Capillary Electrophoresis

On Thursday Dr. Soria, Chris and I went to San Miniato for a lab on capillary electrophoresis. Filippo Carlucci uses this machine to study nucleotides and other proteins for a variety of reasons, including diagnosis of enzymatic disorders. We had fun preparing samples of fish to analyze the degradation of certain proteins over time. I do not know any biology, so the decomposition of ATP to IMP and on was beyond me, but the physics of the capillary machine was quite interesting.

The main component of the apparatus is a silicon capillary with a bore diameter ranging from 50 to 100 micrometers. Since the surface area to volume ration is so high, the capillary can dissipate heat more easily than more standard gel electrophoresis. This means the apparatus can be run with much higher electric potential difference or voltage. Gel electrophoresis is run around 6 volts, while capillary electrophoresis can be run between 5 and 30 kilovolts. The interior of this capillary is made up of silanol (SiOH) groups that, when deprotonated, form a negatively charged tunnel. At this point an electrolyte buffer in pumped into the capillary. The positive ions in the buffer form a layer of charge over the negative silanate. When the voltage is applied across the length of the capillary, these positive ions migrate from the anode to the cathode and carry the whole solution along. This migration is called the electroosmotic flow (EOF), and is dependant on the strength of the voltage, the buffer used, and the quality of the silicon capillary. When a very small amount of the sample is added to the capillary, it is forced to move with the (EOF). The speed that components travel is dependant on their charge to mass ration. Very small very positive components travel the fastest, and will be detected first. Neutral components travel around the speed of the EOF depending on their mass. Negative components are retained in the capillary the longest due to their attraction to the anode. At the outlet end of the apparatus is a small length of exposed capillary, where UV-vis or other forms of detection can be done.

Capillary electrophoresis (CE) is similar to HPLC is its qualitative determination of components. One advantage to CE is that very little carrier solvent used. Also, in CE the detection method is attached to the separation method, unlike HPLC were the detection is separate. This allows for clearer differentiation of the sample components. The disadvantage is that quantitative analysis is not accurate with CE. Part of this comes from limitations of UV-vis detectors for cells shorter that 1 cm; 50 μm in the case of CE. Also, since the components are not traveling at the same as in HPLC, the integrations of the peaks are not very helpful. Another limitation is that CE cannot be used for physical separation. The sample is just too small to make separation meaningful.

http://en.wikipedia.org/wiki/Capillary_electrophoresis