Fahimeh Zarrin

Macromolecule Analysis Based on Electrophoretic Mobility in Air: Globular Proteins

Globular proteins ranging in molecular mass from 5.7 to 669 kDa were separated and analyzed using an aerosol technique based on the electrophoretic mobility of singly-charged molecular ions in air. The ions were produced by electrospraying and drying 100-nm-diameter droplets of a liquid suspension of the proteins, using ionized air to remove the droplet charge due to the spray process. The electrophoretic mobility was measured using a modified commercial continuous-flow differential mobility analyzer operated near atmospheric pressure. An unmodified commercial condensation particle counter was used for detection. The concentrations analyzed ranged from 0.02 to 200 μg of protein/mL of buffer, with a liquid sample flow rate of approximately 50 nL/min. Sampling time of 3 min was used for each complete distribution measured. The electrophoretic mobilities measured were determined entirely from air flow rates, apparatus geometry, and applied potentials. Results were expressed as electrophoretic mobility equivalent diameters using a Millikan formula.

Particle size measurements in the submicron range by the differential electromobility technique: comparison of aerosols from thermospray, ultrasonic, pneumatic and frit-type nebulizers

Abstract The differential electromobility technique was used for the comparison of droplet- and particle-size distributions in the 0.02–0.8 μm range for six nebulization systems often used in inductively coupled plasma (ICP) spectrometry: a thermospray nebulizer coupled to a membrane separator (TNMS); two ultrasonic nebulizers (USNs) used with desolvation; one pneumatic nebulizer (PN) used with and without desolvation; and a frit-type nebulizer. In general, volume concentration (volume of droplets or particles per cubic centimeter of injector gas) increased with NaCl concentration, and it was greater for TNMS followed by USNs compared to other nebulizers. For the desolvated aerosol produced by PN and USN, volume concentration was found to be independent of the temperature (140–180°C) of the heating tube for the desolvation device. As the nebulizer tip temperature in thermospray nebulization was varied from 160 to 240°C, a larger volume concentration of desolvated aerosol was produced. Size distributions shifted towards larger particles with increasing NaCl concentration. The implications of these observations in plasma spectrochemical measurements are discussed.

Laser detectors for capillary liquid chromatography

Abstract The high spatial coherence of cw gas lasers is useful in the development of small volume detectors for capillary liquid chromatography. Three detectors are considered in this manuscript. The first is a very simple, subnanoliter refractive index detector based upon the diffraction of a laser beam by a fluid filled tube. Detection limits of a few nanograms of sugars separated upon a reversed phase Chromatographic column are obtained. The second detector utilizes the crossed-beam thermal lens to produce an absorbance detection limit of 6 × 10−8, measured across the 80-μm square bore flow cuvette. This detector is combined with gradient elution, reversed phase capillary liquid chromatography for sub-femtomole amino acid determination. Last, a light scatter detector provides nearly seven orders of magnitude of dynamic range for capillary hydrodynamic chromatography of sub-micrometer diameter particles.

Measurement of protein rotational motion using frequency domain polarized fluorescence depletion

Polarized fluorescence depletion (PFD) methods (Yoshida, T. M. and B. G. Barisas. Biophys. J. 1986. 50:41-53) are approximately 10(3)-10(4) fold more sensitive than other techniques for measuring protein rotational motions in cell membranes and other viscous environments. Proteins labeled with fluorophores having a high quantum yield for triplet formation are examined anaerobically in a fluorescence microscope. In time domain PFD experiments a several-microsecond pulse of linearly polarized light produces an orientationally-asymmetric depletion of ground state fluorescence in the sample. Monitoring the decay of ground state depletion with a probe beam alternatively polarized, parallel, and perpendicular to the depletion pulse permits the triplet lifetime and rotational correlation time to be resolved and evaluated. We have now explored fluorescence depletion methods in the frequency domain to see whether such measurements could provide simpler and more efficient routine measurements of protein rotational relaxation than previous time domain PFD methods. An acousto-optic modulator (AOM) modulates the intensity of a 514.5 nm argon ion laser beam and a Pockels cell (PC) rotates its plane of polarization. These devices are driven by sinusoidal or square waves in fixed frequency relation, and rigidly phase locked, one to another. The fluorescence emitted from a sample then contains various overtones and combinations of the AOM and PC frequencies. The magnitude and phase of individual fluorescence signal frequencies are measured by a lock-in amplifier using a reference also phase-locked to both the AOM and PC. Specific frequencies permit evaluation of the rotational correlation time of the macromolecule and of the fluorophore triplet state lifetime, respectively. Measurement of bovine serum albumin rotation in glycerol solutions by this method is described.

Laser doppler velocimetry for sub‐micrometer particle size determination

The modulated light scattering signal produced in laser Doppler velocimetry may be used to measure particle size with excellent noise immunity. However, high precision particle size measurement is only possible if particles are constrained to flow through the center of the interference fringe region at a constant velocity. The sheath flow cuvette is used in the biomedical technique of flow cytometry to constrain particles to flow in a thin stream through a laser beam. This cuvette is combined with laser Doppler velocimetry for particle size determination. Polystyrene spheres ranging in size from 45‐nm to over 1‐μm radius may be analyzed with a resolution limited by the inherent size distribution of the standards.