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The compounds were eluted with a gradient elution of mobile phase A (100% acetronitrile) and B (1% acetic acid in deionized water) where A increased from 3%to8%8% in 5 minutes, to 10% by 25 minutes and was maintained at 10% for 20 minutes, then returned to initial condition (3%) in 10 minutes and remained for 5 minutes before next injection.
Hydrocarbons and other non-polar compounds were eluted with petroleum ether (boiling point, 60-80°C 60-80°C
The active compounds were eluted at 5.1 min (S14-1), 6.3 min (S14-2), and 7.9 min (S14-3), respectinely, isocratictic 95% acetonitrile by RP-HPLC.
The extraction solution was pumped into the sample loop and then introduced into the CCC column; the target compounds were eluted with the lower aqueous phase of the two-phase solvent system.
Compounds were eluted from the column with methanol under gravity, and the solvent was removed under low temperature (4°C) under vacuum (Rocket Evaporator System, Genevac, Stone Ridge, NY, USA).
At the second stage, the upper phase of the solvent system of ethyl acetate n-butanol methanol water (3.1:0.6:1.0:2.6, v:v:v:v) wacetate n-butanol methanol waterand HSCCC stationary phase (extracted at 110 °C), and the target compounds were eluted with the corresponding lower phase of the solvent system to separate the moderate polar compounds.
Similar(7)
Lisinopril (LIS) was used as internal standard for the purpose of quantification of FEX and its four related compounds, being eluted at 7.117 min with sharp and symmetric peak.
The volatile compounds are eluted first under isobaric conditions (1500 psi) in less than a minute followed by a linear gradient of the column back pressure (from 1500 to 3500 psi in 5 min) for the elution of the non-volatile compounds up to C40.
By GPC, compounds are eluted in the order of the reverse of their molecular sizes (i.e., largest first).
As depicted in the figure, relatively low affinity compounds are eluted as they are already eluted without the infusion of a competitor.
For longer runs, additional calibrant can be injected using a six-port valve and an infusion pump during the time when no compounds are eluted.
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