Anthocyanins are more stable at low pH (acidic conditions) which gives a red pigment. Meanwhile, the higher the pH value of anthocyanin will provide color fading of the color blue. So as a food colorant, anthocyanin with a low pH or height pH has a significant effect on the food colorant.

You can use ethanol: water (70:30) 0.3% HCl (1W: 4V) is more effective and then you can put it in sonication for 1h and collect it and re-extract for 30 min and mixed together and use rotary evaporation and then use C18

@Ahmed K. Rashwan thank you Sir.

What is C18?

@Carole C Tranchant Thank you for the article. Would go to look that

Chromatography Octadecylsilyl ''C18'' or Macroporous resin

Deya Silviani

@Ahmed K. Rashwan thanks for advice, but we don't have it in our lab. do you know other alternative way?

Anthocyanins are more stable at low pH (acidic conditions) which gives a red pigment. Meanwhile, the higher the pH value of anthocyanin will provide color fading of the color blue. So as a food colorant, anthocyanin with a low pH or height pH has a significant effect on the food colorant.

@Danung Nur Adli thank you. I used acidified solvent metanol is HCl 0,1N as I found a few literature use it. Actually, since I'm trying to detect anthocyanin in biscuit, I assume that processing may degradate it much so I'm doubted about is there any pigmented color still remained

Hello Deya Silviani;

You are using the differential pH spectrophotometric assay for quantifying anthocyanins which is based on the principle quoted by Danung Nur Adli. I presume that in your extract, you are getting other compounds besides anthocyanins that are causing turbidity as Carole C Tranchant pointed out.

Could you please elaborate on the steps that you follow to obtain your extract and perform your measurments? Perhaps this may help us to identify if there is a procedure step that is causing the problem.

Best regards

@Rogelio Valadez -Blanco hello sir, thanks for response

I extracted 1 g biscuit powder with 10 ml metanol-HCl 0,1 N (1:10, w/v), bring it to sonicator 30degree Celcius for 10 minutes, filtered use Whatman 42, centrifuged for 10 minutes, then collected supernatan. I used supernatan for analysis, I didn't vaporated solvent since it hard to do in my lab with very small volume of extract.

Anthocyanin analysis used pH diff method. Each of 1 ml of supernatan of sample added 4 ml buffer pH1 (HCl-KCl) and 4 ml buffer pH 4.5 (sodium citric), vortexed, then read the absorbance in 520 and 700 nm.

Hello Deya Silviani;

Thank you very much for describing your procedure. In general, your extraction method seems appropriate to me, but I suggest to use centrifugation after ultrasound extraction: 1000 rpm or above for 10 min. Then filter the supernatant as described in your procedure, and wash with fresh solvent again. Repeat the procedure three times. This may help to remove suspended particles in your extract.

However, if the procedure above does not reduce the impurities in your extract would mean that the solvent is carrying other compounds such as fatty acids, proteins or carbohydrates. This is to be expected since you are trying to analyze a complex food matrix, which is composed by a mixture of many compounds that can interfere with your determination. So if the procedure above doesn´t work to remove extract turbidity, you will need to use C18 solid-phase extraction (SPE) cartridges in order to remove not wanted compounds (impurities) from the extract.

To understand the main objective of your assay, could you please state what is the source (ingredient) of anthocyanins in the biscuits? I ask you this, because another approach that you can follow is to determine the anthocyanins content of the main anthocyanin-source ingredient, instead of that of the biscuits. The drawback of this apprach is that you will not able to take into account the effect of anthocyanin degradation during cooking.

Apart from the extraction procedure, another consideration that you need to take into account is that the anthocyanin concentration in the biscuit can be reliably measured by the method of analysis used. It can happen that you manage to clean the extract, but the anthocyanin content is too low to be measured by the assay. To have an idea of the concentration of anthocyanin expected in the biscuit, I suggest to analyze first the main anthocyanin-rich ingredient.

Hope this is useful for your research.

Regards

Sorry, in my previos answer, after centrifugation and filtration, you don´t carry out a washing procedure. You simply discard the precipitate and repeat the operation three times.

Regards.

@Rogelio Valadez-Blanco

Thank you so much for brief suggestion, Sir.

It so valuable. Actually, my research already done in the analysis of main source ingredient of antioxidant in this biscuit, which is muntingia calabura fruit.

And the research want to know the effect of processing to this, in this is biscuit processing.

Once again, thank you so much Sir