(T1530-06-33) Gradient Recycling Chromatography for the Preparation of Pure Target Compounds Present in Highly Complex Mixtures

Purpose: One recurrent challenge in the food, biomedical, and pharmaceutical industries is the isolation and high yield/purity preparation of one or a few target compounds that are 1) present in a very complex matrix/mixture and 2) nearly co-elute with undesirable impurities. Conventional batch chromatography, steady state recycling, multi-column counter-current solvent gradient purification, or simulated moving bed chromatography are usually the routine and most successful methods for the preparation of such target compounds. However, they fail for near co-elution scenario (selectivity < 1.03) and cannot deliver both 100% yield and 100% purity levels. The purpose of this work is to deliver a practical solution to the user in order to solve this problem.

Methods: A solution consists of applying gradient twin-column recycling chromatography process (GTCRCP) in order to reach these unmet performances. GTCRCP operates by combining two columns and two switching valves. In a first step it discards all early and late impurities by gradient elution. In a subsequent step, it resolves the target compounds from the most critical impurities by classical isocratic recycling chromatography.

Results: The general GTCRCP principles are described and its successful applications of are demonstrated for 1) the isolation of a polycyclic aromatic hydrocarbon (PAH) compound originally present in a complex PAH mixture in less than 2 hours and 2) the isolation and separation of vitamins D2 and D3 originating from a milk extract in less than 90 minutes. The GTCRCP process is scaled up to 7.8 mm i.d. columns run at a few mL/min, the injection volume of an Arc HPLC system was augmented up to 1 mL/min enabling to prepare about 1 mg of pure target compound impurities (initial mixture concentration ~ 1 mg/L) in less than 12 hours.

Conclusion: The present work demonstrates that it is possible to deliver targeted purification of trace impurities present in very complex mixtures with 100% yield and 100% purity levels. The production rate can be augmented by scaling-up the recycling process to 7.8 mm i.d. columns operated at 5 mL/min and loaded with 1 mL sample volume.


Step 1 of GTCRLC: Elimination of early impurities and of the resolved fraction of nearly co-eluting impurity 1 (blue color) by gradient elution.


Step 4 of GTCRLC: The isocratic recycling chromatography between twin columns 1 and 2 begins in order to baseline resolve the target compound from the unresolved fractions of nearly co-eluting impurities 1 and 2.


Purification of the polycyclic aromatic hydrocarbon (PAH). chrysene (green color) from many early and late eluting PAHs and from nearly co-eluting triphenylene (blue color) and benz[a]anthracene (red color). 7.8 x 150 mm Sunfire-C18 column, T=296 K, 2.5 mL/min acetonitrile/water, 1 mL sample injection volume, 20 isocratic cycles were necessary.