chapter  4
98 Pages

Principles of Online Comprehensive Multidimensional Liquid Chromatography

Two-Dimensional Separations .......................................................... 157 4.3.3 Practical Peak Capacity and Fractional Coverage ............................ 158

4.3.3.1 Combined Effect of Undersampling and Fractional Coverage on Two-Dimensional Peak Capacity .................. 161

4.3.3.2 Analogies to Practical Peak Capacity and Fractional Coverage in Ecology ................... 161

4.3.3.3 Reexamination of the Fractional-Coverage Method of Gilar et al. ...................................................................... 163

4.3.3.4 Assessment of Current Metrics for Practical Peak Capacity and Fractional Coverage ..................................... 165

4.4 Optimization in Online LC × LC ................................................................. 166 4.4.1 Comparison of Isocratic and Gradient Peak Capacity ..................... 167 4.4.2 Optimization of Peak Capacity in Gradient and

Isocratic Elution Chromatography .................................................... 169 4.4.3 Early Studies of Optimization in Online LC × LC .......................... 173

4.4.3.1 Schoenmakers et al. ........................................................... 173 4.4.3.2 Horie et al. .......................................................................... 173 4.4.3.3 Li et al. ............................................................................... 175

Although multidimensional liquid chromatography has a long history, there is little question that interest in the ˆeld was greatly stimulated by the pioneering work of Erni and Frei [1] and, more recently, by Bushey and Jorgenson’s [2] separation of a protein mixture through the use of an ion exchange separation followed by size exclusion chromatography. Unquestionably, work in two-dimensional liquid chromatography (2D-LC) has beneˆted greatly from earlier developments in two-dimensional gas chromatography (2D-GC) [3-6]. A great deal of the current work in 2D-LC is focused on its use as a tool in proteomics wherein an ion-exchange separation of the tryptic peptides of complex mixtures of proteins [7-9] is followed by a reversedphase separation prior to mass spectrometric identiˆcation of the peptides.