Capillary electrophoretic analysis of individual submicrometer size particles has been previously done using custom-built instruments. Despite that these instruments provide an excellent signal-to-noise ratio for individual particle detection, they are not capable of performing automated analyses of particles. Here we report the use of a commercial Beckman P/ACE MDQ capillary electrophoresis (CE) instrument with on-column laser-induced fluorescence (LIF) detection for the automated analysis of individual particles. The CE instrument was modified with an external I/O board that allowed for faster data acquisition rates (e.g. 100 Hz) than those available with the standard instrument settings (e.g. 4 Hz). A series of eight hydrodynamic injections expected to contain 32 ± 6 particles, each followed by an electrophoretic separation at -300 V cm-1 with data acquired at 100 Hz, showed 28 ± 5 peaks corresponding to 31.9 particles as predicted by the statistical overlap theory. In contrast, a similar series of hydrodynamic injections followed by data acquisition at 4 Hz revealed only 8 ± 3 peaks suggesting that the modified system is needed for individual particle analysis. Comparison of electropherograms obtained at both data acquisition rates also indicate: (i) similar migration time ranges; (ii) lower variation in the fluorescence intensity of individual peaks for 100 Hz; and (iii) a better signal-to-noise ratio for 4 Hz raw data. S/N improved for 100 Hz when data were smoothed with a binomial filter but did not reach the S/N values previously reported for post-column LIF detection. The proof-of-principle of automated analysis of individual particles using a commercially available CE system described here opens exciting possibilities for those interested in the study and analyses of organelles, liposomes, and nanoparticles.
Bibliographical noteFunding Information:
Rahul Gupta (Department of Electrical Engineering, University of Minnesota) improved the algorithms for data analysis. Pete Carr provided insightful comments on SOT. E.A. acknowledges support from NIH (1K02-AG21453). This work was supported with NIH R01-GM61969.
- Capillary electrophoresis
- Fluorescence detection
- Individual particle analysis
- Latex microspheres