Enantioseparation of dl -tryptophan by spiral tube assembly counter-current chromatography and evaluation of mass transfer rate for enantiomers

Abstract

Spiral tube assembly counter-current chromatography was successfully applied in enantioseparation of DL-tryptophan using bovine serum albumin as chiral selector. An improved biphasic aqueous-aqueous solvent system 12.0% (w/w) polyethyleneglycol 8000–9.0% (w/w) dibasic potassium phosphate-0.1% ammonia-78.9% water was used as the solvent system for counter-current chromatography, in which bovine serum albumin was predominantly distributed in the lower phase of the two-phase aqueous system. The aqueous-aqueous solvent system gave a very high enantioselectivity for D- and L-tryptophan at α=2.605 along with distribution ratio DD=1.200 and DL=0.461. High peak resolution was obtained for enantioseparation of 2.0 mg of DL-tryptophan by spiral tube assembly counter-current chromatography under room temperature. It was found that 0.1% ammonia added in the aqueous-aqueous solvent system greatly improved the enantioseparations. An unusual extremely broad peak for L-tryptophan was observed during enantioseparations. In order to give an explanation, mass transfer rates of D- and L-enantiomers through the interface between the two phases were measured. It was found that L-tryptophan showed lower mass transfer rate than D-tryptophan. Further discussions were proposed for possible reasons for mass transfer rate difference between the enantiomers.

A Simple Tool for Tubing Modification to Improve Spiral High-Speed Counter-Current Chromatography for Protein Purification

“…In addition, the large molecules have more resistant to break through the interface. These phenomena have been investigated in our previous studies using a simple rotary system and a set of test samples [19, 24]. In order to accelerate the mass transfer rate of protein molecules, one may consider two different ways, i.e., increasing the interface area par volume of the two phases and reducing the thickness of each solvent layer.…”

“…Possibility of further improvement of the system is also explored by modifying the shape of tubing. Performances of flat tubing [20], flat-twisted tubing [19] and cross-pressed tubing [18] are so far tested by manually modifying the shape of tubing with a pair of pliers. However, making these tubing modifications is tedious and time-consuming and also lacks accuracy and reproducibility.…”

Comparison in Partition Efficiency of Protein Separation between Four Different Tubing Modifications in Spiral High-Speed Countercurrent Chromatography

“…In the present studies, tubing modification is further extended to flattened, flat-waving and flat-twisting. Although flattening tubing has been made simply by extruding the tubing through a narrow slit as previously performed [15], the degree of the flattening depends on the opening of the slit and the tubing tends to be stretched according to the applied force and the pulling speed, hence it lacks accuracy and reproducibility. In contrast, positively pushing out the tubing through rotating pressing wheels used in the present study totally eliminates these problems.…”

“…Instead, the performance of the column can be improved by modifying the tube geometry to enhance the mixing and interrupt with the laminar flow of the mobile phase which would produce sample band broadening. In the past several tubing modifications such as cross-pressing [14] and flat-twisting [15] were manually made and tested. Although these modifications substantially improved the partition efficiency, it takes a long tedious process for column fabrication and lacks accuracy and reproducibility.…”

Salts of Tryptophan with p-Toluenesulphonic Acid and Camphorsulphonic Acids

“…The interest in obtaining enantiomerically pure compounds has led to increased research in chiral resolution processes [5,6]. Enantioseparation of DL-tryptophan (DL-TRP) has been reported using techniques such as the use of an ion-exchange membrane [7], ultrafiltration [8] and chromatography [9][10][11]. One of the oldest and most employed crystallisation technique to achieve resolution is the formation of diastereomeric salts using an acidic or a basic chiral resolving agent.…”