HPLC Separation of Polyphenolics

HPLC Separation of Polyphenolics Basic Protocol Reversed-Phase HPLC Analysis of Polyphenolics Separated 11.3.1... 

In reversed-phase HPLC separation of polyphenolics on the basis of polarity, the elution order of polyphenolics may be predicted. The more-polar polyphenolics are generally eluted first under reversed-phase conditions. Glycosylation in flavonoids increases their polarity and therefore their mobility in the re-versed-phase system. The elution order of benzoic acids, hydroxycinnamic acids, and agly-cones of flavonoids can normally be determined on the basis of the number of polar hydroxyl groups and lipophilic methoxyl groups. For additional information about elution order for various classes of polyphenolics, see Background Information. 

The Basic Protocol describes the reversed-phase HPLC analysis of polyphenolic compounds isolated into nonanthocyanin and anthocyanin fractions by solid-phase extraction. The Alternate Protocol describes the HPLC separation of acidic and neutral polyphenolic fractions. Fractionated samples are used because significant amounts of interfering compounds are extracted along with polyphenolics from plant materials. Solid-phase extraction with C18 Sep-Pak cartridges (vnitu.2) is used to selectively eliminate undesired components from crude extracts, and may minimize the effects of sample cleanup or preparation on the integrity of polyphenolics. The isolation and purification step using solid-phase extraction of polyphenolics will make possible the efficient analysis of individual polyphenolics by reversed-phase HPLC. 

REVERSED-PHASE HPLC ANALYSIS OF POLYPHENOLICS SEPARATED INTO NONANTHOCYANIN AND ANTHOCYANIN FRACTIONS... 

Because polyphenolics show chemical complexities and similar structures, isolation and quantification of the individual polyphenolic compounds have been challenging. Many traditional techniques (paper chromatography, thin-layer chromatography, column chromatography) have been used. HPLC, with its merits of exacting resolution, ease of use, and short analysis time, has the further advantage that separation and quantification occur simultaneously. A reversed-phase HPLC apparatus equipped with a diode array detector makes possible the easy isolation and separation of many polyphenolics. For enhanced performance of HPLC separation, the polyphenolics should first be isolated into several fractions to effectively separate the individual polyphenolics (Jaworski and Lee, 1987 Oszmianski and Lee, 1990). 

Solvents used for elution in various cases are different, and the order of use also makes differences. For fractionation of polymeric proanthocyanidins, the solvent combination used is acetone with water which is applied for both Sephadex G-50 and LH-20 [22, 154]. The elution of solvents depends on the solubility of specific phenolic components in specific solvents. Anthocyanins are not soluble in ethyl acetate however, many other polyphenolic compounds are soluble in ethyl acetate. Hence, in the protocol mentioned in the book Handbook of Food Analytical Chemistry-Pigments, Colorants, Flavors, Texture, and Bioactive Food Components for the separation of anthocyanins from the rest of the components in a polyphenolic extract, ethyl acetate is used for elution of the polyphenolic components other than anthocyanins, while the anthocyanins are eluted with acidic methanol [12]. pH is another important property affecting the separation of polyphenols through SPE. Phenolic acids have been reported to be completely ionized at pH 7.0 and un-ionized at pH 2.0 hence, this property is used as the basis for fractionation of neutral polyphenohcs at pH 7.0 and acidic polyphenohcs at pH 2.0, which helps in prevention of interference in-between polyphenohc compounds during HPLC [12]. In certain cases, combination of various SPE methods is used for the fractionation of the extracts, and then the purified extracts are further processed to be analyzed [140, 141, 150]. 

HPLC with UV-Vis detection is the most widely utilized for separation of polyphenols, although other chromatographic methodologies such as GC, CCC, have also been used with success. 

Gu M, Su ZG, Janson JC (2006) The separation of polyphenols by isociatic hydrogen braid adsorption chromatography on a cross-linked 12 % agarose gel. Chromatographia 64 247-253 Klejdus B, Vacek J, Benesova L, Kopecky J, Lapcik O, Kuban V (2007) Rapid-resolutirai HPLC with spectrometric detection for the determination and identification of isoflavraies in soy preparations and plant extracts. Anal Bioanal Chem 389 2277-2285... 

RP-HPLC with gradient elution was employed for the study of the influence of theaflavins and thearubigins on the adsorption of black tea on calcium carbonate. Separation of tea constituents was performed in an ODS column (250 X 4.9mm i.d. particle size 5 im). Aqueous solvent was 1 per cent citric acid, pH adjusted to 2.8 with sodium hydroxide and the organic solvent was ACN. The gradient initiated at 8 per cent ACN, was increased to 31 per cent in 50min. Theaflavins and thearubigins were detected at 460 nm, while total polyphenolics were detected at 280 nm. The flow rate was 1.5 ml/min. The results demonstrated the involvement of theaflavins and thearubigins in the adsorption process [185],... 

RP-HPLC methods have been frequently applied for the investigation of various chemical, biochemical and biophysical processes in in vitro model systems. Thus, the separation of new compounds achieved by enzymatic oxidation of phloridzin was carried out by semi-preparative RP-HPLC. Phloridzin was incubated with a polyphenol oxidase prepared from apple pulp for 6h at 30°C under air agitation. After incubation the suspension was filtered, stabilized by NaF and injected into the RP-HPLC column using diluted acetic acid-ACN gradient. The new compounds were isolated and identified by NMR and MA techniques. The proposed mechanism of the formation of new phloridzin derivatives 3 and 4 is shown in Fig. 2.159. The results illustrate that RP-HPLC can be successfully used for the study of enzymatic processes in model systems [331],... 

C18 solid-phase extraction is used to fractionate polyphenolics for their identification and characterization. This technique can eliminate interfering chemicals from crude extracts and produce desirable results for HPLC or other analytical procedures. To obtain a sufficient volume for all analyses, several separations by solid-phase extraction may be performed. The individual fractions need to be combined and dissolved in solvents appropriate for HPLC analysis. In Basic Protocol 2, the application of a current of nitrogen gas for the removal of water from the C18 cartridge is an important step in the selective fractionation of polyphenolics into non-anthocy-anin and anthocyanin fractions. After the collection of non-anthocyanin polyphenolics, no additional work is necessary to elute anthocyanins bound to the C18 solid phase if anthocyanins are not to be determined. 

In this unit, methods for reversed-phase high-performance liquid chromatography (HPLC) are described for the analysis of polyphenolics. HPLC analysis can be employed in an easy and fast manner to obtain an accurate elucidation and quantification of individual polyphenolic compounds found in plant-based materials. The separation of each polyphenolic is based on the polarity differences among polyphenolics with structural similarities and uses various combinations of mobile and stationary phases. 

Table 11.3.1 Elution Order of Polyphenolic Compounds Separated by HPLC of Nonanthocyanin and Anthocyanin Fractions0...

This fractionation step may be optional. Some samples can be directly analyzed by HPLC after filtration (step 2) without solid-phase extraction. Anthocyanins that can be detected at 280 nm can interfere with the separation of some polyphenolics. If the analyst is interested in nonanthocyanin polyphenolics, and especially if plant materials containing high levels of anthocyanins are being analyzed, this fractionation technique should be utilized. 

Aqueous tea extracts deteriorate rapidly during storage. They develop an undeslreable taste commonly described as "flat" and concurrently turn dark brown in color. Roberts et al. (56) used a modification of the HPLC procedure of Hoefler and Coggon T57) to separate the polyphenols in tea to study this problem. The effluent from the reverse phase acetone gradient was monitored at 380 nm. 

According to the obtained chromatographic profiles, this gradient was then further optimized for more efficient separation. HPLC analysis of the polyphenolic extract of... 

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