Electrospray-condensation particle counter: a molecule-counting LC detector for macromolecules

Area of Science:

• Analytical Chemistry
• Biophysical Chemistry
• Separation Science

Background:

• Macromolecular separations require sensitive detection methods.
• Existing detectors often lack the sensitivity or dynamic range for complex biological samples.
• Electrospray ionization coupled with particle counting offers a potential solution for macromolecular detection.

Purpose of the Study:

• To describe a new detector for macromolecular separations.
• To evaluate the detector's performance in terms of sensitivity, linear range, and flow rate compatibility.
• To validate the detector's mechanism using a predictive Monte Carlo model.

Main Methods:

• Electrospraying the chromatographic effluent followed by neutralization.
• Sweeping the neutralized effluent to a condensation particle counter using an air stream.
• Detecting individual macromolecules via light scattering from condensed droplets.
• Utilizing a Monte Carlo model to simulate electrosprayed droplet behavior.

Main Results:

• The detector counts individual macromolecules (MW > 10,000) reporting counts per second.
• Demonstrated a linear range of 4 orders of magnitude for size exclusion chromatography of proteins.
• Achieved detection limits as low as 0.1 microgram/mL.
• Successfully interfaced with low effluent flows (down to 10 nL/min) without makeup flow.
• Monte Carlo model accurately predicted detector behavior based on droplet size.

Conclusions:

• The described detector provides highly sensitive and quantitative detection of individual macromolecules.
• Its wide linear range and low detection limits are advantageous for macromolecular analysis.
• The detector is compatible with low-flow chromatography systems, enabling direct interfacing.
• The underlying physics of electrospray droplet formation and detection are well-understood and predictable.