examples do exist, in addition to those mentioned above. The technique has been successfully applied to the analysis ofHypericum perforatum(Guttiferae). Online identification of querce- tin, several of its glycosides, and the biflavonoid I5,II8-biapigenin in an extract was possible.106
considered on its own and a suitable procedure has to be developed. In this respect, the flavonoids are no exception.
However, analytical separations of flavonoids are now routine. In quantitative measure- ments, the amounts of the individual components within a particular class of constituent need to be determined. Nowadays, this can easily be achieved through the use of GC, HPLC, and hyphenated techniques.
In HPLC, microbore operation is becoming popular, especially for LC–MS applications, because it allows smaller samples, faster separation times, and lower solvent consumption.
The trend is toward multiple hyphenation techniques like HPLC–UV–MS and HPLC–
UV–NMR. These have an enormous potential for the rapid investigation of plant extracts.100 Multiple hyphenation in a single system provides a better means of identification of com- pounds in a complex matrix.
Applications of LC–NMR are still scarce but the technique will become more widely used.
The main effort for efficient exploitation of LC–NMR needs to be made on the chromato- graphic side, where strategies involving efficient preconcentration, high loading, stop-flow, time slicing, or low flow procedures have to be developed. Microbore columns or capillary separation methods, such as capillary LC–NMR, CE–NMR, and CEC–NMR, will find increased application, one reason being that the low solvent consumption will allow the use of fully deuterated solvents.
Other online HPLC techniques (such as LC–CD or LC–IR) are likely to be exploited. For example, a mixture of diastereoisomeric biflavonoids from the African plantGnidia involucrata (Thymelaeaceae) could not be separated on a preparative scale by HPLC or crystallization.
However, their analytical separation on a C18column was sufficient to run an online LC–CD investigation and provide stereochemical information about the individual isomers.113
5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 minutes
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 mAU
1 2
3 4
5 8
7
6
1112 I.S.
10 9
FIGURE 1.16 Electropherogram of a Passiflorae herba methanol extract. Capillary temperature 358C;
voltage 30 kV; electrolyte buffer 25 mM sodium tetraborate containing 20% MeOH (pH 9.5); UV detection at 275 nm; IS internal standard quercetin 3-O-arabinoside. (From Marchart, E., Krenn, L., and Kopp, B.,Planta Med., 69, 452, 2003. With permission.)
Separation and Quantification of Flavonoids 31
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2 Spectroscopic Techniques Applied to Flavonoids
Torgils Fossen and Øyvind M. Andersen
CONTENTS
2.1 Introduction ... 38 2.2 NMR Spectroscopy... 40 2.2.1 Introduction... 40 2.2.2 NMR Solvents ... 41 2.2.3 NMR Experiments... 42 2.2.3.1 COSY and TOCSY... 43 2.2.3.2 1H–13C Heteronuclear NMR Experiments ... 44 2.2.3.3 Improved Versions of the HMBC Experiment ... 46 2.2.3.4 Nuclear Overhauser Enhancement Spectroscopy ... 46 2.2.3.5 Rotating Frame Overhauser Effect Spectroscopy... 47 2.2.3.6 Two- and Three-Dimensional HSQC-TOCSY, HSQC-NOESY,
HSQC-ROESY, HMQC-NOESY, HMQC-ROESY... 49 2.2.4 Solid-State NMR ... 49 2.2.5 Liquid Chromatography–NMR... 50 2.2.6 NMR Data on Flavonoid Classes ... 52 2.3 Mass Spectrometry... 68 2.3.1 MS Instrumentation and Techniques... 84 2.3.1.1 Electron Impact and Chemical Ionization ... 84 2.3.1.2 Fast Atom Bombardment... 86 2.3.1.3 Matrix-Assisted Laser Desorption Ionization ... 87 2.3.1.4 Electrospray Ionization and Atmospheric Pressure Chemical
Ionization... 88 2.3.1.5 Tandem (MS–MS) and Multiple (MSn) Mass Spectrometry ... 89 2.3.1.6 Mass Analyzers... 90 2.3.2 Coupled Techniques Involving Mass Spectrometry... 91 2.3.2.1 Gas Chromatography Coupled to Mass Spectrometry... 91 2.3.2.2 High-Performance Liquid Chromatography Coupled to Mass
Spectrometry... 92 2.3.2.3 Capillary Electrophoresis Coupled to Mass Spectrometry ... 92 2.3.3 Structural Information... 94 2.3.4 Quantitative Considerations ... 98 2.4 Vibrational Spectroscopy (IR and Raman) ... 100
2.4.1 IR and Raman Spectroscopic Techniques in Studies of Flavonoid
Structures ... 100
37
2.4.2 IR and Raman Spectroscopic Techniques in Studies of Complexes
Involving Flavonoids ... 102 2.4.3 Two-Dimensional IR Analysis... 102 2.4.4 Coupled Techniques Involving Vibrational Spectroscopy ... 103 2.4.5 Near-Infrared Spectroscopy... 104 2.5 Ultraviolet–Visible Absorption Spectroscopy ... 104 2.5.1 Online UV Absorption Spectroscopy in Chromatography... 105 2.5.2 UV–Vis Absorption Spectroscopy on Anthocyanins... 105 2.5.3 UV–Vis Absorption Spectroscopy Involving Flavonoids in Complexes... 108 2.6 Color Measurements Using Commission Internationale de l’Eclairage
Specifications ... 109 2.6.1 Colorimetric Studies on Pure Anthocyanins... 110 2.6.2 Anthocyanin-Based Colors of Plants and Products Derived Therefrom ... 115 2.7 Circular Dichroism Spectroscopy ... 115 2.7.1 Determination of Absolute Flavonoid Configuration ... 116 2.7.2 Circular Dichroism in Studies of Molecular Flavonoid Interaction ... 117 2.8 X-Ray Crystallography ... 117 2.8.1 X-Ray Studies on Flavonoid Structures ... 117 2.8.2 X-Ray Studies on Complexes Involving Flavonoids ... 118 References ... 118
2.1 INTRODUCTION
The purpose of this chapter is mainly to review the different spectroscopic techniques used for flavonoid analysis during the last decade. A typical analysis involving spectroscopic techniques embraces structural elucidation including determination of stereochemical attributes. How- ever, it may also be aimed at tracing specific compounds and presenting quantitative aspects (see Chapter 1), or revealing color depiction. More than 7000 structures in various flavonoid classes have been reported in this book. Nearly half of them have been reported after 1993, which reflect that continual improvements in methods and instrumentation used for separation and struc- tural elucidation have made it easier to use smaller flavonoid quantities to achieve results at increasing levels of precision. Recent attention regarding the variety of flavonoid structures (Chapters 10–17) and their potential properties (Chapters 4–9) has highlighted the need for understanding the physiological functions of individual flavonoids in plants and animals, and their importance to human health. Deciphering biological functions, including pharmaceutical functions, from structural flavonoid information is of increasing importance in our society.
From an analytical point of view, flavonoids may be grouped into various types of monomeric aglycones, bi-, tri-, and oligo-flavonoids including proanthocyanidins, C-alkylated flavonoids, flavonoids with different levels of O-methylation, and flavonoids with one or more saccharide units, which may include various types of acyl substituents (Chapters 10–17). The flavonoids under investigation may be part of complexes, may occur in complex matrices, and some flavonoids like the anthocyanins may exist on a variety of equilibrium forms. A successful characterization will thus follow a specific analytical route designed for the type of flavonoids under investigation, and the sort of information that is looked for. Without reference compounds the characterization of a novel compound will normally require more spectroscopic data than in the determination of a flavonoid that has been reported earlier. The amounts of flavonoids present in most plant tissues are relatively small even though the visual impression is quite striking. Methods for the characterization of individual flavonoids have traditionally reflected the lack of available material, and
sensitive chromatographic and spectroscopic techniques have achieved prominence in the characterization of flavonoids.1–3 Thus, the coupling of instruments performing chro- matographic separations to those providing structural data (hyphenated methods), in particular high-performance liquid chromatography (HPLC) coupled to a diode-array de- tector, and a mass spectrometer or, more recently, a nuclear magnetic resonance (NMR) instrument, has had an enormous impact on structural studies involving flavonoids (see also Chapter 1).
Before a species is analyzed with respect to its flavonoid content, knowledge about earlier reports on the chemistry and flavonoid distribution within the genus and related species may be of value. The most exhaustive source for such information is Chemical Abstracts, and excellent reviews on structures and distribution of flavanoids have been compiled regularly.4–12 Several reviews have recently addressed the general field of flavonoid analy- sis.13–19 Among the earlier reviews in the field, we will particularly recommend consulting Techniques of Flavonoid Identification by Markham2 and Plant Phenolics by Harborne.3 References to review articles on specific spectroscopic techniques applied on flavonoids will be cited under the various spectroscopic methods covered in this chapter. Spectroscopic information of importance is also presented in several other chapters in this book.
In this chapter, examples of the usefulness and recent applications of the different spectroscopic techniques applied on various flavonoids will be presented. Developments in NMR instrumentation including higher fields, high-temperature superconducting probes, low-temperature coils, better radiofrequency technology, as well as improvements of tech- niques and computing power have made NMR spectroscopy (Section 2.2) the most important tool for structural elucidation of flavonoids when these compounds are isolated in the milligram scale. Special effort has been made in this chapter to present assigned1H and13C chemical shifts characteristic for the various flavonoid classes (Table 2.1–Table 2.6), and we present the first report of a 3D heteronuclear single-quantum coherence–total correlation spectroscopy (HSQC–TOCSY) spectrum applied to a flavonoid (Figure 2.6). Advances in mass spectrometry (MS) methodology have been shown to be extremely valuable for flavo- noid analysis during the last two decades, especially the use of mild ionization techniques, which have improved the possibility of recording molecular ions and suppressed the detection limits by several orders of magnitude (Section 2.3). When flavonoid standards are not available, detailed structural information can be obtained by resorting to cone voltage fragmentation (by collision-induced dissociation (CID), tandem MS, etc.) and use of various types of mass analyzers.
Although vibrational spectroscopy (Section 2.4), infrared (IR) spectroscopy and Raman spectroscopy, is not routinely used in most flavonoids studies, the range of potential uses for these methods have been extended considerably by the development of microspectrometers with laser excitation, linked techniques, e.g., liquid chromatography (LC)–Fourier transform IR (FTIR), and two-dimensional (2D) correlation IR. Near-IR (NIR) spectroscopy has been shown to be an effective alternative method to conventional quantitative analysis of flavo- noids in food, plant extracts, and pharmaceutical remedies. Absorption spectroscopy (ultra- violet, UV, or UV–Vis) (Section 2.5) will normally form part of any particular flavonoid analysis during the initial analytical stages; however, during the period of this review only minor advances in methodology were reported. In the flavonoid field, absorption spectros- copy provides most structural information about anthocyanins. Color measurements using CIE (Commission Internationale de l’Eclairage) specifications applied to pure anthocyanins and anthocyanins in plants and products derived thereof, determination of absolute config- uration of flavonoid stereocenters by circular dichroism (CD) spectroscopy, and x-ray dif- fraction studies on solid flavonoid structures have been treated separately in Sections 2.6–2.8, respectively. Abbreviations are listed in Chapter 1.
Spectroscopic Techniques Applied to Flavonoids 39