High-speed Separation Research Articles

Molded continuous poly(styrene- co-divinylbenzene) rod as a separation medium for the very fast separation of polymers Comparison of the chromatographic properties of the monolithic rod with columns packed with porous and non-porous beads in high-performance liquid chromatography of polystyrenes

Gradient elution separations of polystyrene standards in a monolithic molded 50×8 mm I.D. poly(styrene- co-divinylbenzene) rod column and in 50×8 mm I.D. and 30×4.1 mm I.D. columns packed with porous and non-porous poly(styrene- co-divinylbenzene) beads has been carried out. All of these separation media differ in shape and porosity. Excellent separations of eight polystyrene standards were achieved with both the molded monolithic rod and porous beads at moderate flow-rates. However, the monolithic medium proved to be superior for high-speed separations using very steep gradients at a flow-rate of 20 ml/min. Three polystyrene standards were separated in the rod column within 4 s. The separation in the column packed with non-porous beads was poor at flow-rates of 2–8 ml/min, while higher flow-rates led to an unacceptably high back pressure.

High-speed separation of subnanomolar concentrations of noradrenaline and dopamine using capillary zone electrophoresis with laser-induced fluorescence detection.

Capillary zone electrophoresis with laser-induced fluorescence detection has been shown to give rapid separations with high resolution and sensitivity. These advantages are documented for catecholamines analysis in the present work, which shows that separation of subnanomolar concentrations of dopamine and noradrenaline (detection limits of 8.6 x 10(-11) M, corresponding to a detected amount of 143 zeptomoles of each catecholamine) can be performed in 82 s with an efficiency of several million theoretical plates.

Separation Window Diagrams for High-Speed GC

The use of window diagrams for multicolumn optimization in high-speed GC is considered. The distinguishing feature of high-speed GC is the frequent use of low capacity factor values to minimize separation time and maximize the rate of production of theoretical plates. Plots of critical pair relative retention versus column length fraction of either column have previously been used to identify the best separation conditions. For small capacity factor values, relative retention is not a good measure of separation. An alternative separation parameter, relative resolution, is compared to relative retention for optimization of low-capacity-factor separations. This parameter preserves the convenience of requiring only capacity factor values. Application to high-speed separations is illustrated.

Poly(ethyleneoxide) for high-resolution and high-speed separation of DNA by capillary electrophoresis.

Capillary electrophoresis (CE) with polyacrylamide gels has already been demonstrated to allow single-base resolution of single-stranded DNA. However, linear polyacrylamide is not an ideal matrix because of a high viscosity and difficulties in preparing the polymer with well defined pore sizes. Alternatively, poly(ethyleneoxide) (PEO) with a large range of molecular masses from 300,000 to 8,000,000 is available commercially. In addition, it is easy to prepare homogeneous solutions to provide highly reproducible separation performance with sufficient resolution. Single-base resolution of double-stranded DNA between 123 and 124 base pairs can be achieved by the use of homogeneous matrices prepared from PEO (2.5% M(r) 8,000,000), and even better resolution is achieved by using mixed polymer matrices. With further work, it should be possible to change the fractions and the total amounts of polymers to achieve even higher resolution for different samples with different size ranges of fragments. Another advantage of mixed polymer matrices is that relatively high resolution can be obtained while maintaining a relatively low viscosity compared to linear polyacrylamide with identical contents of formamide and urea, which makes it easier to fill these matrices into small capillaries.

High-speed chiral separation of beta-blockers by supercritical fluid chromatography on ChyRoSine-A

High-speed separation of β-blockers have been successfully obtained (analysis time < 2 min) using a short column packed with ChyRoSine-A CSP. Peculiar chromatographic behaviour of β-blockers on tyrosinederived CSPs under supercritical conditions was evidenced by studying the curves of hversus the linear velocity u. Specific solvation of solutes by supercritical carbon dioxide is responsible for the particular conformation of the molecular of β-blocker which enhances chiral discrimination. This was evidenced by using the reciprocality concept leading to a new CSP derived from a propranolol analogue. Thus, it was demonstrated that SFC can be used for the fast chiral separation of β-blockers.

Microparticulation of whey protein: related factors affecting the solubility

Solubility of Simplesse 100, the only whey-based fat substitute, was found to be good, considering the fact that technology for preparation of Simplesse 100 is a sequence of thermal steps. To characterize this phenomen, gel chromatography on Sephadex G-100, Sephacryl S-1000 and SDS-PAGE were used, supported by high-speed separation, UV studies and analytical procedures. Results show that the unusual solubility characteristic of microparticulated whey protein is related to two molecular effects: (1) optimal defolding of protein molecules and (2) stabilization of the defolded status by carbohydrate. Both effects were considered to favour non-covalent bonds, which contribute to the outstanding physico-functional and nutritive properties of microparticles.

High-Speed Separation of Antisense Oligonucleotides on a Micromachined Capillary Electrophoresis Device

A micromachined chemical analysis device based on capillary electrophoresis has been successfully used for very fast size separation of a synthetic mixture of fluorescent phosphorothioate oligonucleotides ranging from 10 to 25 bases in length. The device consists of a channel system which has been formed in the surface of a glass plate by a standard photolithographical process. An integrated volume-defined sample injector allowed for unbiased electrokinetic introduction of sample plugs of 150-μm length (corresponding sample volume, 90 pL) into the separation channel. This well-defined injection procedure, in combination with the application of high electric fields of up to 2300 V/cm, resulted in size separation of single-stranded oligonucleotides in less than 45 s when a separation distance of 3.8 cm was used

Thermal field flow fractionation of polytetrahydrofuran

The possibility to fractionate polytetrahydrofuran (PTHF) samples effectively with Thermal Field Flow Fractionation has been investigated. Retention measurements of four standards of different molecular mass were performed in eight organic solvents. When methylethylketone or dioxane were used no retention was found. Highest retention for a given molecular mass was obtained with the solvents toluene and ethylbenzene. However, in ethyl acetate the highest separation speed was observed. Plate height measurements showed that both the thermal and molecular diffusion coefficients of the PTHF standards are very high in ethyl acetate. According to theory, this combination should result in a high separation speed. A baseline separation of two PTHF standards with molecular masses of 67 000 and 282 300 could be obtained in 8 minutes when ethyl acetate was used as the solvent.

High-Speed Separations on a Microchip

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTHigh-Speed Separations on a MicrochipStephen C. Jacobson, Roland. Hergenroder, Lance B. Koutny, and J. Michael. RamseyCite this: Anal. Chem. 1994, 66, 7, 1114–1118Publication Date (Print):April 1, 1994Publication History Published online1 May 2002Published inissue 1 April 1994https://doi.org/10.1021/ac00079a029RIGHTS & PERMISSIONSArticle Views1359Altmetric-Citations342LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

Rapid, high-resolution high-performance liquid chromatographic analysis of antibiotics

A HPLC column devised for high separation speed combined with highly practical operating features has been found useful for separating antibiotics. Important characteristics involve compromises in packing particle size, column configuration and support-stationary phase combinations. We determined that these columns are useful for rapid, high-resolution separations with unmodified state-of-the-art HPLC equipment without the extra-column band-broadening effects typical of so-called “fast” HPLC columns. The proposed columns feature efficient sterically-protected monofunctional silane stationary phases that provide good separation reproducibility and high column stability. The combination of these unique bonded silanes and a highly purified, less-acidic silica support give superior peak shapes for antibiotic compounds. The proposed column configuration can halve separation times and double peak heights without loss in resolution, compared to widely used analytical columns. Increased mobile phase flow-rates permit even faster separations of antibiotics with only modest loss in resolution and peak heights for trace analyses in biological systems.

Rapid separation of DNA sequencing reaction product using capillary electrophoresis.

The effect of the gel composition, electric field strength, and capillary length on the separation of DNA sequencing reaction product was investigated in order to achieve highspeed DNA sequencing on a large-scale for the Human Genome Project. In this study, we prepared gel-filled capillaries differing in gel concentration (3%T6%T), degree of crosslinking (0%C5%C), and capillary length (15.3 cm36.3 cm). Fluorescencelabeled A-termination sequencing reaction product (M13mp 18 template) was first separated by capillary electrophoresis using the gel-filled capillaries prepared here in electric fields from 200 V/cm to 414 V/cm and then detected at 560 nm by using an on-line argon ion laser (488 nm)-induced fluorescence detector. In conclusion, the use of the short capillary, high concentration gel, and high electric field realizes high-speed separation of DNA sequencing reaction product with high resolution. Under optimum separation conditions, only 10 min is required for sequencing of 250-base DNA.

Capillary gel electrophoresis of biopolymers

Capillary gel electrophoresis is quickly becoming a widely accepted analytical tool to separate and analyze biopolymers such as proteins and DNA. Potential advantages over conventional electrophoretic methods include high-resolution, high-speed separations, detection of small quantities, and the capability for automation and on-line detection. Many successful applications have been developed with this technique such as sodium dodecyl sulfate protein analysis and oligonucleotide, single-stranded and double-stranded DNA separation including DNA sequencing, mutational spectra and anti-sense DNA analysis.

Reproducible and high-speed separation of basic drugs by capillary zone electrophoresis.

The complete separation of a mixture of seventeen basic drugs of different classes was achieved with capillary zone electrophoresis in 11 min. The migration time reproducibility for individual components was between 0.5 and 1.7% relative standard deviation (R.S.D.). Peak detection was achieved by ultraviolet absorption, with peak-area reproducibility ranging from 1.5 to 6.3% R.S.D. The pH of the running buffer was critical in determining the separation of the mixture of basic drugs. The detection of most of these components in urine and plasma is also illustrated.

Separation of non UV-absorbing cations by capillary electrophoresis

A new buffer system for the separation of cations with capillary electrophoresis using indirect UV-detection is described. p-Aminopyridine can be used in the wide pH-range between 3 and 10. High-speed separations of positively charged non-UV absorbing ions are possible. Separation of ions with similar mobilities can be achieved by the addition of complexation reagents. The separation of potassium and ammonium is possible either with the addition of crown ether or at high pH-values. Transition metals can be separated by adding 2-hydroxybutyric acid to the buffer.

A practical approach to high performance capillary electrophoresis with biosynthetic human growth hormone as a model protein

Biosynthetic human Growth Hormone (B-hGH) is a protein comprising 191 amino acids. The molecular weight is 22,125 and the isoelectric point is close to pH 5. Due to the ready availability of closely related analogues B-hGH was used as a model protein thus allowing for the demonstration and evaluation of the high resolution capability of high performance capillary electrophoresis (HPCE). The same apparatus was used throughout the experiments and an optimum signal-to-noise ratio was found at 200 nm. Linearity was observed between peak area, retention time and the hGH concentration or sample introduction time. Baseline separation of hGH, desamido hGH and didesamido hGH was obtained. Examples showing analysis with 1 million theoretical plates per meter, high speed separation, simultaneous analysis of multiple samples, sample stacking, hGH tryptic digest, and hGH lysate are reported. The use of electrophoretic velocities instead of apparent velocities for peak identification is illustrated.

High-speed gas chromatography : Theoretical and practical aspects

The performance of a gas chromatographic (GC) system depends mainly on the column efficiency and for fast separations, on the equipment design. A theoretical and practical study shows the importance of the various phenomena involved in the optimization of a GC system. An original approach, the performance concept, is introduced. Exact and simplified, approximate expressions are given for the calculation of the optimum practical velocity, the optimum column length and the minimum analysis time. The calculated values are in good agreement wit experimental valves from other sources. In the experimental part two cases are distinguished: fast chromatography using conventional equipment and ultra-fast chromatography requiring specially designed instrumentation. The most important considerations for the practical realization of high-speed separations are discussed in detail: column parameters, choice of working conditions and extra-column parts, such as the sampling device, detector, amplifier and data acquisition system. Some examples of extremely fast analyses (analysis times a few of seconds or less) are shown.

Very high speed separation of proteins with a 20-μm reversed-phase sorbent

A five-protein mixture was successfully separated in less than 15 s by reversed-phase gradient elution on a 20-μm polymeric flow-through type chromatographic packing material (POROS R/M). The same mixture could also be separated with far better resolution in less than 60 s on the same column by simply extending the gradient volume. Pressure drop across the column was less than 130 bar, despite a superficial linear velocity of nearly 9000 cm/h. Frontal chromatography at different flow-rates was used to demonstrate that the ability to separate at high speed was due to greatly enhanced mass transport within the particles due to the flow-through (perfusion chromatography) effect.

Instrumentation and strategies for high-speed gas chromatography

Short lengths of capillary column operated at unusually high carrier-gas velocities are used to separate relatively simple mixtures on a timescale of 20 s or less. Two inlet systems are described which can generate the narrow injection bands needed for these elution times. A cryofocusing inlet using a vacuum-pump-operated gas flow direction control is used for compounds generally less volatile than n-pentane. A low-friction gas valve inlet combined with porous-layer open-tubular columns is used for volatile compounds of low molecular mass. Strategies for high-speed separations including high-speed backflush, backflush with recycle, vacuum-outlet operation and selective detection are described. These techniques are used to increase the operational flexibility and the amount of information that can be obtained using high-speed gas chromatography.

Multiwavelength fluorescence detection for DNA sequencing using capillary electrophoresis

Multiwavelength detection of laser induced fluorescence for dideoxynucleotide DNA sequencing with four different fluorophores and separation by capillary gel electrophoresis is described. A cryogenically cooled, low readout noise, 2-dimensional charge-coupled device is used as a detector for the on-line, on-column recording of emission spectra. The detection system has no moving parts and provides wavelength selectivity on a single detector device. The detection limit of fluorescently labeled oligonucleotides meets the high sensitivity requirements for capillary DNA sequencing largely due to the efficient operation of the CCD detector with a 94% duty cycle. Using the condition number as a selectivity criterion, multiwavelength detection provides better analytical selectivity than detection with four bandpass filters. Monte Carlo studies and analytical estimates show that base assignment errors are reduced with peak identification based on entire emission spectra. High-speed separation of sequencing samples and the treatment of the 2-dimensional electropherogram data is presented. Comparing the DNA sequence of a sample separated by slab gel electrophoresis with sequence from capillary gel electrophoresis and multiwavelength detection we find no significant difference in the amount of error attributable to the instrumentation.

High speed DNA sequencing by capillary electrophoresis

A major challenge of the Human Genome Initiative is the development of a rapid, accurate, and efficient DNA sequencing technology. A major limitation of current technology is the relatively long time required to perform the gel electrophoretic separations of DNA fragments produced in the sequencing reactions. We demonstrate here that it is possible to increase the speed of sequence analysis by over an order of magnitude by performing the electrophoresis and detection in ultra thin capillary gels. An instrument which utilizes these high speed separations to simultaneously analyze many samples will constitute a second generation automated DNA sequencer suitable for large-scale sequence analysis.