V2 VITAMIN C IN FRUIT JUICE BY POLAROGRAPHY Last Revised: June 2014 1. PURPOSE This exercise determines the concentration of vitamin C (ascorbic acid) in fruit juice by polarography, by standard addition, where the extra analyte is added to the sample solution in the polarographic cell as a micropipette aliquot of standard solution. 2. REAGENTS & EQUIPMENT 2.1 Fruit juice 2.2 Ethanoate buffer, pH 4.64 (8.2 g sodium ethanoate, 6 mL glacial ethanoic acid/100 mL) 2.3 1000 mg/L ascorbic acid (prepared fresh each session) 2.4 Deoxygenated water (nitrogen bubbled, for dilution) 2.5 200 uL micropipette 2.6 Metrohm polarographic analyser 3. PROCEDURE NOTE – it is essential that contact between the sample (or standard solution) and air (or dissolved oxygen) be minimised. Therefore, ensure that all containers (cartons, flasks etc) are stopped or sealed when not being used. Prework What accurately measurable (by pipette) volume of sample will contain approximately 200 ug of ascorbic acid? Sample Three sample and two recovery check replicates and are to be analysed. Do the third replicate sample after the two RCs. Three standard additions are to be performed on each. 3.1
3.2
3.3
3.4
Clean the polarographic cell with distilled water and dry. Set the Mode to Determination and Load method V2. Pipette 10 mL of deoxygenated water, 1 mL of ethanoate buffer and the calculated volume of sample into the cell and press Start. After the first run is completed, you will be prompted to add the first standard addition aliquot (200 uL of 1000 mg/L standard) to the cell and press Start. Recovery check 3.5 Repeat step 3.3, also adding 200 uL of the 1000 mg/L standard as a recovery check into the cell and proceed as before. 4. REPORT Calculations The instrument should do the standard addition calculations for you, if all peaks are satisfactory. However, you are required to undertake the calculation for one of the datasets manually as below: 
calculate the mass of analyte in 200 uL of 1000 mg/L standard 
plot a standard addition graph for the chosen dataset 
determine the mass of analyte in the sample aliquot from the graph 
calculate the concentration of analyte in the sample 
calculate the average concentration in the sample 
calculate the percentage recovery (use the average masses for sample & recovery check) Discussion 
compare your results to the expected value and account for any difference 
compare your calculation result to the instrument result and comment on any difference 
comment on the recovery check result 
explain the purpose of the ethanoate buffer Questions 1.
If you were to carry this exercise out using normal calibration standards, suggest three suitable concentrations. 2.
What is the reason for minimising contact between air and sample in this exercise? Reference: Metrohm Application Note V73 (see prac webpage) This exercise is based on the project undertaken by Rebekah Livesley in 2007. V2 p2 V2. VITAMIN C IN FRUIT JUICE BY POLAROGRAPHY RESULTS SHEET
Sample (brand & type of juice) Expected conc. PREWORK Volume of sample Diffusion currents Solution Sample Addition 1 Addition 2 Addition 3 Calculated Result Sample 1 Sample 2 RC 1 RC 2 Sample 3