Wybraniec Sławomir, Stalica Paweł, Jerz Gerold, Klose Bettina, Gebers Nadine, Winterhalter Peter, Spórna Aneta, Szaleniec Maciej, Mizrahi Yosef
Faculty of Analytical Chemistry, Institute C-1, Department of Chemical Engineering and Technology, Cracow University of Technology, ul. Warszawska 24, 31-155 Cracow, Poland.
J Chromatogr A. 2009 Oct 9;1216(41):6890-9. doi: 10.1016/j.chroma.2009.08.035. Epub 2009 Aug 18.
Polar betacyanin pigments together with betaxanthins from ripe cactus fruits of Hylocereus polyrhizus (Cactaceae) were fractionated by means of preparative ion-pair high-speed countercurrent chromatography (IP-HSCCC) also using the elution-extrusion (EE) approach for a complete pigment recovery. HSCCC separations were operated in the classical 'head-to-tail' mode with an aqueous mobile phase. Different CCC solvent systems were evaluated in respect of influence and effectiveness of fractionation capabilities to separate the occurring pigment profile of H. polyrhizus. For that reason, the additions of two different volatile ion-pair forming perfluorinated carboxylic acids (PFCA) were investigated. For a direct comparison, five samples of Hylocereus pigment extract were run on preparative scale (900 mg) in 1-butanol-acetonitrile-aqueous TFA 0.7% (5:1:6, v/v/v) and the modified systems tert.-butyl methyl ether-1-butanol-acetonitrile-aqueous PFCA (2:2:1:5, v/v/v/v) using 0.7% and 1.0% trifluoroacetic acid (TFA) or heptafluorobutyric acid (HFBA) in the aqueous phase, respectively. The chemical affinity to the organic stationary CCC solvent phases and in consequence the retention of these highly polar betalain pigments was significantly increased by the use of the more lipophilic fluorinated ion-pair reagent HFBA instead of TFA. The HFBA additions separated more effectively the typical cacti pigments phyllocactin and hylocerenin from betanin as well as their iso-forms. Unfortunately, similar K(D) ratios and selectivity factors alpha around 1.0-1.1 in all tested solvent systems proved that the corresponding diastereomers, 15S-type pigments cannot be resolved from the 15R-epimers (iso-forms). Surprisingly, additions of the stronger ion-pair reagent (HFBA) resulted in a partial separation of hylocerenin from phyllocactin which were not resolved in the other solvent systems. The pigments were detected by means of HPLC-DAD and HPLC-electrospray ionization-MS using also authentic reference materials.
采用制备型离子对高速逆流色谱法（IP-HSCCC），并结合洗脱-挤出（EE）方法以实现色素的完全回收，对来自多根量天尺（仙人掌科）成熟仙人掌果实中的极性甜菜青素色素和甜菜黄素进行了分离。HSCCC分离以经典的“头对尾”模式在水相流动相条件下进行。针对分离多根量天尺中出现的色素谱的分馏能力的影响和有效性，对不同的CCC溶剂系统进行了评估。因此，研究了两种不同的挥发性离子对形成全氟羧酸（PFCA）的添加情况。为了进行直接比较，将五个量天尺色素提取物样品以制备规模（900 mg）在1-丁醇-乙腈-0.7% 三氟乙酸（TFA）水溶液（5:1:6，v/v/v）以及改性系统叔丁基甲基醚-1-丁醇-乙腈-PFCA水溶液（2:2:1:5，v/v/v/v）中运行，水相中分别使用0.7%和1.0%的三氟乙酸（TFA）或七氟丁酸（HFBA）。通过使用亲脂性更强的氟化离子对试剂HFBA而非TFA，显著提高了这些高极性甜菜红素色素对有机固定CCC溶剂相的化学亲和力以及保留率。HFBA的添加更有效地将典型的仙人掌色素叶仙人掌素和量天尺素与甜菜红素及其异构体分离开来。遗憾的是，在所有测试的溶剂系统中，相似的分配系数（K(D)）比率和选择性因子α在1.0 - 1.1左右，这证明相应的非对映异构体，即15S型色素无法从15R-差向异构体（异构体）中分离出来。令人惊讶的是，添加更强的离子对试剂（HFBA）导致量天尺素与叶仙人掌素部分分离，而在其他溶剂系统中它们并未分离。使用标准参考物质，通过HPLC-DAD和HPLC-电喷雾电离-MS对色素进行了检测。
