The Application of Orthogonal Chromatography for the Purification of Synthetic Pharmaceutical Intermediates

Introduction of application technology research center normal phase chromatography and reverse phase chromatography are two common separation modes of flash preparation chromatography, which are widely used in the separation and purification of various organic synthesis products In normal phase chromatography, polar stationary phases (such as those with diol, amino or cyano groups, silica gel, aluminum oxide, etc.) and non-polar mobile phases (such as n-hexane, etc.) are used to separate them according to the molecular polarity Because the positive phase chromatography is based on adsorption effect, it is also called adsorption chromatography In reversed-phase chromatography, nonpolar stationary phase (such as silica gel with C18 group) is used to separate the sample with polar flow These two separation modes are based on different separation mechanisms, so when the two separation modes are combined, they can be called orthogonal chromatography separation mode, so as to obtain higher resolution and better separation effect for complex samples In this paper, a synthetic drug intermediate was used as a sample, sepafflash series of silica gel column and reversed phase C18 column were used together to achieve the high-efficiency separation and purification of the sample, and the target product meeting the purity requirements was obtained, which provided a new idea for the rapid preparation and purification of such complex samples The experimental part of the sample comes from a new drug research company First, take a small amount of sample and dissolve it in methanol for HPLC purity analysis Referring to the HPLC analysis chart of the crude product shown in Figure 1, it can be seen that the purity of the crude product is about 66% Among them, there is one impurity whose retention time is very close to the target product, which puts forward higher requirements for the subsequent separation and purification Fig 1 The HPLC analysis Atlas of the crude product, due to the large polarity of the sample, first consider the use of ordinary silica gel column for separation and purification Take 3 g of the sample and dissolve it in methanol, mix the sample with 6 g of silica gel (the specification is the same as that of silica gel column packing used in flash preparation experiment), remove the solvent with a rotary steamer, and put the solid sample into an ILOK ® empty column used in combination with flash separation column The experimental parameters of flash preparation are shown in Table 1 The flash separation map of the positive phase separation mode is shown in Figure 2 Table 1 Sepabean ® machine t chromatographic column 80 g sepaflash ® standard series normal phase separation column (amorphous silica gel, 40 - 63 μ m, 60 μ m, Order No.: S-5 101-00 80) detection wavelength 220 nm; 254 nm mobile phase flow rate of ethyl acetate 50 ml / min injection volume 3 G elution gradient of crude product 100% isoelution Figure 2 Flash preparation map of crude product on normal phase silica gel column 3 HPLC analysis map of components collected after separation of crude product on normal phase silica gel column Take the collected components for HPLC analysis (as shown in Fig 3) The results show that the purity of the collected components is about 92%, which is a certain gap from the required purity of more than 95% It can be seen from Figure 3 that impurities with approximate retention time with the target can not be separated from the target product In consideration of the orthogonality between the reversed phase and the normal phase chromatography modes, which can provide additional resolution, we take 300mg sample to dissolve in 2.5 after the solvent is removed by rotary evaporation of the collected components separated by the normal phase Ml methanol, and add DMSO properly to make it completely dissolved, then use the way of liquid injection to inject the sample to C18 reversed phase separation column, and the experimental parameters of flash preparation are shown in Table 2 The flash separation map of reverse phase separation mode is shown in Figure 4 Table 2 Sepabean ® machine t column 120g sepaflash ® bonded series C18 reversed-phase separation column (spherical silica gel, 20-45 μ m, 100 μ m, Order No.: sw-52 22-120-sp) detection wavelength: 220 nm; 254 nm mobile phase solvent A: water; solvent B: methanol flow rate: 30 ml / min injection volume: 2.5 ml (300 mg) elution gradient time (CV) solvent B (%) 0.608 80 12 80 12.5 100 HPLC analysis of the collected components shows that the purity of the collected components is about 98%, which meets the preparation requirements and can be used in the further research and development of customers Conclusion the first mock exam shows that the combination of normal chromatography and reversed-phase chromatography can effectively improve the resolution of Flash preparation process, especially for the separation and purification of some complex samples, and accomplish the preparation task which is not completed by ordinary single mode normal chromatography or reversed phase chromatography At the same time, compared with preparative HPLC, flash preparative chromatography has a great advantage in the cost of chromatographic column, which is a cost-effective purification solution for the majority of researchers About sepafflash ® normal phase silica gel column and C18 reverse phase column series products Sepafflash ® standard series normal phase silica gel column and sepafflash ® bonded series C18 reverse phase column series products launched by Santai technology have various specifications (see Table 3 and table 4) Table 3 Sepaflash standard series column parameters (packing: ultrapure infrared silica, 40-63 μ m, 60 μ m) table 4 Sepaflash C18 series reversed-phase column parameters (packing: high efficiency spherical C18, 20-45 μ m, 100 Å) to learn more about the detailed specifications of sepabean ® machine series products, or the ordering information of sepaflash ® series fast separation columns, please visit CBG online store.