Development and optimization of a system for comprehensive two-dimensional liquid chromatography with UV and mass spectrometric detection for the separation of complex samples by multi-step gradient elution

作者：Eggink Mark^*; Romero Wilmar; Vreuls Rene J; Lingeman Henk; Niessen Wilfried M A; Irth Hubertus

来源：Journal of Chromatography A, 2008, 1188(2): 216-226.

DOI：10.1016/j.chroma.2008.02.072

• comprehensive two-dimensional liquid chromatography
• LC x LC
• complex mixtures
• multi-step gradient elution

Comprehensive two-dimensional liquid chromatography (LC x LC) is a powerful tool for the separation of complex biological samples. This technique offers the advantage of simplified automation and greater reproducibility in a shorter analysis time than off-line two-dimensional separation systems. In the present study, an LC x LC system is developed enabling simultaneous UV and MS detection, and which can be easily converted to a conventional reversed-phase LC-UV/MS system. In LC x LC, a 60-min reversed-phase LC separation with a linear solvent gradient in the first dimension is coupled to a second-dimension separation on a mixed-mode cation-exchange/reversed-phase column with a modulation time of 60 s. The isocratic separation in the second-dimension column is optimized by the use of a multi-step gradient where the organic and the ionic modifier are varied independently. Intraday (n = 3) and interday (n = 4) variability of the retention times were evaluated with the complete system and found to be 0.5% and 0.7%, respectively. Good linearity was observed in calibration curves for three different compounds varying in polarity.

• 出版日期2008-4-25