L-ASCORBIC ACID (VITAMIN C) Key words: lactone, enol, oxidation, reduction, L-gulonic acid, D-glucuronic acid.

Transcription

1 L-ASCORBIC ACID (VITAMIN C) Key words: lactone, enol, oxidation, reduction, L-gulonic acid, D-glucuronic acid. Reagents: 1. L-ascorbic acid 2 mmol/l in hydrochloric acid 20 g/l 2. Iron (II) sulphate 0.4 mmol/l 3. Iron (III) chloride 0.4 mmol/l 4. Potassium hexacyanoferrate (III) 0.01 mol/l 5. Sodium 2,6-dichlorophenolindophenolate 1 mmol/l in phosphate buffer (c=30mmol/l), ph 7 6. Concentrated hydrochloric acid l0x diluted by 7. Orange juice water Ascorbic acid Ascorbic acid, γ-lactone of 2-keto-L-gulonic acid or vitamin C is a water-soluble glucose derivative. Vitamin C can be found in fresh fruit and vegetable (mainly potatoes and cabbage), but also in meat. Most plant and animal species synthesize vitamin C from D-glucuronic acid. Ascorbic acid is not synthesized by primates, some species of Caviidae (the capybara, the guinea pig) and some species of birds and fish. All species that do not synthesize ascorbate require it in the diet. Ascorbic acid is a strong reducing agent that is oxidized to dehydroascorbic acid (lactone). Its main cofactor function is reduction of Fe 3+ to Fe 2+ ions in the enzymes prolyl hydroxylase or procollagen-proline-dioxygenase [EC ] and lysyl hydroxylase or procollagen-lysine-5-dioxygenase [EC ]) which posttranslationally hydroxylate prolines and lysines in collagen. Deficiency of vitamin C leads to unstable collagen formation (with no hydroxyl groups on prolines and lysines), which results in defective connective tissue (fragile capillaries resulting in abnormal bleeding), and a disease scurvy (scorbutus). The chemical name for vitamin C, ascorbic acid, is derived from a- (meaning "no") and the Latin name of scurvy (scorbutus). In 1937, the Nobel Prize for chemistry was awarded to Norman Haworth for his work in determining the structure of ascorbic acid and the prize for Physiology or Medicine that year went to Albert Szent-Györgyi for his studies of the biological functions of L-ascorbic acid. The American physician Fred R. Klenner promoted vitamin C as a cure for many diseases in the 1950s by elevating the dosages greatly to as much as tens of grams vitamin C daily by injection. Ascorbate usually acts as an antioxidant. In humans, it is important for iron (Fe 2+ ) uptake from food. In its absence, microcytary anemia is developed. Upon high intake, vitamine C is present in urine. As a reducing agent can affect analysis of some analytes in the urine, mostly reactions that involve hydrogen peroxide (because of its ability to react with oxidants of the reactive oxygen species, such as the hydroxyl radical formed from hydrogen peroxide). It also decomposes

2 diazonium salts used for azocopulation reactions. Fehling s is reduced at low temperatures (the colour intensity depends on ascorbic acid concentration). Analysis of ascorbic acid concentration is needed for determination of ascorbic acid saturation of the organism. Titration is a laboratory method of quantitative analysis used to determine unknown concentration of known substance, which relies on adding a titrant of known molar concentration. A reagent that quantitatively reacts with ascorbic acid as a titrant. When all the ascorbic acid has reacted, the titrant is then in excess, forming a colourful complex with the indicator present in solution. This indicates the end-point of the titration. Calculation of analyzed substance concentration: In the equivalent point (end point of titration), the moles of analyzed substance(1) are equal to moles of the titrant (2) n 1 = n 2 c 1 x V 1 = c 2 x V 2 c 1 = c 2 x V 2 / (V 1 ) V 1 = volume of analyzed substance V 2 = volume of added titrant c 1 = concentration of analyzed substance c 2 = concentration of titrant 1. Reducing properties of L-ascorbic acid L-ascorbic acid (dehydrolactone of L-gulonic acid) is a saccharide derivative. The acidity (pk A = 4.2) and the easy oxidation (E 0 = 0.08 V) of L-ascorbic acid are properties of the two hydroxyl groups adjacent to the double bond. L-ascorbic acid with its oxidised form L-dehydroascorbic acid create a redox system, which participates in biochemical reactions involving nonenzymatic transport of hydrogen atoms. Principle L-ascorbic acid reduces ferric ions (Fe 3+ ) to ferrous ions (Fe 2+ ) (E 0 Fe 3+ /Fe 2+ = 0.77 V), which form a dark blue complex in potassium hexacyanoferrate (III) solution (the blue complex is composed of ferrous, ferric and cyanide ions). The green colour is not evidential! a. Prepare reaction mixtures into three test tubes according to the table 1.

3 Table 1. Test tube N Solution of Fe 2+ (ml) 1 Solution of Fe 3+ (ml) 1 1 Solution of L-ascorbic acid (ml) 1 Distilled water (ml) 1 1 Solution of K 3 [Fe(CN) 6 ] (drops) Colour b. Observe the colour of the reactions, fill in the table Determination of L-ascorbic acid concentration using a redox- dye Principle: The principle is a rapid oxidation-reduction reaction of L-ascorbic acid with the dye 2,6-dichloro-phenolindophenol (E 0 = 0.22 V). This compound is quantitatively reduced into its colourless form. The end-point is a complete turnover of the blue colour of sodium salt of the dye into a pink colour (nonreduced sodium salt of dye is pink at ph below about 5). a. Pipette 2 ml of L-ascorbic acid solution into the Erlenmeyer flask. Titrate with the dye solution until the blue colour of the dye turns into a pink one. b. Perform the titration 3x and calculate the mean value of added dye solution. c. Calculate the concentration of L-ascorbic acid: L-ascorbic acid (mg/l) = M r x c reagent x V reagent V titrated Mr (L-ascorbic acid) = 176,1 d. Fill in the table 2. Table 2. Volume of dye solution added (ml) Mean volume of dye solution (ml) Concentration of ascorbic acid (mg/l) Concentration of ascorbic acid (mmol/l) 3. Determination of vitamin C concentration in orange juice a. Pipette 2 ml of orange juice (10times diluted by distilled water) into an Erlenmeyer flask. b. Titrate with a titrant till a pink colour appears. c. Calculate the concentration of acorbic acid.

4 d. Fill in the table 5. e. Compare your result with the concentration indicated on the manufacturer s label and discuss the usage of preservatives. Table 5. Volume of the titrant (ml) Ascorbic acid concentration in dilluted solution (mg/l) Ascorbic acid concentration in orange juice (mg/l) Concentration indicated on manufacturer s label (mg/l)

5