Separating anthocyanins

Betalains are vacuolar plant pigments. Hence their hydrophilic nature is comprehensible. Although they are slightly soluble in ethanol and methanol, water is the best snited solvent both for stability and solnbility reasons. In contrast to the antho-cyanins, the betalains are even more polar as can be demonstrated by shorter retention times in RP-HPLC and lower solubilities in alcoholic solutions. The varying polarities may also be beneficially used to separate anthocyanins from betalains on an RP-18 solid-phase extraction cartridge (Stintzing, unpublished data). 

Extraction procedures must be adjusted when separated anthocyanins will be tested in biological studies. We have found that the types of acids used for anthocyanin extraction as well as their residual concentrations in the final extract may affect the results obtained from biological tests. The growth inhibitory effect of anthocyanins on HT29 (human colonic cancer) cells may be overestimated if the residual acid in the extract exerts a toxic effect on the cells. Acetic acid residues in anthocyanin extracts showed less toxicity to HT29 cells than hydrochloric acid when samples were prepared under the same extraction procedure and subjected to the same tests on HT29 cells. In addition, the procedure to remove acids affected the acid residual concentration as well in final anthocyanin extracts, with lyophilization being more successful than rotary evaporation. 

CZE is particularly useful for separating anthocyanin dimers or polymeric anthocyanins. Calvo et al. (2004)" separated 13 anthocyanins by CZE including acylated and non-acylated anthocyanins, pyranoanthocyanins, and flavonol derivatives in wine. Saenz-Lopez et al. (2004)" applied CZE to analyze wine aging (1 to 14 yr) as related to monomeric anthocyanins, anthocyanin derivatives, tannins, and fla-vonols. Bicard et al. (1999)" reported the improved detection sensitivity of anthocyanin chemical degradation analysis by CZE. 

This step is optional, depending on how critical it is to separate anthocyanins from other polyphenolics in subsequent analysis. The ethyl acetate fraction is enriched in polypheno-lics such as flavonols, procyanidins, and cinnamates. If analysis of this fraction is desired, a cleaner isolate will be obtained if residual water is removed by passing a nitrogen gas stream through the cartridge for 2 to 3 min before applying ethyl acetate. 

Thoroughly describes the effective fractionation technique of separating anthocyanins and nonan-thocyanins. 

Below is a step-by-step protocol for TLC. In this example the goal is to separate anthocyanins isolated from flower petals. 

There are different methods to separate anthocyanins from seeds. Comparing the extraction efficiency of the different solvents, it appears that the best is 1% HC1 in methanol (Gao and Mazza, 1996 Giirenq and Karaali, 2000). 

The TLC of anthocyanins along with other related phenolic compounds has been reviewed by Harbome (1992). Anderson and Francis (1985) described a method for separating anthocyanins and anthocyanidins from various plant sources by cellulose TLC using the mobile phase HCI-HCOOH-H2O (24.9 23.7 51.4). 

Centrifugal partition chromatography (CPC), a type of CCC that uses discrete partition cells inside a rotor, was used by Renault et al. (1997) to separate anthocyanins from black currant and grape skins. They used two instmments from Sanki Engineering Ltd (Kyoto, Japan) a laboratory model LLB-M and a pilot-scale model LLI-7, both fitted with a stacked disks type rotor. 

Since September 1997, the Journal of Chemical Education (JCE) publishes on a regular base a Classroom Activity that is set separately on a distinct hard sheet, including both the student activity (on one side) and instructor information sheet (on the back side). These activities are designed to actively engage students, while the topics covered are usually coimected with everyday life and apphcations (e.g. acid rain, bath bubblers, toothpastes, water filtration, anthocyanins, latent fingerprints, etc.). Liapi and Tsaparlis (2007) have used three of these activities (acid rain, bath bubblers, toothpastes) in conjunction with two conventional laboratory activities (some properties of acids/some properties of bases) with a class of ninth-grade students in Greece. An evaluation by the students showed a very positive result in favour of the JCE Activities they are connected with life and involve creativity on the part of the students (Fig. 5.2). 

Hong, V. and Wrolstad, R.E., Use of HPLC separation/photodiode array detection for characterization of anthocyanins, J. Agric. Food Chem., 38, 708, 1990. Osmianski, J. and Lee, C.Y., Isolation and HPLC determination of phenolic compounds in red grapes. Am. J. Enol. Vitic., 41, 204, 1990. 

Degenhardt, A., Knapp, H., and Winterhalter, P, Separation and purification of anthocyanins by high-speed countercurrent chromatography and screening for antioxidant activity, J. Agric. Food Chem., 48, 338, 2000. 

Chandra, A., Rana, J., and Li, Y., Separation, identification, quantification, and method validation of anthocyanins in botanical supplement raw materials by HPLC and HPLC-MS, J. Agric. Food Chem., 49, 3515, 2001. 

Supercritical fluid extraction (SEE) is another modern separation technology usually employed to extract lipophilic compounds such as cranberry seed oil, lycopene, coumarins, and other seed oils. Anthocyanins generally and glycosylated anthocyanins in particular were considered unsuitable for SEE due to their hydrophilic properties, since SEE is applicable for non-polar analytes. However, a small amount of methanol was applied as co-solvent to increase CO2 polarity in anthocyanin extraction from grape pomace. New applications of SEE for anthocyanin purification have been reported for cosmetic applications from red fruits. ... 

In many cases, determination of total anthocyanin content may not be enough and information about specific individual pigments will be required. Typically this situation arises in research situations. One analytical method for the quantitative determination of individual anthocyanins involves their separation from a mixture and measurement of each individual pigment. Chromatographic separation of... 

When appreciable amounts of pectin, proteins, lipids, unwanted polyphenols, or other compounds are suspected to be present in anthocyanin-containing extracts, some of them can be precipitated or the anthocyanins may be crystalhzed and separated from the others. Pectin and proteins can be removed by organic solvents such as methanol and acetone in order to reduce their solubility, then precipitated and separated by centrifugation. Gelatin was used to remove proanthocyanidin due to its high molecular weight. Anthocyanins were reported to be precipitated early by lead acetate to achieve isolation from other materials. ... 

Chromatographic separation of anthocyanins plays a major role in analyses of the patterns of phenolic componnds in crnde extracts. ... 

Paper chromatography (PC) and thin layer chromatography (TLC) have been used since the 1940s. Preparative PC on Whatman 3 paper, analytical PC on Whatman 1 paper, and analytical TLC on microcrystalline cellulose, silica gel, or polyamide have been applied with a variety of solvents and the behaviors of anthocyanins have been similar in all media. Two-dimensional TLC allows the separation of several compounds and has been nsed to clarify the anthocyanin compositions of different commodities. ... 

HPLC has proved to be fast and sensitive for the analyses of phenolic plant constit-nents, and is especially useful for the analysis of anthocyanins. The first application of HPLC to anthocyanin analyses was in 1975 by Manley and Shubiak and it has now become the method of choice for the separation of mixtures of anthocyanins and anthocyanidins. HPLC is now used for anthocyanin qualitative, quantitative, and preparative work, offering improved resolution compared to chromatographic procedures previously employed. It also allows for simultaneous rapid monitoring of the eluting anthocyanins. ... 

The most popnlar system is a reversed phase column (Cl8), on a silica base column. However, the use of C18 on a polymer-based column has been reported to provide better resolution, especially for the separation of complex anthocyanin mixtures containing acylated pigments. - Polymer-based columns also show better stability at low pH operating conditions. 

About 15 anthocyanins in bilberries (Vaccinium myrtillus L.) were reported by Ichiyanagi et al. using CZE separation and MS-NMR identification.Anthocyanin analysis of strawberry and elderberry extracts was performed by reverse HPLC at pH 1.8 and CZE using a standard silica capillary and pH 8.0 running buffer. Under these conditions, HPLC had more advantages than CZE in terms of anthocyanin separation in these extracts." ... 

Jackman, R.L., Yada, R.Y., and Tung, M.A., Review separation and chemical properties of anthocyanins used for their qualitative and quantitative analysis, J. Food Biochem., 11, 279, 1987. 

Andersen, O.M., Chromatographic separation of anthocyanin in cowberry (Lingon-berry) Vaccinium vites-idaea L, J. Food ScL, 50, 1230, 1985. 

Hong, V. and Wrolstad, R.E., Use of HPLC separation/photodiode array detection for characterization of anthocyanins, J. Agric. Food Chem., 38, 708, 1990. 

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