# A Spectrophotometric technique to evaluate Vitamin C concentration in Citrus Fruit

Is there a visible spectrophotometric technique that can gage the concentration of vitamin C in solution? I plan on boiling different citrus fruit in water, and taking aliquotes of the boiled solution to determine total leached vitamin C. I’ve found a couple of UV spectrophotometric techniques, but wasn’t certain if those techniques could carry forward using a visible spectrophotometer. Those sources also didn’t specify what the vitamin C concentration range was for a Beer Lambert’s Law assay.

Would spectrophotometry be possible? Or would it be more feasible to perform a redox titration of vitamin C (ascorbic acid) using iodine?

• Yes, it is possible to determine vitamin C by spectrophotometry. It is very tedious and requires sample preparation! I will not recommend it. Aug 9 '20 at 20:10

It is not quite correct of the statement that "ascorbic acid cannot be determined by Beer Lambert's law, because it does not absorb visible light." Although the second part of the statement is true, the highlighted part is incorrect since Beer Lambert's law applies for ultra-violet range of the spectrum. According to Ref.1, ascorbic acid (Vitamin C) absorbs light in UV range of the spectrum:

The result revealed that $$\lambda_\mathrm{max}$$ (peak wavelength of spectra) of L-ascorbic acid at $$\mathrm{pH} \ 2.0$$ was $$\pu{289.0 nm}$$ whereas at neutral $$\mathrm{pH} \ 7.0$$, $$\lambda_\mathrm{max}$$ was $$\pu{295.4 nm}$$. In alkaline $$\mathrm{pH} \ 8.6$$, $$\lambda_\mathrm{max}$$ was $$\pu{295.4 nm}$$ and at $$\mathrm{pH} \ 7.4$$ the $$\lambda_\mathrm{max}$$ of L-ascorbic acid remained the same as $$\pu{295.4 nm}$$.

Thus, if OP has access to a UV/vis spectrometer, assuming no interference from other components in the fruit extract in the UV range, OP can try to determine ascorbic acid percentage in fruits of interest using a direct method with calibration curve of known ascorbic acid concentrations.

In the event of having interferences, there is a better method of getting absorbance in visible region of the spectrum (Ref.2). This is a well-established method, which is called the 2,4-dinitrophenyl hydrazine method (Ref.3).

The method involves oxidation of ascorbic acid to dehydroascorbic acid by bromine water followed by preparation of the corresponding osazone using 2,4-dinitrophenylhydeazine (2,4-DNPH). The osazone upon treatment with 85% $$\ce{H2SO4}$$ forms red colored solution, which absorbs light at $$\pu{521 nm}$$. The procedure was successfully used to determine vitamin C content in various fruits and vegetables (Ref.3). The calibration curve of the red dye prepared from standard ascorbic acid samples is shown in the following diagram (LHS; the plots are from Ref.3):

This method need you to prepare a working solution (5% metaphosphoric acid-10% acetic acid): Fifteen grams of solid metaphosphoric acid were dissolved in mixture of $$\pu{40 mL}$$ of glacial acetic acid and $$\pu{450 mL}$$ of distilled water in a $$\pu{500 mL}$$ volumetric flask.

The standard vitamin C (ascorbic acid) solutions for calibration curve: First, prepare the $$\pu{500 ppm}$$ ($$\pu{500 mg/L}$$) standard stock solution by dissolving $$\pu{0.05 g }$$ of standard crystalline ascorbic acid in $$\pu{100 mL}$$ of working solution. From that stock solution, you can prepare $$5, 10, 15, 20$$, and $$\pu{25 ppm}$$ standard test samples by serial dilution (using working solution to dilute) to prepare the calibration curve.

In addition, you need 10% thiourea solution, 2,4-DNPH solution, and 85% $$\ce{H2SO4}$$ solution in hand.

Procedure: To the equal volumes of filtered fruit solution (prepared by using working solution to extract and dilute) or standard ascorbic acid solutions, add few drops of bromine water until the solution became colored (bromine water color; this is to confirm the completion of the oxidation of ascorbic acid to dehydroascorbic acid). Then few drops of thiourea was added to it to remove the excess bromine and the clear solution was obtained as result. Then 2,4-DNPH solution was added equal volume to all these oxidized ascorbic acid samples (both standards and fruit extract samples) and they are kept in $$\pu{37 ^\circ C}$$ water bath for $$\pu{3 h}$$ (Ref.4). When the coupling reaction of 2,4-DNPH dye with dehydroascorbic acid has been completed, add equal volumes of 85% $$\ce{H2SO4}$$ solution ($$5\times$$ volume of 2,4-DNPH) followed by spectrophotometric determination.

It should be noted that any other compound in the extract also reacts with 2,4-DNPH and 85% $$\ce{H2SO4}$$ solution, if they contain carbonyl groups. For example, common component in fruit is glucose (and other sugar), dye from which are giving absorbance at around $$\pu{450 nm}$$ (see the complete scan of dye prepared by D-glucose on the RHS of the above diagram). As indicated in the diagram, these interference is minimal at $$\pu{521 nm}$$.

Note: This method is good to determine total ascorbic acid content (ascorbic acid + dehydroascorbic acid). The full text of the relevant paper is attached for the convenience.

References:

1. Shaheen A. Maniyar, Jameel G. Jargar, Swastika N. Das, Salim A. Dhundasi, Kusal K. Das, "Alteration of chemical behavior of L-ascorbic acid in combination with nickel sulfate at different $$\mathrm{pH}$$ solutions in vitro," Asian Pacific Journal of Tropical Biomedicine 2012, 2(3), 220–222 (https://doi.org/10.1016/S2221-1691(12)60045-8).
2. R. Riemschneider, M. Z. Abedin, R. P. Mocellin, “Qualitäts und stabilisierung prüfung hitze konservierter Nahrungsmittel unter verwendung von Vit C als kriterium-Mitt 1 (Quality and stability evaluation of food preserved by heat utilizing vitamin C as a criterion),” Alimenta 1976, 15, 171-174.
3. M. Rahman Khan, M. M. Rahman, M. S. Islam, S. A. Begum, “A Simple UV-spectrophotometric Method for the Determination of Vitamin C Content in Various Fruits and Vegetables at Sylhet Area in Bangladesh,” Journal of Biological Sciences 2006, 6(2), 388-392 (DOI: 10.3923/jbs.2006.388.392)(Full Text).
4. Anal Parimal Desai, Shuchi Desai, "UV Spectroscopic Method for Determination of Vitamin C (Ascorbic Acid) Content in Different Fruits in South Gujarat Region," Int. J. Environ. Sci. Nat. Res. 2019, 21(2), IJESNR.MS.ID.556056 (4 pages)(DOI: 10.19080/IJESNR.2019.21.556056)(PDF).

Ascorbic acid cannot be determined by Beer Lambert's law, because it does not absorb visible light. The best way is to perform a redox titration with iodine. Usually a citrus fruit is $$45-50$$ mg ascorbic acid in $$100$$ g juice. But you should know that there is also $$3840 - 6080$$ mg citric acid in $$100$$ g juice.

Ref.: Konrsd Diem, Scientific Tables, CIBA-Geigy SA, Basel, Switzerland, $$1972$$

• At least a similar post about the topic was posted here on ChemSE, too: chemistry.stackexchange.com/questions/84436/…. Equally have a look at publications like the one in J. Chem. Educ. (2016): pubs.acs.org/doi/10.1021/acs.jchemed.6b00344 or the older in 1940: pubs.acs.org/doi/abs/10.1021/ed017p180. Aug 9 '20 at 20:08
• Maurice, yes direct determination is not possible but indirect methods including the official fluorimetric methods exist! Aug 9 '20 at 20:14
• @ Buttonwood. Thank you for the recent reference about the use of iron and 1,10.phenanthroline complex. It was really interesting Aug 9 '20 at 20:23
• The redox titration may not work with real fruit juices because of the juice matrix! Aug 9 '20 at 23:35
• @M. Farooq. You are right. A real fruit juice may contain other reducing substances, apart from ascorbic acid. Carotène for example. But there is only $0.012$ mg carotène in $100$ g juice. I don't know which proportion of proteins are reducing substances. But there is only between $0.5 - 1.1$ mg protein in $100$ g juice. Aug 10 '20 at 13:47