离线全二维逆流色谱-液相色谱分离莪术油成分

Off-line comprehensive two-dimensional countercurrent chromatography-liquid chromatography separation of Curcuma volatile oil

中药挥发油成分复杂,一维色谱分离由于有限的峰容量难以完全分离中药挥发油成分,全二维气相色谱为分离挥发油成分提供了有力的方法,然而气相色谱一般无法用于天然活性成分的筛选。为建立挥发油成分全二维色谱分析新方法,研究建立以液相色谱为基础的全二维色谱分离分析方法。本文主要研究全二维逆流色谱-液相色谱分离莪术油成分的方法,并探讨两种色谱技术之间的正交性,为活性成分筛选提供新的技术支持。通过优化离线全二维逆流色谱-液相色谱分离方法,对全二维色谱峰容量、正交性和空间覆盖率进行度量。优化液相色谱分析条件并筛选逆流色谱分离两相溶剂体系,通过比色法筛选了逆流色谱两相溶剂体系并采用下相为流动相进行梯度洗脱。在290~375 min采用推挤洗脱,莪术油在第一维逆流色谱分离中达到了良好的分离。第二维反相高效液相色谱的流动相组成为乙腈(A)和水(B)。梯度洗脱程序为0~10 min,50%A~65%A;10~14 min,65%A;14~21 min,65%A~85%A;21~25 min,85%A~95%A;25~30 min,95%A~55%A;30~40 min,55%A。在上述条件下进行全二维色谱分析,结果表明该全二维分离体系总峰容量达954个,理论峰容量达到一维色谱的10倍以上,同时具有良好的正交性(线性相关性r=0.17)和空间覆盖率(68.1%)。以上结果表明,离线全二维逆流色谱-液相色谱分离莪术油具有良好的正交性,能显著提高复杂样品的分离效果,为挥发油物质基础研究和活性成分筛选以及二维色谱指纹图谱的建立提供了新的方法与思路。

The chemical constituents of volatile oils used in traditional Chinese medicine are highly complex.Thus,achieving the complete separation of volatile oils by one-dimensional chromatography is difficult owing to the low peak capacity of the technique.Although comprehensive two-dimensional gas chromatography provides an efficient means for separating volatile oils,it cannot be used to screen bioactive components because of its limitations.Therefore,developing a new method to separate volatile oils based on liquid chromatography is of great significance in efforts to obtain new approaches to screen bioactive components in volatile oil.The objectives of the present study are to establish an efficient method for separating the chemical constituents of Curcuma volatile oil using off-line comprehensive two-dimensional countercurrent chromatography-liquid chromatography(CCC-LC)and to investigate the two-dimensional peak capacity,orthogonality,and spatial coverage of this method.Both CCC and LC conditions were optimized.A biphasic n-hexane-methanol-water solvent system was selected via the colorimetric method,and the lower phase was used as the mobile phase in gradient-elution mode:0-55 min,n-hexane-methanol-water(5∶2∶3 v/v/v);55-170 min,n-hexane-methanol-water(5∶3∶2,v/v/v);170-290 min,n-hexane-methanol-water(5∶4∶1,v/v/v).After gradient elution,elution-extrusion elution mode was applied within 290-375 min.Good resolution was achieved by the CCC separation process.The HPLC separation process was carried out with gradient elution using a mobile phase composed of acetonitrile(A)-water(B):0-10 min,50%A-65%A;10-14 min,65%A;14-21 min,65%A-85%A;21-25 min,85%A-95%A;25-30 min,95%A-55%A;30-40 min,55%A.Curcuma volatile oil was separated under the above optimized two-dimensional separation conditions,and the data obtained were drawn into a two-dimensional contour map using Matlab software.The calculated total peak capacity exceeded 954,which was 10 times more than that of one-dimensional chromatography.High orthogonality(r=0.17)and spatial coverage factor(68.1%)were also obtained.Our research provides a new methodology for separating volatile oils used in traditional Chinese medicine as well as an approach for evaluating the quality of traditional Chinese medicinal herbs using two-dimensional chromatographic fingerprints.