Reaction Monitoring of Aliphatic Amines in Supercritical Carbon Dioxide by Proton Nuclear Magnetic Resonance Spectroscopy and Implications for Supercritical Fluid Chromatography Holger Fischer, ² Olle Gyllenhaal, ‡ Jo 1 rgen Vessman, ‡ and Klaus Albert* ,² Institut fu ¨ r Organische Chemie, Universita ¨ t Tubringen, Auf der Morgenstelle 18, D-72076 Tu ¨ bingen, Germany, and AstraZeneca R&D Mo ¨ lndal, S-431 83 Mo ¨ lndal, Sweden In the recent years, it has repeatedly been stated that amines react with CO 2 and can therefore not be chro- matographed under supercritical conditions with CO 2 . The aim of the present work is to elucidate the structural requirements and conditions that can lead to the reaction of an amine analyte with CO 2 and, if this occurs, the structure of the formed product. The use of on-line nuclear magnetic resonance (NMR) spectroscopy with a flow probe for supercritical fluid chromatography (SFC) enables the investigation of these unstable analytes in supercritical mediums. Several alkyl-substituted second- ary benzylamines and some primary aromatic amines were dissolved in supercritical CO 2 and investigated by employing on-line SFC- 1 H NMR spectroscopy. It was found that the condition of carbamic acid formation depends on the steric properties of the substituents of the amine. A 2 -isopropylamino alcohol compound, meto- prolol, was also investigated with the setup. No carbamic acid could be detected with the present conditions. For many years there has been an ongoing debate in the chromatographic community using supercritical fluid chromatog- raphy (SFC) about the reaction of amines with supercritical CO 2 (scCO 2 ) and whether amines can be separated under SFC conditions with CO 2 without complications. 1 A lot of investigations of the separation behavior of aliphatic amines in SFC have been performed in capillaries and in packed columns. In capillary SFC, aliphatic amines showed a similar retention behavior and peak shape. 2,3 Packed column SFC was used for the analysis of metoprolol, an amino alcohol, and analogues with 10% methanol in scCO 2 and diol-silica as support. Triethylamine was used as a basic additive to ensure good peak symmetry. 4 With a similar mobile-phase composition and porous graphitic carbon as support, a baseline elevation was observed for the analyte peak metoprolol. The size of the area of this part of the chromatogram correlated with the temperature of the column oven. Chiral separations of similar analytes were reported and proposed to be enhanced by the aid of CO 2 and a cyclic transient complex formed with CO 2 and the amino alcohol. 5 These sugges- tions were supported by off-line nuclear magnetic resonance (NMR) experiments by bubbling CO 2 through an analyte solution in the NMR probe. 6 With the increasing use of semipreparative SFC, artifacts have been encountered when isopropylamine is used as a basic additive and the collected fractions are evaporated to dryness. 7 The principal reaction of amines with CO 2 to carbamic acids is well known and therefore used for absorption of acid gases. 8 A mixture of piperazine and methyldiethanolamine patented by BASF 9 is widely used in gas absorbers, where the secondary amine piperazine reacts with CO 2 to form the corresponding carbamic acid and derivatives. The stability of aminopropyl silica in scCO 2 was studied by Bayer et al. 10 At 150 bar, 100 °C, and 3-15 h reaction time, the formation of carbamic groups was proposed based on solid-state NMR spectroscopy studies. The new phase showed different and improved selectivity for the analysis of unsaturated triglycerides. Pinkston and Baker developed an ion spray interface for capillary SFC-MS. But due to the presence of CO 2 in the ion source, the M + 44 ion observed from didodecylamine could not be unambiguously assigned to corresponding carbamic acid dervatives. 11 Leitner et al. 12 reported the formation of a white insoluble solid in the iridium-catalyzed enantioselective hydration of imines in * Corresponding author. E-mail: klaus.albert@ uni-tuebingen.de. † Universita ¨ t Tubringen. ‡ AstraZeneca R&D Mo ¨ lndal. (1) Fields, S. M.; Grolimund, K. J. High Resolut. Chromatogr. Commun. 1988 , 11, 727-729. (2) Gyllenhaal, O.; Vessman, J. J. Chromatogr. 1990 , 516, 415-426. (3) Baastoe, M. B.; Lundaners, E. J. Chromatogr. 1991 , 558, 458-463. (4) Gyllenhaal, O.; Vessman, J. J. Chromatogr. 1999 , 839, 141-148. (5) Siret, L.; Bargman, N.; Tambute ´, A.; Caude, M. Chirality 1992 , 4, 252- 262. (6) Bargman-Leyder, N.; Sella, C.; Bauer, D.; Tambute ´, A.; Caude, M. Anal. Chem. 1995 , 67, 952-958. (7) Villeneuve, M.; Lefler, J. L. Lecture 3, 10th Int. Symp. SFC SFE; Myrtle Beach, SC, August 19-22, 2001. (8) Bishnoi, S.; Rochelle, G. T. Chem. Eng. Sci. 2000 , 55, 5531-5543. (9) Appl, M.; Wagner, U.; Henrici, H. J.; Kuessner, K.; Volkamer, F.; Fuerst, E. U.S. Patent 4336233, 1982. (10) Zhang, S.; Nicholson, G.; Schindler, B.; Bayer, E. 18th Int. Symp. Capillary Chromatogr.; Riva del Garda, Italy, 1996; pp 1785-1790. (11) Pinkston, J. D.; Baker, T. R. Rapid Commun. Mass Spectrom. 1995 , 9, 1087. Anal. Chem. 2003, 75, 622-626 622 Analytical Chemistry, Vol. 75, No. 3, February 1, 2003 10.1021/ac020527p CCC: $25.00 © 2003 American Chemical Society Published on Web 01/01/2003