Improving HPLC Separation of Polyphenols
The in-depth study of polyphenols is hampered by their sheer complexity, combined with the inherent limitations of conventional high performance liquid chromatography (HPLC) separation methods. In this paper, the beneficial application of a number of recent developments in HPLC for polyphenol analysis is highlighted. Small particle-packed columns operated at elevated pressures and temperatures are used to improve the speed and/or resolving power of conventional HPLC methods. In addition, comprehensive two-dimensional LC combining hydrophilic interaction chromatography (HILIC) and reversed phase LC (RP-LC) is shown to be a powerful technique to increase the resolving power. Examples of the successful application of these technologies for improved analysis of procyanidins, flavonols and anthocyanins in wine, tea, cocoa and blueberry samples are presented.
The investigation of polyphenols is therefore closely related to the development of suitable analytical techniques for their identification and quantification. High performance liquid chromatography (HPLC), and reversed phase LC (RP-LC) in particular, is most often used for the routine analysis of phenolics, while in recent years the hyphenation of HPLC to mass spectrometry (MS) has become an indispensable tool for the detailed investigation of these compounds.2
The resolving power of chromatographic methods is commonly measured in terms of peak capacity: the number of peaks that can theoretically be resolved with unit resolution using a given separation method. The peak capacity (n p ) for gradient separations can be calculated according to:3
where tg is the gradient run time and w the average (4σ) peak width. Common gradient RP-LC methods typically provide peak capacities in the order of 100–150 (for ~1 h analysis times). Considering that the peak capacity should significantly exceed the number of randomly distributed analytes in a given sample to provide a high likelihood of separation,4 and the fact that typical phenolic extracts contain in excess of 100 species, the limitations of HPLC are evident.
Importantly, in recent years a number of significant developments in HPLC have contributed to deliver improved performance. These include the use of ultra high pressure liquid chromatography (UHPLC), new developments in stationary phase morphologies (monolithic columns and superficially porous particles), high temperature liquid chromatography (HTLC) and multidimensional LC separations. In this contribution we discuss the beneficial application of some of these new technologies to improve the analysis of polyphenols in a variety of samples.
Reagents and materials: Cocoa beans, wine, blueberries and green tea were purchased from a local supermarket. Epicatechin, catechin and uracil standards were from Sigma-Aldrich (Atlasville, South Africa), and anthocyanin standards from Polyphenols Laboratories (Sandnes, Norway). Dimeric procyanidins were isolated by HILIC fractionation from a cocoa extract. Mobile phases were prepared from HPLC-grade solvents (Sigma) and Milli-Q deionized water (Millipore, Milford, Massachusetts, USA).
Sample preparation: Extracts of green tea and cocoa were prepared as reported previously.5,6 For extraction of blueberry anthocyanins, 6.681 g blueberries were frozen and ground under liquid nitrogen and extracted with methanol/water/formic acid (60:37:3), followed by evaporative pre-concentration. Filtered wine samples were directly injected.
Instrumentation: HPLC–UV analyses were performed on an Alliance 2690 HPLC equipped with a photodiode array (PDA) detector (Waters, Milford, Massachusetts, USA). UHPLC experiments were performed on an Acquity UPLC system with PDA detector (Waters). HPLC–ESI-MS analyses were performed on a UPLC system coupled to a Waters Ultima Q-TOF mass spectrometer operated in positive ionization (PI) mode.6 Blueberry anthocyanins were tentatively identified by MS and MS2 spectra and comparison of retention times with literature reports.7 The following columns were used: XBridge C18 (250 × 4.6 mm, 5 µm) and Acquity BEH C18 (50 and 100 × 2.1 mm, 1.7 µm, both Waters), Zorbax SB C18 (50 × 4.6 mm, 1.8 µm, Agilent, Waldbronn, Germany) and Develosil Diol-100 (250 × 1 mm, 5 µm, Nomura Chemical, Japan).
Cocoa procyanidins and green tea flavonols: HPLC analyses at 25 °C were performed using 0.1% formic acid (A) and acetonitrile (B) at a flow rate of 1 mL/min: 0–0.63 min (2%B), 0.63–27 min (2–18%B), 27–45 min (18–25%B), 45–51.75 min (25–100%B), 51.75–56.25 min (100%B). UHPLC analyses were performed on a 100 mm Acquity column at 50 °C using the following gradient at 0.3 mL/min: 0–0.17 min (2%B), 0.17–7.4 min (2–18%B), 7.4–12.48 min (18–25%B), 12.48–14.53 min (25–100%B), 14.53–15.6 min (100%B). UV detection was performed at 280 and 370 nm for procyanidins and flavonols, respectively.
Wine anthocyanins: Mobile phases consisted of 7.5% formic acid (A) and acetonitrile (B). HPLC analyses were performed on the XBridge column at 25 °C and 1 mL/min using the following gradient: 0–1 min (1%B), 1–12 min (1–13.5%B), 12–24 min (13.5–23.5%B), 24–28 min (23.5–28.5%B), 28–35 min (28.5%B). UHPLC analyses were performed on two coupled 100 mm Acquity columns at 50 °C and 0.06 mL/min as follows: 0–3 min (1%B), 3–34 min (1–13.5%B), 34–67 min (13.5–23.5%B), 67–78 min (23.5–28.5%B), 78–98 min (28.5%B). UV detection was performed at 500 nm.
Blueberry anthocyanins: Analyses were performed on the XBridge column using the same mobile phases as for wine anthocyanins. A flow rate of 1 mL/min was used throughout with the following gradients: 0–50 min (1–25%B), 50–55 min (25–40%B) at 25 °C; and 0–50 min (1–20%B), 50–55 min (25–40%B) at 70 °C. A post-column split (1:4) was used prior to eluent introduction into the MS.
HILIC×RP-LC: Conditions were as reported elsewhere.5,6
van Deemter Experiments
For procyanidins, experiments were performed on the XBridge column at 25 °C and on a 50 mm Acquity column at 25 °C and 50 °C, and for anthocyanins on the XBridge column at 25 °C and 70 °C. Mobile phases were as specified for each of these samples above, with the composition varied to ensure similar retention factors at all temperatures. Uracil was used as unretained marker, and all experiments were performed in duplicate. van Deemter curves and kinetic plots were constructed according to reference 8.
Results and Discussion
In fact, this picture is not entirely accurate, since decreasing dp leads to a very important decrease in column permeability. Pressure constraints ultimately limit the column length and therefore efficiency attainable, so that very high efficiencies are easier to achieve for larger particle sizes (a result of the higher permeability of the larger dp phases). Note though that the price to pay for such high efficiencies is a very significant increase in analysis time.
This dilemma has been partially overcome by the availability of a new generation of high-pressure instrumentation. In Figure 2(b) the isocratic efficiency as a function of total analysis time for the procyanidin dimer is compared for the same 5 µm and 1.7 µm columns, the latter operated at pressures up to 1000 bar. Note the significant decrease in analysis time achieved on the UHPLC column for an efficiency of 25000 plates (equal to that provided by a conventional 250 mm, 5 µm column). Moreover, UHPLC clearly offers faster analysis for efficiencies up to N ~80000.
It is relatively straightforward to practically exploit the benefits of UHPLC because HPLC methods may nowadays easily be adapted to UHPLC column formats and instrumentation, essentially providing the same separation mechanism. To date this approach has received comparatively little attention for polyphenol analysis,9,10 with the focus primarily on very fast analysis on short columns, essentially providing the same efficiency as conventional HPLC columns.
However, caution should be exercised in the application of elevated temperature for phenolic analysis. As is well documented in literature, anthocyanins in particular are readily degraded at relatively mild temperatures. Fortunately this aspect may be evaluated quantitively. Using the approach of Thompson et al.15 it at can be demonstrated that analysis times of less than 2 hours at 70 °C do not lead to on-column degradation of anthocyanins.13
Finally, in the context of uni-dimensional HPLC analysis, it should be mentioned that a number of additional developments in column technology in recent years hold some promise for HPLC analysis of polyphenols. These include the use of monolithic16 and superficially porous particles.17 However, limited application of these column formats to polyphenol determination has been reported to date.18
Comprehensive two-dimensional liquid chromatography (LC×LC)
The challenges associated with HPLC analysis of phenolics may be demonstrated using an example: procyanidins (Figure 1) consist of a mixture of monomeric, oligomeric and polymeric compounds composed of flavanol building blocks. As the degree of polymerization (DP) increases, the number of possible isomers increases exponentially (a result of the possible combinations of monomers as well as different inter-flavanol bonds). For example, 48 isomers of dimeric procyanidins containing catechin and epicatechin building blocks are possible.19 Considering that optimized high-efficiency HPLC methods provide peak capacities of maximally 400, no uni-dimensional HPLC method is capable of providing complete separation of higher molecular weight (MW) procyanidins in a single analysis.
Comprehensive two-dimensional HPLC (LC×LC) is a promising method for the separation of such complex samples. LC×LC delivers peak capacities an order of magnitude or more than uni-dimensional LC. In LC×LC, two separation methods are combined in such a manner that the separation in each dimension is retained. As a consequence, the overall peak capacity in a two-dimensional space is (ideally) the product of peak capacities in each dimension. Admittedly, this ideal is only achieved if certain criteria are met, most importantly that both separations are based on different (orthogonal) mechanisms.
Comprehensive 2-D LC may be performed in off-line, on-line or stop-flow modes.19 In on-line LC×LC, fractions from the first dimension are transferred in real time to the second dimension column, usually using multi-port two-position switching valves. This approach provides relatively fast (approximately equal to 1-D HPLC methods) separations, although resolution in the second dimension is sacrificed to meet sampling rate requirements. Off-line LC×LC, by contrast, results in much longer analysis times, but since no constraints are placed on the second dimension separation much higher peak capacities may be obtained. (Stop-flow LC×LC is less often applied, and provides peak capacities and analysis times in between the off-line and on-line approaches).
The analysis of polyphenols represents a severe analytical challenge, and important advances in natural phenolic research hinge on the development of suitable separation strategies. Fortunately, recent developments in HPLC show promise for improving polyphenol analysis.
For very demanding separations, off-line comprehensive 2-D LC offers greatly improved separation power compared with HPLC (>20× higher peak capacities) at similar peak capacity production rates. The choice of the most suitable separation methodology will therefore depend on the requirements for a particular phenolic sample in terms of speed, instrumental simplicity or resolving power.
It should be noted that state-of-the-art mass spectrometric (MS) techniques such as high resolution MS and tandem MS methods, not addressed in the current contribution, represent a complimentary strategy for improving polyphenol analysis. In fact, MS offers an alternative 'separation' dimension (in terms of m/z ratio), increased selectivity for target analysis and a powerful structural elucidation tool. Combined with advanced chromatographic separation, LC–MS will prove an even more powerful tool for detailed phenolic determination.
Clearly, the judicious application of recent advances in HPLC demonstrably offers significant benefits for phenolic analyses, and shows promise for the in-depth investigation of the phenolic compounds found in numerous natural products — an endeavour which, to date, has been hampered by the lack of suitable separation methods.
The authors gratefully acknowledge Stellenbosch University, the Third World Academy of Science (TWAS, 08-077 RG/CHE/AF/AC) and NRF for financial assistance.
André de Villiers is a senior lecturer at the Department of Chemistry and Polymer Science at Stellenbosch University, South Africa. His research activities include fundamental studies and the practical application of HPLC, CE, GC, MS and sample preparation techniques especially to natural product analysis. He is author or co-author of 24 scientific papers, and the recipient of the 2009 Csaba Horvath Award.
Kathithileni M. Kalili is a first year PhD student at Stellenbosch University. Her research focuses on the development of novel comprehensive multi-dimensional high performance liquid chromatography (HPLC) and electromigration methods for the analysis of complex phenolics.
Mareta Malan is a part-time MSc student at Stellenbosch University. She works for Freeworld Coatings Research Centre in Stellenbosch as an analytical chemist where her research is focused on method development for the analysis of surface coatings.
Jeanine Roodman is currently working as a chemical analyst in a food laboratory. She completed her honours degree in chemistry at Stellenbosch University focused on the application of high temperature liquid chromatography to anthocyanin analysis.
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