Chiral separation by high-speed countercurrent chromatoraphy

以牛血清白蛋白（BSA）为手性选择剂，成功地将螺旋管组装逆流色谱法应用于dl -色氨酸的聚合物相体系对映体分离。以牛血清白蛋白为一相手性选择剂的双水相溶剂体系可通过逆流萃取和逆流色谱进行拆分。水双相溶剂体系可以由两种聚合物或一种聚合物和水中的无机盐混合制备，其中白蛋白可以几乎完全分布在两相中的一种，而外消旋小分子在两相中都是自由分配的。过去，牛血清白蛋白被用作手性选择剂，通过逆流分布对dl -色氨酸、dl -犬尿氨酸和氧氟沙星进行对映体分离，通过逆流色谱对dl -犬尿氨酸进行对映体分离。然而，由于逆流分布技术的分离效率较低，以及所报道的溶剂体系对dl -色氨酸的对映体分离因子较低，因此每种情况下的峰分辨率都很差。为了获得更好的峰分辨率，需要提高对映体分离因子和改进逆流色谱技术。逆流色谱法（CCC）属于液-液分配色谱法，它消除了传统液相色谱法中使用固体支撑物可能引起的各种并发症。对于手性分离，可以直接在两相中加入手性选择剂，而不需要昂贵的固定化过程。现代CCC仪器的成本通常低于传统的液相色谱。由于其易于放大的性质，越来越多的论文可用的手性分离逆流色谱法。对于传统的高速CCC，固定相的保留完全由阿基米德螺旋效应提供，通过旋转多层螺旋柱在离心力场（行星运动）。然而，使用这种传统多层线圈分离柱的系统不能保留足够的固定相用于极性溶剂系统，如水-水聚合物相系统。为了解决这个问题，我们的实验室将螺旋形通道的几何形状修改为螺旋形结构，这样系统也可以利用径向离心力来提高固定相的保留。在这里，我们报告了用我们的螺旋管组件CCC对dl -色氨酸的完全对映体分离。发现了一种改进的双水相溶剂体系，对dl -色氨酸有较高的对映选择性。此外，每个色氨酸对映体的传质速率的测定是通过一个简单的旋转装置进行的，以提供对映体分离过程中观察到的l -对映体异常宽峰的解释。

Spiral tube assembly counter-current chromatography was successfully applied in enantioseparation of DL-tryptophan using polymer phase system where bovine serum albumin (BSA) was used as chiral selector. Partition in an aqueous biphasic solvent system for chiral separation using bovine serum albumin as a chiral selector in one phase could be accomplished by counter-current extraction and counter-current chromatography. The aqueous biphasic solvent systems can be prepared by either mixing two polymers, or one polymer and an inorganic salt in water in which albumin could be distributed almost completely in one of the two phases, while the racemic small molecule is freely partitioned in both phases. In the past, BSA has been used as a chiral selector for enantioseparation of DL-tryptophan, DL-kynurenine and ofloxacin by counter-current distribution and enantioseparation of DL-kynurenine by counter-current chromatography. However, peak resolution was very poor in each case because of low separation efficiency of counter-current distribution technique and low enantioseparation factor for DL-tryptophan with the reported solvent systems. In order to achieve better peak resolution, higher enantioseparation factor and improved counter-current chromatography technique are necessary. Counter-current chromatography (CCC) belongs to liquid-liquid partition chromatography, which eliminates the use of solid support which may cause various complications in conventional liquid chromatography. When it comes to chiral separations, chiral selector could be directly added in either of the two phases without expensive immobilizing process. The cost for modern CCC apparatus is usually lower than that for conventional liquid chromatography. And because of its ease-to-be scaled up properties, increasing number of papers became available about chiral separations by counter-current chromatography. For conventional high-speed CCC, the retention of the stationary phase is solely provided by the Archimedean screw effect by rotating the multilayer coiled column in the centrifugal force field (planetary motion). The system using this conventional multilayer coil separation column, however, fails to retain enough of the stationary phase for polar solvent systems such as the aqueous-aqueous polymer phase systems. To address this problem, the geometry of the coiled channel was modified to a spiral configuration in our lab so that the system could also utilize the radially acting centrifugal force to improve the retention of the stationary phase. Here, we report a complete enantioseparation of DL-tryptophan by our spiral tube assembly CCC. And an improved aqueous biphasic solvent system was found which provided a higher enantioselectivity for DL-tryptophan). Furthermore, determinations of mass transfer rate for each tryptophan enantiomer were conducted by a simple rotary device to provide an explanation for an unusual extremely broad peak of L-enantiomer observed during the enantioseparation. The instrument for counter-current chromatography used was a type-J coil planet centrifuge (PC Inc., Potomac, MD, USA) with a 10 cm revolution radius. It holds a spiral tube assembly consisting of multiple sporal layers of coil embedded in a spiral tube support on one side and a counterweight on the other side. The sprial tube support was custom-made by CCBiotech, Maryland, USA. It has 4 spiral grooves, each 2.8 mm wide and ca 5 cm deep with 12 transfer radial grooves. The corner of each spiral was rounded to prevent kinking of the tubing. PTFE polytetrafluoroethylene) tubing of 1.6 mm ID (SW 14) (Zeus Industrial Products, Orangeburg, SC, USA) was flat-twisted and accommodated tightly into the spiral grooves by squashing with a tool which fitted to the radial grooves. Total length of the tubing is estimated as 46 m with 12 spiral layers, and the total capacity is 85 ml. The column temperature was maintained at room temperature (25oC) and the revolution rate of the separation column was set at 800 rpm. The HSCCC separation was performed with a solvent system consisting of 12.0% (w/w) polyethyleneglycol 8000-9.0% (w/w) dibasic potassium phosphate -0.1% ammonia-78.9% water. It was prepared with the following procedures: 72 g of polyethyleneglycol 8000, 54 g of dibasic potassium phosphate, and 2 mL of 30% ammonium hydroxide were dissolved in 474 g of water. The solvent mixture was thoroughly equilibrated in a separatory funnel, and the two phases were separated shortly before use. The enanseparation was initiated by filling the column with the lower phase free of chiral selector. Then, 20 mL of the lower phase containing 3.0 g of bovine serum albumin was pumped into the column, discharging about 20 mL of excess stationary phase without chiral selector from the column outlet. The column therefore contained 65 mL of lower phase with no chiral selector at its outlet. During the separation, a portion of this blank stationary phase was retained in the column to absorb any chiral selector that might be carried over by the mobile phase, thus preventing UV absorbance caused by bovine serum albumin and its contamination into the CCC fractions. The upper mobile phase was pumped into the column at 0.5 mL min-1 while the column was rotated at 800 rpm in the tail-to-head elution mode. The sample solutions were prepared as follows: 2.0 mg of DL-tryptophan was dissolved in 1.0 mL of mixture of upper phase and lower phase containing albumin (1:1, v/v). Sample solution was injected after the hydrodynamic equilibrium was reached, as indicated by a clear mobile phase eluting at the outlet. The effluent from the separation column needs to be diluted by water before entering the detector cell in order to obtain a smooth recording of the chromatogram. The on-line dilution was realized by a MicrokrosTM cross flow syringe filters with hollow fibers membrane (Spectrum laboratories, Inc., CA, USA). The absorbance of the effluent was continuously monitored at 280 nm and 2 mL fractions were collected. Using the aforementioned experiment conditions, the tryptophan racemate was almost completely resolved into two peaks, i.e., first D-tryptophan followed by L-tryptophan which has a high affinity to the BSA chiral selector. Optical purities for both enantiomers were over 96% as determined by chiral HPLC. However, it was noted that the second L-tryptophan peak was extremely broadened such that the ratio of the theoretical plate of first peak over second peak was measure as large as 12. In order to investigate the cause of this unusual phenomenon, the mass transfer rate of L-tryptophan was investigated using a simple rotary device which was reported eariler. The result indicated that the mass transfer rate of L-tryptophan was much lower than that of D-tryptophan apparently due to low transfer rate of BSA-L-tryptophan complex through the interface between the two phases.