Polyacrylamide Gel-filled Capillaries Research Articles

Enantiomeric Separations by Capillary Electrochromatography with Charged Polyacrylamide Gels Incorporating Chiral Selectors

This study examined enantiomeric separation by capillary electrochromatography with charged polyacrylamide gels incorporating chiral selectors, in which electroosmotic flow would be generated. The columns are capillaries filled with a negatively or positively charged polyacrylamide gel, a so-called monolithic stationary phase, incorporating polymeric β-cyclodextrins as chiral selectors. The capillary wall is activated first with a bifunctional reagent to make the resulting gel bind covalently to the inner surface of the fused-silica tubing. Enantiomeric separations of two cationic and a neutral compounds were achieved using negatively charged polyacrylamide gel-filled capillaries incorporating poly β-cyclodextrin and carboxymethyl β-cyclodextrin polymer and a mobile phase of 200 mmol dm−3 Tris–300 mmol dm−3 boric acid buffer (pH 9.0). Enantiomeric separations of twelve anionic compounds were also achieved using positively charged polyacrylamide gel-filled capillaries incorporating poly β-cyclodextrin and a mobile phase of 200 mmol dm−3 Tris–300 mmol dm−3 boric acid buffer (pH 8.1). High efficiencies of up to 240000 plates m−1 were obtained. The within-run reproducibilities of retention time and separation factor were good.

Preparation of polyacrylamide gel-filled capillaries with step gradients and low UV-detection background

Polyacrylamide-filled capillaries with step gradients were designed and prepared with a newly established method, which is also suitable for producing other sorts of capillaries. The resulting capillaries allow the use of any UV-light to approach the most sensitive detection and have the features of fast running speed and high separation efficiency. In addition, the capillaries can be used continuously for more than two weeks.

Preparation of polyacrylamide gel-filled capillaries with step gradients and low UV-detection background

Preparation of polyacrylamide gel-filled capillaries with step gradients and low UV-detection background

Separation of transfer RNA and 5S ribosomal RNA using capillary electrophoresis

Capillary gel electrophoresis and capillary electrophoresis using entangled polymer solutions was investigated for their applicability for the separation of low-molecular-mass RNAs (transfer RNA and 5S ribosomal RNA), with a size range of 70–135 nucleotides, from bacteria. Cross-linked polyacrylamide gel-filled capillaries (3 and 5%) were used for capillary gel electrophoresis. Good resolution was obtained suing gel-filled capillaries only for small tRNAs with lengths to 79 nucleotides, larger tRNAs and 5S rRNA could not be resolved using this method. Buffers containing sieving additives were employed to improve separations of RNA by capillary electrophoresis using entangled polymer solutions. The use of linear sieving polymers in buffers resolved 5S rRNA and tRNAs, even when they possessed only different secondary structure or small differences in length (1–5 nucleotides).

Improving the UV detection sensitivity of condensed polyacrylamide gel-filled capillaries using non-flow buffer-filled capillaries as a detection cell

Capillary coupling techniques are suggested for the connection of a non-flow buffer-filled capillary to gel-filled capillaries as a low-background detection cell. The UV detection sensitivity of the resulting capillaries is improved at least fivefold, as demonstrated by using polyamino acids as test samples. The detection sensitivity can be further improved by using a low-background running buffer. Several ways are suggested to overcome the problems of resolution decrease and baseline drif which were observed after coupling.

Preparation of highly condensed polyacrylamide gel-filled capillaries with low detection background

A method was established for the preparation of capillaries filled partially with highly condensed polyacrylamide gels (including step gradient gels) used for separation and partially with a buffer used for detection. These novel capillaries combine the high resolution of gel-filled capillaries and the low background of buffer-filled capillaries. They can be used for more than 1 week or for more than 50 injections, as demonstrated by the separation of poly-(α,β)- d.l-aspartate. The detection limit tested with diaspartate is about two orders of magnitude lower than that with the totally gel-filled capillaries. Some problems such as baseline drifting arise when using these capillaries but can be overcome by pre-running the capillaries several times with highly concentrated samples.

Preparation of highly condensed polyacrylamide gel-filled capillaries

A fairly quick method has been established for the production of highly condensed polyacrylamide gel-filled capillaries. Void-free capillaries with inner diameters as small as 25 μm and monomer concentrations of up to over 30% T + 5% C can successfully be prepared within 5 h. These capillaries can be used for over 20 injections of poly-(α,β)- d, l-aspartate at 200 V/cm and 25°C, with gels immobilized at the capillary tips after polymerization, and for more than 70 injections with gels immobilized, during polymerization, over a longer section of at least 0.5 cm. The important polymerization conditions in this method are the application of a slight pressure, controlled polymerization directions and the selections of buffer components and/or the concentrations of radicals and catalyst.

The Effect of Electric Fields on the Dispersion of Oligonucleotides Using a Multi-Point Detection Method in Capillary Gel Electrophoresis

Abstract A multi-point detection method is proposed to study the dispersion of oligonucleotides in polyacrylamide gel capillary. In this method a single gel filled capillary is curved into loops, with several detection points aligned with a common detector. In this manner, several electropherograms can be obtained at different migration times during one capillary electrophoresis (CE) run, thus permitting studies of changes in the spatial peak variance σ2 as a function of time. Investigations of polydeoxyadenylic acids in the range of 40–60 bases indicate that the diffusion coefficient of these compounds increases at higher voltages in the polyacrylamide gel-filled capillaries. Using this simple multi-point detection method one can estimate the band broadening originating from sample injection (σ2 inj), which is otherwise difficult to measure.

Native protein separations and enzyme microassays by capillary zone and gel electrophoresis

Native protein separations by capillary gel electrophoresis are achieved using linear acrylamide gel matrices. Polyacrylamide gels with a concentration range of 3.5-5% did not exhibit size separations for native proteins with molecular weights from 20,000 to 47,000. The separation of native proteins in gel-filled capillaries is based solely on the charge of the protein as in normal zonal electrophoresis. Retention of protein activity in the acrylamide matrix was demonstrated by performing enzymatic assays in the gel matrix. Alkaline phosphatase (ALP) and beta-galactosidase assays were conducted in both C18-PF108-modified and polyacrylamide gel-filled capillaries. Enzyme assays were achieved by filling the capillary with an appropriate substrate dissolved in the electrophoresis buffer. The product formed by the reaction of enzyme with substrate was monitored using a standard UV-visible detector. Both constant potential and zero potential modes of analysis were demonstrated. The polyacrylamide gel columns provide the advantages of minimized diffusion and limited band spreading due to the high viscosity of the gel matrix. The lowest detection limit achieved was 5.2 x 10(-20) mol (7.6 x 10(-12) M sample injected) of ALP. The dual enzyme assay of ALP and beta-galactosidase was achieved in gel-filled capillaries simultaneously.

A model for the mobility of single-stranded DNA in capillary gel electrophoresis.

The electrophoretic mobility of single-stranded DNA in denaturing polyacrylamide gel-filled capillaries is analyzed as a function of the applied electric field. The resultant mobility plots are complex functions of the fragment size and electric field. Traditionally, these plots are separated into three mobility regimens corresponding to Ogston sieving, reptation without stretching and reptation with stretching theories. However, none of these theories accurately models the variations in mobility that we observe with electric field strength. As a result, we propose a modification of the Ogston sieving theory which accounts for the stretching of migrating DNA molecules in the direction of the electric field. This theory assumes that the applied electric field in conjunction with the gel matrix distorts the DNA, altering the effective size of the migrating molecule. The stretched DNA offers a smaller cross-section to the gel pore and thus sieves as though it were a smaller molecule. In this modified Ogston theory, the electrophoretic mobility depends only on the applied electric field, the size of the fragment, and constants which are independent of size and field strength. The modified Ogston equation accurately predicts the mobilities of DNA fragments in all three mobility regimes, providing a single, simple model to account for all of the observed behavior.

Stability of capillary gels for automated sequencing of DNA.

Recent interest in capillary gel electrophoresis has been fueled by the Human Genome Project and other large-scale sequencing projects. Advances in gel polymerization techniques and detector design have enabled sequencing of DNA directly in capillaries. Efforts to exploit this technology have been hampered by problems with the reproducibility and stability of gels. Gel instability manifests itself during electrophoresis as a decrease in the current passing through the capillary under a constant voltage. Upon subsequent microscopic examination, bubbles are often visible at or near the injection (cathodic) end of the capillary gel. Gels have been prepared with the polyacrylamide matrix covalently attached to the silica walls of the capillary. These gels, although more stable, still suffer from problems with bubbles. The use of actual DNA sequencing samples also adversely affects gel stability. We examined the mechanisms underlying these disruptive processes by employing polyacrylamide gel-filled capillaries in which the gel was not attached to the capillary wall. Three sources of gel instability were identified. Bubbles occurring in the absence of sample introduction were attributed to electroosmotic force; replacing the denaturant urea with formamide was shown to reduce the frequency of these bubbles. The slow, steady decline in current through capillary sequencing gels interferes with the ability to detect other gel problems. This phenomenon was shown to be a result of ionic depletion at the gel-liquid interface. The decline was ameliorated by adding denaturant and acrylamide monomers to the buffer reservoirs. Sample-induced problems were shown to be due to the presence of template DNA; elimination of the template allowed sample loading to occur without complications.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation of fluorescent oligosaccharide derivatives by microcolumn techniques based on electrophoresis and liquid chromatography

Various aldose oligosaccharides can be quantitatively derivatized into primary amines for subsequent reaction with fluorogenic reagents, such as 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde or 3-benzoyl-2-naphthaldehyde. Capillary electrophoresis (CE) and microcolumn liquid chromatography (LC), coupled with laser-induced fluorescence detection, were evaluated as a means of separating complex oligosaccharide mixtures. Whereas microcolumn LC and open-tubular CE appear confined in their utility to relatively small oligosaccharides, unprecedented results were obtained with polyacrylamide gel-filled capillaries on hydrolysed malto-oligosaccharides and enzymatically degraded samples of chondroitin sulfate and hyaluronic acid.

Problems and achievements in the instrumentation and column technology for chromatography and capillary electrophoresis

Chromatographic and electrophoretic separation methods are compared with regard to miniaturization in capillaries (“open tubular columns”) and with emphasis on the analytical applicability of such techniques. Instrumental problems of detection and sample introduction which are related to the very low sample capacity of capillary systems are discussed together with the procedures for polymeric coating of capillary surfaces as well as porous, small-particle support materials. Surface coatings are also important in capillary electrophoresis in order to control electroosmotic flow and to suppress adsorptivity for analyte molecules. In the micellar electrokinetic chromatography mode of electrophoresis neutral small molecules can also be separated with advantage. Micropacked columns cause less instrumental problems in SFC and LC because of their higher sample capacity. In CGE using polyacrylamide gel-filled capillaries excellent separations of charged, large biomolecules, especially oligonucleotides, can be achieved.