Dear Kavin , I have answered before, have you received it?. 

For pigments separation you can use:

- CC packed with DEAE Sephadex  A-25 and  elution with gradient NaCl 0.3 M until 1.0 M. You can see the coloring fractions, yellow or red, and check them by UV-V. After you join the same fractions, you have to eliminate the NaCl by elution in a Sephadex G-10 CC with water until no reaction with AgN03.

- TLC is possible using Silicagel : PW (phenol: water, 4:1), CMA (chloroform :methanol:17% ammonium , 2:2:1), Bu.HCl (n-butanol: HCl conc., 9:1).

Good luck.

Analysis of pigments betalains

The mixture of juice and methanol (1/5) (v/v) was homogenized by magnetic stirring. The mixture is then filtered through a nylon filter (0.45 μm). Finally, the value of the absorbance of methanolic extracts is obtained using a UV–Visible spectrophotometer (Shimadzu UV–Visible 160 A, Tokyo, Japan). The measurements were made using a quartz cell of 1 cm. The quantities of pigment extracts from fruit juices were evaluated by spectrophotometry according to Lambert Beer’s law: A = log (I10/I) = ε.L.c, after measuring the absorbance at a languid wave corresponding to the maximum absorption of betaxanthin (482 nm) and indicaxanthin (532 nm). The molar extinction coefficients were respectively, of 62,000, L mol−1 cm−1 for betaxanthin and 48,000 L mol−1 cm−1 for indicaxanthin ( Girod and Zryd, 1991). The results are expressed as mg pigment per kg of juice.

The determination of betalain pigments is done in the juice of both prickly pears, red and yellow. The absorbances were measured at the wavelength corresponding to maximum absorption of betaxanthin (482 nm) and indicaxanthin (532 nm). The first remark is the absence of red pigments, indicaxanthin absorbs at 532 nm in the yellow prickly pear. However, yellow pigments (betaxanthin) are present with a relatively large content of an average value of 37.822 mg/kg.

The mean level of the juice of betaxanthin prickly yellow is 37.822 ± 0.151 mg/kg. This value is similar to that found by Stintzing and Scheiber (2003) for Italian fruit. The content in the fruit is about 48.30 mg/kg of juice, lower than that found by Butera et al. (2002), which is 84.20 mg/kg of juice. Betaxanthin content found is much lower than that of the prickly pear juice from Spain, work reported by Fernandez-Lopez and Almela (2001).

Sir/madam I am facing problem while betalain extraction from grain amaranth plant flower. I am using water as solvent because it give higher quantity of betalain pigments. Methanolic extraction has low extraction efficiency in my sample. During extraction I am giving heat treatment to betalain extract for to inactivate betalain oxidase enzyme but this process lead degradation of betalain pigments. Is there any other way to decrease the betalain oxidase activity? How long can I store water extract in freeze at -20 C?

Dear Kavin Soni,

I am trying to do the same by TLC using ethanol/water mixtures with a Bactris species, but I don't how effective it is. I will follow your question waiting for a relevant answer.

Thank you for the question.