7 Table 1. The experimental setup for Flash purifi cation.
Instrument SepaBean machine T Flash cartridge
Wavelength Mobile phase Flow rate Sample loading
12 g SepaFlash silica cartridge (irregular silica, 40 - 63 µm, 60 Å, Order number: S-5101-0012)
254 nm, 280 nm
Solvent A: DCM Solvent B: Methanol 25 mL/min
500 mg (including 250 mg of the sample)
Time (CV) 0 4.0 Gradient
13.5 19.0 23.5 23.8
Solvent A: N-hexane; Solvent B: Ethyl acetate; Solvent C: DCM; Solvent D: Methanol 30 mL/min
3.0 g (including 1.5 g of the sample) Solvent B (%) Time (CV)
0 0
10 10 16
100 0
3.0 6.0
28 100
10.0 20.0 30.0 44.0 44.5 63.0 63.1 85.0 110
Solvent B (%)
0 0
16 16 50 50 80
100 100 / / /
Solvent D (%)
/ / / / / / / / /
0
4.0 4.0
As shown in Figure 3, the sample had a good retention on the silica cartridge when eluted by the DCM/methanol binary solvent system. However, there was no resolution for the components in the sample mixture. Reviewing the synthetic route of the sample and referring to the classic synthetic route of dibenzo-18-crown-6 (as shown in Figure 4), shows that along with the non-polar (lipophilic) target product in the sample mixture, there are excess/unreacted starting materials with strong polarities (such as catechol) or other by-products, resulting in multiple components of diverse polarities. When a simple binary solvent gradient is utilised, the DCM/methanol system is suitable for polar components. However, the selectivity for less polar components when employing the DCM/methanol solvent system is not as good as that of those solvent systems such as n-hexane/ethyl acetate(EA). Therefore, as an improvement to the simple binary solvent gradient, switching between binary solvent systems of different selectivities and continuously eluting the column may offer better resolution for the components.
Diethyl ether DCM
Table 2. The polarity parameter P’ of commonly used solvents. Solvent
P’
25 g SepaFlash silica cartridge (irregular silica, 40 - 63 µm, 60 Å, Order number: S-5101-0025)
N-pentane N-hexane Benzene
0.0 0.1 2.7 2.8 3.1
Solvent
N-propanol THF
Chloroform Ethanol EA
P’
4.0 4.0 4.1 4.3 4.4
Solvent Acetone Methanol
Acetonitrile Acetic acid Water
P’
5.1 5.1 5.8 6.0
10.2
In a conventional confi guration for a binary solvent Flash chromatography system, the mobile phase gradient is generated by a system pump in combination with a binary proportioning valve. When an instrument is used in this confi guration for continuous gradient elution requiring two solvents to be replaced at the same time, the column must be removed from the instrument and then the tubing should be fl ushed with the solvent to be used in the subsequent procedure. Afterwards the column should be re-installed on the instrument for next elution step. These operations will undoubtedly lower the work effi ciency. In this confi guration a quaternary proportional valve is employed to generate a binary gradient with a combination from any two of the four solvent lines, making it possible to continuously switch online between different solvent systems during the elution procedure. Considering solvent miscibility, n-hexane/EA system was employed as the starting eluting solvent and then transitioned to the DCM/methanol system for the continuous elution of the sample. The experimental setup was listed in Table 1. The Flash chromatogram of the sample in this condition was shown in Figure 6.
Figure 6. The Flash chromatogram of the sample by quaternary solvent gradient. Figure 4. The classic synthetic route of dibenzo-18-crown-6.
Referring to Snyder’s Solvent Selectivity Triangle (as shown in Figure 5), commonly used solvents can be roughly divided into eight groups according to their different acidic, basic or dipole properties. Each group of solvents occupies a certain position in this triangle. According to the rule of ‘like dissolves like’, different groups of solvents have different selectivities for the same sample. Therefore, better selectivity and improved resolution for the components in the sample mixture could be obtained by optimising the solvent combination.
As shown in Figure 6, when n-hexane/EA system was employed as the eluting solvent in the fi rst part of the separation, less polar components in the sample were eluted from the column. In the second part of the separation, a DCM/methanol system was utilised as the eluting solvent for the elution of more polar components. Therefore, multiple components of diverse polarity in the sample were separated and collected. The collected fractions were further identifi ed by TLC. A 0.5% potassium permanganate solution was used as the colour developer for TLC analysis. After heating with a heat gun for 5 min, the TLC results show complete separation of the target compounds and byproducts (Figure 7). It can be concluded that the components in the sample were effectively separated and can be used in next step research and development.
Figure 7. The TLC identifi cation results of raw sample and collected fractions.
Conclusion
For complex sample mixtures such as benzocrown derivatives, it is impossible for a conventional binary solvent system to provide satisfactory resolutions of the components in the sample due to the wide distribution range of component polarity. Utilising a quaternary solvent Flash chromatography system for continuous online switching between solvent systems of different polarities, satisfactory purifi cation results were obtained, suggesting a feasible and effi cient solution for the preparative separation of this complex sample mixture.
For further information on detailed specifi cations of SepaBean machine, or the ordering information on SepaFlash series fl ash cartridges, please visit
http://www.santaitech.com/index/.
References Figure 5. The schematic diagram of Snyder’s Solvent Selectivity Triangle.
Looking at the conventional binary solvent system used in the previous experiment: DCM/ methanol system. The solvent polarity table, (as shown in Table 2), shows the polarities of DCM and methanol are 3.1 and 5.1, respectively. As a comparison, the polarities of n-hexane and EA are 0.1 and 4.4, respectively. Comparing these two normal phase solvent systems, it can be speculated that for a complex sample mixture composed of multiple components of diverse polarities, the best could be obtained if one solvent system could be replaced by another one for continuous elution.
1. C. J. Pedersen, J. Am. Chem. Soc., 1967, 89, 7017-7036. 2. C. J. Pedersen, Org. Synth. Coll. Vol., 1988, 6, 395. 3. S. R. Cooper, Crown Compounds: Toward Future Applications, 1992, Chichester: Wiley InterScience. 4. M. Hiraoka, Crown Ethers and Analogous Compounds, 1992, Amsterdam: Elsevier.
5. H., Schneider, A. K. Yatsimirsky, Principles and Methods in Supramolecular Chemistry, 2000, Chichester: John Wiley. 6. P. J. Cragg, A Practical Guide to Supramolecular Chemistry, 2005, Chichester: John Wiley. 7. G. W. Gokel, W. M. Leevy, M. E. Weber, Chem. Rev., 2004, 104, 2723-2750. 8. J. Li, D. Yim, W. Jang, et al, Chem. Soc. Rev., 2017, 46, 2437-2458. 9. U. Olsher, J. Am. Chem. Soc., 1982, 104, 4006-4007. 10. R. A. Bartsch, Y. Liu, S. I. Kang, et al, J. Org. Chem., 1983, 48, 4864-4869.
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