James W. Jorgenson

High-resolution separations based on electrophoresis and electroosmosis

Abstract The related electrokinetic effects of electrophoresis and electroosmosis may be used to achieve high resolution separations. An “instrumental” version of zone electrophoresis carried out in 75 μm I.D. glass capillaries is described. This technique demonstrates excellent resolution of charged substances in a short time. Using virtually the same apparatus, reversed-phase chromatography is performed with electroosmotic pumping of an acetonitrile mobile phase. This technique also produces separations with excellent efficiency. Very low values of reduced plate height are found, which suggests that column packing irregularities are less important when electroosmotic flow is used instead of flow generated by pressure.

Minimizing adsorption of proteins on fused silica in capillary zone electrophoresis by the addition of alkali metal salts to the buffers

Abstract A method for minimizing the adsorption of proteins on fused-silica capillaries in capillary zone electrophoresis has been devised, involving the use of K + concentrations of 0.3 M and above in the operating buffer. The increased ionic strength results in a competition between K + and proteins for cation-exchange sites on the silica surface. The resulting increase in conductivity requires the use of lower voltages and capillaries of smaller diameter to allow adequate heat dissipation. With a voltage of 5 kV applied to a 50-cm capillary filled with a buffer of pH 9 containing 0.25 M potassium sulfate, a separation of five proteins was obtained. Two of these protein bands adsorbed irreversibly without added salt, but showed no apparent adsorption in the presence of 0.25 M potassium sulfate.

Capillary electrophoresis of proteins in buffers containing high concentrations of zwitterionic salts

A method for improving protein separations in capillary zone electrophoresis utilizing high concentrations of zwitterionic buffer additives was examined. Lysozyme and alpha-chymotrypsinogen A were used as test proteins in untreated fused-silica capillaries in buffers of pH ca. 7.0 and 9.0 The zwitterion-containing buffers were compared with buffers containing high ionic salt concentrations and a buffer containing a combination of high ionic salt and high zwitterion concentrations. Over 100,000 theoretical plates were obtained in less than 30 min. for both test proteins in a pH 7 buffer containing both trimethylglycine and potassium sulfate. The advantages and disadvantages of this technique compared with those of other methods used to prevent protein adsorption are discussed.

On-column UV absorption detector for open tubular capillary zone electrophoresis

Abstract A fixed-wavelength on-column UV absorption detector has been developed for open tubular capillary electrophoresis. The detection limits are 15 pg for isoquinoline and 250 pg for lysozyme. Detector response is linear over three to four orders of magnitude for each solute tested. This approach to detector design solves many problems encountered in the use of modified commercial high-performance liquid chromatography detectors. Examples of separations obtained by capillary zone electrophoresis with UV detection are given, including separations in non-aqueous and mixed media.

Liquid chromatography in open-tubular columns. Theory of column optimization with limited pressure and analysis time, and fabrication of chemically bonded reversed-phase columns on etched borosilicate glass capillaries

Abstract A theory is developed which shows that open-tubular columns will have well defined optimum diameters lengths if limits are placed on the available pressure and analysis time. The optimum diameters are shown to be rather insensitive to choice of operating pressure and analysis time, and lie between 1 and 3μm for a wide range of conditions. A method of etching borosilicate glass capillaries to increase surface roughness and column capacity factors is described. Octadecylsilane is chemically bonded to these roughened surfaces and columns are operated in a reversed-phase mode. Detection of solutes is with an on-column fluorescence detector.

Urinary volatile constituents of the house mouse, Mus musculus, and their endocrine dependency

Mouse urine contains a great number of volatile constituents that may be used in chemical communication. Some of these volatiles, identified in this study by combined gas chromatography-mass spectrometry and gas chromatography-Fourier-transform infrared spectroscopy, appear unique to the mouse. Certain urinary volatiles exhibit strong dependence on the sex and endocrine status of the animals, as shown through castration, treatment with an antiandrogen, and hormone supplementation.

Post-capillary fluorescence detection in capillary zone electrophoresis using o-phthaldialdehyde

Abstract A post-capillary fluorescence detection scheme for capillary zone electrophoresis is described using o-phthaldialdehyde (OPA) as the tagging reagent. Use of a coaxial capillary reactor affords mixing of the OPA reagent with migrating zones without excessive zone broadening. The detector is linear over three orders of magnitude and shows detection limits for amino acids and proteins in the femtogram (attomol) range.

Optimization of capillary zone electrophoresis/electrospray ionization parameters for the mass spectrometry and tandem mass spectrometry analysis of peptides

The solution chemistry conditions necessary for optimum analysis of peptides by capillary zone electrophoresis (CZE)/electrospray ionization mass spectrometry and CZE electrospray ionization tandem mass spectrometry have been studied. To maximize the signal-to-noise ratio of the spectra it was found necessary to use acidic CZE buffers of low ionic strength. This not only increases the total ion current, but it also serves to fully protonate the peptides, minimizing the distribution of ion current across the ensemble of possible charge states.The use of acidic buffers protonates the peptides, which is advantageous for mass spectrometry and tandem mass spectrometry analysis, but is problematic with CZE when bare fused silica CZE columns are used. These conditions produce positively charged peptides, and negatively charged silanol moieties on the column wall, inducing adsorption of the positively charged peptides, thus causing zone broadening and a loss in separation efficiency. This problem was circumvented by the preparation of chemically modified CZE columns, which, when used with acidic CZE buffers, will have a positively charged inner column wall. The electrostatic repulsion between the positively charged peptides and the positively charged CZE column wall minimizes adsorption problems and facilitates high efficiency separations. Full-scan mass spectra were acquired from injections of as little as 160 fmols of test peptides, with CZE separation efficiencies of up to 250,000 theoretical plates.

Conformational analysis of membrane proteins in phospholipid bilayer nanodiscs by hydrogen exchange mass spectrometry.

The study of membrane protein structure and enzymology has traditionally been hampered by the inherent insolubility of membrane proteins in aqueous environments and experimental challenges in emulating an in vivo lipid environment. Phospholipid bilayer nanodiscs have recently been shown to be of great use for the study of membrane proteins since they offer a controllable, stable, and monodisperse model membrane with a nativelike lipid bilayer. Here we report the integration of nanodiscs with hydrogen exchange (HX) mass spectrometry (MS) experiments, thereby allowing for analysis of the native conformation of membrane proteins. gamma-Glutamyl carboxylase (GGCX), an approximately 94 kDa transmembrane protein, was inserted into nanodiscs and labeled with deuterium oxide under native conditions. Analytical parameters including sample-handling and chromatographic separation were optimized to measure the incorporation of deuterium into GGCX. Coupling nanodisc technology with HX MS offers an effective approach for investigating the conformation and dynamics of membrane proteins in their native environment and is therefore capable of providing much needed insight into the function of membrane proteins.

Fraction collector for capillary zone electrophoresis

An instrument is described which is capable of collecting fractions from a capillary zone electrophoresis apparatus. The fraction collector is characterized in terms of discretely collecting the separated components of a multi-component sample. In addition, the fraction collector permits the study of the effect of capillary zone electrophoresis on the biological activity of alpha-chymotrypsin.