Effect of temperature on DNA fractionation in slalom chromatography

Slalom chromatography is an alternative chromatographic procedure for the analysis of relatively large double-stranded DNA molecules and is based on a hydrodynamic principle. The retardation of the DNA fragments from the cleavage of the Lambda DNA by the KpnI restriction enzyme was studied using an acetonitrile-phosphate buffer as a mobile phase (flow rate equal to 0.3 ml/min) and a C1 column as a stationary phase at various temperatures. It was shown that the temperature constituted an important parameter for the separation of the DNA fragments in slalom chromatography. The DNA hydrodynamic behavior with the temperature was related to the variation in the fluid viscosity and the modification of the elastic properties of the biopolyrner.     

DNA migration regimes in hydrodynamic chromatography and slalom chromatography: evidence for a transition

Recently, we hypothesized that DNA separation mechanisms in slalom chromatography (SC) and packed column hydrodynamic chromatography (HDC) could be connected and simultaneously observed 'J. Chromatogr. A 886 (2000) 1'. The present paper describes the migration of various circular and linear double-stranded DNAs in a chromatographic system using a C1 stationary phase and an acetonitrile-phosphate buffer mixture as a mobile phase. The migration dependence on the flow rate for the different species showed the relative contribution of both the SC and HDC separation mechanisms. A transition between the SC and HDC regimes was demonstrated for the first time. In addition, the data were analyzed in terms of polymer migration in a dense structure. Three different behaviors were distinguished in relation to the size and the compactness of the nucleic acid.     

Effects of sequence and structure in the separation of nucleic acids using ion pair reverse phase liquid chromatography

Ion pair reverse phase high performance liquid chromatography on non-porous alkylated poly(styrene-divinylbenzene) particles enables the high resolution separation of double stranded DNA fragments. To further understand the separation mechanisms involved in ion pair reverse phase liquid chromatography we have analysed the effects of curved or "bent" DNA fragments with respect to their separation using both gel electrophoresis and ion pair reverse phase liquid chromatography. Size dependent separations of curved DNA fragments that migrate anomalously during gel electrophoresis were observed using ion pair reverse phase liquid chromatography. To further study the sequence effect and resulting changes in hydrophobicity of the duplex DNA, PCR fragments were generated that contain uracil in place of thymine. The resulting fragments were shown to elute with shorter retention times, demonstrating that sequence-specific effects can alter the retention of duplex DNA. The study was extended to the investigation of non-canonical B-DNA structures (Holliday junctions) under various chromatographic conditions, demonstrating that the coaxial stacking of the helices in such structures, in the presence of magnesium causes a change in retention.     

Mobile phase viscosity and velocity dependence on protein retention using nonequilibrium chromatographic techniques

Nonequilibrium chromatography (NEC) is an alternative chromatographic procedure for the separation of macromolecules. The retardation of a protein series is studied using a phosphate buffer as a mobile phase with various concentrations of glycerol fraction (used as a viscosity modifier) at different mobile phase velocities and a C1 column with a very low packing particle diameter as a stationary phase. It is shown that the two factors (viscosity and velocity) of the mobile phase constituted important parameters in the retention mechanism of the proteins in NEC. The retardation velocity domain is divided into two regions. For low velocity regions, the protein retention decreased with a mobile phase velocity increase. This retention is enhanced above a critical value of the mobile phase velocity. The transition between the two well-known NEC methods, slalom chromatography and hydrodynamic chromatography, is clearly visualized for the first time for the protein retention of particular values of the mobile phase velocity.     

Continuous flow fast atom bombardment with packed microcolumns: A comparison of precolumn versus coaxial matrix delivery

The effect of adding glycerol to the mobile phase on the chromatographic separation of peptides has been investigated using a continuous flow fast atom bombardment (CFFAB) interface coupled with commercial packed microcolumns (25 cm × 320 μm.i.d.). In a comparative study using a UV detector, it was found that chromatographic peak broadening progressively increased with increasing percentage of glycerol in the mobile phase. In the liquid chromatographic FAB mass spectrometric analysis, this effect is compounded by the dynamic mixing of the column effluent on the probe. Improvements of 25–155% in the overall separation efficiencies were obtained by introducing the matrix independently to the probe tip via a coaxial arrangement. Application of this coaxial CFFAB is demonstrated by the analysis of peptide mixtures and tryptic digests.     

A viscosity-tunable polymer for DNA separation by microchip electrophoresis

A thermo-responsive separation matrix, consisting of Pluronic F127 tri-block copolymers of poly(ethylene oxide) and poly(propylene oxide), was used to separate DNA fragments by microchip electrophoresis. At low temperature, the polymer matrix was low in viscosity and allowed rapid loading into a microchannel under low pressure. With increasing temperatures above 25°C, the Pluronic F127 solution forms a liquid crystalline phase consisting of spherical micelles with diameters of 17–19 nm. The solution can be used to separate DNA fragments from 100 bp to 1500 bp on poly(methyl methacrylate) (PMMA) chips. This temperature-sensitive and viscosity-tunable polymer provided excellent resolution over a wide range of DNA sizes. Separation is based on a different mechanism compared with conventional matrices such as methylcellulose. To illustrate the separation mechanism of DNA in a Pluronic F127 solution, DNA molecular imaging was performed by fluorescence microscopy with F127 polymer as the separation matrix in microchip electrophoresis. Figure Temperature dependence of the viscosity of 20% w/w Pluronic F127 solution in 1xTBE buffer. Dotted approximates resultant curve.     

Rapid high-performance liquid chromatography of nucleic acids with polystyrene-based micropellicular anion exchangers

Nucleic acids were separated by ion-exchange chromatography on 30 x 4.6 and 100 x 4.6 mm columns packed with a micropellicular anion exchanger made of 3-microns rigid polystyrene-based non-porous microspheres with a covalently bound hydrophilic layer and DEAE functional groups at the surface. The stationary phase particles showed negligible swelling in methanol according to permeability measurements with water and methanol. Nucleic acids and their fragments including synthetic single-stranded oligonucleotides, linear, nicked and supercoiled DNAs as well as DNA restriction fragments were separated in less than 5 min, a time scale that is much smaller than that of conventional high-performance liquid chromatographic analysis for such samples. When only buffer and sodium chloride were used in the eluent for the separation of double-stranded DNA restriction fragments pGEM-3Z/Taq I, electrophoretic analysis of the effluent revealed the presence of smaller fragments in the bands of the larger ones. Upon addition of ethylenediaminetetraacetic (EDTA) salt to the eluent, however, such contamination by shorter fragments was no longer observed. In the absence of EDTA, magnesium chloride in the eluent at a concentration of 1 mM precluded the separation of the restriction fragments under otherwise identical chromatographic conditions.     

Mannitol influence on the separation of DNA fragments by capillary electrophoresis in entangled polymer solutions

A new kind of sieving matrix is presented in this paper to allow satisfactory separation of DNA fragments in a relatively low viscous solution. When a certain amount of mannitol was added to cellulose solution not concentrated enough to separate PGEM-3Zf(+)/HaeIII standards well, a polymer solution with low viscosity but with very good separation effects was obtained. The separation result of this sieving buffer was comparable with those using highly concentrated cellulose solutions. The sieving ability of solutions with different cellulose concentrations and different amounts of mannitol has been investigated. It was proved that 0.5% was the minimum hydroxypropylmethylcellulose (HPMC) concentration that could be used to separate DNA fragments satisfactorily. HPMC solutions with a concentration of less than 0.5% could not separate the standard DNA fragments even in the presence of mannitol. It was found that 6% was the optimized mannitol concentration because either more or less mannitol will lead a decrease of resolution. The principle of the positive influence of mannitol has also been discussed.     

Studying the mechanism of RNA separations using RNA chromatography and its application in the analysis of ribosomal RNA and RNA:RNA interactions

DNA/RNA chromatography presents a versatile platform for the analysis of nucleic acids. Although the mechanism of separation of double stranded (ds) DNA fragments is largely understood, the mechanism by which RNA is separated appears more complicated. To further understand the separation mechanisms of RNA using ion pair reverse phase liquid chromatography, we have analysed a number of dsRNA and single stranded (ss) RNA fragments. The high-resolution separation of dsRNA was observed, in a similar manner to dsDNA under non-denaturing conditions. Moreover, the high-resolution separation of ssRNA was observed at high temperatures (75 °C) in contrast to ssDNA. It is proposed that the presence of duplex regions/secondary structures within the RNA remain at such temperatures, resulting in high-resolution RNA separations. The retention time of the nucleic acids reflects the relative hydrophobicity, through contributions of the nucleic sequence and the degree of secondary structure present. In addition, the analysis of RNA using such approaches was extended to enable the discrimination of bacterial 16S rRNA fragments and as an aid to conformational analysis of RNA. RNA:RNA interactions of the human telomerase RNA component (hTR) were analysed in conjunction with the incorporation of Mg²⁺ during chromatography. This novel chromatographic procedure permits analysis of the temperature dependent formation of dimeric RNA species.     

Effects of DNA topology, temperature and solvent viscosity on DNA retardation in slalom chromatography

Slalom chromatography is a unique size-fractionation method applicable to large DNA molecules [>5 kilobase pairs (kbp)]. The method was first developed by using columns packed with microbeads (diameter, <20 microm) used for high-performance liquid chromatography and by applying a relatively fast flow-rate (>0.3 ml/min). Previous studies suggested that the separation is attributed to a hydrodynamic rather than to an equilibrium phenomenon (J. Hirabayashi and K. Kasai, Anal. Biochem. 178 (1989) 336; J. Hirabayashi, N. Itoh, K. Noguchi and K. Kasai, Biochemistry, 29 (1990) 9515). In the present report, the results of a systematic study on the effects of DNA topology, temperature, and solvent viscosity on DNA retardation are described. Firstly, the behaviour of circular (super-coiled) and linearized forms of charomid DNAs (20-42 kbp) was studied. Circular-form DNA molecules were found to be fractionated size-dependently similarly to linear forms in a flow-rate dependent manner. However, the extent of retardation of the circular form DNA was apparently less than that of the corresponding linear forms. Circular DNAs showed almost the same retardation (e.g., 42 kbp) as DNA fragments (e.g., 20 kbp) having approximately half of the size of the former. This observation indicates that DNA retardation is basically related to physical length, not to mass. Secondly, to study the effect of temperature with special reference to solvent viscosity, we carried out chromatographic analysis at various temperatures ranging from 6 to 65 degrees C in both the absence and presence of sucrose (10 or 20%, w/v). The results showed that it is the solvent viscosity that determines the extent of retardation. Taken together, all of physicochemical parameters that define hydrodynamic properties, i.e., particle size, flow-rate and solvent viscosity, proved to be critical in slalom chromatography as well as the potential physical length of the DNA, thus supporting the concept that slalom chromatography is based on a hydrodynamic principle.     

Contact conductivity detection of polymerase chain reaction products analyzed by reverse-phase ion pair microcapillary electrochromatography

We describe the development of an integrated microelectrophoretic system consisting of a contact conductivity detector mounted on-chip for monitoring the separation of double-stranded (ds) DNA fragments produced via the polymerase chain reaction (PCR) using microcapillary electrochromatography as the separation mode. The separation was carried out in a polymer-based microfluidic device, hot-embossed into poly(methylmethacrylate) (PMMA), whose walls were functionalized to produce a C(18)-terminated surface to act as the stationary phase (open channel format). The carrier electrolyte contained the ion-pairing agent, triethylammonium acetate (TEAA) to allow the separation to be carried out using reverse-phase ion-pair capillary electrochromatography (RP-IPCEC). The microelectrophoretic separations were investigated utilizing various solvent strengths (acetonitrile/water) with 25 mM TEAA to observe the effects on the separation efficiency as well as the chromatographic development time and detector performance. The field strength significantly affected the quality of the separation, with no separation observed at 333 V/cm for a low mass dsDNA sizing ladder, but baseline separation achieved using a field strength of 67 V/cm. It was observed that the solvent strength affected the retention behavior of the polyanionic molecules as well as the electroosmotic mobility. Higher acetonitrile compositions in the run buffer resulted in reduced plate numbers, which produced lower chromatographic resolution. The use of conductivity detection allowed mass detection sensitivities in the range of 10(-21) mol with a separation efficiency of 10(4) plates and the performance of the detector independent of the acetonitrile content used in the carrier electrolyte.     

Chromatographic behaviour and purification of linear lambda phage and plasmid DNA molecules on 2-hydroxyethyl methacrylate-ethylene dimethacrylate-based supports

The HEMA-BIO 1000 support, which is based on a copolymer of 2-hydroxyethyl methacrylate and ethylene dimethacrylate, was used for separation of lambda DNA and its fragments and plasmid pBR322 DNA. The separation of fragments greater than 6.6 kbp was demonstrated according to the slalom chromatography mechanism on column for size-exclusion chromatography in the case of linear lambda DNA fragments. The influence of particle size of column packing, mobile phase rate, and KCl concentration in mobile phase is discussed. The purification of plasmid DNA pBR322 using size-exclusion chromatography was more rapid compared to gel electrophoresis. The presence of salts in the eluate is not disadvantageous. DNA can be recovered from the eluate by ethanol precipitation. Plasmid DNA pBR322 isolated in this way was suitable for different biological applications (cleavage with restrictases, electrotransformation into bacterial cells).     

Study of Monolithic Integrated Micro Gas Chromatography Chip

A monolithic micro gas chromatography (μGC) chip which integrated the micro separation column (μSC) and the micro thermal conductivity detector (μTCD) based on MEMS (Micro-electro-mechanical systems) technique was fabricated. Compared to the state of the art, the μSC with high depth-to-width ratio channels that was coated with mesoporous silica nanoparticles as stationary phase could effectively improve the column capacity and separation performance. Besides, the stable suspending μTCD, which was designed and fabricated in two ports of the μGC chip, could availably enhance the thermal isolation and reliability of the device. The mixture of light hydrocarbons (methane, ethane, propane and butane) could be separated from each other and detected by this monolithic integrated μGC chip, in which the overall analysis and detection time was only 33 seconds, the separation resolution of ethane and propane was 8.34, and the number of theoretical plate was as high as 11420. The monolithic integrated μGC chip has many advantages such as good separation resolution, high column efficiency and short analysis time, and is suitable for portable gas chromatographic field and onsite detection.     

Nonconventional synthesis and characterization of ultrahigh-molar-mass polyacrylamides

In spite of the significant progresses in the field of replaceable sieving matrices for separating DNA in capillary electrophoresis (CE), an intense research activity is still going on to improve the separation of large size DNA sequencing fragments. There are evidences, both from experimental and theoretical sides that the resolution of these fragments, at the single base, requires the use of sieving matrices comprised of long chain linear polymers. In the separation of DNA fragments by CE are of upmost importance: (i) the complete solubility of the polymer, (ii) the linearity of the chain, (iii) the achievement of ultrahigh viscosity in dilute solutions. The aim of this work is the synthesis of ultrahigh-molecular-weight polymers which possess the three requirements mentioned above by employing a nonconventional method. We demonstrate that the sieving performance of polyacrylamide is directly correlated to its intrinsic viscosity.     

毛细管电泳中葡萄糖-羟丙基甲基纤维素体系分离脱氧核糖核酸

葡萄糖作为羟丙基甲基纤维素（Hydroxpropylmethyl cellulose,HPMC)筛分体系的添加剂,可以改善该体系在低浓度时分离脱氧核糖核酸（DNA）的能力。研究了硼酸浓度和pH值对葡萄糖－羟丙基甲基纤维素体系分离性能的影响;并将葡萄糖与其它添加剂如甘油、甘露醇对分离的影响作了比较,葡萄糖特有的环状结构使得其对羟丙基甲基纤维素体系分离能力的提高更为显著。     

Endoglucanase Degradation and Enzyme-Aided Characterization of Cellulose Acetates

Endoglucanases were used as a selective tool to determine the substituent distribution along the chains of partially substituted cellulose acetates. Unsubstituted segments are intensively fragmented while highly substituted segments are only slightly degraded or not affected at all. Two different procedures were developed to perform a preparative separation of the fragments by size exclusion chromatography. One system is based on a direct separation of the fragmentation products using pyridine : water (9 : 1) as an eluent. The isolated fragments can be analysed regarding to their DS values by ATR/FTIR spectroscopy or by hydrolysis and HPLC. The second system is based on deutero-acetylation of endoglucanase fragmented samples. The complete acetylation of all OH groups enables a chromatographic separation in chloroform. Afterwards the isolated fragments can be analysed regarding to their partial DS values by ¹H-NMR spectroscopy.     

Low viscosity DNA sieving matrices for capillary electrophoresis

The rapid development of DNA capillary electrophoresis (CE) technology has increased the demand of new low viscosity sieving matrices with high separation capacity. The high throughput, resolution and automatic operation of CE systems have stimulated the application of the technique to different kinds of DNA analysis, including DNA sequencing, separation of restriction fragments, PCR products and synthetic oligonucleotides. In addition specific methods for PCR-based mutation assays for the study of known and unknown point mutations have been developed for use in CE. The key component for a large scale application of CE to DNA analysis is the availability of appropriate sieving matrices. This article gives an overview of the linear polymers used as DNA separation matrices with particular emphasis on the polymers that combine high sieving capacity, low viscosity and chemical resistance.     

Analysis of mixtures containing free fatty acids and mono-, di- and triglycerides by high-performance liquid chromatography coupled with evaporative light-scattering detection

Commercial monostearates of glycerol, generally used as antistatic agents for thermoplastic polymers, consist of a complex mixture of mono-, di- and trisubstituted glycerides and the corresponding fatty acids. Thus far, the glycerides and the fatty acids have been analyzed separately by high-performance liquid chromatography (HPLC). In fact, the simultaneous analysis of glycerides and fatty acids requires esterification of the acids in order to avoid overlapping of the chromatographic peaks. This paper describes an HPLC method which allows the direct separation and identification, simultaneously and in a single run, of the variously substituted glycerides, and also the corresponding saturated fatty acids that are found as by-products in commercial glycerol monostearates. The procedure is based on the use of a ternary gradient HPLC instrument equipped with an evaporative light-scattering detector; the stationary phase was a reversed-phase RP-8 end-capped (Merck) column; the mobile phase was two consecutive binary gradients consisting of acetonitile-water plus acetic acid (0.1%, v/v) and acetonitrile-methylene chloride. The method is fast and shows high sensitivity and selectivity, being able to separate also the positional isomers of mono- and digycerides in addition to the mixed di- and triglycerides.     

Preparation and evaluation of packed capillary columns for the separation of nucleic acids by ion-pair reversed-phase high-performance liquid chromatography

Oligonucleotides and double stranded DNA fragments were separated in 200 microm I.D. capillary columns packed with micropellicular, octadecylated, 2.1 microm poly(styrene-divinylbenzene) particles by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC). Both the length and the diameter of the connecting capillaries (150 x 0.020 mm I.D.) as well as the detection volume (3 nl) had to be kept to a minimum in order to maintain the high efficiency of this chromatographic separation system with peak widths at half height in the range of a few seconds. Three different types of frits, namely sintered silica particles, sintered octadecylsilica particles, and monolithic poly(styrene-divinylbenzene) (PS-DVB) frits were evaluated with respect to their influence on chromatographic performance. Best performance for the separation of oligonucleotides and long DNA fragments was observed with the PS-DVB frits, whereas the short DNA fragments were optimally resolved in columns terminated by octadecylsilica frits. The maximum loading capacity of 60 x 0.20 mm I.D. columns ranged from 20 fmol (7.7 ng) for a 587 base pair DNA fragment to 500 fmol (2.4 ng) for a 16-mer oligonucleotide. Lower mass- and concentration detection limits in the low femtomol and low nanomol per liter range, respectively, make capillary IP-RP-HPLC with UV absorbance detection highly attractive for the separation and characterization of minute amounts of synthetic oligonucleotides, DNA restriction fragments, and short tandem repeat sequences amplified by polymerase chain reaction.