Thank you for coming back for more in the Colyer lab at Wake Forest University! In my last update, I had completed a lot of data analysis from by injection pressure and voltage tests using pre-column labeling on the Beckman CE. Afterward, I met with Dr. Colyer to discuss and lay out my next series of tests.
One of the first things we decided to do was alter the injection pressure method by varying the time while leaving the pressure constant. This method did make the resulting graph and its resolution much better and of course more accurate. Sometime slight changes can help.
The next big thing was beginning on-column labeling verses what I have been doing with pre-column labeling. With on-column labeling, the dye is pre-mixed with the buffer in the capillary, and then the fructose is injected by itself. Now that I have switched, I had to make a new buffer solution with the SQ-BA dye within it and then run a long condition of my capillary. Here is where things got a little tricky!!! My data was not coming out as expected – a lot of “noise” from the background and low resolution of the major peaks. After pondering on my methods – I had a eureka moment! I made a new fructose in phosphate solution to inject to start with a fresh solution and pre-filtered the buffer before combining my buffer and dye. When one fills the capillary vials, they filter the solutions; however, when I filtered the buffer and dye solution, the dye was being retained on the nylon filter, therefore not binding and separating with the fructose on the capillary. By pre-filtering, I ensured the buffer solution would be filtered while also ensuring the dye remained in solution.
The problems I was experiencing began to subside with the changes mentioned above. Over the next few days, I ran a series of tests including – buffer/dye only, fructose injection, water plug injection, increased fructose concentration injection, and more. Check back with me to see how they all turned out!
Marie Curie stated, “Nothing in life is to be feared, it is only to be understood. Now is the time to understand more, so that we may fear less.” Basically, when you hit a roadblock – do not give up. Instead, step back, ponder on what you are doing, and you might just figure it out.