Pressure refilled polyacrylamide columns for the separation of oligonucleotides by capillary electrophoresis.

The separation of oligonucleotides by capillary zone electrophoresis (CZE) was studied in fused silica separation capillaries filled by linear (noncrosslinked) polyacrylamide (PAA) solutions, introduced into the capillary from the stock by pressure after each analysis. The time-consuming in-capillary polymerization step could thus be avoided, and fast and reproducible repetition of the analyses was assured. The PAA concentrations varied within the range of 3-10% and both the reproducibility of the analyses and the stability of the solution in the capillary, with and without a chemically treated inner wall, were tested. Ferguson plots were used to assess the size selectivity of the separation.     

Size exclusive capillary electrophoresis separation of DNA oligonucleotides in small size linear polyacrylamide polymer solution

The mobility of analytes in capillary electrophoresis using polymer gels and solutions is usually described as having an inverse relationship with the molecular size (mobility decreases as molecular size increases). The most commonly used models for predicting such mobility are the Ogston model and the Reptation model. However, in this study a new separation phenomenon was observed in which the mobility of DNA oligonucleotides increased with molecular size in a capillary electrophoresis phase (CEP) coated capillary column. The polymer system used was a 11% linear polyacrylamide (Mr = 1500) solution. The log-transformed number of base pairs (log N) of three double stranded oligonucleotides had an inverse linear relationship (r2 > 0.9981) with their migration time in the capillary column. Such a relationship is similar to that observed with size exclusive chromatography.     

Determination of therapeutic oligonucleotides using capillary gel electrophoresis

Oligonucleotides have developed into highly versatile and selective therapeutics over the past 20 years. More than five discrete mechanisms of action have been reported and more than 10 different chemical modifications have been used to extend their in vivo half-life and reduce their toxicity. Capillary gel electrophoresis (CGE) has been used extensively for the quantitative analysis of oligonucleotide therapeutics in both preclinical and clinical studies since the 1990s. The success of CGE is based on its extraordinary resolving power, which allows for the simultaneous determination of the parent drug and its metabolites. More recently, capillary gel electrophoresis has seen renewed interest with the emergence of replaceable gels with single-base resolving power and new capillary electrophoresis-mass spectrometry interfaces. This review discusses the bioanalysis of therapeutic oligonucleotides showing the evolution of the field over the past two decades leading to the current new approaches. Included in this review are topics such as different gel types, sample introduction modes, sample extraction procedures, separation conditions and detection methods used in CGE, along with discussions of the successes and limitations associated with each.     

Band broadening of DNA fragments isolated by polyacrylamide gel electrophoresis in capillary electrophoresis

Polyacrylamide gel electrophoresis (PAGE) is used frequently for isolation and purification of DNA fragments. In the present study, DNA fragments extracted from polyacrylamide gels showed significant band broadening in capillary electrophoresis (CE). A pHY300PLK (a shuttle vector functioning in Escherichia coli and Bacillus subtilis) marker, which contained nine fragments ranging from 80 to 4870 bp, was separated by PAGE, and each fragment was isolated by phenol/chloroform extraction and ethanol precipitation. After extraction from the polyacrylamide gel, the peaks of the isolated DNA fragments exhibited band broadening in CE, where a linear poly(ethylene oxide) was used as a sieving matrix. The theoretical plate numbers of the DNA fragments contained in the pHY300PLK marker were >10⁶ for all the fragments before extraction. However, the DNA fragments extracted from the polyacrylamide gel showed decreased theoretical plate numbers (5–20 times smaller). The degradation of the theoretical plate number was significant for middle sizes of the DNA fragments ranging from 489 to 1360 bp, whereas the largest and smallest fragments (80 and 4870 bp) had no obvious influence. The band broadening was attributed to contamination of the DNA fragments by polyacrylamide fibers during the separation and extraction process.     

Formamide modified polyacrylamide gels for DNA sequencing by capillary gel electrophoresis.

Compressions are occasionally found during the separation of DNA sequencing fragments, particularly in G/C-rich regions and in gels operated at room temperature. Addition of at least 10% formamide to urea/polyacrylamide sequencing gels improves the denaturing capacity of the gel, minimizing compressions. Addition of 20% or more formamide decreases the separation rate, theoretical plate count, and resolution for normally migrating fragments. An optimum concentration of 10% formamide improves resolution of compressed regions without degrading the other characteristics of the gel. Operation of gels at room temperature simplifies the engineering associated with automated sequencers based on capillary gel electrophoresis.     

Characterization of single-stranded DNA separation by capillary gel electrophoresis

We have investigated the effect of polymer gel reconditioning, the shape of the capillary, the applied electric field, and the capillary length for single-stranded DNA. The polyethylene oxide gel had deformed under the high electric field causing the degradation of the separation power. By the reintroduction of the fresh polyethylene oxide gel for the next run, one-base resolution was recovered. It turned out that the tip of the capillary at the injection side needed to be clean and symmetric for much improved resolution. Changing DNA motion by the pulsed electric field resulted in the separation of DNA far more than 500 bases.     

Protein separation by capillary gel electrophoresis: A review

Capillary gel electrophoresis (CGE) has been used for protein separation for more than two decades. Due to the technology advancement, current CGE methods are becoming more and more robust and reliable for protein analysis, and some of the methods have been routinely used for the analysis of protein-based pharmaceuticals and quality controls. In light of this progress, we survey 147 papers related to CGE separations of proteins and present an overview of this technology. We first introduce briefly the early development of CGE. We then review the methodology, in which we specifically describe the matrices, coatings, and detection strategies used in CGE. CGE using microfabricated channels and incorporation of CGE with two-dimensional protein separations are also discussed in this section. We finally present a few representative applications of CGE for separating proteins in real-world samples.     

A simple polyacrylamide gel electrophoresis procedure for separation of polyamidoamine dendrimers

A simple, inexpensive, and rapid electrophoresis technique was developed for use as a routine tool for evaluating purity of polyamidoamine (PAMAM) dendrimers. A variety of factors influencing migration of generations 0-7 dendrimers on nongradient polyacrylamide gels were evaluated. The low generation dendrimers were found to be very sensitive to diffusion during or after electrophoresis. The proposed method incorporates steps that minimize diffusion, in order to obtain improved resolution and sensitivity, especially for the lower-molecular-weight dendrimers. This was accomplished by inclusion of a dendrimer fixation step with glutaraldehyde and performing the electrophoresis separation, fixation, staining, and destaining at 4 degrees C. PAMAM dendrimer separation was studied under basic and acidic conditions. Electrophoresis under acidic conditions gave increased resolution and sensitivity over separation at alkaline pH. Oligomers and trailing generations could be clearly separated and visualized under these conditions. The smallest PAMAM dendrimer, generation 0, was visible at 1.5 microg under the optimized acidic conditions. With slight modifications, this technique should be applicable to separation of other water-soluble dendrimers.     

Separation and determination of some stereoisomers by capillary gel electrophoresis with cyclodextrin incorporated in polyacrylamide gel

Capillary gel electrophoresis (CGE) was successfully applied to the separation of optically active isomers and position isomers by incorporating a suitable cyclodextrin chiral selector in polyacrylamide gel. A commercially available ss-cyclodextrin (ss-CD) was used for enantioselectivity towards o-, m- and p-nitrobenzoic acid, o-, m- and p-hydroxybenzoic acid, o-, m- and p-toluic acid and the optical isomers of dansyl-D,L-leucine and R,S-1,1'-binaphthyl-2,2-dihydrogenphosphate. Especially the effect of organic solvents, such as acetonitrile, methanol, dimethylsulphoxide and others were examined in detail. The resolution varied to some extent with the addition of the organic solvent to the polyacrylamide gel and the running buffer solution. The possible mechanism has also been discussed. In addition, quantitative aspects of the separation of stereoisomers using CGE have been studied, showing that both the resolution and accuracy of the determinations were affected by the ratio of the enantiomers.     

Separation and determination of some stereoisomers by capillary gel electrophoresis with cyclodextrin incorporated in polyacrylamide gel

Capillary gel electrophoresis (CGE) was successfully applied to the separation of optically active isomers and position isomers by incorporating a suitable cyclodextrin chiral selector in polyacrylamide gel. A commercially available ß-cyclodextrin (ß-CD) was used for enantioselectivity towards o-, m- and p-nitrobenzoic acid, o-, m- and p-hydroxybenzoic acid, o-, m- and p-toluic acid and the optical isomers of dansyl-D,L-leucine and R,S-1,1-binaphthyl-2,2-dihydrogenphosphate. Especially the effect of organic solvents, such as acetonitrile, methanol, dimethylsulphoxide and others were examined in detail. The resolution varied to some extent with the addition of the organic solvent to the polyacrylamide gel and the running buffer solution. The possible mechanism has also been discussed. In addition, quantitative aspects of the separation of stereoisomers using CGE have been studied, showing that both the resolution and accuracy of the determinations were affected by the ratio of the enantiomers.     

Prediction of migration behavior of oligonucleotides in capillary gel electrophoresis.

The influence of the primary structure (base composition) on the electrophoretic migration properties of single-stranded oligodeoxyribonucleotides in capillary polyacrylamide gel electrophoresis was investigated using homo- and heterooligomers under denaturing and non-denaturing conditions. Homooligodeoxyribonucleotides of equal chain lengths but of different base composition showed significant differences in mobility. In addition, the migration properties of heterooligomers were found to be highly dependent on their base composition. A simple equation is presented for predicting relative migration times using denaturing and non-denaturing polyacrylamide capillary gel electrophoresis. Orange-G was used as an internal standard and as the basis of the relative migration time calculations. Examples are presented using homo- and heterooligomers in the 10-20-mer range to show the correlation of the primary structure and their predicted and observed migration rates.     

HILIC mode separation of polar compounds by monolithic silica capillary columns coated with polyacrylamide

HILIC mode columns were prepared by an on-column polymerization of acrylamide on a monolithic silica capillary column modified with N-(3-trimethoxysilylpropyl)methacrylamide as the anchor group. The products showed HILIC mode retention characteristics with three times greater permeability and slightly higher column efficiency compared to a commercially available amide-type HILIC column packed with 5-μm particles. The selectivity of the monolithic silica-based column was similar to that of the particulate column for each group of solutes towards nucleosides, nucleic bases and carbohydrate derivatives, although a considerable difference was observed in the selectivity for the solute groups. Although the retention of solutes based on the polar functionality was much smaller with the monolithic silica columns, which had a smaller phase ratio, than with the particle-packed column, the former can achieve better separation utilizing the high permeability and higher column efficiencies of a longer column.     

Quantitative capillary gel electrophoresis assay of phosphorothioate oligonucleotides in pharmaceutical formulations

Quantitative capillary gel electrophoresis (QCGE) has been developed for the accurate quantitation of a 21-mer phosphorothioate oligonucleotide, ISIS 2922, and its degradation products in an intravitreal formulation. The electrokinetic mode of injection employed by CGE necessitates formulation of the external reference standard in a sample matrix similar to that of the drug product and the use of an internal standard for improved accuracy and precision. The analytical method detailed in this paper has demonstrated the necessary accuracy, precision, linearity, range, selectivity and ruggedness for use in routine drug product analysis and stability monitoring of phosphorothioate oligonucleotides.     

OFFGEL isoelectric focusing and polyacrylamide gel electrophoresis separation of platinum-binding proteins

In this work a 2D electrophoretic separation procedure able to maintain the integrity of platinum-protein bonds has been developed. The method is based on the use of sequential OFFGEL isoelectric focussing (IEF) and PAGE. A systematic study of the reagents used for PAGE, for OFFGEL-IEF separation, and post-separation treatment of gels (such as enzymatic digestion and sample preparation for MS analysis) was tackled regarding their suitability for the identification of platinum binding proteins using standard proteins incubated with cisplatin. The distribution of platinum in high and low molecular weight fractions (separated by cut-off filters) was determined by ICP-MS, which allows evaluating platinum-protein bond stability under the conditions studied. SDS-PAGE in the absence of β-mercaptoethanol or dithiotreitol preserved the platinum-protein bonds. In addition, neither the influence of the electric field during the electrophoretic separation, nor the processes of fixing, staining and destaining of proteins in the gel did result in the loss of platinum from platinum binding proteins. SDS-PAGE under non-reducing conditions provides separation of platinum-binding proteins in very narrow bands with quantitative recoveries. Different amounts of platinum-bound proteins covering the range 0.3-2.0 μg were separated and mineralised for platinum determination, showing good platinum linearity. Limits of detection for a mixture of five standard proteins incubated with cisplatin were between the range of 2.4 and 13.9 pg of platinum, which were satisfactory for their application to biological samples. Regarding OFFGEL-IEF, a denaturing solution without thiourea and without dithiotreitol is recommended. The suitability of the OFFGEL-IEF for the separation of platinum binding proteins of a kidney cytosol was demonstrated.     

Fast separation of single-stranded oligonucleotides by capillary electrophoresis using OliGreen as fluorescence inducing agent

The fast separation of oligonucleotide (oligos) sizing marker by CE using OliGreen and including effects due to the concentration of separation medium and urea denaturant is presented. OliGreen dye is found to be more sensitive than ethidium bromide (by a factor of about 6 based on S/N considerations) for the oligos' separations. Higher concentration of F127 in 1xTris-boricacid-EDTA (TBE) up to 30% w/v leads to better resolution of oligos separations. The addition of urea into the separation medium decreases the sensitivity. With an optimized running condition, the oligos sizing marker could be successfully separated with 1-base resolution within 1.3 min by using 30% w/v F127/1xTBE solution as the separation medium at an applied electric field of 800 V/cm in a 3 cm long capillary, the fastest capillary gel electrophoresis separation with high resolution reported to date for oligos in the similar size range.     

Ethylbenzene-modified fused-silica columns for capillary electrophoresis

Summary Coated capillaries modified with a hydrocarbon layer have been developed. Modification of the surface with ethylbenzene greatly improved the electrophoretic performance of the capillaries. The column efficiency for basic organic compounds was as high as 378000 theoretical plates per meter on a 50 μm i.d. ethylbenzene-surface-treated fused-silica capillary column. This value did not change during 25 replicate analyses and the capillary columns were very stable against continuous treatment for 30 h with buffer of pH 10 and treatment for 3 h with HCl of pH 1 and NaOH of pH 12. The relative standard deviation of run-to-run, day-to-day, and capillary-to-capillary for coating with the ethylbenzene layer was<2.6%, and reproducibility was good. The separation of four aromatic amines and six pharmacological amines at pH 2.5 is reported.     

Hydrophobic-phase-modified fused-silica columns for capillary electrophoresis

Coated capillaries modified with a hydrophobic layer were developed. Linear hydrocarbons and ethylbenzene modified surfaces greatly improved the electrophoretic performance of the capillaries. The column efficiency for organic compounds reached as high as 327 000 theoretical plate numbers per meter on a 50 microm I.D. linear hydrocarbon (C6) surface treated fused-silica capillary column. This value did not change during 50 repeated analyses and the columns showed strong stability against 0.1 M NaOH and 0.1 M HCl. The relative standard deviation of the run-to-run, day-to-day, and capillary-to-capillary coating with hydrophobic layer showed values of < or =2.5%, and good reproducibility. The separations of four aromatic amines and six pharmacological amines at pH 2.5 is reported.     

Globin chain separation by SDS polyacrylamide gel electrophoresis. Simple screening method for elongated hemoglobin chains

A simple method for the separation of hemoglobin chains from hemolysate or globin, by sodium dodecyl sulfate polyacrylamide gel electrophoresis, is described. The alpha, beta, and gamma chains can be clearly separated from each other. The alpha chain has the highest mobility, the beta chain has a slower mobility than the gamma chain, while the delta chain has about the same mobility as the beta chain. Hemoglobins with elongated chains can easily be detected by this method. Tak-beta, elongated by 11 residues, moves much more slowly than betaA but is much faster than alpha Constant Spring which is elongated by 31 residues. Screening of several individuals with slow-moving hemoglobins using this method led to the finding of a case with Hb Tak-beta thalassemia and other carriers of Hb Tak.