There are several methods available, the classical protocol is using titration with standarized solution of Dichlorophenol-indophenol. This protocol can be consulted in manual by C. Reiss (1993) available on-line.

Reiss, C. 1993. Measuring the amount of asorbic acid in cabbage. Pages 85-96, in Tested studies for laboratory teaching, Volume 7/8 (C. A. Goldman and P. L. Hauta, Editors). Proceedings of the 7th and 8th Workshop/Conferences of the Association for Biology Laboratory Education (ABLE), 187 pages. (http://www.ableweb.org/volumes/vol-7/7-reiss.pdf)

Another relatively easy way is to use UV-spectrofotometry of 2,4-DNPH (2,4-Dinitrophenylhydrazine) treated samples, like it described in Rahman et al. (2007).

Rahman et al. 2007. Analysis of Vitamin C (ascorbic acid) Contents in Various Fruits and Vegetables by UV-spectrophotometry. Bangladesh J. Sci. Ind. Res. 42(4), 417-424, 2007 (http://www.banglajol.info/index.php/BJSIR/article/viewFile/749/789)

Dear Viacheslav Shalisko,

I study on drought stress in vitro condition and i have less leaf sample so i want to use 2,4 DNPH method but Rahman et al. 2007 papers take lots of sample, also i want to know how i prepared ascorbic acid standard curve that is not clear in the Rahman et al. 2007.

Hi Muzafer,

I have used this protocol for fruits and vegetables but I think it could be also applied to leaves.

http://link.springer.com/article/10.1007%2Fs00216-011-5668-x

Good luck!

I have used below mentioned methods in CABBAGE:

Ascorbic acid was determined by the direct colorimetric method as mentioned by Ranganna (1986). This method is based on measurement of the extent to which 2, 6- dichlorophenol indo-phenol solution (dye) is decolorized by ascorbic acid in sample extracts and in standard ascorbic acid solution. Liquid nitrogen frozen sample (2 g) was extracted with 20 ml of 2% m-phosphoric acid. Homogenate was centrifuged at 6000g at 4 0C temperature and supernatant was used for estimation of ascorbic acid. One ml of the extract was mixed with four ml of 2% m-phosphoric acid followed by addition of 10 ml of dye with rapid delivery pipette. The mixture was shaken well and reading was taken within 15-20 seconds. The concentration of ascorbic acid was calculated from a standard curve plotted with known concentration of ascorbic acid.

Ascorbic acid content (mg/ 100g) = [Ascorbic acid content x Extraction volume (ml) x 100]/ Extract volume taken for reaction (ml) x Sample weight (g)

Dr Singh,

I do not have enough leaf sample so i can use this methods.

thank you so much.