In my last update, I had completed the spectral studies with UV/Vis and Fluorescence where I collected data for the excitation and emission wavelengths. I found out my excitation wavelength was 618 nm, which helped me to determine the emission wavelength of 656 nm. With those tasks complete, it was time to start forming my Capillary Electrophoresis method. I met with Dr. Colyer to review some research on using a phosphate buffer instead of a carbonate with the SQBA dye, what size capillary to use, and CE methods. After our discussion, I decided to use a 25 μm i.d. (inside dimensions) x 50 cm effective length (length between the inlet and the detector in a CE) capillary. Since my emission wavelength was 656 nm, a 635 laser to excite the fluorophores in the dye would be best for the CE.
Kathryn, a graduate student in the lab, allowed me to shadow her while she explained her method and how to use the instrument software package. This was very interesting…did not know this could be so complicated while yet so simple! It took me a day to create my method for initial conditioning of the capillary for first-time use (very important to do this right), daily conditioning, and my separation method. Daily conditioning consists of flushing the capillary with Deionized-Distilled (DI) water for 10 minutes followed by a 1.0 M NaOH solution for 10 minutes before another 10 minutes of DI water. Finally the last step is to use my “run” buffer, or phosphate buffer at a pH of 10.0. Now the capillary is ready for separation. Separation methods can vary depending upon what one is looking for.
Let’s not get the cart before the horse…I will let you know more about first CE experiments next time! Check back for more exciting stuff here in the lab!