逆向流層析酸鹼波峰聚集法應用於高效能液相層析可行性之研究

Abstract

近年來，在逆向流層析(Countercurrent Chromatography)之研究領域，發展出一種稱之為酸鹼波峰聚集法的高效率分離方法。分離的過程中，需在分離管柱中注入一過量的有機酸（或鹼），此過量之酸或鹼會在管柱中形成一酸鹼值變化極大的移動介面，樣品分子因為在酸鹼值不同的環境下，於動靜相之分配係數變化極大，從而在流析過程中產生了高度聚集的現象。本研究嘗試將這個新的分離方法用於高效能液相層析（High Performance Liquid Chromatography），實驗過程中使用了兩種不同的靜相，亦即逆相（reversed phase）靜相和離子交換 (ion exchange) 靜相，來測試其可行性。結果發現逆相靜相完全無可行性，至於離子交換靜相，雖然目前尚無法成功達到樣品濃縮的結果，我們卻探討出一些線索，可以提供給後續研究方向。同時，我們發現在探討此種特殊分離技術的分離機制時，要特別注意考慮一個逆向流層析和高效能液相層析最基本不同之處，也就是高效能液相層析之靜相是附著於管柱內之固體填充劑上，而逆向流層析之液體靜相卻是以向心力滯留於分離管柱中。固體填充劑在此特殊的分離過程中，對所加入的酸鹼產生了和在逆向流層析中不同的結果，沒有產生一個酸鹼值變化極大的移動介面，因而無法對樣品分子造成酸鹼聚集的效應，因此對於填充式靜相的效應需要做進一步的瞭解，或有可能證明酸鹼聚集法也可實施於高效能液相層析。

Recently, a new high-efficient separation technique called pH-peak focusing has been developed in countercurrent chromatography (CCC). During the separation process, an excessive amount of organic acid or base is injected in the column. This acid or base creates a moving interface of sharp pH change in the column. The partition coefficients of sample molecules change dramatically under the environment of the different pH; and results in highly focused sample peaks. In this study, we try to transplant the idea from CCC to high-performance liquid chromatography (HPLC). We use two stationary phases, i.e. reversed-phase and ion-exchange, to examine the feasibility. The outcome shows the improbability of using the reversed-phase in this application. As for the ion-exchange stationary phase, we have explored some clues that may be helpful for future study although we are unable to achieve pH-peak-focusing on HPLC presently. As generally known, the stationary phase of HPLC is either coated or bonded to the solid phase packing material while the liquid stationary phase is retained in the column of CCC by centrifugal force without supporting solid. We find that this fundamental difference between CCC and HPLC should not be ignored. The existence of the solid packing prevents the formation of a sharp pH interface that is observed in CCC. This may be responsible for the failure of peak focusing. Accordingly, the effect of the solid packing on peak focusing in HPLC must be further studied to make a breakthrough in this novel research.