Chapter 1: Introduction to Capillary Electrophoresis as a Sample Preparation Tool
1.4 Discontinuous Buffers – a Response to Enrichment Problems in CE
Recently our research group designed a method to create a prolonged pH junction for the enrichment of submicroliter volume, intact proteins. Enrichment factors of over 1000-fold were reported [44]. The discontinuous buffer system used for protein enrichment consisted of two buffers, an acidic buffer and a basic buffer. The buffers were chosen to provide sufficiently constant concentrations of OH- and H+ and little buffer capacity at neutral pH. The term “discontinuous buffers” was given due to the fact that each buffer had exclusive pH regions of buffering capacity and no buffering capacity existed at the interface between the buffers. Previous data demonstrated that the width of the enriched protein band was predominately controlled by the steepness of the pH gradient between the discontinuous buffers, which in turn was determined by the choice of buffers [49]. The buffers used for this method were not the traditional arrangement of buffers that consists of the buffer and a non-buffering counter ion; e.g., sodium acetate or ammonium chloride. In order to adjust the pH values of these buffers, and not introduce any new ions (with unique mobilities) to the system, the discontinuous buffers were prepared only with ammonium hydroxide and acetic acid, one as the buffering ion, the other as the counter ion. The “ammonium buffer” (with acetate as the counter ion) was prepared with 10 mM ammonium hydroxide (pKa 9.25), using acetate as the counter ion to adjust the pH to
9.75. The “acetate buffer” (with ammonium as counter ion) was prepared in a similar manner, with 10 mM acetic acid and ammonium as the counter ion to adjust the pH to 4.25. In the narrow capillary, the surface area of interaction between the discontinuous buffers was very small. Upon voltage application, this pH junction was sustained during the entire experiment by the OH- and H+ fluxes respectively from the catholyte and anolyte, and this neutralization reaction has given this pH junction the name, Neutralization Reaction Boundary (NRB) [48].
Under normal CE conditions, the EOF would flush the contents out of the capillary in a matter of minutes if the buffer was acidic, or even in seconds if the buffer was basic. However, the inner-capillary wall for our discontinuous buffers system was modified with the semi-permanent, zwitterionic, phospholipid bilayer coating, DLPC, which suppressed the EOF allowing the pH junction to remain in the capillary for well over ten minutes. Since the coating was zwitterionic, neither cationic nor anionic analytes were retained on the wall. Under basic conditions with the zwitterionic coating, the EOF was cathodic (towards the cathode) but slow, while under acidic conditions, the EOF was reversed, but with an even slower mobility. A schematic of a typical experiment with discontinuous buffers is displayed in Figure 1.4 and began with an entire capillary filled with the basic buffer. A slow, forward EOF was observed. As the bulk solution moved towards the cathode, the acidic buffer was drawn into the capillary, leading to an even greater suppression of the forward EOF. The pH junction passed the detector before the slow forward effect under the basic conditions balanced the even slower, reversed effect of the acidic conditions. The net result was a suppressed EOF that allowed for prolonged enrichment and carried the pH junction past the detector.
When amphoteric molecules, such as proteins, with isoelectric points between the two buffer pH values (i.e. 4.25 > pI > 9.75) were introduced, they possessed opposite net charges on either side of the neutralization reaction boundary, and as a result were trapped and enriched at the pH junction. The experimental design is depicted in Figure 1.4 A using myoglobin as a model protein (pI 7). Myoglobin was prepared in the ammonium buffer and was injected to fill the entire capillary (internal volume of 0.95
discontinuous buffers allows an entire capillary full of sample to be enriched. The sample loading is not even limited to one capillary volume, as it has been demonstrated that the sample can be prepared in both the ammonium and acetate buffers, (filling the capillary and in the reservoir vials) leading to 2000-fold enrichment [49]. When the acidic and basic buffers were placed at the anode and cathode, respectively, a sharp pH junction was formed at the interface between the acetate buffer reservoir and the myoglobin- ammonium buffer at the capillary inlet (Figure 1.4 A). Proteins stacked at this pH junction as zwitterions (Figure 1.4 B). A cathodic, suppressed EOF slowly pushed the entire capillary content forward towards the detector (Figure 1.4 C). Thus, an entire capillary full of sample was enriched into a sharp band which was mobilized by the EOF to the detector.
Figure 1.4. Schematic of protein enrichment using discontinuous buffers. A step-pH
junction was created by acetate and ammonium under voltage application. The myoglobin molecules (circles) inside the capillary electromigrated either as cations (in acetate) or anions (in ammonium) toward the junction and were trapped at the junction.
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EOF+
Ammonium Buffer pH 9.75 Acetate Buffer pH 4.25 +-
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EOF+
Ammonium Buffer pH 9.75 Acetate Buffer pH 4.25 Detector-
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Myoglobin in ammonium buffer Myoglobin collects at pH junction-
EOF+
Ammonium Buffer pH 9.75 Acetate Buffer pH 4.25EOF carries pH junction past detector
A
B
Until this point, discontinuous buffer experiments were conducted with pre- purified protein standards, free of contaminating salts [44,49,50]. Thus, to prepare this discontinuous buffer method for real biological samples, the effect of the background matrix, such as ionic salts and buffering ions, must be investigated. The challenges and successful adaptation of discontinuous buffers to samples containing these contaminating ions are discussed in the following two chapters.