Preparation of linear polyacrylamide-coated capillaries - Study of the polymerization process and its effect on capillary electrophoresis performance

27th Scientific Meeting of the Group-of-Chromatography-and-Related-Techniques of the Spanish-Royal-Society-of-Chemistry JUL 08-10, 1998 LUGO, ITALYThe effect of different parameters controlling the characteristics of linear polyacrylamide coatings deposited on the inner wall of fused-silica capillaries and their influence on capillary electrophoresis (CE) performance of these coated columns is investigated. To carry out this study, a reproducible procedure to obtain capillaries with similar extent of modification of the surface silanols with 7-oct-1-enyltrimethoxisilane was first approached. Next the polymer attachment to the silica wall, via covalent linkage to the silyl reagent grafted onto the silica, was investigated. In this way, by using columns with a similar silylation extent, differences in CE performance observed among capillaries coated under diverse conditions could be assigned to the characteristics of the polyacrylamide layer. It is demonstrated that the characteristics and reproducibility of these polymeric coatings depend on the adequate control of both the temperature of polymerization and the degassing of the polymerizing dissolutions used. More interestingly, it is also demonstrated that the quantities of monomer (acrylamide), initiator (ammonium persulfate) and activator (N,N,N9,N9-tetramethylethylenediamine), and the ratio among them used in the preparation of the coating polymer have a large influence on the performance of CE columns. The optimum conditions for preparing the polyacrylamide coatings are discussed. The applicability of these linear polyacrylamide-coated capillaries to the separation of basic and acidic proteins in free zone CE is demonstrated. Besides, the use of these coated columns in capillary gel electrophoresis for the separation of DNA fragments is shown.We thank Professor J. San Roman, Dr. A. Gallardo, and Dr. B. Vazquez (Institute of Polymers, CSIC, Spain) and Dr. M. de Frutos (Institute of Organic Chemistry, CSIC) for fruitful discussions. This work was supported by a CYCIT project MAT96-9564 (Spain).Peer reviewe

Whey proteins eluted in reversed phase gradients

The behavior of bovine whey proteins in Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) systems has been investigated. The Linear Solvent Strength (LSS) model has been applied to the separation of these proteins studying how their retention time and band broadening change when different gradi- ent parameters are modified. From our results it is deduced that the LSS model describes the behavior of the whey proteins in RP- HPLC. Also, it seems that ts(the retention time for non-retained solutes) depends on the size of these proteins. The good fit observed between experimental data and the equations deduced from the LSS model allows the prediction of a gradient shape that permits a rapid analysis of the above mentioned proteins.This work was supported by CICYT (project ALI94-0737).Peer reviewe

Behavior of Whey Proteins in Hydrophobic Interaction Chromatography

The authors thank J. Gavilanes (Universidad Complutense, Madrid, Spain) performing the CD studies. This work was supported by ClCYT (project ALI 94-0737)The authors thank J. Gavilanes (Universidad Complutense, Madrid, Spain) performing the CD studies. This work was supported by ClCYT (project ALI 94-0737).Peer reviewe

Analysis of Whey Proteins by Capillary Electrophoresis Using Buffer-Containing Polymeric Additives

This paper shows the possibilities of capillary electrophoresis for analysis of the major whey proteins (β-lactoglobulin A, β-lactoglobulin B, α-lactalbumin, and BSA) in standard samples and raw and UHT milks. A new separation buffer, consisting of Tris-boric acid, SDS, and polyethylene glycol 8000, was used for the separation. Good migration time reproducibility was achieved for the proteins of standard samples and those of real samples analyzed on the same day and on 3 different d. Area reproducibility for the protein peaks ranged from 2.7% for β-lactoglobulins to 17.9% for BSA, including standard and real samples. Also, the concentration of the major proteins determined in the milks studied agrees quite well with the values obtained by HPLC. Furthermore, analysis time can be fourfold smaller using capillary electrophoresis than that usually obtained in HPLC.Peer reviewe

Use of detergents and high contents of organic solvents for simultaneous quantitation of ionic and nonionic drugs by electrokinetic chromatography

Buffers containing high percentages of organic solvents, typically 50% of acetonitrile and /or methanol, together with sodium dodecyl sulfate (SDS) are employed for the separation and quantitation by electrokinetic chromatography (EKC) of analytes found in a nasal spray. Solutes consist of benzalkonium chloride, a family of highly positive compounds, and 2-phenylethanol and beclomethasone dipropionate, which are electrically neutral and poorly soluble in aqueous buffers. It is observed that the effect of both concentration of SDS and temperature on the separation depends on the organic solvent used and the solute nature. It is also observed that SDS–solute interaction for neutral and cationic compounds are weaker in the presence of high contents of acetonitrile than in methanol. Concentration of SDS, temperature, and organic solvent nature and content, allow one to modify the selectivity of the separation when neutral and ionic species have to be simultaneously determined. The optimization of EKC conditions enables the analysis of compounds in less than 5 min. A one-step sample treatment consisting of centrifugation of the nasal spray solved in acetonitrile, together with the referenced optimum separation conditions enable the reproducible quantitation of the analytes. Relative standard deviation values of inter-day migration times lower than 2.45% are obtained (R.S.D. ), while R.S.D. values for inter-day peak areas were lower n512 n512 than 6.32%. Ó 1998 Elsevier Science B.V. All rights reserved.The authors thank Glaxo Wellcome S.A. (Aranda de Duero, Spain) for the donation of standards and Beconase samples, as well as for the information supplied about its pharmaceutical formulation. This work was supported by the Commission of the European Communities (Training and Mobility of Researchers, contract No. ERBFMBICT950003) and by a DGICYT project (No. PB94-02818-C02-02).Peer reviewe

Amino acids determination using capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection

Free amino acids have been derivatized on-capillary with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and analyzed using a laboratory-made capillary electrophoresis apparatus with laser-induced fluorescence detection. Several parameters that control on-capillary derivatization of amino acids, including pH, mixing time, reaction time, concentration of the derivatization reagents (potassium cyanide and FQ) and solvent of FQ, as well as the temperature of mixing and reaction were optimized. Repeatabilities better than 1.8% for migration time and 7.8% for peak height were obtained. Assay detection limits for the different amino acids ranged from 23 nM for glycine to 50 nM for lysine and glutamic acid. The methods developed were applied to the analysis of several amino acids in pharmaceutical preparations and plasma samples. Results showed a good agreement with those obtained using an amino acid autoanalyzer for the same samples.M.T.V. acknowledges the Spanish Ministry of Education and Science for a pre-doctoral grant. This work has been supported by TIC project 2003-01906. Authors acknowledge M.J. Garcia of Center of Diagnosis of Molecular Diseases (Autonoma University, Madrid, Spain) for providing the plasma samples.Peer reviewe

On-capillary derivatization and analysis of amino acids in human plasma by capillary electrophoresis with laser-induced fluorescence detection: Application to diagnosis of aminoacidopathies

A capillary electrophoresis with laser-induced fluorescence detection method for the analysis of free amino acids (AA) in human plasma was developed. A mixture of 16 AA was on-capillary derivatized with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and separated inside the capillary in less than 30 min using 70 mM borax-3.5 mM SDS pH 9.3 as running buffer. Four plasma samples from a healthy donor and patients suf- fering from phenylketonuria, propionic acidemia, and tyrosinemia type II were studied. Repeatabilities calculated as intra-day RSD (n = 3) values for the AA involved in these aminoacidopathies (glycine, phenylalanine, and tyrosine) were in the range of 0.3 to 1.2% for migration time and 3.7 to 8.2% for peak height. Reproducibilities calculated as inter-day RSD (n = 4) values for the same AA were between 0.7 and 1.4% for migration time and 4.7 and 9.1% for peak height. A fast qualitative analysis allowed the identification of the corresponding disease by comparing the electrophoretic profiles from the patient and the healthy donor and noting the increased level of the specific AA accompanying each individual disease. The results of the quantitative analysis for glycine, phenylalanine, and tyrosine in the plasma samples studied using the devel- oped method showed a good agreement with those provided by the Center of Diag- nosis of Molecular Diseases using a standard method for AA analysis.The authors acknowledge Dr. Maria Jose Garcia (Auton- oma University, Madrid, Spain) for providing plasma sam- ples and CICYT-Spain (project TIC 2003-01096) and to Ramon Areces Foundation (Spain) for financial support. M.T.V. acknowledges the Spanish Ministry of Education and Science for a pre-doctoral grant.Peer reviewe

CE methods for analysis of isoforms of prostate-specific antigen compatible with online derivatization for LIF detection

Prostate-specific antigen (PSA) is the usual biomarker for prostate cancer (PCa). However, its lack of selectivity has lead to the search for new biomarkers. PSA glyco- sylation seems to depend on the pathophysiological conditions of the individual. Thus, methods to separate PSA isoforms (peaks) to study their role as PCa markers are needed. In this work, CE methods for PSA isoforms separation, based on the use of different dynamic coatings, are developed using UV detection. Three complementary CE methods allowing the separation of 8 or 9 PSA isoforms are selected. The longest method takes only 17 min, while the shortest one separates 9 isoforms in o 8 min. Depending on the isoforms of interest for their use as PCa biomarker, the CE method to be used can be chosen or various of them can be combined. A remarkable aspect of these methods is that the BGEs employed are devoid of compounds with primary amino groups, making the CE methods compatible with fluorescent on-column derivatization through amino residues. As a proof-of-concept, a preliminary result shows that LIF detection of labeled PSA analyzed by one of the three developed methods permits detection of glycoprotein isoforms.Peer reviewe

Treatments of fused-silica capillaries and their influence on the electrophoretic characteristics of these columns before and after coating

Making coated columns in capillary electrophoresis (CE) is a laborious task involving many preparation steps such as etching, leaching, dehydration, silylation and coating of the inner wall of fused-silica capillaries. In this work we demonstrate, by testing more than 250 columns, that it is possible to follow up the influence of the different steps on both the electrophoretic behavior of CE columns and the reproducibility of their preparation. This study was done by carrying out triplicate measurements of electroosmotic flow values from columns at four different pH values after each step. The effectiveness of the coatings was also investigated by injecting a test-group of basic proteins. It is demonstrated that etching the columns with sodium hydroxide followed by a leaching treatment with hydrochloric acid provides higher reproducibility than by either leaching or etching alone. Dehydration of the capillary tubing affects the yield of the subsequent silylation reaction while the best results seem to be obtained by dehydrating the columns overnight at 1608C. The silylation degree achieved is demonstrated to also depend on the time of reaction and the concentration of silyl reagent. Moreover, a conclusive demonstration about the effect of both silylation and polymerization reactions on the final coating performance is given. Ó 1998 Elsevier Science B.V. All rights reserved.Thanks are given to Dr. P. Martin-Alvarez (In- stituto de Fermentaciones Industriales, CSIC, Spain) and Dr. M. de Frutos (Instituto de Quimica Organica General, CSIC) for fruitful discussions. This work was supported by a CYCIT project (MAT96-0564).Peer reviewe

Use of immunodotting to select the desorption agent for immunochromatography

In immunochromatography, a technique of increasing use, the sample containing the antigen (Ag) to be purified or determined is introduced into a chromatographic column containing an antibody (Ab) bound to the packing material. The antigen is retained based on antigen – antibody recognition. To reuse the immunocolumn for subsequent assays, the antigen has to be eluted without causing irreversible damage of antibodies. Selection of conditions for performing immunochromatography is usually made by trial and error. This way of working is time consuming and it may ruin the column. In this article, the feasibility of using immunodotting to select the conditions to be employed in one immunochromato- graphic assay is shown. An immunodotting method is developed to select the best desorption agent for an enzyme-linked immunoaffinity chromatography (ELIAC) assay to determine h-lactoglobulin (h-LG). The effect of several factors on the immunodotting performance is studied. The way of performing solvent exchange to treat the antibody with different solutions considered as potential desorption agents to check their effect is shown. Effectiveness of the solution chosen by immunodotting (4 M MgCl2 in 20 mM Tris, pH 5.9) as desorption agent is demonstrated by immunochromatographic assays. The immunodotting and solvent exchange methods developed should be useful to choose solvents and conditions for any other kind of assay.A.P. acknowledges the Spanish Ministry of Science and Technology for a predoctoral grant. Dr. E. Camafeita is acknowledged for performing MALDI – TOF assay. This work has been supported by Spanish CICYT (project AGL2000-1480).Peer reviewe