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    Home > Sepaflash ® C18 reversed phase separation column applied to the separation and purification of enantiomers

    Sepaflash ® C18 reversed phase separation column applied to the separation and purification of enantiomers

    • Last Update: 2019-12-23
    • Source: Internet
    • Author: User
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    The existence of chiral molecules is a common phenomenon in nature Chirality comes from the Greek hand Just like people's left and right hands can't be completely overlapped, they can only mirror each other, and chirality molecules can't be completely overlapped no matter how they rotate For example, the simplest amino acid, alanine, has two forms, namely: (s) - alanine and (R) - alanine, as shown in Figure 1 Because the four groups connected to the central carbon atom are arranged in different order in space, they have different spatial structures Figure 1 Chemical structure formula of alanine enantiomer In 1848 Louis Pasteur, a French chemist and biologist, discovered two different forms of tartaric acid, marking the discovery of chiral characteristics of organic molecules It was not until more than a century later that people realized that chirality not only plays a key role in the life characteristics of animals and plants, but also plays an important role in the fields of pharmacy, agriculture and other chemical industries [1-3] All proteins, peptides, amino acids, nucleotides, carbohydrates and some alkaloids and hormones are chiral compounds [4,5] At present, about 60% of the drugs used in clinic are chiral compounds, and about 88% of the chiral compounds used in treatment are racemic drugs Chirality has become the focus of academic research and pharmaceutical development [6-8] Figure 2 Classification of isomers In stereochemistry, chiral molecules belong to the category of isomers, that is, the molecules with the same formula and different spatial structure are chiral to each other As shown in Figure 2, isomers can be divided into structural isomers and stereoisomers In stereoisomers, they can be divided into enantiomers and diastereomers The enantiomers are also called optical isomers A pair of enantiomers is a mirror image which can not overlap each other The enantiomers have exactly the same physical properties, but may have essentially different biological effects For the resolution of enantiomers, it is necessary to rely on the chiral environment provided by the chiral column or use supercritical fluid chromatography (SFC) to carry out [9-11] Diastereomers refer to stereoisomers with two or more chirality centers but not mirror relation, including cis / trans isomers, constructors, racemates and optical isomers with diasteresm The enantiomers not only have different optical properties, but also have different physical and chemical properties Therefore, the enantiomers can be separated by chromatography or recrystallization In this application case, the sample comes from the Synthesis Laboratory of a new drug R & D company, which is a pair of enantiomers obtained in the synthesis process Conventional normal phase separation can not achieve the purpose of purification, and reversed-phase high-pressure preparation is not suitable due to its sample size and cost Therefore, the application engineer of Santai technology tried to use sepabean ® machine t with sepaflash ® C18 reversed phase separation column to separate and purify the sample, and successfully obtained the target product meeting the purity requirements, providing a cost-effective solution for the rapid preparation and purification of such non enantiomers Experiment part 1 Sample information in this application case, the structural formula of the sample is shown in Figure 3, with two chiral centers in the molecular structural formula, and the structural formulas of the two possible configurations are listed in Figure 3 Figure 3 Two possible configurations of sample molecules 2 Preliminary analysis of the sample the preliminary purity analysis of the sample was carried out by HPLC (see Figure 4) According to the analysis of the HPLC chromatogram, the two components in the sample have very similar retention time, which challenges the preparation and separation of the subsequent flash In order to improve the separation degree, it is a simple and easy method to use multiple flash columns in series (refer to the application case "multiple separation columns in series to improve the separation degree in compound purification" issued by Santai technology before) In the subsequent flash separation experiment, we adopted the setting of two separation columns in series and controlled the sample volume Figure 4 HPLC analysis of crude product 3 The flash separation and purification parameters of flash preparation and purification samples are shown in Table 1 Table 1 Parameters setting of flash preparation and purification experiment Instrument sepabean ® machine t separation column 25g sepaflash ® C18 reversed phase separation column, two in series (spherical silica gel, 20-45 μ m, 100 μ m, order No.: sw-5222-025-sp) detection wavelength 220 nm; 254 nm mobile phase solvent A: water; Solvent B: acetonitrile flow rate 15 ml / min injection volume 0.75 ml (100 mg) elution gradient time (min) solvent B (%) 0 20 56 83 95 83 100 87 100.5 100 105 100 results and discussion Figure 5 Two columns in series Schematic diagram of flash preparation mode As shown in Figure 5, two sepaflash ® C18 reversed phase separation columns are used in series to improve the separation of sample components The flash preparation map of the sample is shown in Figure 6 Figure 6 Flash separation spectrum of the sample The purity of the two components collected was analyzed by HPLC (see Figure 7) The results showed that the purity of the two components after purification was over 98%, which met the purity requirements Figure 7 HPLC analysis spectrum of the two components after purification In this case, two 25 g sepaflash ® C18 reversed-phase columns were used to separate and purify 100 mg sample successfully In the subsequent amplification of the method, if the flash column with the same packing size of 330 G is used, the single sample loading amount can reach about 600 mg Compared with the chiral separation column or preparative HPLC, this method undoubtedly has the advantages of higher flux and lower cost Conclusion sepabean ® machine produced by Santai technology and sepaflash ® series C18 reversed-phase separation 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 ordering information of flash purification column, please visit chembeango online store: https://store.chembiango.com/ And, J Mod Med Chem , 2013 , 1: 10-36 6.   D.Burke, D J Henderson, Brit J Anaesth , 2002 , 88: 563-576 7.   M.C Nunez, M E Garcia-Rubino, A Conejo-Garcia, et al, Curr Med Chem , 2009 ,16: 2064-2074 8.   L.A Nguyen,H He, C Pham-Huy, Int JBiomed Sci , 2006 , 2: 85-100 9.   Y.Zhang, D R Wu, D B Wang-Iverson, et al, DrugDiscov Today , 2005 , 10:571-577 10. E.R Francotte , J Chromatogr A , 2001 , 906: 379-397 11. A.Calcatgerra, I D’Acquarica, JPharmaceut Biomed , 2018 , 147:323-340.
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