以全管柱偵測法探討液相層析之波峰壓縮效應

Abstract

樣品甫注入液相層析管柱時，會產生波峰壓縮效應，另一種波峰壓縮效應則發生於梯度沖提時，於兩溶劑強度不同之移動相交界處。本研究以全管柱偵測(whole-column detection, WCD)系統觀察以上兩種波峰壓縮效應，全管柱偵測系統可直接觀察梯度沖提過程中的以空間與以時間為座標軸之波峰變化。 在管柱入口處樣品波峰寬度和滯留因子k有很高的相關性，在管柱入口約一公分處，樣品波峰在空間座標之標準差和1/(1+k)成線性關係，在填充效率較好之管柱中，使用溶劑強度較小之移動相沖提，會產生較大的k值，當樣品分子位移至管柱末端時，其波峰寬度比使用溶劑強度較大之移動相沖提可能會較狹窄；然而，使用填充效率較差之管柱，以較大k值移動相沖提，當樣品波峰位移至管柱末端時，波峰寬度則可能會較寬。以連續式沖提，其理論板數之增加會超過使用階梯式沖提之理論板數增加。在本研究中，我們以樣品移動速率為基礎來計算梯度沖提之理論板數，和傳統方式計算理論板數相比，其結果顯示有大幅的改善，在某些實驗中有高達四倍的改進。

Peak compression occurred when a sample solution was injected into a liquid chromatographic column. Another compression phenomenon took place at the interface of the two mobile phases with different solvent strengths under a gradient elution. We examined both effects using a whole-column detection (WCD) system which enabled direct observations of both the spatial and temporal peak profiles during the elution. The sample peak width at the column inlet was highly related to the retention factor k. The peak standard deviation on the spatial coordinate was linearly proportional to 1/(1+k) at the column position ~1 cm. The peak width might stay narrower at the column outlet for a chromatographic run of a large k compared with a run of a small k with the use of a column with good efficiency. However, the peak width of a large k might become wider at the column outlet with the use of a low-efficiency column. The plate number increase of the continuous gradient prevailed over that of the stage-wise gradient. Plate numbers for gradient elutions were calculated in this study based on analyte moving rates. The results gave significant improvements, up to four folds in some experiments, compared with the use of conventional equations.