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This paper describes the use of fluorosurfactants as buffer additives for capillary electrophoretic separation of proteins and peptides. Due to fluorosurfactant bilayer formation at the capillary inner wall, the surface charge can be adjusted and even reversed. If the running buffer pH is kept at a level where the proteins have the same sign of charge as the wall, electrostatic repulsion will be obtained. The protein wall adsorption can therefore be reduced and the separation performance can be noticeably increased. The separation performance can also be further improved by including mixtures of different types of fluorosurfactants in the running buffer. The buffer system can accordingly be adapted for a certain separation problem. Mechanisms for the use of fluorosurfactants for wall deactivation in capillary electrophoretic protein separations is discussed in the present work and some examples of applications are also presented.

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A new laser-induced fluorescence (LIF) detector for multicapillary electrophoresis is presented. The detection principle is based on waveguiding of the emitted fluorescence from the point of illumination to the capillary ends by total internal reflection (TIR) and imaging of the capillary ends. The capillaries themselves thus act as liquid core waveguides (LCWs). At the illumination point, the capillaries are arranged in a planar array, which allows clean and efficient illumination with a line-focused laser beam. The capillary ends are rearranged into a small, densely packed two-dimensional array, which is imaged end-on with high light collection efficiency and excellent image quality. Wavelength dispersion is obtained with a single prism. Intercapillary optical crosstalk is less than 0.5%, and rejection of stray light is very efficient. The detector is applied to four-color DNA sequencing by gel electrophoresis in a 91-capillary array, with simple fluorescein and rhodamine dyes as fluorophores. Since the imaged two-dimensional array is so compact, the detector has a high potential for very large-scale multiplexing.

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The present paper describes a new technique to suppress evaporation of solvent from very small volumes. Vials (15 nl) on a silicon microchip were filled with water, and covered with a thin, flowing film of a volatile liquid (e.g., octane). Water evaporation was greatly reduced. At 37 degrees C, no appreciable loss of water could be observed over a period of 90 min. At 95 degrees C, most of the water sample was left in the vial for more than 3 min. The applicability of the method is demonstrated by a tryptic digest, where the resulting peptide fragments were analyzed by capillary electrophoresis. In addition, a discussion of the possibilities and limitations of some alternative methods is included in the paper, as well as an outlook on future developments of the liquid lid concept.

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A new laser-induced fluorescence detector for capillary electrophoresis (CE) is described. The detector is based on transverse illumination and collection of the emitted fluorescent light via total internal reflection along the separation capillary. The capillary is coated with a low refractive index fluoropolymer and serves as a liquid core waveguide (LCW). The emitted light is detected end-on with a CCD camera at the capillary exit. The observed detection limit for fluorescein is 2.7 pM (550 ymol) in the continuous-flow mode and 62 fM in the CE mode. The detector is applied to DNA sequencing. One-color G sequencing is performed with single-base resolution and signal-to-noise ratio approximately 250 for peaks around 500 bases. The signal-to-noise ratio is approximately 50 for peaks around 950 bases. Full four-color DNA sequencing is also demonstrated. The high sensitivity of the detector is suggested to partly be due to the efficient rejection of scattered laser light in the LCW. The concept should be highly suitable for capillary array detection.

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In an earlier report (Litborn, E., Emmer, A., Roeraade, J., Anal. Chim. Acta 1999, 401, 11-19, we described a technique for performing chemistry in chip-based vials. A major problem, solvent evaporation, was partially remedied by using a closed humidity chamber. In this paper we report an improved technique for performing parallel reactions in open, 15 nL volume, chip-based vials. The evaporation of solvent from the reaction fluid was continuously compensated by addition of solvent via an array of microcapillaries. The suitability of the method was demonstrated by performing eight separate peptide maps of myoglobin in parallel, using the three enzymes trypsin, alpha-chymotrypsin and endoproteinase Glu-C. The total amount of myoglobin utilized to perform the eight digests was less than 100 pmol. The corresponding amount of enzymes was ca. 0.1 pmol per reaction. In order to evaluate the operating limits of the technique, a study of the evaporation of solvents from a series of vials with proportionally smaller volumes operated at different temperatures was performed. The results showed that the concept for continuous compensation of solvent evaporation should be applicable to reaction volumes down to 30 pL.

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A novel method for the manufacture of detection windows in polyimide-coated fused-silica capillary columns is described. Concentrated fuming nitric acid at room temperature is utilized for stripping of the coating from a defined section of the capillary. The technique is particularly suitable for columns that have a heat sensitive coating on the inner wall or for columns that contain an immobilized hydrogel sieving-matrix. A column holder for the simultaneous preparation of detection windows in an array of more than 100 capillaries is also reported.

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Extracts prepared from Antarctic krill (Euphausia superba), mainly consisting of acidic proteolytic enzymes, have been studied with capillary electrophoretic techniques. Approximately 50 repeatable peaks were obtained with capillary zone electrophoresis on an untreated fused-silica capillary using a phosphate buffer containing anionic and cationic fluorosurfactant additives as separation medium. A faster separation was achieved on a polyvinyl alcohol coated capillary. Quantitative variations of individual proteins regarding different krill enzyme batches were noted. In the krill samples trypsin-like serine proteinase, carboxypeptidase A and carboxypeptidase B were tentatively identified.

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In this work, we studied the behavior of electrophoretic columns, having an inner diameter (ID) of 2-10 microm, filled with a cross-linked polyacrylamide gel matrix. The usefulness of these columns for DNA sequencing is discussed. Evaluation of column performance included tests of gel stability and migration time reproducibility. Confocal laser-induced fluorescence (LIF)-detection was employed utilizing a 488 nm argon ion laser for separations of C- and T-terminated DNA Sanger fragments. Reducing the inner diameter of the column from 50 microm to 10 microm resulted in an approximately eightfold increase in lifetime, under conditions in which the columns were subjected to a field strength of 1000 V/cm. The 10 microm ID columns were utilized for separation of Sanger fragments, and adequate detection sensitivity was obtained by stacking of the fragments from a deionized sample solution. A linear algorithm for retention data synchronization between individual electropherograms was employed to provide a route towards a reliable automated base calling protocol.

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The performance of poly(N-acryloylaminopropanol) (poly AAP) gel columns, proved to be stable during electrophoresis at elevated temperature, was investigated. The column manufacturing procedure included the preparation of a coating of the inner wall of the fused silica capillary column with linear poly(AAP). Then, a mixture of the AAP monomer, the cross-linker dihydroxyethylenebisacrylamide (DHEBA) and linear poly(AAP) was introduced into the column and in situ polymerized (for preparation of linear gel columns, the addition of DHEBA was omitted). The poly(AAP) columns were first evaluated by electrophoresis of oligonucleotides at room temperature and at 50 degrees C, utilizing 260 nm UV-absorbance detection. In a further evaluation of column performance, samples of T-terminated DNA Sanger fragments from the bacteria Moraxella were separated at 200 V/cm electrical field strength, utilizing a 488 nm argon ion laser and a confocal optical setup for laser-induced fluorescence (LIF) detection. A temperature increase from 25 degrees C to 50 degrees C effectively released a compression of DNA bands. However, for cross-linked poly(AAP) gel columns, the elevated temperature resulted in a considerable reduction of the DNA sequence reading length. When a linear poly(AAP) column was utilized, no detrimental effect of elevated temperature on the separation could be observed.

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In this study we report the development and performance of a system for continuous-flow extraction of dissolved and colloidal analytes in an aqueous matrix. Initial studies, using a classical segmented-flow extraction procedure, showed poor extraction efficiency for the hydrophobic colloidally dispersed analytes. Insufficient contact between the extractant and the colloidal constituents seems to be the primary reason for poor extraction. Improved performance is obtained when mechanical energy is added to the system, to effect a forced contact between the sample and the solvent. This was accomplished by injecting the extractant, with a high velocity, into the continuous flow of analyte through a narrow-bore nozzle. In this way, the solvent stream is dispersed into fine droplets with high kinetic energies. A region of intense turbulence is created, which was studied by high-speed photography using pulsed laser fluorescence. Comparison with classical flow extraction, using a model sample of colloidal wood resin compounds in water, showed that the dissolved components extracted well with both systems, while an extraction enhancement of up to 9 times was experienced with colloidal triglycerides.

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Atomic force microscopy (AFM) has been used to probe the surface of a capillary after coating with "soft" polymers, notably polyacrylamides. The aim was the investigation of the efficiency of coverage of the silica surface, so as to reduce or eliminate the electroosmotic flow (EOF), particularly noxious in the separation of macromolecules. The quality of such coating is strongly dependent on two variables: temperature and pH. In the first case, progressively higher temperatures produce open silica patches, where no polymer seems to be bound. The transition from coated to largely uncoated surfaces occurs at 50 degrees C. Also the pH of the polymerizing solution strongly affects the coating efficiency. Since in all coating procedures the monomer solution is not buffered, addition of accelerator (TEMED, N,N,N'N'-tetramethylethylendiamine) induces polymer growth at pH 10-11. These pH values generate hydrolysis of the siloxane bridge anchoring the bifunctional agent (Bind Silane, onto which the polymer chain should grow) to the wall. Thus, coating and de-coating occur simultaneously. Low temperatures during polymer growth (typically 10 degrees C) and buffered solutions (pH 7, titrated after TEMED addition) ensure a most efficient and thorough coating, with virtual elimination of EOF: well coated capillaries exhibit residual EOF values, at pH 10, of the order of 10(-7) cm2 V-1 s-1 vs. a standard value for uncoated capillaries of the order of 10(-4) cm2 V-1 s-1. The AFM data have been fully confirmed by direct measurement of EOF in coated and uncoated capillaries under an electric field.

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An attempt was made to separate the isoenzymes and subunits of pig liver esterase by capillary zone electrophoresis. This enzyme is a complex mixture and is strongly adsorbed on a fused-silica capillary. However, by simply adding a cationic fluorosurfactant to the running buffer, adsorption was significantly reduced. The effects of adding a zwitterionic and a neutral fluorosurfactant were also investigated. Large changes in the elution pattern were observed when using different combinations of these additives. Mixtures of different fluorosurfactants added to the running buffer can therefore be utilized in strategies for optimization of the separation selectivity.

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Continuous zone electrophoretic separations in channels have been demonstrated. This new technique has the potential to continuously sample and separate analytes from volume-limited microenvironments. A small-bore capillary is used to electrophoretically sample, but not separate, a mixture of dansylated amino acids. The capillary is coupled to a quartz channel structure in a manner which allows continuous injection of the sampled material into the channel. The channel functions to continuously separate the sampled material via electrophoresis. A laser-induced fluorescence detection scheme, which involves two fiber optic arrays situated at the channel exit, monitors eluting analytes. A continuous separation of dansylated amino acids on the time scale of a few minutes demonstrates the utility of the technique. Sampling has been performed continuously up to 400 s, and initial detection limits are approximately 30 microM.

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