Capillary electrochromatography (CEC) on columns 25 and 40 cm long, 100 μm i.d., and packed with 3 μm Hypersil ODS was evaluated for the separation of triglycerides in different vegetable oils. The mobile phase optimized for isocratic elution of triglycerides via micropacked liquid chromatography (micro-LC) and composed of acetonitrile/isopropanol/n-hexane in the ratio 57/38/5 could be applied by adding 50 mM ammonium acetate. The influence of the voltage and the temperature on the resolution was studied. The possibilities of peak collection in CEC for further elucidation of the triglyceride structure are illustrated with the identification of triolein in a salad oil by electrospray mass spectroscopy (ESMS). © 1997 John Wiley & Sons, Inc. J Micro Sep 9: 409–419, 1997

No Abstract.

The proteome of the human nucleolus was investigated in a single analysis using off-line strong cation exchange chromatography and microfraction collection combined with HPLC-chip/MS. The analysis was conducted either as a 1-D workflow with HPLC-chip alone or as a 2-D workflow. Two hundred and six unique proteins were identified in the International Protein Index human database corresponding to 2024 unique tryptic peptides identified in the 2-D analysis. In contrast, only 34 proteins and 151 corresponding tryptic peptides were found by applying a 1-D separation strategy. This clearly indicated that the complexity of the samples required the combination of more than one orthogonal separation technique. Stringent database search criteria, including reversal of sequences and therefore better exclusion of false-positive identifications, were applied for reliable protein identification.

A systematic investigation of the influence of the perimeter shape of open and particle packed fused silica capillaries on chromatographic properties such as resistance to flow and dispersion of solutes propelled through these channels has been conducted. Verifications of these uncommon experiments with existing theoretical treatments are presented and the insights transferred to a novel polymer chip design with integrated facilities for complex separations. A comparison of the chromatographic performance of a real life proteomics sample on this chip with a capillary column of “similar” dimensions is presented.

To fulfil the increasing demand for faster and more complex separations, modern HPLC separations are performed at ever higher pressures and temperatures. Under these operating conditions, it is no longer possible to safely assume the mobile phase fluid properties to be invariable of the governing pressures and temperatures, without this resulting in significantly deficient results. A detailed insight in the influence of pressure and temperature on the physico-chemical properties of the most commonly used liquid mobile phases: water–methanol and water–acetonitrile mixtures, therefore becomes very timely. Viscosity, isothermal compressibility and density were measured for pressures up to 1000bar and temperatures up to 100°C for the entire range of water–methanol and water–acetonitrile mixtures. The paper reports on two different viscosity values: apparent and real viscosities. The apparent viscosities represent the apparent flow resistance under high pressure referred to by the flow rates measured at atmospheric pressure. They are of great practical use, because the flow rates at atmospheric pressure are commonly stable and more easily measurable in a chromatographic setup. The real viscosities are those complying with the physical definition of viscosity and they are important from a fundamental point of view. By measuring the isothermal compressibility, the actual volumetric flow rates at elevated pressures and temperatures can be calculated. The viscosities corresponding to these flow rates are the real viscosities of the solvent under the given elevated pressure and temperature. The measurements agree very well with existing literature data, which mainly focus on pure water, methanol and acetonitrile and are only available for a limited range of temperatures and pressures. As a consequence, the physico-chemical properties reported on in this paper provide a significant extension to the range of data available, hereby providing useful data to practical as well as theoretical chromatographers investigating the limits of modern day HPLC.

Microanalytical separation techniques including capillary liquid chromatography, capillary electrophoresis and capillary electrochromatography are suitable for detection of diagnostically important changes in the metabolic profiles of biological fluids. A prototype instrument was employed to serve as an integrated platform for the analysis of urine sample from patients suffering from cancer cachexia. The instrument provides for convenient, rapid and efficient multidimensional approach towards method development which would facilitate simultaneous analysis of complex biological mixtures by the above techniques.

A method based on capillary zone electrophoresis is presented for the determination of the purity of commercial dimeric cyanine dyes (TOTO, YOYO, BOBO, all -1 and -3 species, LOLO-1, POPO-1) that are common as fluorescent probes for nucleic acid staining. These dyes are tetracharged cations, and have a strong tendency to interact with negatively charged centres, where they are rapidly adsorbed, especially from aqueous solutions. Thus anionic sites at the capillary wall must be avoided, and aqueous buffers are not suitable. The method introduced here avoids both complications, using non-aqueous N,N-dimethylacetamide as solvent, and suppressing the dissociation of silanol groups at the capillary surface due to selection of acidic separation conditions (20 mmol/l perchloric acid as background electrolyte). The present method enables the determination of the purity of all 10 dyes in less than 15 min. The selectivity of the method allows separation of at least five main and differentiating a number of unresolved minor contaminants as demonstrated in detail for TOTO-3 as an example. Quantitation (with 100% normalisation of the peak areas) of nine lots of this dye results in a purity between 33 and 87%.

The proteome of the human nucleolus was investigated in a single analysis using off-line strong cation exchange chromatography and microfraction collection combined with HPLC-chip/MS. The analysis was conducted either as a 1-D workflow with HPLC-chip alone or as a 2-D workflow. Two hundred and six unique proteins were identified in the International Protein Index human database corresponding to 2024 unique tryptic peptides identified in the 2-D analysis. In contrast, only 34 proteins and 151 corresponding tryptic peptides were found by applying a 1-D separation strategy. This clearly indicated that the complexity of the samples required the combination of more than one orthogonal separation technique. Stringent database search criteria, including reversal of sequences and therefore better exclusion of false-positive identifications, were applied for reliable protein identification.

A systematic investigation of the influence of the perimeter shape of open and particle packed fused silica capillaries on chromatographic properties such as resistance to flow and dispersion of solutes propelled through these channels has been conducted. Verifications of these uncommon experiments with existing theoretical treatments are presented and the insights transferred to a novel polymer chip design with integrated facilities for complex separations. A comparison of the chromatographic performance of a real life proteomics sample on this chip with a capillary column of “similar” dimensions is presented.

Benzodiazepines, namely flunitrazepam and its three major metabolites, were successfully separated by microemulsion electrokinetic chromatography. Separation was achieved using an untreated fused-silica capillary (48 cm (effective length 40 cm) × 50 νm) at 25 kV; detection was performed by UV at 220 nm. The microemulsion system consisted of 70 mM octane, 800 mM 1-butanol, 80 mM sodium dodecyl sulfate (SDS) and 10 mM borate buffer, pH 9. Very high efficiencies (up to 400 000 plates) and resolution better than 3 were achieved. Since this technique is not compatible with mass spectrometry (MS) detection, a capillary electrochromatographic (CEC) method was developed to separate flunitrazepam and its metabolites. The effects of mobile phase composition and pH as well as voltage and temperature were systematically investigated. The optimized CEC method allowed the baseline separation of the investigated compounds. For the on-line coupling of CEC with electrospray ionization-mass spectrometry, the column was connected to a void fused-silica capillary using a Teflon connection. This configuration was found efficient and suitable for hyphenation of commercial CEC and MS instrumentation using commercially available CEC columns.

A review is presented of the most important recent applications of capillary electrochromatography (CEC) for the analysis of acidic, basic, and neutral compounds, of biomolecules, environmental substances, natural products, pharmaceuticals, and chiral compounds. Packed-column CEC (packed-CEC), open-tubular (OT-CEC), as well as pressure-assisted CEC (pseudo-CEC) are hereby considered. Papers published between July 1999 and April 2001 were taken into account. Applications before July 1999 have been reviewed in Electrophoresis 1999, 20, 3027 135 135–3065.

LC–fluorescence and LC–MS methods have been previously reported for use in decoding bead-based combinatorial libraries. We present the use of capillary electrochromatography (CEC) for highly selective decoding in combination with laser-induced fluorescence (LIF) detection for high sensitivity. The results are compared to prior data obtained using HPLC with fluorescence detection. The use of CEC shows promise for miniaturization and multiplexing for future applications, and the use of LIF detection can allow for detection at sub-pmol amounts.

Small-volume chromatographic columns are only able to generate narrow peaks when flow rates, injection volume and instrument components, such as detector, connecting tubing and fittings, are matched to the peak dispersion from the column. Criteria for the proper design of chromatographic instrumentation are therefore derived from a general model on total dispersion. The performance of such a system is then experimentally evaluated from applications run on narrow-bore, small-volume columns. In order to achieve flow rates that match the dimensions of such columns, a new concept for electronic flow control (EFC) is introduced. A theoretical optimization of column efficiency and throughput is discussed and the results verified with practical examples on short, narrow-bore columns packed with small, porous and superficially porous particles. For complex sample mixtures, the concept of peak capacity is introduced and applied to orthogonal separation principles in multiple chromatographic dimensions through column switching techniques.

Absorption spectrophotometry has been and still is the industry standard for detection in HPLC. Limit of Detection (LOD) and Linear Dynamic Range (LDR) are the primary performance requirements and have driven continuous improvement of spectrophotometric HPLC detectors. Recent advances in HPLC column technology have led to low flow-rate HPLC such as Capillary HPLC and Nano-flow HPLC, and put higher demands on optical HPLC signal detection. However, fundamental principles in spectrophotometric HPLC detection have not been reviewed for many years. In particular the relationship between the detector’s Signal-to-Noise Ratio (SNR) and band broadening needs to be re-evaluated. In this work a new quantitative model is presented which allows the calculation of the trade-off made between chromatographic resolution and SNR in spectrophotometric HPLC detection. Modern optics flow cells based on total internal reflection are included and compared to conventional flow cells.