Study On The Isolation Of Minor Impurities By A Twin-column Recycling Chromatography With Solvent Gradient

Impurities,reducing the purity of Active Pharmaceutical Ingredients(APIs)in the active pharmacological substance,are the focus of the safety and efficacy on the clinical medication.Generally,extraction,distillation and other separation process reach not the threshold of trace impurities which set by the pharmacopoeia,and the chromatographic operation is the best choice.Some key impurities such as homologues,isomers,enantiomers,etc.,have low resolution with APIs due to similar molecule structure and physicochemical property.In the process scale,the band broadening and nonlinear adsorption is obvious owing to feeding overload.Therefore,trace impurities are difficult to be isolated by preparative batch chromatography.Recycling chromatography is suitable for separation of APIs and key impurities because of concise equipment and excellent efficiency.However,the band broadening,caused by non-ideal factors such as dead volume on extra-column,mass transfer resistance,longitudinal diffusion,eddy diffusion,etc.and nonlinear adsorption on column,is adverse for separation of impurities.So,weakening and offsetting band broadening have become a key point to need to be resolved in recycling chromatography.In this paper,solvent gradient was introduced into twin-column recycling chromatography.When the proportion of weak solvents in the downstream solvent increased,the sorption of the molecules on the stationary phase became stronger.And then,the migratory velocity reduced gradually among the flow direction,that is,the velocity of the band tail was faster than the front,contributing to a compression to weaken or even offset band broadening.What the band was neither spreading nor narrowing would overcome greatly the negative effect of band broadening.A twin-column recycling chromatography with solvent gradient was applied to isolate impurities of Orlistat and special fermented product.The effects of cycle times,feeding amounts and solvent gradient were investigated.For the reversed phase chromatography which separated impurities of Orlistat,the operation with solvent gradient could make the band narrower and higher and increase the concentration of molecules on the stationary phase which enhanced competition adsorption.The displacement effects caused by competitive adsorption was beneficial to the removal of pre-impurities but the Tag-Along effects was not conducive to the removal of the post-impurities.The larger the solvent gradient,the better the removal of the pre-impurities and the worse the removal of the post-impurities.In this way,an ultraviolet detector,installed midway between the two columns,could show a combined signal which could monitor the migration of band and control the switch manually.The purpose was to eliminate the influence from fluctuation of experimental condition such as flow rate,column temperature and desorbent composition.The suitable condition was optimized:the dimension of chromatographic column was 2 × 4.6mm × 250mm with packing C18 stationary and mobile phase was 86/14 methanol/water(v/v).the concentration of material was 20mg/mL,the feeding volume was 18mL and the solvent gradient was 0.5%.After 8 cycles at 135min,the yield was 91.36%and the productivity was 375 g·L-1·day-1.For the normal phase chromatography which separated impurities of special fermented product,the migratory velocity of the band was related to the feeding amounts,solvent gradient and flow rate.There was direct influence in band width,cycling time,solvent consumption and yield.In addition,the band was severely tailed caused by a strong non-linear adsorption so that higher concentration at the band front and lower at the tail.Consequently,the idea which switched valve as soon as the band tail to be eluted to downstream in the reversed phase operation was change to ensure the band front not leakage from the downstream outlet.At the same time,a large amount of post-impurities could be removed when eluted the band tail after switching.The suitable condition was optimized:the dimension of chromatographic column was 4 ×10mm × 100mm with packing silica gel stationary and mobile phase was 94/6 n-hexane/tetrahydrofuran(v/v).the concentration of the material was 150mg/mL,the feeding volume was 8mL and the solvent gradient was 3.9%.After 6 cycles at 108min,the yield was 88.01%and the productivity was 744 g·L-1·day-1.