (1,1,2-trichloroethane) for 20 ml of tetraglyme. With regard to the desorption stage, the effect of tetraglyme on the air/water partition of organic compounds was investigated through the measurement of air/tetraglyme-water partition coefficients Kat-w for 2-31% (v/v) tetraglyme in water. Finally a clean-up procedure for tetraglyme was evaluated. Analysis of a blank tetraglyme-water (17:83, v:v) mixture by gas chromatography-flame ionization detection/mass spectrometry showed minor background signals. None of the target compounds were detected.     

Retention indices as identification tool in pyrolysis-capillary gas chromatography

Pyrolysis-capillary gas chromatography (Py-cGC) represents important method to identify the analytes in the mixture after thermal degradation. This combines high effective analyte separation on-line coupled with thermal degradation process that depends on analyte structure. System of retention indices has been used for identification of the analytes after on-line pyrolysis and chromatographic separation. The pyrolysate composition has been studied during thermal degradation of polymethylmethacrylate (PMMA) at different pyrolysis temperatures and chromatographic column conditions. Homologues series of n-alkanes have been used for calculation of pyrolysate Kováts retention indices (I) and compared with mass spectrometric (MS) data of pyrolysate model mixture. To identify PMMA thermal degradation products the high density polyethylene (HDPE) as additive standard producing triplets of the olefin homologous series during co-pyrolysis has been used. These homologous series enable to calculate programmed temperature retention indices (ITPGC) to identify the analytes present in the pyrolysate. Calculated I values were compared with published I values databases to identify analytes yielded at different pyrolysis temperatures.     

Analysis of catechins in extracts of Cistus species by microemulsion electrokinetic chromatography

A microemulsion electrokinetic chromatographic (MEEKC) method was developed for the separation of six catechins, specific marker phytochemicals of Cistus species. The MEEKC method involved the use of sodium dodecyl sulfate (SDS) as surfactant, heptane as organic solvent and butan-1-ol as co-solvent. In order to have a better stability of the studied catechins, the separation was performed under acidic conditions (pH 2.5 phosphate buffer). The effects of SDS concentration and of the amount of organic solvent and co-solvent on the analyte resolution were evaluated. The optimized conditions (heptane 1.36% (w/v), SDS 2.31% (w/v), butan-1-ol 9.72% (w/v) and 50 mM sodium phosphate buffer (pH 2.5) 86.61% (w/v)) allowed a useful and reproducible separation of the studied analytes to be achieved. These conditions provided a different separation profile compared to that obtained under conventional micellar electrokinetic chromatography (MECK) using SDS. The method was validated and applied to the determination of catechin and gallocatechin in lyophilized extracts of Cistus incanus and Cistus monspeliensis.     

Determination of pesticide residues in red wines with microporous membrane liquid–liquid extraction and gas chromatography

A sample pretreatment method based on microporous membrane liquid-liquid extraction (MMLLE) was developed for the subsequent gas chromatographic determination of pesticides in wine. MMLLE provided efficient and selective extraction with enrichment factors in the range 3-13. The gas chromatographic separation was carried out using on-column injection and flame ionization detection. The method was linear, repeatable and sensitive. The limits of quantification were better than 0.006 mg/L for all the analytes except for iprodione (0.37 mg/L). The method was applied to the determination of pesticides in several red wines of different origin.     

Determination of betaine, choline and trimethylamine in feed additive by ion-exchange liquid chromatography/non-suppressed conductivity detection

An ion chromatography method with non-suppressed conductivity detection was developed for the simultaneous determination of betaine, choline and trimethylamine in feed additive. The analytes and the common inorganic cations were well separated by means of cation-exchange chromatography using a 4.5 mmol/L methanesulfonic acid solution containing 10% (v/v) acetonitrile as eluent and an IonPac SCS1 column (250 mm x 4 mm i.d.) as the separation column. The effects of the different chromatographic parameters on the separation were also investigated. Detection limits of betaine, choline and trimethylamine were 0.076, 0.044 and 0.041 mg/L. The relative standard deviations (RSDs) of the retention time and peak area were less than 0.30% and 0.88%, respectively. The recoveries were between 93.2% and 112.6%. The method is suitable for use as a routine method in production quality control of feed additive.     

Improving analysis of apolar organic compounds by the use of a capillary titania-based column: Application to the direct determination of faecal sterols cholesterol and coprostanol in wastewater samples

This article reports a new procedure for the direct determination of faecal sterols coprostanol and cholesterol in wastewater samples as tracers of human sewage contamination. The method combines in-tube solid-phase microextraction (IT-SPME) for analyte enrichment and capillary liquid chromatography (LC) for separation with diode array detection for identification and quantification. A titania-based polymeric capillary column and a conventional octadecyl silica (ODS) capillary column were evaluated and compared for their ability to separate the analytes. The titania-based column allowed the separation of the analytes in much shorter chromatographic times and with better chromatographic profiles, which in turn resulted in better detectability. In addition, IT-SPME allowed the direct injection into the chromatographic system of sample volumes as large as 200 μL, thus making unnecessary off-line clean-up and concentration steps. In such a way, the tested compounds could be directly analysed in less than 10 min, the limits of detection (LODs) being 10 and 1.2 μg/L for coprostanol and cholesterol, respectively. The reliability of the proposed method was tested by processing several wastewater samples.     

A simple micellar electrokinetic capillary chromatographic method for the quantitative analysis of organic expectorants

A simple and rapid micellar electrokinetic capillary chromatographic method is described for the separation and quantification of five expectorant drugs, including ambroxol (AMB), bromhexine (BRM), carbocysteine, guaiacol and guaifenesin. The drugs were separated in a mixed solution of phosphate buffer (35 mM; pH 3.90) and acetonitrile (75:25, v/v) with sodium dodecylsulfate (120 mM) as the micellar source, and the separated drugs were directly monitored with UV detector (200 nm). Several key parameters affecting the separation and analysis of the drugs were studied and optimized. Based on the corrected peak-area ratios of the drugs to an internal standard (7-hydroxy-4-methyl-coumarin) versus the concentration of the drugs, the established method is applicable to quantify AMB and BRM each over 20-150 microM, carbocysteine over 100-1500 microM, guaiacol and guaifenesin each over 10-150 microM. The detection limits (S/N=3; 0.5 psi, 5 s injection) of the method for the analytes are in the range of 3.0-5.0 microM (except that of carbocysteine at 30 microM). The precision (relative standard deviation) and accuracy (relative error) of the method for the intra-day (n=3) and inter-day (n=5) analyses of the analytes at three levels are all below 4%. The method is speedy with a run time of about 6 min for the analysis of the five analytes. Application of this method to the analysis of AMB and BRM in pharmaceutical preparations or AMB in the urine of a dosed subject proved simple and effective.     

Optimization via experimental design of an SPE-HPLC-UV-fluorescence method for the determination of valsartan and its metabolite in human plasma samples

A chemometric approach was applied for the optimization of the extraction and separation of the antihypertensive drug valsartan and its metabolite valeryl-4-hydroxy-valsartan from human plasma samples. Due to the high number of experimental and response variables to be studied, fractional factorial design (FFD) and central composite design (CCD) were used to optimize the HPLC-UV-fluorescence method. First, the significant variables were chosen with the help of FFD; then, a CCD was run to obtain the optimal values for the significant variables. The measured responses were the corrected areas of the two analytes and the resolution between the chromatographic peaks. Separation of valsartan, its metabolite valeryl-4-hydroxy-valsartan and candesartan M1, used as internal standard, was made using an Atlantis dC18 100 mm x 3.9 mm id, 100 angstroms, 3 microm chromatographic column. The mobile phase was run in gradient elution mode and consisted of ACN with 0.025% TFA and a 5 mM phosphate buffer with 0.025% TFA at pH 2.5. The initial percentage of ACN was 32% with a stepness of 4.5%/min to reach the 50%. A flow rate of 1.30 mL/min was applied throughout the chromatographic run, and the column temperature was kept to 40+/-0.2 degrees C. In the SPE procedure, experimental design was also used in order at achieve a maximum recovery percentage and extracts free from plasma interferences. The extraction procedure for spiked human plasma samples was carried out using C8 cartridges, phosphate buffer (pH 2, 60 mM) as conditioning agent, a washing step with methanol-phosphate buffer (40:60 v/v), a drying step of 8 min, and diethyl ether as eluent. The SPE-HPLC-UV-fluorescence method developed allowed the separation and quantitation of valsartan and its metabolite from human plasma samples with an adequate resolution and a total analysis time of 1 h.     

Understanding chiral molecular micellar separations using steady-state fluorescence anisotropy, capillary electrophoresis, and NMR

Chiral separations employing four diastereomers of poly sodium N-undecanoyl leucylvalinate (p-SULV) as chiral selectors are probed by use of MEKC, steady-state fluorescence anisotropy, and NMR. By employing diastereomers and thus altering the stereochemistry of a single amino acid in a systematic way, one may control the enantiorecognition ability of the chiral selector. As a result, one can gain a better understanding of the mechanisms of chiral recognition for the two classes of neutral or anionic chiral analytes studied. Evaluation of the chiral interactions leading to chiral separations confirmed our earlier observation of a strong relationship between the selectivity (alpha) observed using a chromatographic separation technique (MEKC) and that determined from the spectroscopic parameter, beta. A linear alpha versus beta relationship was observed for the molecular micelle p-(L)-SULV with all eight analytes included in this study. However, as we earlier predicted, different groups of analytes had different slopes, i.e., values of m, suggesting different chiral separation mechanisms. Evaluation of the data allowed a grouping of the analytes according to the primary site of chiral interaction with the leucine or valine moiety of molecular micelle chiral headgroup.     

Microemulsion electrokinetic chromatography for the separation of retinol, cholecalciferol, delta-tocopherol and alpha-tocopherol

A microemulsion electrokinetic chromatographic method was used to separate fat-soluble vitamins. The separation of retinol, cholecalciferol, and delta- and alpha-tocopherol was performed using a microemulsion containing 0.75% (v/v) n-heptane, 30 mM bis(2-ethylhexyl)sodium sulfosuccinate (AOT), 5% (v/v) 1-butanol, 15% (v/v) 1-propanol and 15% (v/v) methanol in 20mM boric acid-sodium borate buffer. The effect of the different microemulsion constituents was studied, including the type and concentration of surfactant, buffer, oil and co-surfactants. The presence of methanol in the microemulsion was found to be necessary to achieve the separation of the tocopherols. Detection was carried out at 200, 265 and 325 nm for the tocopherols, cholecalciferol and retinol, respectively. Calibration curves and precision data were obtained for each analyte. Good linear relationships were found between the analytical signal and the analytes concentration in the 25-500 mg L(-1) for retinol and cholecalciferol, and 25-300 mg L(-1) for tocopherols ranges. The precision of the method afforded relative standard deviations in the 4.0-10% range.     

Hydrophilic interaction chromatography separation mechanisms of tetracyclines on amino-bonded silica column

Effects of mobile-phase variations on the chromatographic separation on amino-bonded silica column in hydrophilic interaction chromatography (HILIC) were investigated for four zwitterionic tetracyclines (TCs): oxytetracycline, doxycycline, chlortetracycline, and tetracycline. A mixed-mode retention mechanism composed of partitioning, adsorption, and ion exchange interactions was proposed for the amino HILIC retention process. Buffer type and pH significantly influenced the retention of TCs, but showed similar separation selectivity for the tested analytes. Experiments varying buffer salt concentration and pH demonstrated the presence of ion exchange interactions in TCs retention. The type and concentration of organic modifier also affected the retention and selectivity of the analytes, providing direct evidence supporting the Alpert retention model for HILIC. The retention time of the analytes increased in the following order of organic modifiers: tetrahydrofuran < methanol < isopropanol < acetonitrile. The linear relationships of logk' versus %water (v/v) curve and logk' versus logarithm of %water (v/v) in the mobile phase indicated that TCs separation on the amino phase was controlled by partitioning and adsorption. The developed method was successfully utilized in the detection of TCs in both river water and wastewater samples using solid-phase extraction (SPE) for sample cleanup.     

Comparison of the chromatographic behavior of monolithic capillary columns in capillary electrochromatography and nano-high-performance liquid chromatography

Porous monoliths based on N,N-dimethylacrylamide (DMAA) or methacrylamide (MAA) were prepared inside fused silica capillaries as stationary phases for nano-chromatography. The columns were characterized in terms of flow rate and backpressure and showed, e.g. differences as a function of the salt concentration added to the polymerization mixture. When the columns were investigated for the separation of uncharged (polar hydroxylated aromatic compounds) and charged (amino acids) analytes under pressure driven conditions (pLC), differences to the previously observed behavior under voltage driven conditions (CEC) were observed. Whereas the non-charged analytes showed similar behavior in both cases--thus, corroborating the previous assumption of a mainly chromatographic separation mode driven by hydrophilic interactions in CEC--the charged amino acids did not. Assuming that the separation was governed by chromatographic phenomena in the pLC mode and by both chromatographic and electrophoretic effects in the CEC mode, the experiments allowed deconvoluting the two contributions. In particular, the charged amino acids appeared to interact with the stationary phases mainly by electrostatic interactions modified by some hydrophilic effects.     

Fast screening of stabilizers in polymeric materials by flow injection–tandem mass spectrometry

In the present study, the applicability of rapid flow injection–triple quadrupole mass spectrometry for simultaneous qualitative screening of different classes of stabilizers in polymeric materials is demonstrated. Electrospray ionization and atmospherical pressure chemical ionization were compared, whereby the latter yielded generally poorer detection limits and only single charged ions that were for some analytes beyond the mass range of the quadrupole mass spectrometry. Positive electrospray ionization allowed the interference-free monitoring of multiple reaction monitoring transitions selective for 36 commonly used stabilizers without chromatographic separation. Real polymer samples were extracted by toluene and the method allowed the detection of analytes down to 0.00001–0.025 wt% depending on the stabilizer.