Aharon S. Cohen

Use of complexing agents for selective separation in high-performance capillary electrophoresis. Chiral resolution via cyclodextrins incorporated within polyacrylamide gel columns

Abstract The incorporation of a complexing agent within a polyarcylamide gel column provides a general means of manipulating the selectivity of a capillary electrophoresis separation. As an example of this approach, chiral resolution of dansylated amino acids by high-performance capillary electrophoresis has been achieved by adding β-cyclodextrin within the gel matrix. A retention model has been developed which can be used for selectivity optimization. Various parameters, such as concentration of cyclodextrin, addition of methanol to the buffer, and temperature, have been examined in terms of their influence on retention and selectivity. From this study high-performance separations have been developed. Efficiencies as high as 100 000 plates in 15 cm have been achieved.

Separation of DNA restriction fragments by high performance capillary electrophoresis with low and zero crosslinked polyacrylamide using continuous and pulsed electric fields

This paper presents results on the separation of DNA restriction fragments by high performance capillary electrophoresis (HPCE). Capillaries containing polyacrylamide with low amounts of crosslinking agent (i.e. 0.5% C) were first studied. The greater molecular accessibility offered with columns of low crosslinking, relative to higher crosslinked gels (e.g. 5% C), permitted high efficiency separations of double stranded DNA fragments up to 12,000 base pairs in length. Capillaries containing no crosslinking agent, i.e. linear polyacrylamide, were then examined. Ferguson plots (i.e. log mobility vs. %T) were used to assess the size selectivity of linear polyacrylamide capillaries. In another study, it was determined that the relative migration of DNA species was a strong function of applied electric field and molecular size. Lower fields yielded better resolution than higher fields for DNA molecules larger than about 1000 base pairs, albeit at the expense of longer separation time. Based on these results, we have examined pulsed field HPCE and have demonstrated the use of this approach to enhance separation.

High-performance sodium dodecyl sulfate polyacrylamide gel capillary electrophoresis of peptides and proteins

High-performance capillary sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has been developed for the separation and molecular weight (MW) determination of peptides and proteins. In this work, acrylamide was polymerized in fused-silica capillaries of 75 microns I.D. and 10 or 20 cm length. On-line UV detection was employed by placing the capillary in the optical path of the detector. Rapid separations of myoglobin fragments and of a mixture of standard proteins were demonstrated with efficiencies of the order of 40,000 plates for a column, 20 cm in length. Linear plots of log MW vs. mobility for the SDS protein complexes were observed. Variation in the percentage monomer composition (% T) of the polyacrylamide yielded the expected common intercept of log mobility vs. % T, confirming the size separation mechanism in the gel capillaries. The separation of the A and B chains of insulin with a 10-cm column in less than 10 min was also demonstrated.

High-performance capillary electrophoresis in the biological sciences

High-performance capillary electrophoresis (HPCE), the instrumental approach to electrophoresis, is a method undergoing rapid development at the present time. There is a high expectation that HPCE will become a widely applicable tool within the biochemical community. This review presents principles and instrumentation followed by typical applications of HPCE in the biological area. In the instrumentation area, particular attention is paid to current and future detectors, including laser-induced fluorescence and HPCE-mass spectrometry. In the applications section, both peptide or protein and oligonucleotide high-resolution separations are described for open-tube and polyacrylamide gel capillary columns. The use of isoelectric focusing and electrokinetic chromatography to manipulate separation is also presented. Future directions of methodology are suggested, and it is predicted that in the next few years HPCE will become a complementary tool to liquid chromatography and slab gel electrophoresis.

Separation and analysis of DNA sequence reaction products by capillary gel electrophoresis

This paper demonstrates the potential of capillary gel electrophoresis with laser induced fluorescence detection as a tool for DNA sequence determination. Both synthetic oligonucleotides and single-stranded phage DNA were utilized as templates in the standard chain termination procedure. Primer molecules were tagged at the 5 end with the fluorescent dye, JOE. First, baseline resolution of a dA extended primer from 18 to 81 bases long, a total of 64 fragments, was observed. A second synthetic template was designed to yield alternating stretches of dA and dT extensions of the primer. Thirdly, the sequence reaction products from a synthetic oligonucleotide template containing all four bases was analyzed in four independent runs, one for each of the four base-specific reactions. In all cases, the expected number and patterns of peaks were observed by capillary gel electrophoretic analysis. Finally, separation of sequence reaction products generated with single-strand M13mp18 phage DNA as template exhibited baseline resolution of fragments differing in length by a single nucleotide and from 18 to greater than 330 bases total length.

Use of peltier thermoelectric devices to control column temperature in high-performance capillary electrophoresis

Abstract A temperature control system for high-performance capillary electrophoresis is described utilizing Peltier thermoelectric devices. The thermoelectric devices heat or cool an alumina block which has a high thermal conductivity for efficient heat removal from the external capillary column wall. The heat dissipation characteristics of this device are compared to natural convection and fan cooling by observing the stability of current with time and the dependence of current on applied field. A linear relationship of electroosmotic flow velocity with increasing field is found, indicative of a constant temperature at the inside wall of the capillary. Also, the change in peak shape of horse heart myoglobin as a function of increasing temperature is shown to demonstrate the importance of temperature control in obtaining optimum efficiency.

Rapid separation of DNA restriction fragments using capillary electrophoresis

Open-tube capillary electrophoresis has been applied to the separation of restriction fragments of DNA with a Tris-borate buffer containing 7 M urea and 0.1% sodium dodecyl sulfate. The importance of sample pretreatment and of the injection of heated samples has been demonstrated. In one separation, a DNA restriction fragment mixture from 72 to 23,130 base pairs (DRIgestTM III) (molecular weight range from 4.6.10(4) to 1.5.10(7] has been electrophoresed in 10 min on a column of 15 cm effective length. Over 600,000 plates have been obtained for individual peaks. Several of the peaks have been identified, by spiking slab gel electrophoretically purified components. Other examples of restriction fragment separations are illustrated in this paper. The results of this study when further validated with full characterization of individual species, open up the possibility of rapid restriction enzyme mapping.

Separation of DNA fragments by capillary electrophoresis using replaceable linear polyacrylamide matrices.

The use of low percent (1.5-6% T) replaceable linear polyacrylamide (LPA) network matrices for rapid separation of double-stranded DNA fragments was explored. Separations of fragments ranging from 20 to 23,000 base pairs were readily achieved. Typically, 4 x 10(6) theoretical plates/m were obtained in less than 30 min. Short separation times under 2 min were also possible, using the DNA intercalating dye, ethidium bromide, along with high electric fields. The high resolving power of linear polyacrylamide was demonstrated in the separation of two fragments which differ by a single base pair (123/124 base pairs) using 6% T LPA and ethidium bromide intercalation. This LPA composition allowed for the possible single base-pair resolution of dsDNA fragments up to 300 base pairs in length. Several concentrations of the linear polyacrylamide for different ranges of fragment lengths have been employed. In addition, replaceable LPA offers the advantage of a fresh separation matrix for each run, thus overcoming column stability problems and minimizing needs for sample cleanup. Electro-osmotic flow was substantially reduced using stable capillary coatings, which were required for obtaining high efficiencies and good reproducibility.

Identification of DNA molecules by pre-column hybridization using capillary electrophoresis

Abstract In this paper, we demonstrate the feasibility of using gel-filled capillary electrophoresis to perform Southern blotting with on-line detection and to study parameters that affect the hybridization of DNA molecules in solution. A fluorescence-tagged oligonucleotide was used as the probe for hybridization in solution with complementary DNA molecules prior to electrophoresis. The reaction mixture was subject to capillary electrophoresis in a polyacrylamide gel-filled capillary, and the hybridized species was identified on-line by UV absorption or laser-induced fluorescence detection. The effects of probe concentration and annealing temperature were studied.

9.2. Biotechnological products

Publisher Summary To choose the correct biological system and process steps for expression or production of these biopharmaceuticals it is essential to understand the structural features of the biopolymers that are important to the desired therapeutic activity. A vast number and variety of analytical tools are involved in the structural studies of the products of modem biotechnology such as HPLC in various modes, capillary electrophoresis, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopic techniques, circular dichroism (CD), X-ray diffraction and light scattering studies. Therapeutic biopolymers include monoclonal antibodies (MAbs), interleukins (IL-s), interferons (IFN-s), growth factors (GF-s), tissue plasminogen activator (tPA), erythropoietin (Epo), tumour necrosis factor (TNF) and blood clotting factors. These proteins may be produced by hybridoma cell lines, by genetically engineered cells or by natural producer lines. The end-product of the cell culture or fermentation contains the recombinant product together with impurities which consist of product related degradation products, fragments, or aggregates and contaminants such as host cell proteins, DNA etc.