Elution Temperature Research Articles

DNA Sequence Fragment Containing C to A Mutation as a Convenient Mutation Standard for DHPLC Analysis.

Denaturing high performance liquid chromatography (DHPLC) is a high throughput approach for screening DNA sequence variations. To assess oven calibration, cartridge performance, buffer composition and stability, the WAVE Low and High Range Mutation Standards are employed to ensure reproducibility and accuracy of the chromatographic analysis. The purpose of this study was to provide a cost-effective homemade mutation standard for DHPLC analysis. DHPLC was performed to evaluate different elution temperatures of a 374 bp DNA fragment with C>A mutation at position of 59 to achieve a peak profile similar to the Low Mutation Standard. In order to verify the reproducibility of the homemade mutation standard using DHPLC, 15 different experiments were performed to compare the homemade mutation standard, the WAVE Low Range Mutation Standard with a positive DNA control sample. We identified a comparable elution temperature and a peak profile with the WAVE Low Range Mutation Standard. This study confirmed the reproducibility of the peak profile of our homemade mutation standard compared to the Low Mutation Standard using DHPLC analysis.

Characterisation of capillary ionic liquid columns for gas chromatography–mass spectrometry analysis of fatty acid methyl esters

Due to their distinct chemical properties, the application of ionic liquid (IL) compounds as gas chromatography (GC) stationary phases offer unique GC separation especially in the analysis of geometric and positional fatty acid methyl ester (FAME) isomers. Elution behaviour of FAME on several commercialised IL capillary columns including phosphonium based SLB-IL59, SLB-IL60, SLB-IL61 and SLB-IL76 and imidazolium based SLB-IL82, SLB-IL100, and SLB-IL111 as well as a general purpose column SLB-5ms, were evaluated in gas chromatography–mass spectrometry (GC–MS) analysis. The phases were further characterised by using a linear solvation energy relationship (LSER) approach according to the equivalent chain length (ECL) index of FAME. Among all tested IL columns, elution temperatures of saturated FAME increased as their McReynolds’ polarity value decreased, except for IL60. ECL values increased markedly as the stationary phase polarity increased, particularly for the polyunsaturated FAME. The LSER study indicated a lowest l/e value at 0.864 for IL111, displaying phase selectivity towards unsaturated FAME, with higher peak capacity within a carbon number isomer group. s and e descriptors calculated from LSER were validated by excellent correlation with dipole moments and lowest unoccupied molecular orbital (LUMO) energies, with R2 values of 0.99 and 0.92 respectively, calculated using GAUSSIAN.

Separation of Fatty Acid Methyl Esters by GC-Online Hydrogenation × GC

The separation of fatty acid methyl esters (FAME) provided by a 200 m × 0.25 mm SLB-IL111 capillary column is enhanced by adding a second dimension of separation ((2)D) in a GC × GC design. Rather than employing two GC columns of different polarities or using different elution temperatures, the separation in the two-dimensional space is achieved by altering the chemical structure of selected analytes between the two dimensions of separation. A capillary tube coated with palladium is added between the first dimension of separation ((1)D) column and the cryogenic modulator, providing the reduction of unsaturated FAMEs to their fully saturated forms. The (2)D separation is achieved using a 2.5 m × 0.10 mm SLB-IL111 capillary column and separates FAMEs based solely on their carbon skeleton. The two-dimensional separation can be easily interpreted based on the principle that all the saturated FAMEs lie on a straight diagonal line bisecting the separation plane, while the FAMEs with the same carbon skeleton but differing in the number, geometric configuration or position of double bonds lie on lines parallel to the (1)D time axis. This technique allows the separation of trans fatty acids (FAs) and polyunsaturated FAs (PUFAs) in a single experiment and eliminates the overlap between PUFAs with different chain lengths. To our knowledge, this the first example of GC × GC in which a chemical change is instituted between the two dimensions to alter the relative retentions of components and identify unsaturated FAMEs.

Reversal of elution order in a single second dimension by changing the first column nature in comprehensive two-dimensional gas chromatography

This paper explains how one single stationary phase can involve two different elution orders for linear alkanes, cyclic alkanes, aromatics and phenols using comprehensive two-dimensional gas chromatography. For this purpose, a coal-derived middle distillate was injected in nonpolar×semipolar and polar×semipolar configurations implying the same second dimension stationary phase (trifluoropropyl). Results show that even if the same column is utilised as a second dimension, the group-type elution order is reversed from one combination to the other. This can be explained as follows:for the polar×semipolar combination, each fraction eluting from the first dimension contains species that differ so much in terms of boiling points, that volatility plays a key role in the second isothermal separation. This is exemplified by the separation of a phenol and demonstrated using the proportional relationship between retention times, vapour pressures and activity coefficients. Moreover, van’t Hoff plots (plots of lnk vs. 1/T) demonstrated the influence of the elution temperature from the first dimension on the second dimension separation. Therefore, available choice of stationary phase's combinations is much higher considering that one single column leads to very different retentions for similar compounds. Finally, this can explain why a reverse orthogonality approach is usually proficient for the separation of polar compounds.

Ultra‐high performance liquid chromatographic method for the determination of polycyclic aromatic hydrocarbons in a passive environmental sampler

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous compounds released in the environment by different sources. The aim of the present work was to validate a solid-phase extraction (SPE) and a rapid ultra-high performance liquid chromatographic (UHPLC) method for the analysis of PAHs in a passive environmental sampler, namely a Dacron® (the commercial name of a synthetic fiber based on polyethylene terephthalate) textile. The elution temperature was optimized to improve the resolution of early-eluted compounds, namely acenaphthene (Ac) and fluorene (F). The UHPLC method lasts about 10 min and showed good linearity for all the 16 PAHs considered, with regression coefficients over 0.99. Recoveries, limits of detection (LODs), and limits of quantification (LOQs) of the SPE method were well within the performance criteria fixed by the Regulation n. 836/2011, namely 0.3 and 0.9 μg/kg, respectively.

Predictions of comprehensive two-dimensional gas chromatography separations from isothermal data

Two-dimensional retention times and peak widths in temperature programmed comprehensive two-dimensional gas chromatography were predicted using a theoretical model developed from experimental data obtained under isothermal conditions. A Matlab program was written to calculate the following parameters: dead time (tM), elution temperature (Te), retention time (tR), hold-up width (wM) and peak width (wR). The two-dimensional retention times of a sample mixture that contained n-alkanes and polyaromatic hydrocarbons (PAHs) were predicted and were observed to be in excellent agreement with experimentally determined values. The relative deviation between the model and the experimental data was less than 2 and 7% for the primary and secondary retention times, respectively. The relative deviation of peak width was less than 7 and 10% in the primary and secondary dimensions, respectively. The advantage of this model was its simplicity, informed entirely from experimental data, with no reliance on theoretical parameters. This prediction model would be useful for optimizing GC×GC separation conditions and for confirming compound identifications of components that are chromatographically resolved but that have nearly identical mass spectra.

Comparison of Separations of Fatty Acids from Fish Products Using a 30‐m Supelcowax‐10 and a 100‐m SP‐2560 Column

Commercial fish oils and foods containing fish may contain trans and/or isomerized fatty acids (FA) produced during processing or as part of prepared foods. The current American Oil Chemists' Society (AOCS) official method for marine oils (method Ce 1i-07) is based on separation by use of poly(ethylene glycol) (PEG) columns, for example Supelcowax-10 or equivalent, which do not resolve most unsaturated FA geometric isomers. Highly polar 100-m cyanopropyl siloxane (CPS) columns, for example SP-2560 and CP Sil 88 are recommended for separation of geometric FA isomers. Complementary separations were achieved by use of two different elution temperature programs with the same CPS column. This study is the first direct comparison of the separations achieved by use of 30-m Supelcowax-10 and 100-m SP-2560 columns for fatty acid methyl esters (FAME) prepared from the same fish oil and fish muscle sample. To simplify the identification of the FA in these fish samples, FA were fractionated on the basis of the number and type of double bonds by silver-ion solid-phase extraction (Ag⁺-SPE) before GC analysis. The results showed that a combination of the three GC separations was necessary to resolve and identify most of the unsaturated FA, FA isomers, and other components of fish products, for example phytanic and phytenic acids. Equivalent chain length (ECL) values of most FAME in fish were calculated from the separations achieved by use of both GC columns; the values obtained were shown to be consistent with previously reported values for the Supelcowax-10 column. ECL values were also calculated for the FA separated on the SP-2560 column. The calculated ECL values were equally valid under isothermal and temperature-programmed elution GC conditions, and were valuable for confirmation of the identity of several unsaturated FAME in the fish samples. When analyzing commercially prepared fish foods, deodorized marine oils, or foods fortified with marine oils it is strongly recommended that quantitative data acquired by use of PEG columns is complemented with data obtained from separations using highly polar CPS columns.

Hemicellulose sugar recovery from steam-exploded wheat straw for microbial oil production

There are currently few successful examples of using straw hemicellulose as a carbon source in the fermentation industry. In this paper, hemicellulose hydrolysates were recovered from steam-exploded wheat straw (SEWS) and used to produce microbial oil. The effects of the steam explosion treatment conditions, the elution temperature and the ratio of elution water to SEWS on sugar recovery were examined. A broth with 3.8 g l −1 of reducing sugar and 22.3 g l −1 of total soluble sugars was obtained with a 10-fold excess (w/w) of water at 40 °C to wash the SEWS treated under steam explosion conditions at 200 °C for 5 min. This broth was used to produce microbial oil by the oleaginous fungus Microsphaeropsis sp., which was able to secrete xylanase to degrade oligosaccharides from straw hemicellulose and accumulate microbial oil. Under optimized conditions, the oil concentration was 2.6 g l −1. The yield of oil from sugar consumed was 0.14 g g −1. The microbial oil produced by this research could be used as feedstock for biodiesel production because the microbial oil was primarily composed of neutral lipids. This research establishes a novel protocol for microbial oil production from straw hemicellulose.

Mutations in CDON, Encoding a Hedgehog Receptor, Result in Holoprosencephaly and Defective Interactions with Other Hedgehog Receptors

Holoprosencephaly (HPE), a common human congenital anomaly defined by a failure to delineate the midline of the forebrain and/or midface, is associated with diminished Sonic hedgehog (SHH)-pathway activity in development of these structures. SHH signaling is regulated by a network of ligand-binding factors, including the primary receptor PTCH1 and the putative coreceptors, CDON (also called CDO), BOC, and GAS1. Although binding of SHH to these receptors promotes pathway activity, it is not known whether interactions between these receptors are important. We report here identification of missense CDON mutations in human HPE. These mutations diminish CDON's ability to support SHH-dependent gene expression in cell-based signaling assays. The mutations occur outside the SHH-binding domain of CDON, and the encoded variant CDON proteins do not display defects in binding to SHH. In contrast, wild-type CDON associates with PTCH1 and GAS1, but the variants do so inefficiently, in a manner that parallels their activity in cell-based assays. Our findings argue that CDON must associate with both ligand and other hedgehog-receptor components, particularly PTCH1, for signaling to occur and that disruption of the latter interactions is a mechanism of HPE.

Separation of 125IBZM and 125ILIS using polyacrylamide-acrylic acid resin

Radioiodination of both S(−)BZM and LIS was carried out using n-bromosuccimide(NBS) as a mild oxidizing agent. The factors affecting on the radiochemical yield such as pH, reaction time, substrate concentration and oxidizing agent have been studied. The chromatographic separation of both 125IBZM and 125ILIS was carried out using HPLC and poly(acrylamide-acrylic acid) resin P(AAm-AA). The copolymer was prepared by a template polymerization of AA in aqueous solution on PAAm as a template polymer and in the presence of N,N-methylenebisacrylamide (NMBA) as a crosslinker using gamma rays as initiator. The purifications of tracers were carried out using prepared resin compared with TLC and HPLC. The effects of pH buffer, variable elution volumes, flow rate and temperature on the separation process of the resin efficiency have been studied.

Droplet-based Extraction of Hepatitis B Virus DNA in a Capillary

A novel capillary droplet-based DNA-extraction method was developed, using the principle of water-in-oil droplet formation in a highly hydrophobic polytetrafluoroethylene capillary. By sequentially introducing droplets containing a sample or reagents, all the steps related to Hepatitis B virus DNA extraction from blood, including sample injection, DNA binding, magnetic bead rinsing, and DNA elution were continuously implemented in the capillary. Experimental results showed that Proteinase K lysis and elevated elution temperature facilitated higher extraction efficiency. Under optimized conditions, an extraction was completed within 25 min with microliter level consumption of the sample or reagent. The droplet-based method achieved an extraction efficiency of about 60%, which was comparable to that of conventional methods, with the relative standard deviation of extraction efficiency for the droplet-based method being relatively higher. In summary, the herein-developed droplet-based DNA-extraction method is simple, fast, and economic, thus having the potential to be integrated into a micro-total analysis system for nucleic acids.

Use of different sample temperatures in a single extraction procedure for the screening of the aroma profile of plant matrices by headspace solid-phase microextraction

This study proposes a new approach to the optimization of the extraction of the volatile fraction of plant matrices using the headspace solid-phase microextraction (HS-SPME) technique. The optimization focused on the extraction time and temperature using a CAR/DVB/PDMS 50/30 μm SPME fiber and 100 mg of a mixture of plants as the sample in a 15-mL vial. The extraction time (10–60 min) and temperature (5–60 °C) were optimized by means of a central composite design. The chromatogram was divided into four groups of peaks based on the elution temperature to provide a better understanding of the influence of the extraction parameters on the extraction efficiency considering compounds with different volatilities/polarities. In view of the different optimum extraction time and temperature conditions obtained for each group, a new approach based on the use of two extraction temperatures in the same procedure is proposed. The optimum conditions were achieved by extracting for 30 min with a sample temperature of 60 °C followed by a further 15 min at 5 °C. The proposed method was compared with the optimized conventional method based on a single extraction temperature (45 min of extraction at 50 °C) by submitting five samples to both procedures. The proposed method led to better results in all cases, considering as the response both peak area and the number of identified peaks. The newly proposed optimization approach provided an excellent alternative procedure to extract analytes with quite different volatilities in the same procedure.

High performance liquid chromatography of slightly soluble brominated indigoids from Tyrian purple

Attempts at identification of mollusc species producing Tyrian purple from archaeological material are usually done with high performance liquid chromatography in the reversed-phase system, but the peaks obtained are often wide and asymmetric. This is due to the low solubility of the indigoids and their brominated derivatives in the mobile phase, especially 6,6′-dibromoindigotin, which is soluble in only few, particular solvents. Our study focused on improving both symmetry and peak height for more precise quantification. The influence of various factors was evaluated: stationary phase characteristics, mobile phase composition, elution gradient parameters and temperature on the peak shape of the main components of Tyrian purple. The best results were obtained using highly retentive, but moderately bonded ODS stationary phases (about 2.8 μmol m −2), percolated with gradient of acetonitrile with acidified aqueous mobile phases (0.1% strong acid) at elevated temperatures (70 °C). The upper quantification limit for 6,6′-dibromoindigotin was improved by over 350%, between standard and optimised systems. Using them, the detection and quantification of trace Tyrian purple components (less than 0.15%) aside from major indigoids becomes possible. Consequently, for the first time, the new analogues of brominated and unbrominated indirubins were found in the shellfish purple from Hexaplex trunculus.

A new procedure for the determination of distillation temperature distribution of high-boiling petroleum products and fractions

The distribution of distillation temperatures of liquid and semi-fluid products, including petroleum fractions and products, is an important process and practical parameter. It provides information on properties of crude oil and content of particular fractions, classified on the basis of their boiling points, as well as the optimum conditions of atmospheric or vacuum distillation. At present, the distribution of distillation temperatures is often investigated by simulated distillation (SIMDIS) using capillary gas chromatography (CGC) with a short capillary column with polydimethylsiloxane as the stationary phase. This paper presents the results of investigations on the possibility of replacing currently used CGC columns for SIMDIS with a deactivated fused silica capillary tube without any stationary phase. The SIMDIS technique making use of such an empty fused silica column allows a considerable lowering of elution temperature of the analytes, which results in a decrease of the final oven temperature while ensuring a complete separation of the mixture. This eliminates the possibility of decomposition of less thermally stable mixture components and bleeding of the stationary phase which would result in an increase of the detector signal. It also improves the stability of the baseline, which is especially important in the determination of the end point of elution, which is the basis for finding the final temperature of distillation. This is the key parameter for the safety process of hydrocracking, where an excessively high final temperature of distillation of a batch can result in serious damage to an expensive catalyst bed. This paper compares the distribution of distillation temperatures of the fraction from vacuum distillation of petroleum obtained using SIMDIS with that obtained by the proposed procedure. A good agreement between the two procedures was observed. In addition, typical values of elution temperatures of n-paraffin standards obtained by the two procedures were compared. Finally, the agreement between boiling points of polar compounds determined from their retention times and actual boiling points was investigated.

Development of chromatographic separation of some biological materials using poly(acrylamide-acrylic acid-dimethylaminoethyl methacrylate) resin

The aim of the present study is to develop of chromatographic separation of some biological materials using poly(acrylamide-acrylic acid-dimethylaminoethyl methacrylate) resin P(AAm-AA-DMAEMA). The copolymer was prepared by a template copolymerization of DMAEMA and AA in aqueous solution on polyacrylamide (PAAm) as a template polymer and N,N-methylenebisacrylamide (NMBA) as a crosslinker using gamma rays as initiator. Some biological materials such as alpha-fetoprotein (AFP), bovine serum albumin (BSA) and tri-iodothyronine (T3) were labeled using 125I and the purifications of all tracers were carried out using prepared resin compared with Sephadex. Factors affecting were studied to reach the most efficiency purification including pH buffer, variable elution volumes, flow rate and temperature. The radiochemical purity percent was determined using paper electrophoresis and immunoreactivity of tracer was checked. The efficiency of purification that obtained using the prepared resin is nearly equivalent to that of Sephadex.

GC separation of amino acid enantiomers via derivatization with heptafluorobutyl chloroformate and Chirasil‐L‐Val column

Heptafluorobutyl chloroformate (HFBCF), a recently introduced derivatization reagent, was examined in enantioseparation of amino acids (AAs) by GC. Twenty proteinogenic AAs, plus ornithine, cystine and 4-fluorophenylalanine (internal standard) were treated with the reagent and separation properties of the derivatives were assessed on a Chirasil-Val capillary column. Nineteen AA enantiomers were efficiently separated in 43 min except proline, arginine and cystine. The HFBCF derivatives of the studied DL-AAs show improved separation over other chloroformate-based derivatives hitherto reported. A combination of the improved and faster separation with a simple derivatization protocol, involving an immediate one-step reaction-extraction in two-phase aqueous-organic medium, and low elution temperatures extend application of HFBCF to chiral AA analysis.

Gas chromatographic retention in uncoated fused silica capillaries

Understanding of retention in uncoated fused-silica capillaries is of interest due to increased attention on precolumn backflushing in capillary GC. Uncoated capillaries offer several advantages as precolumns compared to coated precolumns. In order to examine the possibility of predicting elution temperatures of alkanes from uncoated capillaries a priori, several sizes of deactivated but uncoated fused-silica capillaries were evaluated under various operating conditions. Retention was found to depend on dimensionless ramp rate (degrees C/t(M)), sample loading (capacity), flow mode, and column dimensions (probably related to surface area).

Use of FT‐IR Spectrometry for On‐Line Detection in Temperature Rising Elution Fractionation

AbstractSummary: Temperature rising elution fractionation (TREF) has become a popular analytical technique that is able to determine the chemical composition distribution (CCD) of an ethylene/α‐olefin copolymer. An infrared (IR) detector is commonly used in TREF detection to measure the concentration of the polymer solution exiting the column as a function of elution temperature. The chemical composition of the eluting polymer at a given elution temperature can be predicted from the relationship between comonomer content and TREF elution temperature pre‐established through 13C nuclear magnetic resonance (NMR) analysis of TREF fractions. In this article, a Fourier transform infrared (FT‐IR) spectrometer has been coupled with a TREF instrument to provide a more powerful tool for characterizing complex olefin copolymers. The Partial Least Squares (PLS) technique is used when analyzing the FT‐IR spectra of the eluting polymer solutions. The power of on‐line FT‐IR detection in TREF is demonstrated using a few complex copolymer systems, such as ethylene‐octene copolymer, polystyrene grafted ethylene‐vinyl acetate copolymer and ethylene‐methyl acrylate copolymer.

Successive solution fractionation mechanisms for high‐density polyethylene

AbstractBACKGROUND: Preparative fractionation techniques are currently used in order to obtain large amounts of polyethylene fractions. Preparative successive solution fractionation (SSF) and temperature rising elution fractionation (TREF) are compared as regards obtaining, at a multi‐gram scale, low‐dispersity fractions of high‐density polyethylene (HDPE). The operative separation mechanisms during a SSF of a broad HDPE, which are not yet totally elucidated, are also studied in this work.RESULTS: SSF and TREF approaches lead to the separation of HDPE macromolecules according to their molar masses. If very homogeneous fractions (dispersities from 1.1 to 1.3) are isolated in TREF at the lowest elution temperature, the collected mass is too low. At higher elution temperatures, the fractions have too broad a molar mass distribution (dispersities from 2.7 to 3.7). With the SSF procedure, dispersities are not as low as for the first TREF fractions. But, the relative weight fraction is better distributed between the different extraction temperatures. The molar mass distribution exhibits a dispersity of around 1.9.CONCLUSION: The SSF method is the most suitable way to obtain large gram amounts of low‐dispersity (ca 2) HDPE fractions over a wide molar mass range. Complementary gram‐scale rheological characterization is thus possible enabling a better comprehension of the SSF mechanism. Liquid–liquid demixing is the main mechanism in SSF, but its relative importance depends on polymer characteristics and solvent quality. Copyright © 2008 Society of Chemical Industry

Gas chromatography‐mass spectrometry with supersonic molecular beams

Gas chromatography-mass spectrometry (GC-MS) with supersonic molecular beams (SMBs) (also named Supersonic GC-MS) is based on GC and MS interface with SMBs and on the electron ionization (EI) of vibrationally cold analytes in the SMBs (cold EI) in a fly-through ion source. This ion source is inherently inert and further characterized by fast response and vacuum background filtration capability. The same ion source offers three modes of ionization including cold EI, classical EI and cluster chemical ionization (CI). Cold EI, as a main mode, provides enhanced molecular ions combined with an effective library sample identification, which is supplemented and complemented by a powerful isotope abundance analysis method and software. The range of low-volatility and thermally labile compounds amenable for analysis is significantly increased owing to the use of the contact-free, fly-through ion source and the ability to lower sample elution temperatures through the use of high column carrier gas flow rates. Effective, fast GC-MS is enabled particularly owing to the possible use of high column flow rates and improved system selectivity in view of the enhancement of the molecular ion. This fast GC-MS with SMB can be further improved via the added selectivity of MS-MS, which by itself benefits from the enhancement of the molecular ion, the most suitable parent ion for MS-MS. Supersonic GC-MS is characterized by low limits of detection (LOD), and its sensitivity is superior to that of standard GC-MS, particularly for samples that are hard for analysis. The GC separation of the Supersonic GC-MS can be improved with pulsed flow modulation (PFM) GC x GC-MS. Electron ionization LC-MS with SMB can also be combined with the Supersonic GC-MS, with fast and easy switching between these two modes of operation.