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At least 631 records · Page 35Linked to original sources
PubMed2006

Onset of non-linearity in zonal elution separators: the concept of effective analyte concentration.

When an analyte injected in a zonal separation method (chromatography, capillary zone electrophoresis, field-flow fractionation) is not highly diluted in the carrier fluid, the retention ratio, R--or ratio of the cross-sectional average migration velocity of the analyte to that of the carrier fluid--depends on the local concentration, c, of the center of mass of the analyte zone, and the zone migration occurs in non-linear conditions. Because the zone broadens as it moves along the separator, R varies continuously from the inlet to the outlet of the separator. That concentration, c(eff), for which R(c(eff)) is equal to the length-averaged apparent retention ratio, R(app), is called effective concentration, and that distance, z(eff), from the separator inlet, for which c(z(eff)) is equal to c(eff), i.e. for which R(z(eff)) is equal to R(app), is called effective position. Assuming that near the onset the non-linear behavior, R(c), is a linear function, values of R(app), c(eff) and z(eff) have been computed in a wide range of operating conditions which are typical of situations encountered in capillary zone electrophoresis, liquid chromatography, or field-flow fractionation. Computations have been performed both in presence and in absence of the dispersion arising from the concentration dependence of the analyte migration rate (called thermodynamic dispersion in chromatography or electromigration dispersion in capillary zone electrophoresis). It is found that, whatever the range of analyte concentration covered from inlet to outlet of the separator, c(eff) is always close to two times the analyte concentration, c(out), at the outlet of the separator, and z(eff) between one-fourth and one-third of the separator length. As c(out) is easily determined from the peak recorded by a concentration-sensitive detector, a simple pragmatic expression is given for the estimation of c(eff). This effective concentration is the appropriate concentration to be used for comparing predictions of theoretical models of R(c) with experimental retention data. This is of particular interest for validating such models in field-flow fractionation.

Chemical Fractionation↗
PubMed2006

Preparation and evaluation of a chiral stationary phase covalently bound with a chiral pseudo-18-crown-6 ether having a phenolic hydroxy group for enantiomer separation of amino compounds.

In order to develop a chiral stationary phase (CSP), which has even higher separation ability than the corresponding commercially available crown ether based CSP (OA-8000 having a pseudo-18-crown-6 ether with an OMe group as a selector), chemically bonded type CSP having a phenolic OH group on a crown ring was developed. Normal mobile phases with or without acid additive can be used with this OH type CSP in contrast to the conventional OMe type CSP which has a neutral chiral selector. Enantiomers of 25 out of 27 amino compounds, including 20 amino acids, 5 amino alcohols, and 2 lipophilic amines, were efficiently separated on a column with this CSP. Nine amino compounds out of 27 were separated with better separation factors than the corresponding OMe type CSP. It is noteworthy that the chromatography on this CSP exhibited excellent enantiomer-separations for amines and amino alcohols when triethyl amine was used as an additive in the mobile phase. Comparison of enantiomer separation ability on this OH type of CSP and on the OMe type of CSP and correlation between the enantioselectivity in chiral chromatography and that of the corresponding model compounds in solution imply that the chiral separation arose from chiral recognition in host guest interactions.

Amines↗
PubMed2006

Evaluation of the separation characteristics of application-specific (volatile organic compounds) open-tubular columns for gas chromatography.

The solvation parameter model is used to characterize the separation characteristics of two application-specific open-tubular columns (Rtx-Volatiles and Rtx-VGC) and a general purpose column for the separation of volatile organic compounds (DB-WAXetr) at five equally spaced temperatures over the range 60-140 degrees C. System constant differences and retention factor correlation plots are then used to determine selectivity differences between the above columns and their closest neighbors in a large database of system constants and retention factors for forty-four open-tubular columns. The Rtx-Volatiles column is shown to have separation characteristics predicted for a poly(dimethyldiphenylsiloxane) stationary phase containing about 16% diphenylsiloxane monomer. The Rtx-VGC column has separation properties similar to the poly(cyanopropylphenyldimethylsiloxane) stationary phase containing 14% cyanopropylphenylsiloxane monomer DB-1701 for non-polar and dipolar/polarizable compounds but significantly different characteristics for the separation of hydrogen-bond acids. For all practical purposes the DB-WAXetr column is shown to be selectivity equivalent to poly(ethylene glycol) columns prepared using different chemistries for bonding and immobilizing the stationary phase. Principal component analysis and cluster analysis are then used to classify the system constants for the above columns and a sub-database of eleven open-tubular columns (DB-1, HP-5, DB-VRX, Rtx-20, DB-35, Rtx-50, Rtx-65, DB-1301, DB-1701, DB-200, and DB-624) commonly used for the separation of volatile organic compounds. A rationale basis for column selection based on differences in intermolecular interactions is presented as an aid to method development for the separation of volatile organic compounds.

Chromatography, Gas↗
PubMed2006

Separation and identification of phytosiderophores and their metal complexes in plants by zwitterionic hydrophilic interaction liquid chromatography coupled to electrospray ionization mass spectrometry.

A sensitive method for the separation of different phytosiderophores (PS) of the mugineic acid (MA) family, and the candidate ligand for intracellular metal transport in plants nicotianamine (NA), and respective metal complexes in plants by zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) coupled to electrospray ionization mass spectrometry (ESI-MS) is described. Separation of mugineic acid, 2'-deoxymugineic acid (DMA), 3-epi-hydroxymugineic acid (epi-HMA), nicotianamine, Fe(III)-DMA, Fe(III)-NA, M(II)-DMA, and M(II)-NA complexes (M(II)=Zn(II), Cu(II), Ni(II), and Fe(II)), was achieved within 22 min on the ZIC-HILIC column by using a gradient elution with a mobile phase consisting of ammonium acetate and acetonitrile at pH 7.3, at a flow rate of 0.15 mL/min. The on-line coupling to ESI-MS in the negative ionization mode enables the detection of these compounds in the micromol/L range, which is the relevant concentration range in real plant samples. DMA-complexes of Fe(III), Zn, and Cu in wheat root, and an NA-complex of Ni in Arabidopsis were detected and identified by the proposed method. Even in the case of partial coelution of some divalent metal complexes, the identification is possible by their distinct mass spectra. The stability of metal complexes during separation was checked by injecting ethylenediaminetetraacetic acid (EDTA) after each run of metal-phytosiderophore complexes. Good stability of divalent-phytosiderophores, except for Fe(II)-complexes, was observed. During gradient separation, Fe(III)-complexes are partly dissociated (<20%), but a good sensitivity of Fe(III)-DMA in real plant samples is still achieved. In order to avoid instability problems with the separation of Fe-complexes, an isocratic separation is proposed, which allows the separation of ferrous and ferric complexes in 2 min.

Chromatography, Liquid↗
PubMed2005

The influence and utility of varying field strength for the separation of tryptic peptides by ion mobility-mass spectrometry.

The influence of field strength on the separation of tryptic peptides by drift tube-based ion mobility-mass spectrometry is reported. Operating the ion mobility drift tube at elevated field strengths (expressed in V cm(-1) torr(-1)) reduces separation times and increases ion transmission efficiencies. Several accounts in the literature suggest that performing ion mobility separation at elevated field strength can change the selectivity of ion separation. To evaluate the field strength dependant selectivity of ion mobility separation, we examined a data set of 65 singly charged tryptic peptide ion signals (mass range 500-2500 m/z) at six different field strengths and four different drift gas compositions (He, N2, Ar, and CH4). Our results clearly illustrate that changing the field strength from low field (15 V cm(-1) torr(-1)) to high field (66 V cm(-1) torr(-1)) does not significantly alter the selectivity or peak capacity of IM-MS. The implications of these results are discussed in the context of separation methodologies that rely on the field strength dependence of ion mobility for separation selectivity, e.g., high-field asymmetric ion mobility spectrometry (FAIMS).

Amino Acid Sequence↗
PubMed2005

On-line combination of capillary isoelectric focusing and capillary non-gel sieving electrophoresis using a hollow-fiber membrane interface: a novel two-dimensional separation system for proteins.

A novel two-dimensional (2D) separation system for proteins was reported. In the system, a piece of dialysis hollow-fiber membrane was employed as the interface for on-line combination of capillary isoelectric focusing (CIEF) and capillary non-gel sieving electrophoresis (CNGSE). The system is similar equivalent to two-dimensional polyacrylamide gel electrophoresis (2D PAGE), by transferring the principal of 2D PAGE separation to the capillary format. Proteins were focused and separated in first dimension CIEF based on their differences in isoelectric points (pIs). Focused protein zones was transferred to the dialysis hollow-fiber interface, where proteins hydrophobically complexed with sodium dodecyl sulfate (SDS). The negatively charged proteins were electromigrated and further resolved by their differences in size in the second dimension CNGSE, in which dextran solution, a replaceable sieving matrix instead of cross-linked polyacrylamide gel was employed for size-dependent separation of proteins. The combination of the two techniques was attributed to high efficiency of the dialysis membrane interface. The feasibility and the orthogonality of the combined CIEF-CNGSE separation technique, an important factor for maximizing peak capacity or resolution elements, were demonstrated by examining each technique independently for the separation of hemoglobin and protein mixtures excreting from lung cancer cells of rat. The 2D separation strategy was found to greatly increase the resolving power and overall peak capacity over those obtained for either dimension alone.

Animals↗
PubMed2004

Experimental design as a tool when evaluating stationary phases for the capillary electrochromatographic separation of basic peptides.

Two different capillary electrochromatography (CEC) stationary phases, Hypersil phenyl and Hypersil C(18), have been characterised with respect to their ability to separate the four basic peptides H-Tyr-(D)Ala-Phe-Phe-NH(2) (TAPP), H-Tyr-(D)Ala-Phe-NH(2) (TAP), H-Phe-Phe-NH(2) (PP) and H-Phe-NH(2) (P). Optimal separation conditions were first established separately for the two phases by applying experimental design in a stepwise procedure. The first step comprised a study to acquire basic knowledge about the variables, their influence on the response and their respective experimental domains for each of the two stationary phases. The second step was screening the significant variables and the third step was an optimisation with response surface modelling (RSM) to locate the optimum separation conditions for each stationary phase. The experimental procedure was identical for both stationary phases, but their respective experimental domains were different. The response functions were peak resolution and peak efficiency. This procedure enables specific optimal experimental conditions to be identified for each of the two stationary phases. The optimal conditions identified for the separation on the phenyl stationary phase were to use 50% ACN, 20% 50 mM Tris(hydroxymethyl)aminomethane (TRIS) pH 7.5, 30% H(2)O as BGE, operating at 20 degrees C and 20 kV high voltage. For the C(18) stationary phase optimal separation was achieved using a BGE with 80% ACN, 20% 30 mM TRIS pH 8.5, again operating at 20 degrees C and 20 kV high voltage. Results show that the phenyl stationary phase is better suited for the separation of basic, hydrophilic peptides.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2006

Separation of poly(amidoamine) (PAMAM) dendrimer generations by dynamic coating capillary electrophoresis.

The separation of compounds possessing amino groups (peptides, proteins, polyamino compounds) by capillary zone electrophoresis suffers from the interaction (sticking) of these solutes with the capillary wall. This sticking can result in the absence or incomplete separation of compounds or even in their retention in the capillary. Polyamidoamine (PAMAM) dendrimers are a class of spherical polymers with primary amino groups at the surface. These compounds can be separated reasonably well at acidic pH but not at neutral pH. A new method based on the dynamic coating of the capillary was developed for the separation of these compounds at pH 7.4. The method comprises separation in a fused-silica capillary (57 cm total length, 50 cm to the detector, ID 75 microm) and a background electrolyte consisting of a Tris-phosphate buffer (50 mmol/L, pH 7.4) and 0.05% (w/v) polyethyleneimine. This system is suitable for the separation of 7 generations of dendrimers (generations 0-6). The dynamic coating agent (polyethyleneimine) also improves the separation at acid pH.

Dendrimers↗
PubMed2005

Separation of charged colloids by a combination of pulsating lateral electric fields and poiseuille flow in a 2D channel.

Separation of colloidal particles of different sizes is becoming increasingly important due to rapid developments in the area of proteomics, genetic engineering, drug discovery, etc. In particular, there is a need to accomplish these separations on a microscale in 'lab-on-a-chip' devices. In this paper, we propose a new method for accomplishing separation of charged colloids of different sizes in a microchannel. This method involves a combination of pulses of lateral electric fields and Poiseuille flow in the axial direction. We develop a model for this separation technique and obtain closed form solutions for the mean velocity and the dispersion coefficient for a pulse of molecules introduced into the channel. These expressions are then utilized to determine the channel length and the separation time. For reasonable value of design constants, the proposed technique can separate molecules of different sizes that have diffusivities of 10(-10) and 0.5 x 10(-10) m2/s in 15.7 s in a 3.7 mm long channel. The length and the time increase to 5.45 cm and 231 s if the ratio of the diffusivities is reduced from 2 to 1.2, i.e., the latter diffusivity is increased to 0.835 x 10(-10) m2/s, while keeping all the other parameters the same. If the diffusivities are about 10(-9) m(2)/s, the length and the time for separation are 1 cm and 17.5 s for D1/D2=2, and 16 cm and 269 s for D1/D2=1.2.

Journal Article↗
PubMed2007

Ionic separation efficiency of a novel electric-field-assisted membrane module comprising an array of microchannel units.

The ionic separation efficiency of a novel membrane module comprising an array of microchannel units is analyzed. Under the Debye-Hückel approximation, we derive a semianalytical expression for the ionic separation efficiency. Analyses reveal that the effects of the size of the microchannel, the fixed charge density in the membrane layer, and the permittivity of the membrane layer on ionic separation efficiency depend strongly on the valence type of electrolyte in treated water. Under the condition of a symmetric electrolyte, the ionic separation efficiency is found to be about unity and unresponsive to variation of system parameters. If the valence of the cation is higher than that of the anion, the ionic separation efficiency is larger than unity, and decreases to unity as the size of the microchannel increases. In contrast, if the valence of the cation is lower than that of the anion, the ionic separation efficiency is smaller than unity and increases to unity as the size of the microchannel increases. Under the latter two conditions, the effects of both fixed charge density in the membrane layer and permittivity of the membrane layer on the ionic separation efficiency are found to be reversed.

Journal Article↗
PubMed2004

The application of independent component analysis to the multi-channel surface electromyographic signals for separation of motor unit action potential trains: part II-modelling interpretation.

The purpose of this article was to investigate whether or not FastICA can separate identical motor unit action potential trains (MUAPTs) of the 8-channel surface electromyographic (sEMG) signals constructed by an sEMG model into the independent components. Firstly, we have examined how much the increase of motor units (MUs) in the simulated sEMG signals influenced the performance on the separation of MUAPTs by kurtosis. The decreased trend of mean kurtosis on both sEMG signals and their independent components were observed as MUs were increased. These data suggested that the separation performance decayed when MUs were increased. Secondary, the differences between the independent components and the principal components have been also applied to the simulated sEMG signals with or without time delay between the sEMG channels. FastICA could successfully separate identical MUAPTs with no time delay but principal component analysis (PCA) could not do so. Against it, both FastICA and PCA could not separate MUAPTs with some time delay. In conclusion, our results suggested that FastICA could separate identical MUAPTs with no time delay into the independent components by FastICA, which might offer a new technique for the separation of interfered MUAP waveforms based on statistical properties of sEMG signal distributions.

Action Potentials↗
PubMed2004

Comparison of the miniaturised techniques capillary electrochromatography and capillary liquid chromatography for the chiral separation of chlorthalidone.

The aim of this study was to compare the miniaturised techniques, capillary electrochromatography (CEC) and capillary liquid chromatography (CLC), for the chiral separation of chlorthalidone. In both cases, hydroxypropyl-beta-cyclodextrin was used as a chiral selector in the mobile phase, while an achiral stationary phase was used. Earlier, this separation was already optimised in CEC. Now, the separation was optimised in CLC. The influence of the organic modifier content and the cyclodextrin concentration on the separation was studied by means of a central composite design. Optimal separation conditions were determined, after response modelling, from the response surface contour plots. When these conditions were compared with those of the CEC optimisation, we can see the potential of using CLC as a chiral separation technique since less chiral selector was used, faster separations were obtained and better repeatability was observed in comparison with its electrical-driven counterpart.

Chlorthalidone↗
PubMed2005

Investigation of hydrocyclones for the separation of shredded fridge plastics.

The recycling of fridges produces a mixed plastic product of limited value. In order to maximise its value, the separation of the individual polymers that include high impact polystyrene (HIPS), acrylonitrile butadiene styrene (ABS), polyvinyl chloride (PVC) and polyethylene (PE) must take place. The effectiveness of a hydrocyclone system for the separation of these plastics, using both water and calcium chloride solutions, has been investigated. In addition a qualitative procedure for the determination of the HIPS content of a mixed ABS/HIPS product, by selective dissolution in R-limonene, has been developed. It was found that the effective density of separation depended on the particle size and aspect ratio. As the particle size and aspect ratio decreased, the separation became more efficient and the offset between separation density and hydrocyclone medium density decreased. This suggests that, for efficient density separation, closely sized, fine plastic fractions are required. Using these criteria, it was found that the removal of high density plastic such as PVC was readily achieved using a hydrocyclone. A partial separation of HIPS from ABS was found to be possible, taking advantage of a small density difference, using a hydrocyclone medium density of 1035kgm(-3).

Calcium Chloride↗
PubMed1995

Poly(ethylene glycol)-induced and temperature-dependent phase separation in fluid binary phospholipid membranes.

Exclusion of the strongly hygroscopic polymer, poly(ethylene glycol) (PEG), from the surface of phosphatidylcholine liposomes results in an osmotic imbalance between the hydration layer of the liposome surface and the bulk polymer solution, thus causing a partial dehydration of the phospholipid polar headgroups. PEG (average molecular weight of 6000 and in concentrations ranging from 5 to 20%, w/w) was added to the outside of large unilamellar liposomes (LUVs). This leads to, in addition to the dehydration of the outer monolayer, an osmotically driven water outflow and shrinkage of liposomes. Under these conditions phase separation of the fluorescent lipid 1-palmitoyl-2[6-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) embedded in various phosphatidylcholine matrices was observed, evident as an increase in the excimer-to-monomer fluorescence intensity ratio (IE/IM). Enhanced segregation of the fluorescent lipid was seen upon increasing and equal concentrations of PEG both inside and outside of the LUVs, revealing that osmotic gradient across the membrane is not required, and phase separation results from the dehydration of the lipid. Importantly, phase separation of PPDPC could be induced by PEG also in binary mixtures with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), for which temperature-induced phase segregation of the fluorescent lipid below Tm was otherwise not achieved. In the different lipid matrices the segregation of PPDPC caused by PEG was abolished above characteristic temperatures T0 well above their respective main phase transition temperatures Tm. For 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DMPC, SOPC, and POPC, T0 was observed at approximately 50, 32, 24, and 20 degrees C, respectively. Notably, the observed phase separation of PPDPC cannot be accounted for the 1 degree C increase in Tm for DMPC or for the increase by 0.5 degrees C for DPPC observed in the presence of 20% (w/w) PEG. At a given PEG concentration maximal increase in IE/IM (correlating to the extent of segregation of PPDPC in the different lipid matrices) decreased in the sequence 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC) > DPPC > DMPC > SOPC > POPC, whereas no evidence for phase separation in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) LUV was observed (Lehtonen and Kinnunen, 1994, Biophys. J. 66: 1981-1990). Our results indicate that PEG-induced dehydration of liposomal membranes provides the driving force for the segregation of the pyrene lipid. In brief, phase separation of PPDPC from the matrix lipid could be attributed to the diminishing effective size of the phosphatidylcholine polar headgroup resulting from its partial dehydration by PEG. This in turn would allow for enhanced van der Waals interactions between the acyl chains of the matrix lipid, which then caused the exclusion of PPDPC due to the perturbing bulky pyrene moiety. Phase separation in DMPC/PPDPC liposomes was abolished by the inclusion of 25 mol % cholesterol and to a lesser extent by epicholesterol.

Chemical Phenomena↗
PubMed2000

Enantiomer separations by nonaqueous capillary electrophoresis using octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin.

The newest member of the single-isomer isomer sulfated cyclodextrin family, octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin (ODAS-gamma-CD) was used for the first time as a resolving agent for the nonaqueous capillary electrophoretic separation of the enantiomers of 26 weak base pharmaceuticals in an acidic methanol background electrolyte. The solubility limit of ODAS-gamma-CD at room temperature proved to be 55 mM in this background electrolyte, which afforded good, fast enantiomer separations for most of the basic drugs tested. For all the bases studied, the effective mobilities and separation selectivities were found to follow the predictions of the charged resolving agent migration model of electrophoretic enantiomer separations. The effective mobilities of the weakly binding weak bases remained cationic throughout the entire 0 to 45 mM ODAS-gamma-CD concentration range; separation selectivities increased as the ODAS-gamma-CD concentration was increased. The effective mobilities of the moderately binding weak bases became anionic in the 2.5 to 45 mM ODAS-gamma-CD concentration range; separation selectivities first increased as the effective mobilities approached zero, then decreased again as the ODAS-gamma-CD concentration was increased further. The effective mobilities of the strongly binding weak bases became anionic in the 0 to 2.5 mM ODAS-gamma-CD concentration range; separation selectivities decreased as the ODAS-gamma-CD concentration was increased above 2.5 mM.

Cyclodextrins↗
PubMed2001

Semi-preparative gas chromatographic separation of all-trans-perhydrotriphenylene enantiomers on a chiral cyclodextrin stationary phase.

Enantiomers of all-trans-perhydrotriphenylene (PHTP) were separated by gas chromatography using heptakis(6-O-tert.-butyldimethylsilyl-2,3-di-O-methyl)-beta-cyclodextrin (TBDMS-beta-CD) as the chiral selector. Conditions for semi-preparative separations were established using a 2 m x 2 mm I.D. packed column and subsequently extended to a 1.8 m x 4 mm I.D. column which enabled separations on a mg scale. The column packing was TBDMS-beta-CD dissolved in SE-54 coated on Chromosorb P AW-DMCS 80-100 mesh. Optimization of the chromatographic conditions (oven temperature, carrier gas flow, and column load) with respect to better efficiency and peak retention resulted in a system capable of separating up to 10 mg of the racemate per day. Purities of separated enantiomers were determined by capillary gas chromatography. Yields and purities of the fractions obtained by single- and double-step separations are compared. Highly enriched enantiomers with purities of up to 99.6% (99.2% ee) were obtained by a single separation step.

Chromatography, Gas↗
PubMed2001

Optimizing separation conditions for polycyclic aromatic hydrocarbons in micellar electrokinetic chromatography.

We report the separation of polycyclic aromatic hydrocarbons (PAHs) using 0.1% poly(ethylene oxide) (PEO) in micellar electrokinetic chromatography (MEKC). In the presence of PEO, adsorption of PAHs on the capillary wall was reduced, leading to better resolution and reproducibility. Effects of tetrapentylammonium iodide (TPAI), dextran sulfate (DS), methanol, and sodium lauryl sulfate (SDS) on the separation of PAHs were elucidated. In terms of resolution and speed, DS, compared to TPAI, is a better additive for separation of PAHs. When using 0.1% PEO solution containing 45% methanol, 50 mM SDS, and 0.02% DS, separation of 10 PAHs containing 2 to 5 benzene rings was accomplished in less than 12 min at 15 kV in a commercial CE system. The method has also been tested for separating seven PAHs with high quantum yields when excited at 325 nm using a He-Cd laser. Unfortunately, separation of the seven PAHs was not achieved and sensitivity diminished under the same conditions. To optimize sensitivity, resolution and speed, a stepwise technique in MEKC has been proposed. The seven PAHs were resolved in 35 min at 15 kV when separation was performed in 0.1% PEO solution containing 35 mM SDS, 40% methanol and 0.02% DS for 2 min, and subsequently in 0.1% PEO solution containing 20 mM SDS, 50% methanol, and 0.02% DS.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2002

Micropreparative fractionation of DNA fragments on metathesis-based monoliths: influence of stoichiometry on separation.

Applying Grubbs' first generation benzylidene-type catalyst Cl2Ru(PCy3)2(CHPh) in ring opening metathesis polymerization (ROMP) of norborn-2-ene (NBE) and 1,4,5,8,8a-hexahydro-1,4,5,8, exo, endo-dimethanonapthalene (DMN-H6), various monoliths were prepared within the confines of silanized borosilicate columns (100x3 mm I.D.) and investigated for the micropreparative separation of pBR322 DNA-Hae III restriction fragments ranging in size from 51 to 587 base pairs (bp), as a sample of double-stranded (ds) DNA. The approach to good resolution of dsDNA on monolithic columns entailed the modulation of the polymer morphology in terms of structure and porosity to suit such an analysis. Structural variations were achieved by changing the relative ratios of comonomers (NBE+DMN-H6) at the expense of porogens, and by increasing the DMN-H6 to NBE mass ratio. For dsDNA separations, eluents comprised 0.1 M aqueous triethylammonium acetate, pH 7.0, and acetonitrile. Alternatively, methanol was introduced in this study as a less polar gradient former. In terms of column evaluation, each column prepared was first tested in the separation of 5'-phosphorylated oligodeoxythymidylic acids [p(dT)(12-18)], since good separation of oligodeoxynucleotides indicates the potential liability of the column tested for dsDNA analysis, and vice versa. It was noted that monoliths with combinations of 25:25:40:10, 28:28:35:9, and 30:30:32:8 (as weight% of NBE/DMN-H6/2-propanol/toluene) showed good resolution of p(dT)(12-18). Moreover, they demonstrated good separation of the first 12 fragments (51-267 bp) of the pBR322 DNA-Hae III digest; however, reduced resolution in the separation of the last five highest molecular mass fragments (434-587 bp) was experienced. The best separation of these fragments was accomplished on a 25:25:40:10 NBE/DMN-H6/2-propanol/toluene combination at a flow-rate of 2 ml/min, a temperature of 50 degrees C, and a gradient of 4-10% acetonitrile in 1 min, then 10-16% in 14 min. The total amount of pBR322 HaeIII digest that may be fractionated on these systems is 0.5-2.5 microg.

Chromatography, High Pressure Liquid↗