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

Abstract

A comprehensive 2-D LC x LC system was developed for the separation of phenolic and flavone antioxidants, using a PEG-silica column in the first dimension and a C(18) column with porous-shell particles or a monolithic column in the second dimension. Combination of PEG and C18 or C8 stationary phase chemistries provide low selectivity correlations between the first dimension and the second dimension separation systems. This was evidenced by large differences in structural contributions to the retention by -COOH, -OH and other substituents on the basic phenol or flavone structure. Superficially porous columns with fused core particles or monolithic columns improve the resolution and speed of second dimension separation in comparison to a fully porous particle C(18) column. Increased peak capacity and high orthogonality in different 2-D setups was achieved by using gradients with matching profiles running in parallel in the two dimensions over the whole 2-D separation time range. Multi-dimensional set-up combining the LC x LC separation on-line with UV and multi-channel coulometric detection and off-line with MS/MS technique allowed positive peak identification. The Coularray software compensates for the effects of the baseline drift during the gradient elution and is compatible with parallel gradient comprehensive LC x LC technique. Furthermore, it provides significant improvement in the sensitivity and selectivity of detection in comparison to both UV and MS detection. The utility of these systems has been demonstrated in the analysis of beer samples.

Effects of the gradient profile, sample volume and solvent on the separation in very fast gradients, with special attention to the second-dimension gradient in comprehensive two-dimensional liquid chromatography

“…Fast gradient separations within a fixed time improve at high linear flow velocities on short efficient columns, such as sub-2 m columns at very high operation pressures. Similar results can be achieved at high flow rates allowed by the pressure limits with standard HPLC instrumentation (to 400 bar) using monolithic columns [13][14][15], or columns packed with fused-core porous-shell particles, which however usually provide better efficiencies at high flow rates [11,14,16].…”

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;…”

Strategies in the Analysis of Plant Flavonoids

“…In these systems, it is preferable to use the more polar of the two columns in the first dimension and the more retentive (usually a bonded alkylsilica) in the second dimension, so as to suppress band broadening connected with the sample transfer (Jandera, Hajek, and Cesla, 2010). Hajek et al (2008) combined a polyethylene glycol (PEG) column in the first dimension with different alkylsilica stationary phases in the second dimension for the separation of 27 antioxidant polyphenols including various flavonoids, concluding that monoliths and SPP columns provided lower hold-up time and improved resolution in the second dimension than totally porous columns of similar length. However, the better permeability of monolithic columns allowed using higher flow rates, thus leading to significant reduction in the 2D separation time compared with particle-based columns.…”