RESUMEN
In the separation of strongly polar antioxidant compounds from natural products using high-speed counter-current chromatography that is target-guided by 2,2-diphenyl-1-picrylhydrazyl high-performance liquid chromatography experimentation, low adsorption ability is encountered due to the strong polarity of the target compounds. In this study, a strategy of novel partition coefficient value calculation was proposed for overcoming this problem. The partition coefficient value was expressed as the ratio of the antioxidant activities of the upper phase and the lower phase. This strategy was used in high-speed counter-current chromatography with a hydrophilic organic/salt-containing aqueous two-phase system for bioassay-guided separation of strongly polar antioxidant compounds from Lycium barbarum L. The antioxidant activity was determined via the radical scavenging activity method using 2,2-diphenyl-1-picrylhydrazyl radicals. A hydrophilic organic/salt-containing aqueous two-phase system of 95% EtOH - sat. (NH4)2SO4 (1:1.8, v/v) was successfully used to separate Lycium barbarum L. extract. Five fractions were collected via high-speed counter-current chromatography separation. The antioxidant activity of the third fraction was the highest. Three compounds were separated via MCI gel column chromatography and Sephadex LH-20 column chromatography from the third fraction, and their antioxidant activities were determined. The antioxidant activities of the three compounds were higher than that of the third fraction. These results demonstrate that this strategy can be used to separate strongly polar antioxidant compounds from natural products.
Asunto(s)
Antioxidantes , Distribución en Contracorriente/métodos , Lycium/química , Extractos Vegetales/química , Antioxidantes/química , Antioxidantes/aislamiento & purificación , Compuestos de Bifenilo , Interacciones Hidrofóbicas e Hidrofílicas , PicratosRESUMEN
The essential attributes of a solvent system for separation polar compounds on CCC are polarity, selectively and performance. Here, hydrophilic organic/salt-containing aqueous two-phase system (HO/S TPS) was evaluated as an alternative solvent system for CCC separation of polar compounds. Polarity measurements based on Rohrschneider-Snyder parameter was developed as quantitative assessing the polarity of HO/S TPS and comparing with an organic/aqueous system. All investigated 1-butanol/ethanol/saturated ammonium sulfate solution/water (BEAsWat) and 1-butanol/ethanol/saturated dipotassium hydrogen phosphate solution/water (BEDhpWat) systems with polarity values of organic phase from 4.5 to 6.8, were more polar than chloroform/methanol/water (1/1/1). The considerable water content of BEAsWat and BEDhpWat (0/1/1/1/) was 45.4 and 42.6% (w%) of hydrophilic organic phase, and 66.4 and 51.2% (w%) of salt-containing aqueous phase, respectively, closed to conventional aqueous two-phase system. Therefore, the polarity of HO/S TPS is in the middle of organic/aqueous and aqueous two-phase system. The LogKC values of twenty four polar compounds as model mixture confirmed that the polarities of HO/S TPSs were matched to that of the polar compounds and shown to be a very selective technique capable of separating positional isomers. Moreover, BEAsWat and BEDhpWat systems can be easily retained in CCC column with suitable elution mode. The hydrodynamic behavior reversion of HO/S TPS on hydrodynamic CCC was observed and was tentatively explained based on the density difference. Finally, caffeoylquinic acid isomers and dihydroxybenzoic acid isomers were successfully separated with HO/S TPS on CCC, respectively. Those results demonstrate that HO/S TPS on CCC is a performant and stable way to separate polar compounds from natural products.
Asunto(s)
1-Butanol/química , Sulfato de Amonio/química , Cloroformo/química , Distribución en Contracorriente/métodos , Etanol/química , Metanol/química , Fosfatos/química , Compuestos de Potasio/química , Cromatografía Líquida de Alta Presión/métodos , Interacciones Hidrofóbicas e Hidrofílicas , Extractos Vegetales/química , Solventes , Agua/químicaRESUMEN
Hydrophilic organic/salt-containing aqueous two-phase system composing of ethanol, water and ammonium sulfate for separation polar compounds was investigated on multilayer coil associated with J-type HSCCC devices. Compared to the classical polar solvent system based on 1-butanol-water or PEG1000-ammonium sulfate-water, the water content of upper phase in ethanol-ammonium sulfate-water systems was from 53.7% to 32.8% (wt%), closed to PEG1000-ammonium sulfate-water aqueous two-phase systems and higher than 1-butanol-water (22.0%, wt%). Therefore, the polarity of ethanol-ammonium sulfate-water is in the middle of 1-butanol-water and PEG-ammonium sulfate-water system, which is quite good for separating polar compounds like phenols, nucleosides and amino acids with low partition coefficient in 1-octanol-water system. The retention of stationary phase in four elution mode on type-J counter-current chromatography devices with multilayer coil column changed from 26% to 71%. Hydrodynamic trend possess both intermediate and hydrophilic solvent system property, which closely related to the composition of solvent system. The applicability of this system was demonstrated by successful separation of adenosine, uridine guanosine and cytidine.