Extension of separation range in capillary isoelectric focusing for resolving highly basic biomolecules

The non-availability of commercial carrier ampholytes in the pH range greater than 11 has contributed to difficulties in focusing and resolving highly basic proteins/peptides using capillary isoelectric focusing (cIEF). Two different approaches, involving the use of N,N,N′,N′-tetramethylethylenediamine (TEMED) and ampholyte 9–11, are investigated for their effects on the extension of separation range in cIEF. The addition of TEMED into pharmalyte 3–10 not only prevents the peptides/proteins from focusing in sections of the capillary beyond the detection point, but also extends the separation range to at least isoelectric point (pI) 12. The combination of ampholyte 9–11 with pharmalyte 3–10 surprisingly provides baseline resolution between bradykinin (pI 12) and cytochrome c (pI 10.3). The sample mixture, containing bradykinin, the high-pI protein calibration kit (pI 5.2–10.3), and cytochrome c digest, is employed to demonstrate the cIEF separation of proteins and peptides over a wide pH range of 3.7–12.