Optimization of separation in two-dimensional high-performance liquid chromatography by adjusting phase system selectivity and using programmed elution techniques

Multidimensional LC×LC analysis of phenolic and flavone natural antioxidants with UV‐electrochemical coulometric and MS detection

“…First dimension gradient: 5% B 0 -2 mL; 5 -45% B 2 -6 mL; 45% B 6 -7 mL (the total gradient time of 70, 140 or 280 min corresponds to the 7 mL volume of eluate from the first dimension PEG column). The 2-D gradients were matched to the total time of the first dimension separation and was optimized as described elsewhere [20], according to the retention of the second dimension column. After the end of each second dimension run, both columns were equilibrated with the initial gradient mobile phases for 15 min.…”

“…However, there is still partial correlation of the retention in the two dimensions under isocratic conditions, which is the reason for imperfect orthogonality of the PEG-C18 2-D systems. We have found earlier that using parallel gradients of ACN in buffered mobile phases for 2-D LC6LC separations of samples containing both phenolic acids and flavone compounds can largely improve the orthogonality of the two systems [19,20]. Further, gradient elution significantly increases peak capacity in both the first and in the second dimensions, as it allows covering a broad retention range and it results in narrower bandwidths in comparison to isocratic separations.…”

“…Two parallel gradients in the first and in the second dimension, spanning over the whole 2-D separation time offer an alternative to fast second dimension gradient in comprehensive LC6LC [19,20]. In this approach, the second dimension separation time can be utilized more efficiently, as the re-equilibration to the initial conditions is not necessary for each transferred fraction, but can be accomplished after the end of the whole 2-D separation, at the same time as the re-equilibration of the first dimension column.…”

“…In this approach, the second dimension separation time can be utilized more efficiently, as the re-equilibration to the initial conditions is not necessary for each transferred fraction, but can be accomplished after the end of the whole 2-D separation, at the same time as the re-equilibration of the first dimension column. Matching parallel gradients with increasing elution strength in the two dimensions decreases the retention of later eluting compounds in the second dimension, improves the separation efficiency, provides a more regular coverage of the available space in the second dimension and increases the 2-D peak capacity in comprehensive LC6LC, especially for the systems with partially correlated retention under isocratic conditions [19,20].…”

Theory and Practice of UHPLC and UHPLC–MS

“…On the one hand, for NP  RP separation of carotenoids, either silica or cyanopropyl (CN) stationary phases have been investigated (Dugo et al , 2008d(Dugo et al ,e, 2009cCacciola et al, 2012Cacciola et al, , 2016a, whereas for triacylglycerol (TAG) separation, an Ag  RP combination turned out to be useful for this task Dugo et al, 2006b,c;Van der Klift et al, 2008;Hu et al, 2013;Wei et al, , 2015Costa et al, 2015;. On the other hand, for RP  RP separation of phenolic antioxidants, the effect of several stationary phase chemistries (C8, C18, CN, Diol, F 5 , NH 2 , PEG, Phenyl, RPAmide, and other laboratory-made stationary phases) on the separation selectivity and resolution has been investigated (Blahovà et al, 2006;Cacciola et al, 2006Cacciola et al, , 2007aCacciola et al, ,b,c, 2011Hyilompolo, 2007, 2008;Pol et al, 2007, Pol andDugo et al, 2008cDugo et al, , 2009aJandera et al, 2008aJandera et al, ,b, 2012Hájek et al, 2008Cesla et al, 2009;Kalili and de Villiers, 2009, 2013aZhou et al, 2009;Russo et al, 2011;Fu et al, 2012;Hájek and Jandera, 2012;Kalili et al 2013Kalili et al , 2014Montero et al, 2013aMontero et al, ,b, 2014Montero et al, , 2016aKrol-Kogus et al, 2014;Krauze-Baranowska et al, 2014;Leme et al, 2014;Tanaka et al, 2014;…”