Electroanalytical technique: Polarographic determination of ascorbic acid in fruits and juices

Objective : To determine the amount of ascorbic acid in sample juices using polarography. Introduction : Analytical chemistry has been an important field especially in quality control of the manufactured product. For example food or packaged drinks could be analyzed for contaminants and for essential nutrients like vitamin. It is useful in analyzing the amount of certain substances which in other words the nutritional value of food. Ascorbic acid is a strong antioxidant and it is very important in human diet because human cannot synthesise ascorbate.Therefore,human obtain the ascorbic acid from the vegetables or fruits especially the citrus fruits(eg, orange,lemon,kiwi). It is important to be able to quantitatively analyse the amount of ascorbic acid in various fruits,drinks or beverages in the market to ensure consumers could obtain the better dietary intake. It is reported that manufacturer would add in more vitamin C content than the stated nutritional value in their products because ascorbic acid gets oxidized very easily.

ascorbic acid There are various analytical methods being carried out to perform the analysis for example spectrophotometry method and electroanalytical method. Other conventional methods are time-consuming and not easy to be carried out. Ascorbic acid oxidizes electrochemically at surface of Dropping Mercury Electrode(DME).In this mini project, polarography is chosen as the method for the analysis because it is faster,easy ,reliable, and could detect substances with low concentration range from10-1 M to 10-4M with good accuracy. The analysis sample were obtained directly from fruit juice and packaged beverage and the standard addition technique and calibration method were carried out to determine ascorbic acid content in the samples.

At higher pH, ascorbic acid could even reduce atmospheric oxygen therefore it is better to prepare ascorbic acid in low pH values. Apparatus :Volumetric flask 100ml x 4, ,mercury waste bottle,beaker, 20 l micropipette and caps,pipette,pipette pump,glass rod,magnetic bar,tissue paper,spatula. Instrument : polarographic instrument, pH meter,computer,printer.

Material : lime juice, Sunkist fruit juices, concentrated acetic acid(17mol),13 mol phosphoric acid, boric acid powder, distilled water,ascorbic acid. Procedure: (a) preparation of supporting electrolyte (Britton Robinson Buffer pH 3.1 to 3.45) 1.) equal parts of 0.04M acetic acid(0.24ml of concentrated 17M acetic acid was diluted with distilled water in 100ml volumetric flask),0.04M phosphoric acid(0.27ml of 13 mol phosphoric acid was diluted with distilled water in 100ml volumetric flask),and 0.04 M boric acid (0.2598g of boric acid powder was diluted with distilled water in 100ml volumetric flask)were mixed together in a beaker. 2.) 0.2 mol of sodium hydroxide solution was prepared dissolving 0.8g of sodium hydroxide salt with distilled water in 100 ml volumetric flask. 3.) The beaker with acids was put on the calibrated pH meter and the pH is slowly adjusted to 3.45 with the prepared sodium hydroxide solution. (b)preparation of stock ascorbic solution 1.) The stock solution was freshly prepared not long before analysis of sample was carried out. 2.) 0.0317 g of ascorbic acid salt was dissolved using 100ml distilled water in a 100ml volumetric flask. (c)Analysis of sample Two samples were used, lime juice from the lime and the Sunkist fruit juices.

(i)Method of standard addition 1. Using a 20 l micropipette,80l (micropipetted x 4)of sample solution was added into 20 ml of the prepared supporting electrolyte in polarographic cell to get the analysis sample. 2. The analysis sample is deaerated and analysed using differential pulse polarographic instrument with anodic scan from -0.15V to 0.20V. 3.step 2 was carried out twice and the polarogram(current generated versus voltage apllied ) was recorded in same axes. 4. 40 l of aliquots of ascorbic acid stock solution was added successively into the analysis sample in polarographic cell and after each addition,steps 2 and 3 were repeated. 5. A standard addition curve with 4 determination points was obtained by plotting the instrumental response (the peak current) against the volume of ascorbic acid added to the solution. (ii) Method of calibration curve 1. 20 ml of supporting electrolyte was deaerated and was analysed using polarography instrument with anodic scan from -0.15v to 0.20v. 2. Step 1 was repeated twice and a polarogram was recorded. 3. 80 l of ascorbic acid stock solution was added into the supporting electrolyte and step 1 and 2 were repeated twice. 4. 40 l of aliquots of ascorbic acid stock solution were added successively twice into the calibration solution and after each addition step 1 and 2 were repeated. 5. A standard ascorbic acid calibration curve with 4 determination points was obtained by plotting the instrumental response (current) against the concentration of ascorbic acid in the solution. 5.Analysis sample juice was then analysed by using 20 ml of supporting electrolyte added with 80 l of the sample juice using polarography instrument. 6. the result was compared to the calibration curve to obtain the concentration of ascorbic acid in the sample juice.

Result and data analysis: (a)Preparation of supporting electrolyte Acetic acid solution, CH3COOH Concentration of concentrated acetic acid used Volume of concentrated acetic acid used Volume of the acetic acid solution prepared = 17 mol dm-3 = 0.24ml = 100ml

Phosphoric acid solution, H3PO4 Concentration of phosphoric acid used Volume of phosphoric acid used Volume of the phosphoric acid solution prepared = 13 mol dm-3 = 0.27ml = 100ml

= 0.0351M Boric acid solution,H3BO3 Mass of boric acid salt used Relative molecular mass of boric acid Concentration of boric acid solution prepared = 0.2598g = 61.83 g mol-1 = = 0.0420 M pH of the supporting electrolyte prepared = 3.45

(b) preparation of stock ascorbic solution Mass of ascorbic acid salt used Relative molecular mass of ascorbic acid Volume of ascorbic acid solution prepared Concentration of ascorbic acid stock solution prepared = 0.0317 g =176.12 gmol-1 = 100ml = 0.1 dm3 = = 1.7999 x 10-3 M OR Concentration of stock ascorbic acid in ppm = 31.7mg/0.1 L = 317 ng /L = 317ppm (c) Analysis of sample (i)Method of standard addition Sample juice
analysis sample

= lime juice
volume of ascorbic acid added /l concentration of vitamin C added ppm 0 12680 25360 38040 Peak current (ip)/nA reading 1
0.495

reading 2
0.258

mean
0.3765

1 2 3 4

0 40 80 120

0.947 1.69 1.737

0.865 0.647 1.115

0.906 1.168 1.426

figure 1: Peak current versus amount of ascorbic acid added (sample = lime juice)
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 10000 20000 30000 40000 amount of vitamin C added / ng y = 3E-05x + 0.4576 Series1 Linear (Series1)

From figure 1, the linear graph obtained has the equation as followed y = 3E-05x + 0.4576 ------------------------------------------------------------------------(1) The principle of standard addition method : The x-axis is expressed in units of added analyte (ascorbic acid). The y-axis response current reflects the actual total amount of analyte (ascorbic acid) in each analyte sample. So, the response is linear in total analyte concentration = [Unknown] + [added]. So, when the total analyte concentration is 0, the response is 0. The line is extrapolated back to a point at which response = 0. We will get the ascorbic acid concentration as x-intercept when response = 0 But in order to get the response in 0 with the concentration is 0,we will have to remove x-intercept value of the analyte ascorbic acid due to the presence of the unknown sample ascorbic acid which was placed in every container has that value of concentration units. [Analyte]total = [added] + [unknown]. 0 = - x-intercept value / concentration of ascorbic acid added + [unknown], Thus [unknown] = x-intercept To get value of x-intercept, y = 0 and solve for the x From equation 1, 0 = 3E-05x + 0.4576 x-intercept = -15253.33 [unknown] = value of x-intercept = 15253.33 ng

ip ,nA

So,concentration of unknown ascorbic acid in sample = 15253.33ng/80l = 190.67ppm

Sample juice
analysis sample

= Sunquick fruit juice

volume of ascorbic acid added /l concentration of vitamin C added ppm 0 12680 25360 38040 Peak current (ip)/nA reading 1

reading 2

mean

1 2 3 4

0 40 80 120

0.782 1.551 1.923 0.346

0.558 0.738 1.877 4.297

0.670 1.145 1.900 2.322

figure 2: Peak current versus amount of ascorbic acid added (sample = sunquick juice)
2.5 2 y = 5E-05x + 0.6526

ip/nA

1.5 1 0.5 0 0 10000 20000 30000 40000 Series1 Linear (Series1)

amount of vitamin C added / ng y = 5E-05x + 0.6526 ------------------------------------------------Equation (2) To get value of x-intercept, y = 0 and solve for the x From equation 1, 0 = 5E-05x + 0.6526 x-intercept = -13052 [unknown] = value of x-intercept = 13052 ng So,concentration of unknown ascorbic acid in sample = 13052ng/80l = 163.15ppm

(ii) Method of calibration curve Solution Volume Total volume addition of solution, of stock Vtotal = (Vbuff ascorbic + Vadd) /L acid, Vadd /L 20000 0 Concentration of standard ascorbic acid, Cstd /ppm
Peak current (ip)/nA reading 1

reading 2

average

Blank

0.0000
0.448 0.298 0.373

20080 1.26295 Standard 1 80 20120 1.8907 Standard 2 120 20160 2.51587 Standard 3 160 *concentration of standard ascorbic acid added(eg standard 1) =317*80/20080 = 1.26295
Figure 3: ascorbic acid Calibration curve
0.8 0.7 0.6 0.5 ip/nA 0.4 0.3 0.2 0.1 0 0 0.5 1 1.5 2 2.5 3 Series1 Linear (Series1) y = 0.12x + 0.3012

0.247 0.649

0.370 0.702

0.3085 0.6755

Cstd /ppm

Sample

Peak Current, Ip /nA Reading 1 Reading 2 1.763 Average

Concentration of ascorbic acid in analyte solution, Cdet / ppm 6.8608

Sunquick Juice

0.486

1.1245

Concentrati on of vitamin C in sample, Cx/ ppm 1722.06

1.335 2.085 1.71 11.74 2946.74 Lime Juice Concentration of ascorbic acid in analyte solution is determined by substituting the peak current value into the calibration curve equation. Concentration of ascorbic acid in sample solution is determined calculation as shown: Eg sunquick : 6.8608*20080/80 =1722.06ppm

Diccussion The analyte here is the ascorbic acid (also known as vitamin C) in various samples like fruits or packaged drink. Ascorbic acid is needed in human diet.It is a very good antioxidant ,meaning it is a very good reducing agent in which it itself is easily oxidized electrochemically to dehydroascorbic acid at the surface of the dropping mercury electrode. Ascorbic acid can be determined by differential pulse polarography by analyse the anodic wave due to the oxidation of the enediol system. In fact, the forward reaction is almost irreversible.

From the experimental data obtained,the concentration of the ascorbic acid in the same juice sample is quite different for different methods of analysis. Method of standard addition is a better method than calibration curve in determination of ascorbic acid or others materials since it eliminates matrix effect but the line of standard addition graph must be linear over the concerned concentration range . Error in determining unknown sample concentration is due to the careless preparation of spiked ascorbic acid where the points do not fall onto the same regression line. In contrast, A calibration curve method can be used to find the concentration of an unknown sample of ascorbic acid by measuring the response of the unknown sample under the same conditions as used for the standards (the solutions used in establishing the calibration line). The response of the unknown, peak current, is then set inserted into the regression line formula and concentration of unknown is calculated. But, it is not possible to prepare a set of calibration solutions that mimic the environment in which the sample analyte is found(biological source eg in fruit juices matrix) since interactions between the analyte and other materials in the matrix may change the observed peak current causing the calibration curve prepared with standard ascorbic acids are not applicable for the unknown fruit sample. Therefore,to analyze a sample when these matrix effects are present(like those in fruit juices), the standard addition method is prefered.

Before each polarographic determination is carried out ,the nitrogen gas is allowed to run through the analysis sample in the polarographic cell, function as to: (i)eliminate oxygen present which might oxidize ascorbic acid beforehand (ii) remove oxygen since oxygen could get reduced at DME and develop interfering waves or response (iii) to mix up the solution with the spiked material,forming homogenous analyte sample.

Conclusion Concentration of ascorbic acid in ppm (i)standard addition method : lime 190.67ppm Sunquick fruit juice 163.15ppm (ii)calibration curve method : lime 2946.74ppm Sunquick fruit juice 1722.06ppm

Reference 1. J.Heyrovsky and P.Zuman, Practical polarography,Academic Press (1968) 2. PAR Application Brief A-4,ascorbic acid and fumaric acid in fruit juice(1975)