Analysis of Vitamin C by ~igh-pressureLiquid Chromatography David E. Goodney Willamette University, Salem, OR 97301
Primates, including human beings, cannot synthesize vitamin C (L-ascorbic acid) and must rely on their diets to provide adequate amounts. T h e main sources of dietary vitamin C are fresh fruits and veeetahles. In addition, many packaged foods are "fortified" k i t h additional vitamin C. Vitamin C is also known to be easily oxidized to the less potent dehydroascorbic acid. Thus vitamiu C remains a popular topic in the scientific literature and methods for the determination of vitamin C in many types of food have been reported (1-3). Vitamin C frequently appears in the popular literature because of controversies over its effectiveness against ailments ranging from the common cold to cancer. The standard methods for analysis of ascorbic acid are the 2,6-dichloroindophenol titration based on the reducing ahility of ascorhic acid, or the o-phenylenediamine fluorescence method which requires oxidation of ascorbic acid to dehydroascorbic acid ( I ) . I n recent sears several authors have reported high-pressure liquid chromatographic (HPLC) methods for ascorbic acid analysis (see references in review l experiments (4-7) articles (2, 3)). I n this ~ o u r n a several have been reported to determine the vitamin C in a variety of samples, by a variety of methods except HPLC. I report an HPLC method for the analysis of ascorbic acid that can be used with a varietv of beveraee s a m ~ l e s .The HPLC analysis is a modification of the procedure-reported hv Dennisou e t al. (8)usine isocratic elution and UV absorbance detection. method is rapid, reproducible, and sensitive enough for ascorbic acid in the concentration range investigated. HPLC with UV detection provides a convenient method of analysis of ascorhic acid in natural and processed juices because the ascorbic acid is separated from potential interferences, and it absorbs strongly in the UV while most other constituents of juice do not. The ascorhic acid is also separated from dehydroascorhic acid, but the dehvdroascorbic acid does not absorb stronelv enoueh to be The extent of oxidation of as&& acid in a s a m ~ l eis determined hv reducine the dehvdroascorhic acid and measuring the resulting "total ascorbic acid".
Typical Student Results
Sample Orange Juice (Frozen Concentrate) Orange Juice (Campus Foods) Vitamin C Enriched Beverage (Powdered) Tomato Juice (Canned) Grapefruit Juice (Fresh) Grapefruit Juice (Fresh) Lemonade (Frozen)
AsCOrblc Acid wg/m~ 25.6 . .. ' . 189
. .
315
Total Ascorbic Acid wglm~ 39.9 3124 342
107 295
'
32.2
47.1
27.1
27.0
~ h k
Experimental A Beckman model 342 HPLC with 4.6-mm X 25-cm Ultrasil-NHz column was used for all the analyses. The mobile phase was a 4050 mixture of methanol: 0.25% KH2P04 huffer (KHzPOdHzO + HIPO, adjusted to pH 3.5 before mixing). Detection was done by UV absorption at 244 nm. At a flow rate of 1.25 mLImin., the retention time for ascorbic acid was 7 min (Fig. 1).Sample size was 20 pL. Standards for the HPLC analysis of ascorbic acid must be carefully prepared because of their low concentrations, and the possibility of oxidation. The standard solution is prepared hy weighing 1g of ascorbic acid to the nearest 0.1 mg and dissolving it in a 1-L volumetric flask using distilled water that has heen deoxygenated by bubbling nitrogen through it. The standard should be prepared on the day it is to be used and stored in the dark. Dilutions of the standard in the 25-250 pg1mL range are used to generate a calibration curve of peak height versus concentration for quantitative analysis. Approximately 3 mL of each standard are filtered through a 0.45-pm filter prior to injection into the chromatograph. Quantitative analysis of ascorbic acid can be done on samples of fresh,canned, frozen, or powdered beverage. Students bring to class their own sample of beverage containingvitamin C and, if necessary,
Time (M,",
Chmmatograms of solutions containing vitamin C. (A) Standard solution containing 101 pglmL of ascorbic acid. (B) Diluted sample of vitamin C enriched beverage. (C) Beverage sample after reduction. Note the different absorbance scale on A compared with Band 0.
prepare it according to package instructions. Exposure to light is minimized. The beverage is diluted by pipeting 5 mL into a 50-mL volumetric flask and filling to the mark with deoxvrcenated water. Approximately 3 mL of t
