Kathithileni M. Kalili

Recent developments in the HPLC separation of phenolic compounds.

Phenolic compounds represent a class of highly complex naturally occurring molecules that possess a range of beneficial health properties. As a result, considerable attention has been devoted to the analysis of phenolics in a variety of samples. HPLC is the workhorse method for phenolic separation. However, conventional HPLC methods provide insufficient resolving power when faced with the complexity of real-world phenolic fractions. This limitation has been traditionally circumvented by extensive sample fractionation, multiple analysis methods and/or selective detection strategies. On the other hand, there is an increasing demand for improved throughput and resolving power from the chromatographic methods used for phenolic analyses. Fortunately, during the last decade, a number of important technological advances in LC have demonstrated significant gains in terms of both speed and resolution. These include ultra high-pressure liquid chromatography (UHPLC), high-temperature liquid chromatography (HTLC), multi-dimensional separations as well as various new stationary phase chemistries and morphologies. In recent years, these technologies have also found increasing application for phenolic analysis. This review seeks to provide an updated overview of the application of recent advances in HPLC to phenolic separation, with the emphasis on how these methodologies can contribute to improve performance in HPLC analysis of phenolics.

Off-line comprehensive 2-dimensional hydrophilic interaction × reversed phase liquid chromatography analysis of procyanidins

The development of an off-line comprehensive 2-dimensional liquid chromatography (2-D-LC) method for the analysis of procyanidins is reported. In the first dimension, oligomeric procyanidins were separated according to molecular weight by hydrophilic interaction chromatography (HILIC), while reversed phase LC was employed in the second dimension to separate oligomers based on hydrophobicity. Fluorescence, UV and electrospray ionisation mass spectrometry (ESI-MS) were employed for identification purposes. The combination of these orthogonal separation methods is shown to represent a significant improvement compared to 1-dimensional methods for the analysis of complex high molecular weight procyanidin fractions, by simultaneously providing isomeric and molecular weight information. The low correlation (r(2)<0.2100) between the two LC modes afforded a practical peak capacity in excess of 2300 for the optimal off-line method. The applicability of the method is demonstrated for the analysis of phenolic extracts of apple and cocoa.

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

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 rooibos ( Aspalathus linearis ) phenolics

Rooibos tea is an unique beverage prepared from unfermented and fermented plant material of the endemic Cape fynbos plant, Aspalathus linearis. The well-known health-promoting benefits of rooibos are partly attributed to its phenolic composition. Detailed investigation of the minor phenolic constituents of rooibos is, however, hampered by the limitations associated with conventional HPLC methods used for its analysis. In this study, the applicability of comprehensive two-dimensional liquid chromatographic methods for the in-depth analysis of rooibos phenolics was investigated. Phenolic compounds were separated according to polarity by hydrophilic interaction chromatography (HILIC) in the first dimension, whilst reversed-phase liquid chromatography (RP-LC) provided separation according to hydrophobicity in the second dimension. Ultraviolet photodiode array and electrospray ionisation mass spectrometry were used to identify phenolic compounds. Comprehensive HILIC × RP-LC demonstrated its applicability for the analysis of a diverse range of phenolic compounds in unfermented and fermented rooibos samples, in which large qualitative differences in the phenolic composition were established. The combination of these orthogonal separations provided a significant improvement in resolution, as exemplified by practical peak capacities in excess of 2000 and 500 for off-line and on-line methods, respectively.

Systematic optimisation and evaluation of on-line, off-line and stop-flow comprehensive hydrophilic interaction chromatography×reversed phase liquid chromatographic analysis of procyanidins, part I: theoretical considerations.

Comprehensive two-dimensional liquid chromatography (LC×LC) provides significantly improved separation for complex real-life samples. LC×LC can be performed in one of three different ways, using on-line, off-line, or stop-flow configurations. We have previously shown how off-line comprehensive hydrophilic interaction chromatography (HILIC)×reversed-phase liquid chromatography (RP-LC) provides a powerful separation system for procyanidins (PCs), one of the most complex fractions of natural phenolics. In the current contribution, a systematic approach for the optimisation and evaluation of each of the LC×LC methodologies is presented using HILIC×RP-LC analysis of PCs as application. Optimisation was performed using the peak capacities of individual one-dimensional separations measured for different gradient times and flow rates and their combination in each of the three LC×LC modes by taking into account the effects of first dimension under-sampling, the degree of orthogonality between the two dimensions and additional band broadening associated with stop-flow analysis. The performance of all three methods is compared in terms of practical peak capacities, analysis times and peak production rates. One-dimensional LC provided the best performance for separations requiring relatively low peak capacities, whereas the on-line LC×LC system was advantageous for required practical peak capacities up to ~600. For higher resolution, the off-line or stop-flow systems should be used. Especially noteworthy is the fact that, due to slow diffusion of PCs, the contribution of stop-flow to first dimension band broadening was negligible for stop-flow times of up to 15min. In a separate contribution, the experimental verification of the findings of this study will be reported.

Toward Unraveling Grape Tannin Composition: Application of Online Hydrophilic Interaction Chromatography × Reversed-Phase Liquid Chromatography–Time-of-Flight Mass Spectrometry for Grape Seed Analysis

Despite the significant importance of tannins in viticulture and enology, relatively little is known about the detailed chemical composition of these molecules. This is due to challenges associated with the accurate analytical determination of the highly structurally diverse proanthocyanidins which comprise tannins. In this contribution, we address this limitation by demonstrating how online comprehensive two-dimensional liquid chromatography (LC × LC) coupled to high resolution mass spectrometry (HR-MS) can be exploited as a powerful analytical approach for the detailed characterization of grape seed tannins. Hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RP-LC) were employed in the two dimensions to provide complementary information in terms of separation according to hydrophilicity and hydrophobicity, respectively. Online coupling of HILIC × RP-LC with fluorescence detection and electrospray ionization MS delivered high resolution analysis in a practical analysis time, while allowing selective detection and facilitating compound identification. Time-of-flight (TOF) MS provided high acquisition rates and sensitivity coupled to accurate mass information, which allowed detection of procyanidins up to a degree of polymerization (DP) of 16 and a degree of galloylation up to 8 in a red grape seed extract. This analytical methodology promises to shed new light on these important grape constituents and potentially on their evolution during wine production.

Systematic optimisation and evaluation of on-line, off-line and stop-flow comprehensive hydrophilic interaction chromatography × reversed phase liquid chromatographic analysis of procyanidins. Part II: Application to cocoa procyanidins

Procyanidins present a severe analytical challenge due to their structural complexity and diversity. Comprehensive two-dimensional hydrophilic interaction chromatography×reversed-phase liquid chromatography (HILIC×RP-LC) provides a highly efficient separation system for procyanidins. In the first part of this contribution a systematic approach for the optimisation and evaluation of HILIC×RP-LC analyses in on-line, off-line and stop-flow modes was presented. The three systems were compared in terms of peak capacity, the number of peaks produced per unit time as well as the total analysis time required to carry out such analyses by taking under-sampling, degree of orthogonality and stop-flow band-broadening into account. In this paper, the experimental verification of these findings using cocoa procyanidins as an application is presented. The results show that while optimisation procedures based on theoretical considerations remain largely valid in practice, several important experimental considerations should also be taken into account to achieve maximum performance in all three modes of HILIC×RP-LC. On-line analysis provides an effective tool for the screening of procyanidin content within reasonable times, provided that under-sampling of first dimension peaks is minimised. Off-line- and stop-flow HILIC×RP-LC analyses on the other hand are more suited for the detailed analysis of complex procyanidin fractions, with the latter being shown to be a promising automated alternative providing the same performance as the off-line approach. Experimental data presented verify the conclusion that stop-flow operation has a negligible effect on first dimension band broadening under the optimised experimental conditions used.

Comprehensive two-dimensional liquid chromatography coupled to the ABTS radical scavenging assay: a powerful method for the analysis of phenolic antioxidants

AbstractThe on-line combination of comprehensive two-dimensional liquid chromatography (LC × LC) with the 2,2′-azino-bis(3-ethylbenzothiazoline)-6 sulphonic acid (ABTS) radical scavenging assay was investigated as a powerful method to determine the free radical scavenging activities of individual phenolics in natural products. The combination of hydrophilic interaction chromatography (HILIC) separation according to polarity and reversed-phase liquid chromatography (RP-LC) separation according to hydrophobicity is shown to provide much higher resolving power than one-dimensional separations, which, combined with on-line ABTS detection, allows the detailed characterisation of antioxidants in complex samples. Careful optimisation of the ABTS reaction conditions was required to maintain the chromatographic separation in the antioxidant detection process. Both on-line and off-line HILIC × RP-LC–ABTS methods were developed, with the former offering higher throughput and the latter higher resolution. Even for the fast analyses used in the second dimension of on-line HILIC × RP-LC, good performance for the ABTS assay was obtained. The combination of LC × LC separation with an on-line radical scavenging assay increases the likelihood of identifying individual radical scavenging species compared to conventional LC–ABTS assays. The applicability of the approach was demonstrated for cocoa, red grape seed and green tea phenolics. FigureOn-line HILIC×RP-LC–ABTS analysis of cocoa proanthocyanidins

Kinetic optimisation of the reversed phase liquid chromatographic separation of proanthocyanidins on sub-2μm and superficially porous phases

Phenolic compounds, and proanthocyanidins in particular, are important natural molecules which are of significant importance due to their sensory and biological activities. The analysis of proanthocyanidins in natural products is very challenging due to their complex nature. In this study, the kinetic performance of a range of recently developed C18 columns, including sub-2 μm fully porous and 2.6 μm superficially porous particle-packed columns, was evaluated for improved proanthocyanidin analysis. The kinetic plot method was employed to compare the ultimate performance limits of each column in terms of efficiency and speed for different maximum pressures and temperatures using representative proanthocyanidins comprising a range of molecular weights and functionalities as test analytes. By combining plate height data with relevant parameters such as column permeability and mobile phase viscosity, plots of practically attainable efficiencies as a function of analysis time for specific experimental configurations were obtained and performance limits for all investigated supports could accurately be compared. Both fully- and superficially porous particles provided significant speed and/or efficiency gains compared to conventional 5 μm particle packed columns. Analyte properties, particle size and packing quality as well as analysis temperature were all found to have a significant influence on the performance of the presently investigated chromatographic supports. For smaller compounds, higher optimal linear velocities and better performance in the low-efficiency range were observed, while the lower diffusion coefficients of larger procyanidins resulted in lower optimal linear velocities and better performance in the high-efficiency regime. Faster analyses become possible at higher temperatures due to decreased eluent viscosity and faster mass transfer, which was especially beneficial for larger compounds and resulted in dramatic improvement in efficiency. A possible explanation of the abnormal behaviour of oligomeric procyanidins is presented. Our findings indicate that new column formats, when used under optimal conditions, significantly improve the speed and/or efficiency of reversed phase liquid chromatographic analyses of proanthocyanidins.

Fast method development of rooibos tea phenolics using a variable column length strategy

The development of a method for the separation of standard compounds of the 15 main phenolics found in rooibos tea is presented. The separation of these compounds in a single HPLC analysis is particularly challenging due to the similarity of rooibos phenolics. As a result, multiple methods are often required to analyze all major phenolics in rooibos tea samples. The method development process is significantly enhanced in this study by using the recently introduced automated column coupler in combination with the variable column length strategy. This strategy consists of performing the initial scouting runs, wherein the best separation conditions are determined, on a short column and subsequently fine-tuning the separation on longer columns to benefit from their higher separation performance. It is demonstrated that the method development process can further be expedited by operating each column length at the maximum pressure, in this case 1000 bar. Although this holds in general, it is even more the case for the presently considered sample, since the selectivity of the sample is more pressure- than temperature-dependent. Applying the optimized method to unfermented and fermented aqueous rooibos tea extracts in combination with Q-TOF mass spectrometry, some 30 phenolic compounds are tentatively identified.