Sample retention is very weak. How to prepare it? ——A case of sepaflash ® reversed phase purification column used for impurity removal

Qian Meiyuan Xu Bo applied technology research center about a week ago, Xiaoqian, the application engineer of Santai technology preparation and purification laboratory, received a preparation task A new drug research and development company needs to prepare and purify a sample The polarity of the sample is very large, and it is very weak on the reversed-phase HPLC analytical column In view of the nature of the sample, Xiaoqian first considered to use the normal phase separation mode to separate and purify it However, the client provided the HPLC analysis map of the sample, and the sample was analyzed in the reverse phase mode In order to ensure the consistency of the analysis conditions, the reverse phase separation mode can only be considered in this preparation and purification Firstly, the samples were analyzed by HPLC to confirm the retention time and separation conditions The sample has been dissolved in methanol solvent, so it can be analyzed directly on the HPLC analyzer The HPLC analysis map of the original sample is shown in Figure 1 Figure 1 The HPLC analysis of the original sample according to the HPLC analysis of the original sample provided by the customer, combined with the analysis results of the original sample in the analysis laboratory, it can be confirmed that the retention time of the target product is 1.630min (detection wavelength 220nm), and this sample is very weak on the reversed-phase C18 analysis column However, the separation of the target product and other impurities is very high by observing the HPLC analysis spectrum, which suggests that we can use flash preparation to remove the impurities in the sample, so as to obtain the target product with high purity After LC-MS analysis (as shown in Figure 2), the two target peaks with retention time of 1.16 min and 1.51 min on the tic map contain target substances with molecular weight of 232.2, so they are collected in the subsequent flash preparation Figure 2 The experimental parameters of flash preparation and purification of LC-MS analysis Atlas of the original sample are shown in Table 1: Table 1 Sepabean ® machine chromatographic column 25 g sepabelash ® bonded series C18 reversed-phase purification column (spherical silica gel, 20-45 μ m, 100 μ m, order No.: sw-52 22-025-sp) detection wavelength 220 nm; 254 nm mobile phase Solvent A: water; solvent B: methanol flow rate 20 ml / min injection volume 2.5 ml (30 mg) elution gradient time (min) solvent B (%) 0 25 29 5 34 72 35 92 50 92 Fig 3 Flash preparation separation map of sample is shown in Fig 3, because the particle size is 20-45 The results show that the separation efficiency of the sample is high and the separation degree of the target product and other impurities is satisfactory HPLC analysis of the target product (as shown in Figure 4) shows that the purity of the target product is more than 99.5%, which meets the customer's requirements for the purity of the target product Figure 4 The HPLC analysis Atlas of the target product summarizes the preparation case of this sample In the future, the idea should be expanded in the preparation and purification of the sample It should not be entangled in the retention strength of the sample on the stationary phase As long as the target product has sufficient separation degree with other impurities, then the target product can be quickly obtained by using flash purification system 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/.