26
Sample purifi cation by Flash chromatography
The sample was purified by a Flash chromatography system (SepaBean machine 2) according to the parameters as shown in Table 1.
Table 1. The experimental setup for Flash purifi cation. Instrument
SepaBean machine 2 Flash cartridge
12 g SepaFlash C18AQ cartridge (spherical silica, 20 - 45 µm, 100 Å, Order number: SW- 5222-012-SP(AQ))
Wavelength Mobile phase
254 nm; 220 nm
Solvent A: water Solvent B: methanol
Flow rate Sample load 15 mL/min
0.5 mL (100 mg) Time (CV) 0
Gradient
20 21 30
Results and Discussion
The sample used in this post was highly polar and soluble in water. Due to its high polarity and strong retention on normal phase silica cartridges normal phase separation was impractical and excluded. In a reversed phase separation mode, if a conventional C18 RP Flash cartridge was used, the sample will be eluted from the stationary phase quickly since the organic phase ratio was more than 10% in the initial mobile phase. Furthermore, there is still certain amount of salt impurities in the collected fractions, leading to a bad desalting result. Therefore, an C18AQ cartridge was utilised to purify the sample.
A step gradient was set for the Flash chromatography. To ensure that the salt impurities with a MW very close to the target product are fully removed, pure water was used as the mobile phase to fl ush the cartridge for about 20 column volumes (CV). As shown in Figure 5, the sample was fully retained on the C18AQ cartridge when pure water was used as the mobile phase. Next, methanol in the mobile phase was directly increased to 100% and the gradient was
Solvent B (%) 0 0
100 100
Figure 5. Flash chromatogram of the sample on a C18AQ cartridge.
Compared to a linear gradient, the use of step gradient has the following advantages:
• Solvent usage and run time for sample purifi cation is reduced.
• The target product elutes in a sharp peak, which reduces the volume of collected fractions and thus facilitates the following rotary evaporation as well as saving time.
• The collected product is in methanol which is easily evaporated, thus drying time is reduced.
In conclusion, for the purifi cation of a sample which is strongly polar or highly hydrophilic, a SepaFlash C18AQ cartridge combiined with a Flash chromatography system (SepaBean machine) is undoubtedly a fast and effi cient solution.
References
1. Przybyciel M, Majors RE (2002). Phase collapse in reversed-phase liquid chromatography. LCGC North Am. 20 (6): 516–523.
2. Majors RE (2013). The top 10 HPLC and UHPLC column myths. LCGC North Am. 31 (7): 522-537.
3. Bidlingmeyer BA, Broske AD (2004). The role of pore size and stationary phase composition in preventing aqueous-induced retention time loss in reversed-phase HPLC. J Chromatogr Sci. 42: 100-106.
4. Nagae N, Enami T and Doshi S (2002). The retention behavior of reversed-phase HPLC columns with 100% aqueous mobile phase. LCGC North Am. 20 (10): 964-972.
5. Majors RE, Przybyciel M (2002). Columns for reversed-phase LC separations in highly aqueous mobile phases. LCGC North Am. 20 (7): 584-593.
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maintained for 10 CVs. The target product was eluted out from 22.5 to 24 CVs. In the collected fractions, the aqueous sample solution was replaced with methanol. The methanol was easily removed by rotary evaporation in the subsequent step, which facilitates following research on the purifi ed sample.
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