Background

Ascorbic acid (AA) or vitamin C is widely known as an antioxidant and a free radical scavenger. It is also important in helping to produce collagen, a protein needed in the development and maintenance of bones, cartilage, joint linings, skin, teeth, gums and blood vessels. It’s reputed antiviral properties to prevent the common cold remains controversial.

AA is a water-soluble vitamin found mainly in fruits and vegetables, particularly green leafy veggies, citrus fruits, tomatoes, guavas, melons and berries. Because these were lacking in the diet of early day sailors, scurvy was common, often resulting in death. It is a white crystalline powder with a molecular formula of C₆H₈O₆ and a F.W. of 176.12. The recommended daily intake is 200 – 500 mg per day [ref. 1,2].

The oxidative mechanism for AA, as proposed by Ruiz [ref. 3], for pH 8 or less is shown in Figure 1. The electron transfer involves the deprotonated anion (step 1) that is oxidized in a one electron, one proton reaction (step 2) to the radical anion. A subsequent fast one electron irreversible oxidation takes the anion to dehydroascorbic acid (DHAA, step 3). It is electro-inactive. DHAA is rapidly protonated and then dehydrated to 2,3-diketogluconic acid (steps 4 and 5).

Figure 1. Proposed mechanism for the electrooxidation of ascorbic acid in acid or neutral solution [ref. 3].

Cyclic voltammetry (CV) is a convenient electrochemical method to examine the oxidation of AA at a glassy carbon (GC) electrode that has been “activated” to different degrees. That is, the peak height (Ip) and the peak potential (Ep) are very dependent on the state of the GC surface. Besides contamination, the surface may contain carbon-oxygen functionalities from prolonged exposure to air and moisture. With polishing, surface contaminants and functionalities may be minimized to give a “clean” carbon surface at which AA undergoes a relatively fast electron transfer process, albeit the deprotonated anion being the electroactive species. The 1^st pKa of AA is 4.17 and the 2^nd is 11.57. Thus, the parent AA is the predominant species in solution at pH 2 where experiments will be run. Since steps 1-3 are fast on the time scale of scan rates used, and the product of reaction 3 is irreversible, only the forward 2 electron, 2 proton CV wave is observed. The reverse cathodic wave, attributable to the reduction of the radical anion (product of step 2) is seen only at high scan rates where the time window competes favorably with the kinetic rate (~ 10³ s^-1) of its removal. You may wish to run CVs at high scan rates (e.g., 10 V/s or more) if your potentiostat has the capability to scan at these high rates to see if you can “capture” this radical anion. Details of AA’s electrochemical oxidative mechanism are reported in references 3 and 4. Example CV waves at three different levels of GC electrode activity are shown in Figure 2.

Figure 2. CV of ascorbic acid oxidation at different levels of GC electrode activation. Trace A is an electrode used without any pretreatment; B is the same electrode after a cursory polish with 0.5 μm alumina and washed with pure water; C is the electrode after light polishing on Grit 0000 paper followed by polishing with 0.5 μm alumina on a glass plate and washed with pure water.

The CV with the lowest Ep value (i.e., trace C in Figure 2) has a large background current indicative of ahigher effective surface area, possibly due to micro-graphitic particles on the surface left from the polishing process. One of the most pristine surfaces is produced by a vacuum heat treatment that gives rise to a well-defined CV with a peak at ~ 240 mV [ref. 4].

In biological fluid samples, like urine, the presence of uric acid interferes due to its oxidative potential being similar to AA. Liquid chromatographic with electrochemical detection (LCEC) is an effective method to separate and determine AA and uric acid independently. Cyclic voltammetry has been proposed as a method to evaluate the antioxidant capacity of AA in biological samples such as blood plasma, tissue homogenates and plant extracts [refs. 5-7].

In this experiment CV scans will be conducted after various pretreatments of GC electrode. When a reproducible method is found, calibrations with known AA solutions will be run prior to determining amount of AA in a fruit juice sample.