Polyphenolics HPLC separation

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

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. 

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. 

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

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). 

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. 

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],... 

Removing Interfering Polyphenols. The most troublesome problem encountered was high polyphenol content. If not removed, this dark-colored wood extractive decreased resolution during chromatographic separations, quickly render expensive chromatographic media (e.g., HPLC columns) nearly useless, and often precipitated enzymes during subsequent purification steps (14,15), We found it best to remove the bulk of this material prior to the first concentration step. 

In earlier times, thin-layer chromatography (TLC), polyamide chromatography, and paper electrophoresis were the major separation techniques for phenolics. Of these methods, TLC is still the workhorse of flavonoid analysis. It is used as a rapid, simple, and versatile method for following polyphenolics in plant extracts and in fractionation work. However, the majority of published work now refers to qualitative and quantitative applications of high-performance liquid chromatography (HPLC) for analysis. Llavonoids can be separated. 

In this protocol, polyphenolics are fractionated into neutral and acidic fractions to prevent interference among polyphenolics in HPLC analysis. Phenolic acids are completely ionized at pH 7.0 and un-ionized at pH 2.0. This property allows for fractionation of neutral polyphenolics at pH 7.0 and acidic polyphenolics at pH 2.0. Two individually preconditioned Cl8 cartridges, one for neutral polyphenolics and the other for acidic polyphenolics, are used for this separation (Figure 11.2.2). 

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. 

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

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. 

Reversed-phase HPLC can separate polyphenolics of extracts on the basis of polarity. HPLC easily produces better resolution among chemically similar compounds in extracts than conventional chromatographic methods. The operating temperature of the column during reversed-phase HPLC analysis should be controlled for data reproducibility. A change in temperature produces only a minor effect, however, on band spacing in reversed-phase HPLC and produces essentially no effect in normal-phase HPLC (Lee and Widmer, 1996). A range of ambient temperatures is widely used, and elevated temperatures are often applied. The retention times of the peaks are dependent upon the type of column and the combination of various solvents used in the method. 

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