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 μl
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 μl micropipette,80μl (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 μl 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 μl of ascorbic acid stock solution was added into the supporting electrolyte and step
1 and 2 were repeated twice.

4. 40 μl 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 μl 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 = 17 mol dm-3

Volume of concentrated acetic acid used = 0.24ml

Volume of the acetic acid solution prepared = 100ml

► Phosphoric acid solution, H3PO4

Concentration of phosphoric acid used = 13 mol dm-3

Volume of phosphoric acid used = 0.27ml

Volume of the phosphoric acid solution prepared = 100ml

= 0.0351M

►Boric acid solution,H3BO3

Mass of boric acid salt used = 0.2598g

Relative molecular mass of boric acid = 61.83 g mol-1

Concentration of boric acid solution prepared =

= 0.0420 M

►pH of the supporting electrolyte prepared = 3.45

(b) preparation of stock ascorbic solution

► Mass of ascorbic acid salt used = 0.0317 g

Relative molecular mass of ascorbic acid =176.12 gmol-1

Volume of ascorbic acid solution prepared = 100ml = 0.1 dm3

Concentration of ascorbic acid stock solution prepared =

= 1.7999 x 10-3 M

OR

Concentration of stock ascorbic acid in ppm = 31.7mg/0.1 L

= 317 ng /μL

= 317ppm

(c) Analysis of sample

(i)Method of standard addition

►Sample juice = lime juice

analysis sample volume of concentration of Peak

ascorbic vitamin C added current
(ip)/nA
acid added ppm reading 1 reading 2 mean
/μl
1 0 0 0.495 0.258 0.3765
2 40 12680 0.947 0.865 0.906
3 80 25360 1.69 0.647 1.168
4 120 38040 1.737 1.115 1.426
 figure 1: Peak current versus amount of ascorbic acid
added (sample = lime juice)
1.6
1.4
1.2
y = 3E-05x + 0.4576
1
0.8
ip ,nA

Series1
0.6
0.4 Linear (Series1)
0.2
0
0 10000 20000 30000 40000
amount of vitamin C added / ng

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

►Sample juice = Sunquick fruit juice

analysis sample volume of concentration of Peak

ascorbic vitamin C added current
(ip)/nA
acid added ppm reading 1 reading 2 mean
/μl
1 0 0 0.782 0.558 0.670
2 40 12680 1.551 0.738 1.145
3 80 25360 1.923 1.877 1.900
4 120 38040 0.346 4.297 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 Series1
Linear (Series1)
0.5

0
0 10000 20000 30000 40000
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/80μl

= 163.15ppm
(ii) Method of calibration curve

Solution Volume Total volume Concentration Peak

addition of solution, of standard current
of stock Vtotal = (Vbuff ascorbic acid, (ip)/nA
ascorbic + Vadd) /µL Cstd /ppm reading reading average
acid, Vadd 1 2
/µL
Blank 0 20000 0.0000
0.448 0.298 0.373
Standard 1 80 20080 1.26295 0.247 0.370 0.3085
Standard 2 120 20120 1.8907 - - -
Standard 3 160 20160 2.51587 0.649 0.702 0.6755
*concentration of standard ascorbic acid added(eg standard 1) =317*80/20080 = 1.26295

Figure 3: ascorbic acid Calibration curve

0.8
0.7
y = 0.12x + 0.3012
0.6
0.5
ip/nA

0.4
Series1
0.3
Linear (Series1)
0.2
0.1
0
0 0.5 1 1.5 2 2.5 3

Cstd /ppm

Sample Peak Current, Ip /nA Concentration of Concentrati

ascorbic acid in on of
Reading Reading Average analyte solution, vitamin C
1 2 Cdet / ppm in sample,
Cx/ ppm
Sunquick Juice 0.486 1.763 1.1245 6.8608 1722.06
Lime Juice 1.335 2.085 1.71 11.74 2946.74
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)