Two-Step Separation of Nostotrebin 6 from Cultivated Soil Cyanobacterium (Nostoc sp.) by High Performance Countercurrent Chromatography
Abstract
:1. Introduction
2. Results and Discussion
2.1. HPLC/DAD-ESI-HRMS Analysis of the Crude Extract
2.2. Optimization of HPCCC Conditions
System no. | Composition | Relative Proportions of Solvents (v/v/v/v) | Phase Volume Ratio | Settling Time (s) | Partition Coefficient (K) |
---|---|---|---|---|---|
1 | n-hexane–ethyl acetate–MeOH–water | 6:4:5:5 | 0.72 | 18 | 0.10 |
2 | n-hexane–ethyl acetate–MeOH–water | 5:5:5:5 | 0.67 | 19 | 0.21 |
3 | n-hexane–ethyl acetate–MeOH–water | 4:5:4:5 | 0.70 | 17 | 0.85 |
4 | n-hexane–ethyl acetate–MeOH–water | 3:5:3:5 | 0.82 | 23 | 1.47 |
5 | n-hexane–ethyl acetate–MeOH–water | 2:5:2:5 | 0.94 | 24 | 1.62 |
6 | n-hexane–ethyl acetate–MeOH–water | 1:5:1:5 | 1.00 | 23 | 3.53 |
7 | EtOAc–water | 5:5 | 0.86 | 20 | 12.40 |
8 | EtOAc–n-BuOH–water | 4:1:5 | 0.91 | 60 | 23.71 |
9 | EtOAc–n-BuOH–water | 3:2:5 | 0.99 | 38 | 16.57 |
10 | EtOAc–n-BuOH–water | 2:3:5 | 1.09 | 55 | 17.53 |
System no. | Partition coefficient (K) | Separation factor (α) | Density difference (LP−UP g/mL) | |
---|---|---|---|---|
K a | K b | |||
3 | 0.85 | 0.90 | 1.06 | 0.09 |
3 c | 0.51 | 0.80 | 1.57 | 0.12 |
2.3. Separation of Nostotrebin-6 by HPCCC and Structural Identification
3. Experimental
3.1. Reagents
3.2. Cultivation of Nostoc sp. str. Lukešová 27/97
3.3. Preparation of the Crude Extract and Sample Solution
3.4. Apparatus
3.4.1. Countercurrent Chromatography
3.4.2. HPLC/DAD-ESI-HRMS
3.5. Selection of the Two-Phase Solvent Systems for HPCCC
3.6. Preparation of the Two-Phase Solvent System and Sample Solution for HPCCC Separation
3.7. HPCCC Separation Procedure
3.8. HPLC/DAD-ESI-HRMS Analysis and Identification of HPCCC Peak Fractions
3.9. Structural Identification
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Cheel, J.; Kučerová, P.; Garrard, I.; Ignatova, S.; Hrouzek, P.; Kopecký, J. Two-Step Separation of Nostotrebin 6 from Cultivated Soil Cyanobacterium (Nostoc sp.) by High Performance Countercurrent Chromatography. Molecules 2014, 19, 8773-8787. https://doi.org/10.3390/molecules19078773
Cheel J, Kučerová P, Garrard I, Ignatova S, Hrouzek P, Kopecký J. Two-Step Separation of Nostotrebin 6 from Cultivated Soil Cyanobacterium (Nostoc sp.) by High Performance Countercurrent Chromatography. Molecules. 2014; 19(7):8773-8787. https://doi.org/10.3390/molecules19078773
Chicago/Turabian StyleCheel, José, Petra Kučerová, Ian Garrard, Svetlana Ignatova, Pavel Hrouzek, and Jiří Kopecký. 2014. "Two-Step Separation of Nostotrebin 6 from Cultivated Soil Cyanobacterium (Nostoc sp.) by High Performance Countercurrent Chromatography" Molecules 19, no. 7: 8773-8787. https://doi.org/10.3390/molecules19078773