Hello,

This reference may help for pigments. For ascorbate I have used HPLC-MS, so I cannot help there.

Lichtenthaler, H. K., Wellburn, A. R. (1983): Determinations of total

carotenoids and chlorophylls a and b of leaf extracts in different

solvents. Biochem. Soc. Trans. 11, 591–592.

I do not advise analysis of ascorbic acid by spectrophotometer because it does not give reproducible results as much as HPLC. However, if you have to use spectrophotometric method , The procedure I used in my Ph. D. dissertation as follows:

Notes: Important issues here, sample should be immediately extracted using metaphosphorric acid and absorbance should be read as soon as possible.  Another point is you have to have a spectrophotometer with stable absorbance reading . I suggest also do recovery test to make sure you extract as much as ascorbic as from tissue and compare results with the literature values reported for that specific fruit or  vegetable. With this method you measure  only L-ascorbic acid not total vitami C.

Vitamin C content (reduced ascorbic acid only) was analyzed by a modification of the procedures described by Morell (1941) and Pearson (1976). All reagents were prepared daily and filtered through Whatman #4 paper with buchner funnel and stored in an amber glass bottle under refrigeration until use. Metaphosphoric acid -acetic acid solution (MA) was used for extraction. Hammett and Miller (1982), Jenkins and Moore (1956) and Wu et al. (1991) analyzed ascorbic acid content of sweetpotatoes using Morell’s (1941) method, except they substituted oxalic acid with metaphosphoric acid in the extraction solution. Preliminary studies showed low recovery of ascorbic acid with oxalic acid; therefore analysis of ascorbic acid was conducted with the MA solution instead of oxalic acid. Ascorbic acid is quite stable against oxidation in the MA solution (Egberg et al., 1973). The MA solution is also stable under refrigeration for 7 to 10 days (AOAC, 1984), while oxalic acid needs to be prepared fresh and kept under dark (Cooke and Moxon, 1981). 

The MA solution was prepared by shaking 60 g of metaphosphoric acid (Sigma; St. Louis, Mo) pellets in 160 mL glacial acetic acid and 800 mL deionized water followed by dilution to 2000 mL with additional deionized water. A 2,6-dichloroindophenol sodium salt (Sigma; St. Louis, Mo) (DCIP) solution was used as a redox reagent. It was prepared by dissolving 12 mg of DCIP salt and 10.1 mg sodium bicarbonate (Mallinckrodt Inc.; Paris, Ky.) in 1000 mL of deionized water. Standards of 1 to 5 mg ascorbic acid/100 ml MA solution were prepared from a stock solution of 100 mg ascorbic acid /100 ml MA solution by diluting with MA solution. Ascorbic acid was obtained from Fisher Scientific (Fair Lawn, N.J.). Exactly 10.00 g of tissue was homogenized in 50 ml of MA solution using a Virtishear homogenizer. Samples were immediately filtered through Whatman #4 paper. The residue and original container were washed with additional MA solution and made to a final volume to 100 ml. Vitamin C was quantified spectrophotometrically using a DU Series 65 Spectrophotometer (Beckman Instruments Inc.; Fullerton, Calif.).  The spectrophotometer was adjusted to zero using 3 mL of MA solution at 520 nm. The absorbance of DCIP dye (2.045 mL) plus MA solution (0.955 mL) was read after exactly 15 sec. This value was noted as L1, which represents absorbance of DCIP dye. Then, the instrument was again adjusted to zero using the sample extract (0.273 mL) plus MA solution (2.727 mL). The absorbance of the MA solution (0.682 mL) plus the sample extract (0.273 mL) plus DCIP dye (2.045 mL) was recorded at the end of 15 sec as the L2 value, which represents decrease in absorbance of DCIP dye due its reaction with ascorbic acid.  L1 and L2 values for each standard were obtained by the same procedure as the samples.  Absorbance values of L1-L2 versus concentration of standard ascorbic acid solutions were plotted to construct a standard curve of ascorbic acid (Appendix I). The concentration of vitamin C in the samples was calculated from the least square equation of the standard curve and expressed as mg ascorbic acid per 100 g fresh weight. The percent recovery was determined by taking two 10.00 g samples from the same root. One portion was spiked with 1.00 g ascorbic acid and the other was not. Both samples were prepared and analyzed using the procedure described above. The percent recovery was calculated from the difference between the spiked and non-spiked sample. Four individual root replicates were used and percent recovery was 90% ±1.29.

Hi, you may modify this to measure ascorbic acid in fruits and vegetables:

Szeto, Y.T. & Benzie, I.F.F. Total antioxidant power and ascorbic acid content of aqueous extracts of fresh fruits and vegetables: implications for dietary planning and food preservation. British Journal of Nutrition (2002) 87 (1) 55-59.

There are kits for ascorbic acid that are accepted and standard, http://www.r-biopharm.com/wp-content/uploads/items/l-ascorbic-acid-4059/Ascorbic-acid_EN_10409677035_2011-05.pdf. Chlorophyll is done by Arnon , or as above but Porra et al. has got better confidence, http://www.chem.ucla.edu/dept/Faculty/merchant/pdf/Porra_1989.pdf. All test are done with an unexpensive VIS spectrophotometer, ther is no need for UV.

Thanks for all your answers.

I will look into each one and let you know if I need more details.

The ascorbic acid content in the fruits and vegetables can be estimated by macerating the sample with agent like metaphosphoric acid.

(Link- http://www.fssai.gov.in/Portals/0/Pdf/15Manuals/FRUITS%20AND%20VEGETABLES.pdf)

Any other thoughts on using UV spec for chlorophyll analysis?

You can try out the following method for chlorophyll estimation from fruits.

http://cse.naro.affrc.go.jp/mnagata/pigment2.pdf

@Sarang: Thanks