peak at 4.2 min, nobody can tell me what it is, ideas
Method: C18 150×4.6, 5µm. 0.1% TFA in water / acetonitrile, 20→45% over 25 min. 214nm, 20µL injection. Semaglutide, reseller vial, nothing exotic. Main peak at 11.8 min as expected. There is a reproducible 0.9% peak at 4.2 min that I cannot place. It survives a blank subtraction, so it is not the mobile phase, and it…
4.2 min on that gradient is very early — you are basically still at 20% organic. That is where you find things that barely retain on C18: salts, small polar fragments, excipients, and occasionally the preservative if the vial was reconstituted.
Was it? Because benzyl alcohol elutes early and 0.9% of total area would not be surprising if you injected a reconstituted sample rather than redissolving powder in mobile phase.
oh no. it was reconstituted. with bacteriostatic water.
There it is. That is your benzyl alcohol. Redissolve dry powder in mobile phase and the peak will vanish.
ran it. gone. completely gone. i have spent four evenings on a preservative. edit: leaving this up in full because somebody else will do this and at least now they can find it.
Adding it to the c/hplc wiki under "early peaks that are not your product". This is the third time in two years and the first time anyone wrote it down.
the blank did not catch it because the blank was mobile phase, not bac water. run a bac-water blank as well when the sample was reconstituted.