On-flow gas chromatographic method for the determination of the enantiomer interconversion energy barrier

A novel on-flow gas chromatographic (GC) method is developed for the determination of the kinetic rate constants and interconversion energy barrier of thermally labile enantiomers. The validity of the developed method is approved by the study of interconversion of 1-chloro-2,2-dimethylaziridine enantiomers on an achiral column. The overall experiments are performed in a series of three columns placed in two independently heated GC ovens. The racemate of the 1-chloro-2,2-dimethylaziridine is injected and separated in the first chiral column at 60 degrees C in which the interconversion of enantiomers is suppressed. Separated enantiomers are then transferred into the achiral column, where the enantiomers are interconverted at a selected temperature under the current carrier gas flow. Effluent from this column is transferred into the second chiral column, where the native enantiomers and those originated by the on-flow interconversion on an achiral column are again separated at 60 degrees C. Chromatograms obtained by monitoring the effluents from the second chiral column are used to determine the peak areas of the original and the newly interconverted enantiomers. The corresponding peak areas and the interconversion times are used to calculate the interconversion rate constants and energy barriers of the 1-chloro-2,2-dimethylaziridine enantiomers. The apparent energy barriers of the enantiomers of 1-chloro-2,2-dimethylaziridine are equal for both enantiomers within a 95% confidence interval and independent of the polarity of the stationary phase of the column in which the interconversion of enantiomers occur.     

Analysis of 1-nitropyrene in air particulate matter standard reference materials by using two-dimensional high performance liquid chromatography with online reduction and tandem mass spectrometry detection

1-Nitropyrene (1-NP) is enriched in diesel exhaust particulate matter (DPM) relative to other sources of particulate matter (PM), and has been proposed as a marker for DPM. However, in ambient air, 1-NP concentrations are typically in the low pg/m(3) range. Therefore, collection of large volume air samples coupled with extensive sample clean-up procedures has been required to achieve adequate detection limits to measure 1-NP in ambient samples. We report here an improved LC-MS/MS method suitable for the detection and quantification of 1-NP in low volume ambient PM samples. The method involves ultrasonic extraction of ambient PM in organic solvent, concentration of the sample under reduced pressure, and two-dimensional HPLC analysis of the extract. 1-NP is isolated on the first HPLC column, then converted to 1-aminopyrene (1-AP) via online reduction in a column packed with a Pt/Rh catalyst. The 1-AP containing fraction from the first column is refocused on a trapping column, then eluted through a second HPLC column prior to MS/MS detection. Deuterated (d(9)) 1-NP (1-dNP) is added to each sample prior to extraction as an internal standard for quantification of 1-NP. The accuracy and precision of the assay, as applied to ambient particulate standard reference materials are 110+/-5.7% for SRM 1650b, 116+/-7.1% for SRM 2975, 108+/-5.8% for SRM 1649a, and 53+/-9.2% for SRM 1648. The analytical limit of detection was 152 fg on column, and analytical limit of quantitation 221 fg on column. To our knowledge, the sensitivity of this method is comparable with GC-NICI-MS methods while having the advantage of considerably less extensive sample preparation. This method is an approximately 10-fold improvement in sensitivity over HPLC methods utilizing fluorescence and/or chemiluminescence detection.     

Chromatographic methods for product-profile analysis and isolation of oligosaccharides produced by heparinase-catalyzed depolymerization of heparin.

A two-dimensional chromatography method for monitoring the formation of oligosaccharides produced by heparinase-catalyzed depolymerization of heparin is reported. In the first step of the two-dimensional method, the depolymerized heparin is size-fractionated by high-performance gel permeation chromatography (HPGPC). The size-uniform fractions are then separated on the basis of charge by strong anion-exchange (SAX) HPLC on a high resolution CarboPac PA1 column. To demonstrate application of the two-dimensional product-profile analysis method, data are presented for the heparinase I-catalyzed depolymerization of heparin in the absence and presence of histamine, a ligand that binds site-specifically to heparin. Results from the two-dimensional analysis indicate that histamine alters the extent of depolymerization and the product-profiles for the tetrasaccharide and hexasaccharide fractions. The use of CarboPac PA1 columns for the semi-preparative scale separation of oligosaccharides in size-uniform fractions isolated from depolymerized heparin by low-pressure (gravity flow) GPC is also reported. The semi-preparative scale CarboPac PA1 column gives high resolution and excellent reproducibility after repeated use over an extended period of time, making it possible to reliably combine fractions from multiple separations. The oligosaccharides are eluted from the CarboPac PA1 column with a NaCl gradient at relatively low pH (3 or 7) where they are stable. An efficient two-step procedure is described for desalting oligosaccharides separated     

Specific assay for unconjugated dehydroepiandrosterone in human plasma by capillary gas chromatography with electron-capture detection.

A specific and sensitive method for the determination of unconjugated dehydroepiandrosterone in plasma is described. After extraction and purification of the extracts on a Celite column, the iodomethyldimethylsilyl ether derivative of dehydroepiandrosterone was isolated on an aluminium oxide column and assayed by gas chromatography with electron-capture detection. The method is sensitive: sample volumes of 0.5-1 ml are sufficient for the determination of dehydroepiandrosterone in plasma of normal male and female subjects aged 1-80 years. The assay is highly specific and has the potential to be used as a reference method for the determination of unconjugated dehydroepiandrosterone in biological samples.     

Comparative Study of Packed and Capillary Column Chromatography for the Characterization of Vacuum Gas Oils

Gas chromatographic methods are of widespread use in the petroleum industry for assessing petroleum and derivatives quality. The feedstocks for catcracking processes are very sensitive to the n-paraffin contents, which can be accurately determined using capillary column chromatography. Nevertheless, packed column chromatography does have potential advantages, viz. sometimes shorter analysis times, lower costs, higher column capacity, for vacuum gas oil analysis. This paper presents a method for such a determination using a Dexsil-300 packed column. The results obtained (n-alkane contents, carbon number distribution) agree fairly well with those obtained on a 30 m × 0.25 mm DB-1 fused silica capillary column.     

Mixed mode chromatography via column switching for the simultaneous HPLC analysis of lonic and non-lonic nucleic acid constituents

Summary A method is presented for the separation of purine and pyrimidine nucleotides, nucleosides and bases in one analysis. This method uses both the ion-exchange and the reversed-phase modes for the analysis of these compounds. The modes are coupled through automatic switching valves. The use of these valves permits the isolation of either column during the analysis. The nucleotides are separated on an anion-exchange column and the nucleosides and bases on a C-18 column. Total analysis time is approximately one hour, and a very small volume of a sample can be analyzed. The technique is flexible and mobile phase conditions for one column can be altered without affecting the resolution on the other column. The method is applicable to biological matrices.     

Capillary column gas chromatographic method using electron-capture detection for the simultaneous determination of nicardipine and its pyridine metabolite II in plasma

A rapid, specific and direct method based on capillary column gas chromatography with electron-capture detection is described for the simultaneous determination of nicardipine, a new calcium antagonist, and its pyridine metabolite II in human plasma. In this method, the nicardipine, its pyridine metabolite II and internal standard are extracted from the plasma and then partially purified by acid-base partitioning prior to the final injection onto the capillary column gas chromatograph for quantification by means of an electron-capture detector. The quantification limit of the method is 1 ng/ml of plasma for both nicardipine and its pyridine metabolite II. The coefficients of variation for nicardipine and the pyridine metabolite II at concentrations of 1-50 ng/ml are less than 7% and less than 9% (n = 4), respectively. The method has been validated against a previously developed high-performance liquid chromatographic method (sensitivity 5 ng/ml).     

Quantitation of trace betamethasone or dexamethasone in dexamethasone or betamethasone active pharmaceutical ingredients by reversed-phase high-performance liquid chromatography

Adequate separation is essential for the quantitation of trace amounts of dexamethasone that are typically found in betamethasone active pharmaceutical ingredients and vice versa. In this paper, we describe three simple and efficient high-performance liquid chromatography methods from which true baseline separations between betamethasone and dexamethasone are achieved even when the concentration ratios between these two epimers are larger than 2000:1. One method is developed on a 5 cm ACE C8 column that uses water and acetonitrile as the mobile phase and 20 mM beta-cyclodextrin as the mobile phase additive. The resolution factor between betamethasone and dexamethasone is 3.3. The second method is developed on a 10 cm ACE C8 column that uses water and acetonitrile as the mobile phase, in which the resolution factor between the epimers is 2.7. The third method is developed on a 10 cm ACE C8 column using water and tetrahydrofuran as the mobile phase, in which the resolution factor between the epimers is 3.1. Preliminary validation studies are carried out for the second and third methods.     

适用于质谱分析的在线固相萃取除盐方法

建立了一个简单、快速、有效的适用于质谱或液相色谱-质谱联用的在线固相萃取(SPE)高通量除盐方法。方法分为单柱和双柱模式,借助于包含双梯度泵(上样泵/分析泵)、自动进样器和配有十通切换阀的柱温箱的高效液相色谱系统,完成样品的自动化在线除盐。单柱模式通过上样泵实现在SPE柱上进样和除盐,被分析物则保留在SPE柱上;除盐完成后,通过阀切换利用分析泵洗脱富集在SPE柱上的被分析物。双柱模式则在单柱模式基础上增加了1根SPE柱,在色谱管理软件控制下2根SPE柱轮流工作,高效率完成样品的在线除盐。该方法在结合质谱分析蛋白质、多肽等领域具有较好的应用前景。     

Establishing an alternative method for the quantitative analysis of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans by comprehensive two dimensional gas chromatography-time-of-flight mass spectrometry for developing countries

Comprehensive Gas Chromatography-Time-of-Flight Mass Spectrometry (GC×GC-TOFMS) methodology has been refined for the analysis of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in samples with different matrices. This is specifically for application in developing countries where access to gas chromatography-high resolution mass spectrometry (GC-HRMS) and highly skilled personnel is limited. The method, using an Rxi-5 Sil MS column in the first dimension ((1)D) coupled with an Rtx-200 column in the second dimension ((2)D), was used to quantify PCDDs and PCDFs in different environmental sample matrices. The results were compared with those obtained using GC-HRMS and good agreement was observed. The limit of detection (LOD) for the method (300fg on column for spiked soil samples) was determined using an Rxi-XLB ((1)D) column coupled with an Rtx-200 column ((2)D). Preliminary South African sample results are also discussed. Isomer specificity for different tetrachloro dibenzo-p-dioxins (TCDDs) and tetrachloro dibenzofurans (TCDFs) was investigated using a commercial standard. Adequate resolution was achieved. The method as described has great attraction for developing countries being both financially and operationally favourable.     

Evaluation of Fumonitest Immunoaffinity Columns

Abstract

A method for the determination of fumonisins B1 and B2 in corn was developed. The method involves sample extraction with methanol:water (80:20) and the use of a commercially available Fumonitest column for sample cleanup. The capacity, selectivity, column-to-column and lot-to-lot reproducibility of the Fumonitest columns were evaluated. The total capacity of the column was found to be 1.2 μg fumonisin. Both fumonisins B1 and B2 had an equal affinity toward the Fumonitest column, with the sample matrix demonstrating little effect on the column performance. The maximum sample size was 0.5 g for samples containing total fumonisins of less than 2 ppm. After elution from the immunoaffinity column, fumonisins B1 and B2 were reacted with naphthalene-2, 3-dicarboxaldehyde (NDA) to produce a highly fluorescent derivative, 1-cyano-2-alkyl-benz[f]isoindole (CBI). The derivatives were then separated from the sample matrix on a reverse phase C-18 column with a mobile phase consisting of acetonitrile:water:acetic acid (55:45:1) Average recoveries of fumonisins B1 and B2 from corn samples spiked at a level of 1000 ng (500ng B1 + 500ng B2)/g were 85.4 and 87.1%, respectively. The detection limit for B1 and B2 was estimated to be 10 and 4 ppb, respectively. The coefficient of variations for fumonisins B1 and B2 were determined to be 10.2% and 10.6%, respectively.     

Direct separation of regio- and enantiomeric isomers of diacylglycerols by a tandem column high-performance liquid chromatography

A novel HPLC-based method for direct separation of the three isomers of mono-acid diacylglycerols (DAGs), i.e., 1,2-DAG, 2,3-DAG and 1,3-DAG, has been established. The method employs a tandem column system, in which two different columns (a conventional silica gel column and a chiral stationary phase column) are connected in series. Two isomeric mixtures of DAGs (i.e., dicapryloylglycerol and dioleoylglycerol) and lipase-catalyzed reaction mixtures were successfully resolved on the tandem column HPLC system without any derivatization prior to the analysis. According to the established analytical method, stereoselectivity of two lipases toward mono-acid triacylglycerols in ethanolysis reaction was investigated. The tested enzymes were immobilized Candida antarctica lipase B (CALB) and Rhizomucor miehei lipase (RML). Analyses of the enantiomeric purity of 1,2-DAG and 2,3-DAG, generated as intermediates during the reaction, revealed that CALB and RML have sn-3 and sn-1 stereopreference, respectively.     

Quantitation of 6-(pyridin-3-yl)quinolin-2(1H)-one, a cardiac stimulant, and its N-oxide metabolite in dog plasma by high-performance liquid chromatography.

A method is described for the determination of UK-57,400 (I), a 6-substituted quinoline cardiotonic, and its pyridyl-N-oxide in dog plasma. The analytes are selectively retained from plasma (1 ml) on a solid-phase extraction column and eluted with 1 ml of methanol. After evaporation to dryness, the residue is reconstituted in 100 microliters of the mobile phase. Chromatography is carried out on a Spherisorb 5 microns phenyl high-performance liquid chromatography column, with ultraviolet detection. Calibration curves are linear for concentrations from 10 to 100 ng ml-1. For I, the coefficients of variation at highest and lowest concentrations are 1 and 14%, respectively, while the corresponding figures for the pyridyl-N-oxide metabolite are 4 and 10%, respectively. Sample recovery from extraction is greater than 90%. The limit of detection is 4 ng ml-1 for both analytes.     

Measurement of Chelating Agent Levels in Media with High Concentrations of Interfering Metal Ions

Abstract

An ion-exchange method for the measurement of levels of chelating agents in media with high levels of metal ions has been developed. The test solution is passed through a specially prepared ion-exchange column, which both removes metal ion interferences, and places the chelating agent in the copper form. The copper levels in the column effluent are then measured by flame atomic absorption methods, the copper levels corresponding in a 1:1 ratio to the levels of chelating agent in the sample. The detection limit for this method is on the order of 1 ppm copper equivalent chelating capacity, and is applicable in situations where the interfering metal ions are at concentrations in the range of 10^?2 M. The method is reproducible in the pH ranges of 4 to 9 Precision and     

Optimal design of affinity membrane chromatographic columns

A method for the optimal affinity membrane column design, based in the solution of the Thomas kinetic model for frontal analysis in membrane column adsorption, is presented. The method permits to choose suitable membrane operating conditions, column dimensions and processing time, to maximize the throughput when an operating capacity restriction in the range of 80–95% of the column capacity is used. Two basic design charts were obtained by computer simulation, for residence and processing time calculation, respectively. These charts can be used and manipulated in a wide range of operational conditions, provided that four design specifications related to column axial and radial Peclet numbers, length and pressure drop, are fulfilled. The application of the method was illustrated using experimental data and a simple analytical procedure. The implications of the method and results on the design and optimization of affinity membrane chromatographic columns are discussed.     

氯硅烷的气相色谱分析

采用涂邻苯二甲酸二乙酯的气相色谱柱对用化学气相沉积法制备ＳｉＣ纤维的主要原料——氯硅烷直接进样分析,在较短的时间内即可得知每批原料的差异。实验表明,方法操作简便、快速,重复性和精密度较好,重复６次的变异系数均＜１％。可用于ＳｉＣ纤维原料的质量监测。     

Application of ion-exchange separations to determination of trace elements in natural waters-IX: simultaneous isolation and determination of uranium and thorium

A method is described for the determination of uranium and thorium in samples of natural waters. After acidification with citric acid the water sample is filtered and sodium citrate and ascorbic acid are added. The resulting solution of pH 3 is passed through a 4-g column of Dowex 1 x 8 (citrate form) on which both uranium and thorium are adsorbed as anionic citrate complexes. Thorium is eluted with 8M hydrochloric acid and separated from co-eluted substances by anion-exchange in 8M nitric acid medium on a separate 2-g column of the same resin in the nitrate form. After complete removal of iron by washing with a mixture consisting of IBMK, acetone and 1M hydrochloric acid (1:8:1 v v ) and treatment of the resin with 6M hydrochloric acid, the uranium is eluted from the 4-g column with 1M hydrochloric acid. In the eluate thorium is determined spectrophotometrically (arsenazo III method) while fluorimetry is employed for the assay of uranium. The procedure was used for the determination of uranium and thorium in numerous water samples collected in Austria, including samples of mineral-waters. The results indicate that a simple relationship exists between the uranium and thorium contents of waters which makes it possible to calculate the approximate thorium content of a sample on the basis of its uranium concentration and vice versa.     

Targeted multidimensional gas chromatography for the quantitative analysis of suspected allergens in fragrance products

Two approaches are described and compared for the analysis of suspected allergens (SAs) in fragrance products, which are defined by the Scientific Committee of Cosmetics and Non-Food Products (SCCNFP). The first consists of a comprehensive two-dimensional gas chromatography (GCxGC) experiment using both a "conventional" non-polar/polar column combination and an "inverse" polar/non-polar column set. The second approach uses a targeted multidimensional gas chromatography (MDGC) system employing a Deans type pneumatic switch and a longitudinally modulated cryogenic system (LMCS). It was found that the conventional and inverse column sets complement each other well, providing identification of SAs present. Compounds well retained on the second dimension of one column set were the first to be eluted from the other. In some instances SAs co-eluting with matrix components on the second dimension for a given column set were clearly resolved on the other, although this has the disadvantage of requiring two analytical runs. Targeted MDGC with a non-polar/polar column set, successfully separated all SAs identified within a fragrance product. The instrument is set up in a similar fashion to a GCxGC system though with longer second dimension ((2)D) column, a cryogenic trap at the beginning of the second column, and a pneumatic switch coupling both columns. The data are easier to process than for a GCxGC experiment. The targeted MDGC method has the capacity to deliver far greater efficiency to targeted regions of a primary separation than a GCxGC experiment, whilst still maintaining overall run times similar to those of a conventional one-dimensional (1D) GC experiment. Cryogenic focussing at the beginning of the (2)D column delivers enhanced sensitivity, accurate (2)D retention times and narrow peak widths; these are responsible for an increased resolution obtained from the fast, relatively short ( approximately 5m) (2)D column. The two column set GCxGC analysis provided a quick and effective means to qualitatively determine the presence of six SAs in a commercially available air freshener, however all were not adequately resolved from matrix components. In contrast, quantitation was straightforward using the targeted MDGC method.     

Determination of clozapine in human serum by capillary gas chromatography

A gas chromatographic method using a fused-silica wide-bore capillary column and a nitrogen-specific detector for the determination of the antipsychotic agent clozapine in human serum is described. This method was found to be suitable for the determination of serum levels down to 1-2 ng/ml. The sensitivity, precision and accuracy of this method are adequate for studies on pharmacokinetics and bioavailability.