Universal liquid chromatographic methods : III. Sensitive, low-wavelength gradients with a novel injection-loading ion-pairing technique

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This paper extends to the ion-pairing mode the “universal methods” previously reported for the reversed-phase mode. These methods offer savings of laboratory and customer time and cost as they often allow “first run problem solving”, reduce method development for each new problem and allow different separation problems to be run in sequence with no system changes. Major and minor components of unknown wide-polarity mixtures are analyzed with the near-universal detection possible at 210 nm. A new technique of injection loading an ion-pairing (IP) agent with an automated liquid chromatograph into simple buffered eluents is introduced. Full gradients from aqueous buffers to acetonitrile are achieved at 0.1 a.u.f.s. and 210 nm without ghost peaks or baseline shifts (less than 5%). Additioally, in three consecutive 12-min runs, the three liquid chromatographic modes (with different selectivities), viz., cation-pairing, anion-pairing and simple reversed-phase, can be explored in order to optimize the separation of a particular sample. The charge of the individual components at the eluent pH can be determined by the movement of peaks in consecutive runs. Other advantages of injection loading are that the pump and column life are preserved, only small amounts of clean ion-pairing agents are required and the effect of new ion-pairing agents on a sample can be investigated in one run. We also present a new technique for achieving flat 210-nm UV baselines. This is accomplished by bubbling a mixture of nitrous oxide (UV absorbing) with helium of a composition that varies the aqueous background UV absorption to match the second gradient eluent, acetonitrile.