Solid-state UV laser-induced fluorescence detection in capillary electrophoresis.

Two solid-state UV lasers were applied to the laser-induced fluorescence (LIF) detection of various groups of compounds after separation by capillary electrophoresis. These lasers are thermoelectric-cooled, highly compact, and inexpensive. Such lasers provide few mW of quasi-continuous wave (CW) power which are sufficient and stable for LIF detection. Native fluorescence detection of tryptophan-containing proteins and peptides and related indoles was achieved at the nM level with the laser operating at 266 nm. Detection of fluorescamine-labeled amino acids and peptides was also possible at the nM level with the laser operating at 355 nm. Amino acids at a concentration as low as 10 ng/mL could be labeled with fluorescamine. Solid-state UV-LIF detection of the tryptic digest of cytochrome c after fluorescamine derivatization was demonstrated.

A simple two-dimensional high performance liquid chromatography/high performance capillary electrophoresis set-up for the separation of complex mixtures.

A two-dimensional high performance liquid chromatography/capillary electrophoresis (HPLC/CE) instrumental set-up was assembled from commercially available equipment. Fractions of the effluent from the HPLC system are collected into microtiter plates with a microfraction collector. The fractions are then dried under vacuum at room temperature, reconstituted, and analyzed by capillary zone electrophoresis (CZE). This method allows the collection of samples by time, drops, or external signal (peaks). Any size or type of HPLC or CE column can be used with no limitation on the amount of sample injected into the HPLC. Any CE detection, laser-induced fluorescence (LIF), mass spectrometry (MS), ultraviolet (UV) or other, can be used. This set-up is practical, simple, robust and allows the separation of complex mixtures. Preliminary results show the utility of this system for the analysis of protein digest.

Peptide mobility and peptide mapping in capillary zone electrophoresis. Experimental determination and theoretical simulation.

The electrophoretic mobilities of 58 peptides that varied in size from 2 to 39 amino acids and varied in charge from 0.65 to 7.82 are presented. The measurements were conducted at 22 degrees C using a 10% linear polyacrylamide-coated column and a 50 mM phosphate buffer at pH 2.5. Excellent separation of peptides and highly reliable peptide maps of protein digests are routinely obtained using these experimental conditions. The electrophoretic data were used to test existing theoretical models that correlate electrophoretic mobility with physical parameters. The results indicate that the Offord model that correlates electrophoretic mobility with the charge-to-size parameter q/M2/3 offers the best fit of our reliable experimental data. Furthermore, we also obtained the capillary zone electrophoretic profile of the endoproteinase Lys-C digests of a peptide sequencing standard, melittin, and horse myoglobin under the same experimental conditions as described above. The resulting peptide maps were compared with corresponding theoretical simulation.

High-speed electrophoretic separation of DNA fragments using a short capillary.

Capillary electrophoresis using a replaceable gel buffer was applied to the separation of DNA fragments. A short effective length capillary (1-2 cm) at low electric field allowed the separation of a 20-1000 bp ladder in 1 min. Although similar separation speed was achieved with a longer capillary at high field, the resolution of larger fragments was degraded. The short effective length capillaries were able to separate the wildtype and mutant PCR products of the TGF-beta1 gene in under 45 s.

The effect of column length, applied voltage, gel type, and concentration on the capillary electrophoresis separation of DNA fragments and polymerase chain reaction products.

This work examines the effect of different parameters on migration time, resolution, and speed of analysis of DNA fragments and PCR products. These parameters include column length, applied voltage, gel type and concentration, and buffer ionic strength. Our results indicate that 1 cm capillary at an applied voltage of 185 V/cm, filled with commercial gel, was adequate for the separation of small DNA fragments in under 1 min. Resolution of large fragments is directly proportional to column length at the same field strength. Also, resolution of large fragments is higher (better) at lower field strength at constant column length. Analysis is fastest (high throughput) using a short capillary and moderate field strength (200 v/cm). CE using a single short capillary (2-7 cm) is comparable to slab gel in throughput, but more economical. The Sigma DNA buffer and hydroxyethyl cellulose liquid gel gave equivalent results in terms of resolution and reproducibility. The Sigma DNA replaceable gel gave reproducible results when used as received or diluted at 60%. In our hands hydroxyethyl cellulose gave more reproducible results than polyacrylamide gel.

Electrokinetic chromatography without electroosmotic flow.

This review summarizes the various aspects of conducting electrokinetic chromatography in coated columns with suppressed electroosmotic flow. The specific features of the technique will be presented and the potential applications explored. The equations of migration, resolution and zone spreading for neutral solutes will be presented, compared, and contrasted with those of conventional electrokinetic chromatography in bare-silica columns. The principle of separation is the same in electrokinetic chromatography with or without electroosmotic flow; however, there are many significant differences that will be highlighted.

High-speed screening of polymerase chain reaction products by capillary electrophoresis.

In an effort to develop capillary electrophoresis (CE) for high-throughput polymerase chain reaction (PCR) molecular diagnostics, a method was developed to rapidly screen small PCR products of similar molecular weights. The assay of interest required the separation of two PCR products (375 and 400 bp) in an assay of TGF-beta 1 knockout mice to determine the genotype of neonates. Using a commercially available CE instrument, the two PCR products were separated in 12 min with a replaceable gel buffer, a 20-cm effective length DB-17 capillary, and 185 V/cm field strength. With the coinjection of a 20-bp ladder, the sizes of the PCR products were determined from the electropherogram without using a calibration plot and curve-fitting program. Faster separation was obtained using the combination of a short effective length capillary and high field strength. The two PCR products were separated in 82 s with a 7-cm effective length capillary and 556 V/cm. A 60% buffer further reduced the separation time in about a minute. This high-speed separation, with minimum postrun data processing, is highly desirable for the high-throughput screening of PCR products using a single-capillary CE system.

Electrokinetic chromatography in suppressed electroosmotic flow environment: use of a charged cyclodextrin for the separation of enantiomers and geometric isomers.

Electrokinetic chromatography (EKC), with negatively-charged cyclodextrins (NCDs) added to the buffer, was conducted in polyacrylamide-coated columns under suppression of electroosmotic flow. The equations of migration and resolution for neutral solutes in this mode of chromatography, which for brevity we term NCD-EKC, are presented. The chiral sulfated cyclodextrin, beta-CD-SBE (IV), used in this study is anionic over the entire pH range accessible to capillary electrophoresis, and the coated columns are stable and provide reproducible performance in the pH range 2.5-8.8. Optimum separation was obtained in the pH range where the solutes are neutral. The incorporation of an alkyl spacer between the sulfate ion and the rim of the cyclodextrin allows an unhindered approach and inclusion of neutral solutes in the cyclodextrin cavity. Solute migration time is inversely proportional to the concentration of the chiral selector. Separation (relative migration time difference) increases with decreasing chiral selector concentration and approaches a maximum, beyond which further decreases in chiral selector concentration result in broad peaks and loss of resolution. A chiral selector concentration of 1% in a 10 mM phosphate buffer produced excellent separation of amino acids and dipeptide enantiomers. In addition to being chiral selectors, cyclodextrins are also known as shape selectors. NCD-EKC is particularly suited for the separation of positional isomers of hydrophobic solutes. The separation of aflatoxin isomers and chlorophenol congeners is presented. In the separation of chlorophenols the more hydrophobic trichlorophenols eluted first and the least hydrophobic, phenol, eluted last.

Micellar electrokinetic chromatography in zero-electroosmotic flow environment.

Micellar electrokinetic chromatography (MEKC) is conducted in polyacrylamide-coated capillaries under almost complete suppression of electroosmotic flow. The equations of migration and resolution for neutral solutes in this mode of MEKC operation are presented. The technique is termed reversed-flow MEKC (RF-MEKC) because, in contrast to MEKC in bare-silica capillaries (N-MEKC), solute migration order is reversed and solute migration time is inversely proportional to micelle concentration. This presents an advantage for the high-efficiency separation of extremely and moderately hydrophobic solutes in a short analysis time. Examples of the separation of polycyclic aromatic hydrocarbons, aflatoxins and dansylated-amino acids are presented using sodium dodecyl sulfate (SDS) surfactant. Polycyclic aromatic hydrocarbons are separated using a relatively low micelle concentration. The detection sensitivity for these compounds is enhanced in two ways. First, the peaks are sharp because of the short analysis time and the inertness of the column surface. Second, the fluorescence background and Joule's heating are minimal because of the low concentration of SDS and other additives needed to affect the separation. While N-MEKC is mainly conducted with basic buffers, RF-MEKC can be conducted in basic as well as acidic media as illustrated in the separation of 15 dansylated-amino acids at pH 4.2.

Separation of tryptophan and related indoles by micellar electrokinetic chromatography with KrF laser-induced fluorescence detection.

Micellar electrokinetic chromatography (MEKC) was applied for the separation of tryptophan and related indoles. Using a 5 mM sodium borate buffer (pH 9.2) containing 50 mM sodium dodecyl sulfate and 5% acetonitrile, eleven indoles were baseline separated in under 17 min. Most of the indoles were detected at the nM level by native fluorescence using KrF laser-induced fluorescence (LIF), which was approximately 100 times more sensitive than UV absorption detection at 200 nm. Preliminary results show that the MEKC-LIF with direct sample injection is a feasible method for assessing indole profiles in diluted urine and serum.

Production of brefeldin-A.

Fermentation conditions are described for the production of the antitumor antibiotic 7-(S)-brefeldin-A (brefeldin-A) in liquid culture by Eupenicillium brefeldianum, (B.Dodge) Stolk and Scott, ATCC 58665. An analytical hplc method was developed which allowed rapid quantitation of the compound during fermentation. A kilogram of brefeldin-A was isolated from a fermentation at the 6800-liter scale.

Enantiomeric separation of amino acids using micellar electrokinetic chromatography after pre-column derivatization with the chiral reagent 1-(9-fluorenyl)-ethyl chloroformate.

Direct enantiomeric separations of some racemic amino acids derivatized with 9-fluorenylmethyl chloroformate were obtained using cyclodextrin-modified micellar electrokinetic chromatography (CD/MEKC) with a buffer made up of 5 mM sodium borate (pH 9.2), 150 mM sodium dodecyl sulfate (SDS) and 40 mM gamma-CD. Alternatively, enantiomeric separations were also achieved indirectly using MEKC after pre-column derivatization with (+)-1-(9-fluorenyl) ethyl chloroformate (FLEC). Using either a 10 mM sodium phosphate (pH 6.8) or a 5 mM sodium borate buffer (pH 9.2), each of which contained 25 mM SDS and 10-15% of acetonitrile, FLEC-derivatized serine, alanine, valine, methionine, leucine, phenylalanine, tryptophan, and their diastereomeric pairs were all separated: the L-isomers migrated faster than the corresponding D-isomers. However, when (-)-FLEC was used for derivatization, the D-isomers migrated faster than the corresponding L-isomers. Also, the diastereomers of aspartic acid, glutamic acid, and proline were resolved using a 10 mM sodium citrate buffer (pH 4.4). Using KrF (248 nm) laser-induced fluorescence, the detection limit of (+)-FLEC derivatized DL-amino acids was obtained at the nM level, which was about 100 x more sensitive than UV absorption at 200 nm. Analyte concentrations as low as 3 x 10(-8) M (DL-Val) could be derivatized with (+)-FLEC.

Separation of estrogens by micellar electrokinetic chromatography.

Capillary electrophoresis of the sex hormone estrogens using different buffer components was investigated. Free zone electrophoresis with 10 mM phosphate buffer (pH 11.5) or 10 mM phosphate buffer with 10-20% methanol was not effective in separating the ten estrogens used in this study. However, nine estrogens were resolved by micellar electrokinetic chromatography using a 10 mM borate buffer (pH 9.2) containing 100 mM sodium cholate. In addition, some estrogens were partially separated using sodium dodecyl sulfate (SDS) micellar buffers; however, the addition of modifiers such as organic solvents or cyclodextrins improved resolutions significantly. Using a 10 mM phosphate buffer (pH 7.0) containing 50 mM SDS and 20% methanol, or a 10 mM borate buffer (pH 9.2) containing 50 mM SDS and 20 mM gamma-cyclodextrin, all ten of the tested estrogens were separated. However, the cyclodextrin-modified buffer allowed faster separation.

Micellar electrokinetic chromatography of the anti-human immunodeficiency virus (HIV) agent michellamine B with absorption and laser-induced fluorescence detection.

Micellar electrokinetic chromatography (MEKC) was applied to the separation of the anti-HIV agents, michellamines A and B, and two other structurally related monomers found in the extract of the Ancistrocladus plants. Using buffers containing either 10 mM sodium phosphate (pH 7.0), 50 mM sodium deoxycholate and 10-20% acetonitrile or 5 mM sodium phosphate (pH 7.0), 20 mM sodium dodecyl sulfate and 25% acetonitrile allowed baseline separations of the four components in the mixture in less than 10 min. The MEKC methods gave sharper peaks and better resolution compared to high-performance liquid chromatography. For MEKC separation of the plant extracts, UV absorption detection provided adequate sensitivity; however, higher sensitivity could be achieved with UV laser-induced fluorescence detection (LIF). Using the sodium dodecyl sulfate-containing buffer and LIF, the limit of detection for michellamine B was approximately 2 ng/mL. The sensitivity was degraded approximately 100-fold when using the deoxycholate buffer because of high background fluorescence. Preliminary results show that MEKC with LIF is feasible for the sensitive detection of michellamine B in serum.

High-performance liquid chromatography and micellar electrokinetic chromatography of taxol and related taxanes from bark and needle extracts of Taxus species.

High-performance liquid chromatography (HPLC) and micellar electrokinetic chromatography (MEKC) were applied for the separation of taxol, cephalomannine, and baccatin III in crude extracts from the needle and bark of Taxus species. The chromatogram of the bark extract was cleaner than that of the needle allowing a more reliable detection of taxol and cephalomannine in the bark extract. However, HPLC quantitation of taxol in the needle extract would be difficult due to coeluting taxinines. Nevertheless, this was not a problem in the MEKC experiment. In comparison to HPLC, MEKC offered baseline resolution of taxol from taxinines in the needle extract, less solvent waste, a smaller sample requirement, and the simultaneous detection of taxol, cephalomannine and baccatin III in a relatively simpler electrophoretic run.

Analysis of nitrate and nitrite in water and urine by capillary zone electrophoresis.

A capillary zone electrophoresis method for the separation and analysis of nitrate and nitrite in water and urine was developed. No interference in the electropherogram from other anions is observed by using a polyacrylamide-coated column with a modified phosphate buffer at pH 3 for the separation, and UV absorption at 214 nm for the detection. The method does not require sample pretreatment or the use of organic solvents. The limit of detection for each analyte (S/N = 3), using a 75 microns I.D. capillary, is 0.5 microgram/ml. Urine samples require 40-fold dilution in order to maintain migration time reproducibility to within 1% relative standard deviation.

Micellar electrokinetic chromatography of hydroxyproline and other secondary amino acids in biological samples with laser-induced fluorescence detection.

Micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) was used for the rapid and sensitive detection of hydroxyproline in serum and hydrolyzed urine that were pre-column derivatized with 9-fluorenylmethyl chloroformate (FMOC). The application of the combined o-phthalaldehyde (OPA)/FMOC derivatization in MEKC for the selective detection of secondary amino acids in biological samples is investigated.

Separation of pyridinecarboxylic acid isomers and related compounds by capillary zone electrophoresis. Effect of cetyltrimethylammonium bromide on electroosmotic flow and resolution.

The effect of the addition of cetyltrimethylammonium bromide (CTAB) to the buffer system in capillary electrophoresis on electroosmotic flow (EOF) is examined. At a CTAB concentration of 2.5 x 10(-4) M, EOF is anodal (flow towards the positive detector column end). With bare silica columns, anodal EOF first increases with increasing pH, up to a maximum in the pH range 4-6 depending on CTAB concentration, then decreases as pH is further increased. Optimum resolution of pyridinecarboxylic acid isomers is obtained at pH 2.7 with a 10 mM phosphate buffer and 30 mM CTAB. Using the same buffer system, optimum resolution for hydroxy-substituted pyridinecarboxylic acid isomers is obtained at pH 7.5. The use of CTAB results in a dramatic improvement in peak shape. Preliminary results, using an excimer laser operated at 248 nm, show that the fluorescence intensity of isonicotinic acid is substantially enhanced with the addition of 0.3% hydrogen peroxide to the phosphate buffer system.

Laser-induced fluorescence detection of 9-fluorenylmethyl chloroformate derivatized amino acids in capillary electrophoresis.

Laser-induced fluorescence (LIF) was applied to the detection of 9-fluorenylmethyl chloroformate (FMOC-Cl) derivatized amino acids separated by capillary electrophoresis. Fluorescence excitation was provided by a pulsed, KrF laser operating at 248 nm. A limit of detection of 5 x 10(-10) M was obtained for FMOC-alanine (S/N = 2). Separation of FMOC-derivatized proline, hydroxyproline, and sarcosine was achieved with a 20 mM borate buffer (pH 9.2), and the separation of FMOC-derivatized amino acid standard mixture was obtained using a 20 mM borate buffer (pH 9.2) containing 25 mM sodium dodecyl sulfate.

Taxol: quantitative internuclear proton-proton distances in CDCl3 solution from nOe data: 2D nmr ROESY buildup rates at 500 MHz.

Quantitative nmr internuclear proton-proton distance measurements obtained by observation of the initial buildup rates of nOe's in 2D ROESY spectra of taxol [1] in CDCl3 are reported. A comparison to the X-ray crystal structure of taxotere [2] is made, and the results are discussed in terms of previous studies of structure-activity relationships.