Separation of linear gramicidins using carbon dioxide-containing mobile phases

Packed-column supercritical-fluid chromatography (pSFC) is presented as a novel method for separating and analyzing gramicidin samples. By use of methanol-modified carbon dioxide as a mobile phase the pentadecapeptides gramicidin A (gA), gramicidin B (gB), and gramicidin C (gC) are readily separated and eluted from a PRP-1 poly(styrene-divinylbenzene) column. Although optimum separation conditions are typically achieved near a column temperature of 40 degrees C, a column pressure of 11 MPa, and 30% methanol modifier, pressure and modifier gradients around these values are also found to improve the overall separation time. Measurements indicate that the mobile phase solubility of gramicidin under these conditions is 5.0+/-0.4 microg mL(-1). Collection of individual peaks during chromatography achieved analytical-scale isolation of 2 microg refined gC from 20 microg injected gramicidin D. Further, supercritical-fluid extraction of 200 microg gramicidin D from a Chromosorb 102 support packed into the vessel produced 57 microg gA in 90% purity. The results establish that carbon dioxide-based mobile phases can be successfully used for the separation of individual gramicidin species.