Off‐line comprehensive two‐dimensional hydrophilic interaction×reversed phase liquid chromatographic analysis of green tea phenolics

Abstract

The separation of green tea phenolic compounds by comprehensive 2-D LC (LCxLC) has been studied. An off-line LCxLC system was designed based on hydrophilic interaction chromatography in the first dimension and gradient RP-LC using a C18 column in the second dimension, to separate compounds based on polarity and hydrophobicity, respectively. Green tea was found to contain a complex composition of low molecular weight phenolic compounds, identified by fluorescence, diode-array and negative ESI-MS detection systems. The LCxLC system was characterized by a very high practical peak capacity (>2100, in approximately 22.5 h) due to the low degree of correlation (r(2)<0.2) between the selected separation mechanisms. The proposed methodology demonstrated its suitability for the analysis of various groups of phenolic compounds including proanthocyanidins, phenolic acids, flavonols and flavonol derivatives, all of which cannot be separated in a single analysis by conventional 1-D HPLC methods.

Comprehensive two-dimensional liquid chromatographic analysis of anthocyanins

“…Various combinations of modes such as have been applied to the analysis phenolic compounds in a wide variety of samples, including NP-LC×RP-LC [146][147][148], HILIC×RP-LC [130,137,138,149,150] and RP-LC×RP-LC [144,[151][152][153][154][155][156]. The same separation mode may be applied in both the dimensions, as is the case with RP-LC×RP-LC, which has been demonstrated by Mondello and co-workers [144,151,153,156].…”

“…HILIC×RP-LC has been studied in detail by Kalili et al [138,149,150] for the analysis of diverse phenolic compounds in natural products. These authors also hyphenated on-line HILIC×RP-LC with fluorescence and MS detection for the analysis of grape procyanidins [137] .…”

“…Off-line HILIC×RP-LC was utilised by Kalili et al [130,138,149,150], for the analysis of various phenolic compounds such as procyanidins, phenolic acids, flavanols etc., in a range of samples which included cocoa, apples, green tea and rooibos tea. Much higher peak capacities were obtained compared to the on-line configuration, however at longer analysis times.…”

“…UV is by far the most common detection method in the LC×LC analysis of phenolics [144,159,162], although the selectivity of fluorescence for procyanidins has been exploited in combination with LC×LC [130,137,138,149,150], and several studies have used MS in combination with LC×LC [137,138,163]. Hyphenation of LC×LC with MS is expected to find increasing application in phenolic analysis.…”

Theory and Practice of UHPLC and UHPLC–MS

“…It allows independent optimization of the two separation systems; the fractions collected from the first column can be pretreated before injection onto the second column, such as using evaporation to dryness of manually collected HILIC fractions before introduction to a C 18 column in the second dimension . Off-line coupling of HILIC and RP-LC was shown to provide a powerful separation system for procyanidins (Kalili and de Villiers, 2010) and flavonoids (Beelders et al, 2012). Another recent example reports an off-line RP-HILIC method coupled with MS for impurity profiling of infusion solutions (Schiesel et al, 2012).…”