以全管柱偵測法探討液相層析之分離

Abstract

液相層析法是一種可以應用到各科學部門的有力分離方法，由於傳統偵測方式之限制，導致層析理論方面有時會有錯誤之陳述，本研究以全管柱型偵測系統觀察層析管柱內的樣品流動訊號，藉著獲得空間及時間的實驗數據，來對部分有疏失的層析理論進行修正。 根據實驗結果顯示，本研究可釐清數項容易被忽略或者誤導的層析觀念：第一，以15組不同組成之動相沖堤樣品，使獲得15組滯留因子（k）的實驗結果，層析圖譜中，k=7.27的波峰寬度約為k=0.54樣品的4.6倍，然而，兩組分析物在管柱內的空間分佈寬度卻幾乎相同，此結果讓我們清楚地了解，層析圖譜上影響波峰變寬的真正原因是樣品釋放出管柱的速度，換句話說，層析圖譜上波峰寬度的變化並不能直接反應出樣品在管柱內的真正空間分佈寬度；第二，藉著階梯式梯度沖堤的實驗，讓我們了解在非線性層析條件中，傳統計算理論板數以及板高的公式，不能用來處理非線性條件的實驗數據；第三，藉著觀察階梯式梯度沖堤非線性層析實驗之管柱內樣品遷移情形，讓我們更清楚的知道梯度沖堤條件對分離實驗的影響，並成功的觀察到分析物在管柱內所產生的滯留順序交錯現象。 藉由本研究室所設計研發之全管柱型偵測系統的應用，使我們更全面性地觀察樣品在層析管柱中的分離行為，進而成功地修正一些容易被層析操作者所忽略的錯誤層析觀念。

Liquid chromatography is a powerful separation method that finds applications to all branches of science. There are, however, some mistaken statements in chromatographic theory caused owing to the limitation of using conventional detection techniques. We designed a whole-column detection (WCD) system which enabled monitoring of whole on-column chromatographic peaks during the elution process on both spatial and time coordinate in order to clarify the shortcomings. In the first part, a single-compound sample was eluted under mobile phases of fifteen different compositions, generating chromatograms of 15 different retention factors (k). The bandwidth of the peak (k=7.27) increased by almost 4.6 folds compared with the peak (k=0.54). However, the bandwidths of these two peaks remained almost the same on the spatial coordinate in the column. The peak width was found linearly dependent on the solute releasing speed from the column outlet to the attached post-column detector. In other words, the peak widths obtained using the post-column detector cannot reflect the true widths in the column. In the second part, stepwise gradient elutions were carried out. The theoretical plate number and the plate height were erroneously obtained using the traditional equations. It revealed the incompetence of the theoretical plate number and plate height definition while non-linear chromatographies are performed. In the third part, solute migrations were clearly monitored in the column. Peak crossover phenomenon was observed during the entire migration course using the WCD system. In conclusion, we fabricated a WCD system in this study to comprehensively observe the elution process in liquid chromatography. Several misconceptions were clarified by the spatial data. The detailed on-column observations obtained using the WCD system may further provide useful information to uncover hidden aspects which may have been ignored by chromatographers.