The Purification of Hydrophilic Compounds by SepaFlash® HILIC Cartridges

Reverse phase chromatography (RPC) is the first choice for the separation of most conventional samples, which is generally better than other chromatographic models in the universality, and the satisfactory resolution also makes reverse phase chromatography the most widely used purification method However, reversed-phase chromatography is not very good for the separation of hydrophilic compounds HILIC column (see Fig 1) has not only suitable retention time, but also satisfactory resolution Fig 1 stationary phases of C18 and Hilic columns Sepaflash ® HILIC column and sepabean ® machine of Changzhou Santai Technology Co., Ltd In combination with its superior separation performance, this paper compares sepaflash ® HILIC column [view specific parameters] with sepaflash ® C18 column [view specific parameters] in the reverse phase mode for the separation effect of two strong polar compounds The experimental results show that sepaflash ® HILIC column has excellent separation effect, which provides a set of efficient and practical solutions for the separation and purification of these compounds In this experiment, the specifications of the fast separation column used in this paper are 25gsepaflash ® HILIC column and 25gsepaflash ® C18 column The sample is a mixture of cytosine and vitamin C (see Figure 2 for the structural formula) Under the condition of the same sample amount, the rapid liquid chromatography system sepabean ® machine is used (see Fig 3) for separation and purification, the experimental results clearly show the different selectivity of the two columns for high polarity compounds Figure 2 two strong polar compounds (A: cytosine; B: Vitamin C) Figure 3 Sepabean ® rapid liquid preparation chromatographic system The experimental parameter setting of machine [application for trial] is shown in Table 1: the detection wavelength of sepabean ® machine chromatographic column 25g sepaflash ® HILIC column 25g sepaflash ® C18 column is 220nm, 254 nm mobile phase A: 200 mM ammonium acetate (pH 9.0) B: acetonitrile flow rate 15 ml / min sample amount 10 mg mixture elution gradient% solution B time / (min) 95 0 95 5 80 10 70 13 70 30 Results and discussion according to the separation spectrum, we can accurately measure the retention time and peak width The corresponding resolution can be calculated by the formula R = 1.18 (t 2 - t 1 )/ [(W 1/2 ) 1 + (W 1/2 ) 2 ] Where T 1 and T 2 They are the retention time of the adjacent peaks, (W 1 / 2) 1 and (W 1 / 2) 2 are the width of peak 1 and peak 2 measured at half peak height According to the formula, the resolution of the adjacent peaks in Fig 4 and Fig 5 is calculated respectively The resolution of the adjacent peaks in sepafflash ® C18 column is R ≈ 1.53, and the resolution of the adjacent peaks in sepafflash ® HILIC column is R ≈ 5.97 It can be seen from the calculation of the separation that the separation of sepaflash ® HILIC column is much higher than that of sepaflash ® C18 column Fig 4 sepaflash ® C18 column separation spectrum of mixture Fig 5 sepaflash ® HILIC column separation spectrum of mixture experimental results show that sepaflash ® HILIC column showed excellent selectivity and successfully preserved and separated the mixture of cytosine and vitamin C It can be seen from the separation spectrum that even if the separation is carried out on sepaflash ® C18 reversed-phase column with up to 95% aqueous solution mobile phase, the two strong polar compounds are only weakly retained, and the mixture sample has not been baseline separated Compared with the separation results of C18 reversed-phase column, we can see that the mixture sample has not only a suitable retention time, but also an ideal separation degree on sepaflash ® HILIC column Therefore, the method in this paper provides a set of efficient and practical solutions for the rapid separation and purification of strong polar compounds If you need specific information about the products used in this article, please click sepabean ® machine and Sepaflash ® hydrophilic column.