Exposed
How much of a molecule's polarity can a cell membrane actually see?
A journey through supercritical CO₂ to measure it. The method is experimental polar surface area, or EPSA, read from a retention time on the Nexera UC.
In plain terms: EPSA measures how much polar surface a molecule shows to the outside, which helps predict whether a drug can slip through cell membranes. Supercritical fluid chromatography (SFC) is chromatography that uses supercritical CO₂ as its main mobile phase.
Scroll to pressurizeFirst, make
the solvent.
Squeezed at room temperature, CO₂ condenses to a liquid near 5.7 MPa, and the pump delivers it at 20.4 MPa, the pressure the back-pressure regulator holds. In the 40 °C column oven it passes 31.1 °C with the pressure already above 7.38 MPa, and the boundary between liquid and gas disappears. The fluid is now supercritical: dense like a liquid, quick like a gas. Methanol with 20 mM ammonium formate is mixed in as the modifier.
On paper,
278 Ų.
This is cyclosporin A, a cyclic peptide of eleven residues. Topological polar surface area (TPSA) adds up the contribution of every nitrogen and oxygen, each lit here in amber.
By that count it sits far past 140 Ų, the line beyond which oral drugs tend to stop crossing membranes. Yet patients absorb it from a capsule.
In solution,
it hides.
counted
The ring folds. Four internal hydrogen bonds pair N–H with C=O, and the lipophilic side chains close over the polar core. What faces outward is mostly hydrocarbon. EPSA measures only that surface: 62 Ų.
Press and hold to see what TPSA countsPolarity
becomes time.
Nine reference compounds ride the CO₂ through the column, with ritonavir and cyclosporin A alongside. They run from lidocaine (47 Ų) to furosemide (230 Ų). The more polar surface a molecule shows, the longer the stationary phase holds it.
Two ways
to see.
The PDA reads absorbance at 254 nm. The LCMS-2050 reads ions. Cyclosporin A barely absorbs UV, so the PDA misses it, but the mass spectrometer finds it at m/z 1201 [M−H]⁻. Either retention time gives EPSA.
Time becomes
area.
Every morning,
prove it.
EPSA comes straight from retention time, so any drift in the system becomes an error in Ų. Open Solution runs diclofenac at startup. It passes only if it elutes between 2.100 and 2.200 min.
On 2 February the modifier salt was halved from 20 to 10 mmol/L. Diclofenac came out at 2.237 min and the run failed. On the calibration line, a 0.11 min shift is worth about 6 Ų, so results from that day could not be trusted.
Between users the instrument conditions itself, idles at low flow, and shuts down when left alone.