以全管柱偵測法探討液相層析之波峰跳動現象

Abstract

在層析的過程中，將沖堤能力較弱的移動相變換為沖堤能力較強的移動相，便會加快固定相將樣品釋放到移動相的速度。若樣品已經有一部分流出管柱時感受到移動相的沖堤能力增強，則管柱末端填充的固定相會加速釋出樣品，隨後被管柱外的UV/Vis偵測器偵測到，造成層析圖譜中波峰特殊的轉折甚至跳動。 本研究利用全管柱偵測法(Whole-column detection)配合管柱後端連接的UV/Vis偵測器，來觀察階梯式沖堤(stepwise elution)時不同時間改變移動相對管柱末端樣品的影響。全管柱偵測法可以得到管柱中樣品濃度的空間分布，以補足管外偵測器所無法觀察到的管內樣品實際的流動狀況。 實驗中發現，樣品在沖堤力強的移動相到達之前便先流出管柱的話，則層析圖譜的波峰高度會受到壓縮效應(compression effect)及擴散效應(diffusion effect)影響，移動相沖堤能力改變較不明顯者會受擴散效應影響較大，反之，若移動相沖堤能力改變較劇烈者會受到壓縮效應影響較強。 另外，樣品在部分流出管柱時遭遇移動相之改變，移動相沖堤能力改變較不明顯者，層析圖譜中波峰產生轉折，若移動相沖堤能力改變較劇烈者，則圖譜中波峰有跳動的現象。上述觀察結果正好印證了白書禎教授於尾論(Tailography)中的計算推導。

In the chromatographic process, the release of sample from stationary phase will speed up when the mobile phase of weak strength changed to the mobile phase of strong strength. If the solvent strength of the mobile phase became stronger when a part of sample had already flowed out of the column, the sample would be released faster from the stationary phase packed in the end of the column. Then, that would cause a shoulder or jump on the chromatogram monitored using a post-column detector. In this research, we used whole-column detection (WCD) and a UV/Vis detector to examine the influence of stepwise elutions at which the mobile phase was changed at different time when the sample appeared at the end of the column. The WCD was used to obtain the spatial distribution of sample which was not available in using the post-column detector. It is suggested that both the compression effect and diffusion effect would affect the peak shape. Which effect dominated was decided by the difference of solvent strengths of the two mobile phases. When the difference of the solvent strengths was large, the compression effect dominated, and vice versa. In addition, there would appear a shoulder on the chromatogram if the strength of second solvent was not much greater than that of the first solvent. However, there would be a signal jump if the second solvent possessed a much greater strength than that of the first. This observation matches the tailography theory proposed by Pai in recent years.