The Application of SepaFlash® Reversed-Phase Cartridges in the Purification of Very Polar and Low Soluble Samples

Qian Meiyuan, Xu Bo Applied Technology Research Center recently, Xiaoqian, purification engineer of Santai technology preparation and purification laboratory, received a sample preparation and purification task entrusted by a pharmaceutical company, which requires the preparation and purification of a sample with great polarity and difficult to dissolve in water Based on the nature of the sample, Xiaoqian first thought of the normal phase separation mode, but after the purification of a small amount of samples, Xiaoqian found that the sample was too strong to be eluted on the normal phase separation column, so it seems that the normal phase mode is not feasible It doesn't matter You can also try the reverse phase separation mode, so Xiaoqian took 50 mg of sample and dissolved it in 5 In ML acetonitrile / methanol / water / DMF (V: V: V: v = 23:23:2:2), the sample is completely dissolved by ultrasonic heating, and then the elution gradient is set according to the HPLC analysis conditions of the sample, and the gradient setting is adjusted in real time according to the peak conditions in the actual preparation and separation process The experimental conditions for preparation and purification are shown in Table 1 Table 1 Sepabean ® machine chromatographic column 25 g sepaflash ® bonded series C18 reversed-phase purification column (spherical silica gel, 40-60 μ m, 120 μ m, Order No.: sw-5231-025-sp) detection wavelength 220 nm; 254 nm mobile phase solvent A: water; solvent B: acetonitrile flow rate 20 ml / min injection volume 5 ml (50 mg) elution gradient time (min) solvent B (%) 0 5 20 35 23 35 26 26 40 30 40 43 60 45 90 50 90 Figure 1 Preparation and separation of small sample As shown in Figure 1, from the separation results of the pre experiment, the target product can be effectively separated from impurities, and the separation degree is high, which can meet the requirements of the target and impurities collection respectively Then Xiaoqian carried out the amplification experiment She dissolved 1g of sample in 50ml of methanol / acetonitrile / water / DMF (V: V: V: v = 23:23:2:2) The sample was completely dissolved by ultrasonic heating, and then set the parameters for the preparation and purification experiment, as shown in Table 2 Table 2 Sepabean ® machine column 330 g sepaflash ® bonded series C18 reversed-phase purification column (spherical silica gel, 40-60 μ m, 120 μ m, order No.: sw-5231-330-sp) detection wavelength 220 nm; 254 nm mobile phase solvent A: water; solvent B: acetonitrile flow rate 55 ml / min injection volume 50 ml (1 g) elution gradient time (min) solvent B (%) 0 10 2 12 10 12 12 23 27 36 27 38 30 41 30 55 50 62 50 64 90 70 90 Figure 2 Preparation and separation map after sample volume amplification The preparation of the separation map is shown in Fig 2 According to the analysis map, after the sample loading is increased by 20 times, the corresponding proportion of the separation column is enlarged, but the separation result is not consistent with the expectation due to the solvent problem of the sample Due to the strong absorption of DMF in the UV detection band, it will interfere with the detection of samples, so a high proportion of methanol and acetonitrile are used in the preparation of sample solution Under this condition, a large number of samples were quickly eluted with the eluting solvent, which failed to obtain effective retention and separation In addition, the effective column bed height of 330 g purification column did not increase in proportion to that of 25 g purification column, so the column efficiency of the former decreased more than that of the latter, which also led to the failure of effective separation of target products and impurities in the separation map After discussing with his colleagues, Xiao Qian decided to give up the idea of "one step in place" After dissolving a small amount of samples with DMF solvent, he prepared and purified the samples several times Xiaoqian weighed 600 mg of sample and dissolved it in 6 ml DMF After the sample was completely dissolved by ultrasonic heating, the sample separation and purification experiment was started The experimental parameters were set as shown in Table 3 Table 3 Sepabean ® machine column 120 g sepaflash ® bonded series C18 reversed phase purification column (spherical silica gel, 40-60 μ m, 120 μ m, order No.: sw-5231-120-sp) detection wavelength 220 nm; 254 nm mobile phase solvent A: water; solvent B: acetonitrile flow rate 40 ml / min injection volume 6 ml (600 ml Mg) elution gradient time (min) solvent B (%) 0 10 15 26 25 26 50 52 90 62 90 Fig 3 The improved sample preparation and separation map Xiaoqian used the same experimental parameter settings to inject a total of 6.5g samples several times, and Fig 3 shows the sample preparation and separation map for two times It can be seen from the analysis spectrum that after reducing the sample volume, the sample has been effectively separated and purified, the separation degree of target product and impurity is good, and the separation reproducibility is good After HPLC analysis, the purity of the target product collected after purification is 98.4%, recovery rate is 89.4%, impurity purity is 88.1%, recovery rate is 74.8% The HPLC analysis map before and after sample separation is shown in Figure 4 Figure 4 HPLC analysis of samples before separation, target products and impurities after separation and purification To sum up this sample preparation and purification case, we can refer to the idea in this case when we separate and purify this kind of sample which has great polarity and is difficult to dissolve in water, that is, after a small amount of organic phase solvent is used to solvent the sample, inject the sample several times, and then complete the preparation and purification of all samples Sepabean ® machine produced by Santai technology and sepaflash series reverse phase purification column can provide users with fast and efficient sample preparation and purification solutions, and help laboratory researchers easily complete various sample purification tasks To learn more about the detailed specifications of sepabean ® machine, or the flash column ordering information used with it, please visit CBG online store: https://store.chembergo.cn/.