Application Bulletin 98/5 e

Determination of ascorbic acid (vitamin C) and its compounds

Branch
Bi-voltammetric determination
General analytical chemistry; organic chemistry;
pharmaceutical industry; food, stimulants, beverages, with iodine
flavours; biochemistry, biology;
Instruments
• Titrator with MET mode
Keywords • 10 mL burette
Ascorbic acid; titration; bi-voltametric titration; photometric • Stirrer
titration; polarography; vitamin C; ISO 6557/2; branch 1;
branch 3; branch 4; branch 7; branch 8; 6.0309.100;
6.1115.000 Electrode
Double Pt-sheet electrode 6.0309.100

Summary
In addition to the natural occurrence, ascorbic acid (vitamin Reagents
C) is added to food and beverages as an antioxidant. It can • Volumetric iodine solution, c(I2) = 0.01 mol/L
also be found in various pharmaceutical products.
• Glyoxal, w(C2H2O2) = 40%
Ascorbic acid, as well as its salts and esters, can be
• Sodium hydroxide solution, c(NaOH) = 1 mol/L
determined by titration or by means of polarography. The
determination is based on the oxidation of ascorbic acid to • Sulfuric acid, w(H2SO4) = 25%
.
dehydroascorbic acid. • Disodium thiosulfate pentahydrate, Na2S2O3 5 H2O
• Acetic acid, c(CH3COOH) = 2 mol/L
HO HO
O O O O
HO - 2 H+ - 2 e- HO Solutions

HO OH O O Titrant c(I2) = 0.01 mol/L

Glyoxal solution w(C2H2O2) = 40%
200 mL w(C2H2O2) = 40% is
For the titrimetric determination bi-voltametric or photometric
adjusted with c(NaOH) = 1 mol/L
equivalence point indication can be used. The bi-voltametric
to pH = 7.0.
indication is independent of inherent coloration of the sample.
The solution has to be stored in a
The polarographic method is the most selective of the
dark bottle in a refrigerator.
described methods, since other reducing or oxidizing
substances do not interfere.
Standard
Disodium thiosulfate Disodium thiosulfate pentahydrate
pentahydrate is dried at 120 °C for 2 h and
cooled down in a desiccator for at
least 1 h.

Sample preparation
• Drinks, fruits and vegetable juices can be analyzed
directly.

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 Application Bulletin 98/5 e
Determination of ascorbic acid (vitamin C) and its compounds

• For tablets and other vitamin preparations, a diluted

solution in degased dist. water is prepared. Thereof an f: Titer of the selected titrant
aliquot is used for the titrimetric determination. ms: Mass of standard in mg
• Foods, stimulants and animal feeds are extracted using VEP1: Titrant consumption until the first equivalence
the appropriate procedures. point in mL
2: Stoichiometric factor
cI2 : Concentration of the selected titrant in mol/L;
Analysis here c(I2) = 0.01 mol/L
MA: Molar mass of the analyte; here 248.18 g/mol
Titer
Approximately 50 mg Na2S2O3 ∙ 5 H2O is weighed into a Sample
titration beaker with an accuracy of 0.1 mg, 5 mL VEP1 × cI2 × f × MAA ×1000
βAA =
c(CH3COOH) = 2 mol/L is added and the solution is diluted to VS
approximately 80 mL with deionized water. The solution is
then titrated with c(I2) = 0.01 mol/L until after the first βAA: Mass concentration of ascorbic acid (AA) in
equivalence point. mg/L
VEP1: Titrant consumption until the first equivalence
Sample point in mL
50 mL sample is pipetted into a titration beaker, 2 mL glyoxal cI2 : Concentration of titrant in mol/L
solution is added and the mixture is stirred briefly and allowed f: Correction factor («titer») without unit
to stand for 5 min. After the addition of 5 mL sulfuric acid MAA: Molar mass of ascorbic acid in g/mol; here
w(H2SO4) = 25% it is titrated with c(I2) = 0.01 mol/L until after 176.13 g/mol
the equivalence point. 1000: Conversion factor
VS: Sample volume in mL

Parameters
Example determination
Titer
Mode MET Ipol
Vol. increment 0.1 mL
Signal drift 50 mV/min
Max. waiting time 26 s
I(pol) 1 μA
EP criterion 30 mV
EP recognition greatest

Sample
Mode MET Ipol
Vol. increment 0.1 mL
Signal drift 50 mV/min
Max. waiting time 26 s Fig. 1: Titration curve of the bi-voltametric ascorbic acid
determination with iodine
I(pol) 1 μA
EP criterion 30 mV
EP recognition greatest Comments
• The addition of the glyoxal solution ensures that no SO2
is determined together with the ascorbic acid.
• This method does not determine ascorbic acid alone.
Calculation
Under the given conditions other oxidizable compounds
Titer will also be determined. For a more selective titration
ms 2,6-dichlorophenol indophenol (DPIP) can be used as
f=
VEP1 × 2 × cI2 × MA titrant.

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 Application Bulletin 98/5 e
Determination of ascorbic acid (vitamin C) and its compounds

References
• Schweizerisches Lebensmittelbuch – Method 703.1
Determination of ascorbic acid in fruit and vegetable
juices, iodometric

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Bi-voltametric determination
Sample preparation
with 2,6-Dichlorophenol- • Drinks, fruits and vegetable juices can be analyzed
indophenol directly.
• For tablets and other vitamin preparations, a diluted
Instruments
solution in degased dist. water is prepared. Thereof an
• Titrator with MET mode
aliquot is used for the titrimetric determination.
• 10 mL burette
• Foods, stimulants and animal feeds are extracted using
• Stirrer the appropriate procedures.

Electrode Analysis
Double Pt-sheet electrode 6.0309.100
Titer
1 to 3 mL standard solution and 50 mL oxalic acid solution
Reagents
are added into a titration beaker. The solution is then titrated
• 2,6-Dichlorophenol-indophenol sodium salt dehydrate, with c(DPIP) ≈ 0.001 mol/L until after the first equivalence
DPIP point.
• Sodium hydrogen carbonate, NaHCO3
Sample
• Oxalic acid dihydrate, C2H2O4 ∙ 2 H2O
The sample containing 0.05 to 0.5 mg ascorbic acid is
pipetted into the titration beaker. 50 mL oxalic acid solution is
Solutions added. The solution is then titrated with c(DPIP) ≈
Titrant c(DPIP) ≈ 0.001 mol/L 0.001 mol/L until after the equivalence point.
Approx. 330 mg DPIP is dissolved
in about 250 mL hot dist. water Parameters
(50 – 60 °C) containing 100 mg
NaHCO3, transferred into a Titer
1000 mL volumetric flask and Mode MET Ipol
made up to the mark with dist. Stirring rate 4
H2O.
Start volume 1 mL
The solution can be stored up to
Pause 30 s
one week in a brown glass bottle
in a refrigerator. Vol. increment 0.05 mL
The titer of this solution has to be Signal drift 30 mV/min
determined daily. Max. waiting time 32 s
Oxalic acid solution w(H2C2O4) = 2% I(pol) 1 μA
28 g oxalic acid dihydrate is EP criterion 30 mV
weighed into a 1 L volumetric
EP recognition greatest
flask, dissolved in dist. H2O. The
flask is then filled up to the mark
with dist. H2O.

Standard
Ascorbic acid β(ascorbic acid) = 0.5 g/L
standard 50.0 mg dried ascorbic acid is
weighed into a 100 mL volumetric
flask and made up to the mark
with the oxalic acid solution.
This solution has to be freshly
prepared.

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 Application Bulletin 98/5 e
Determination of ascorbic acid (vitamin C) and its compounds

Sample Example determination

Mode MET Ipol
Stirring rate 4
Start volume 1 mL
Pause 30 s
Vol. increment 0.05 mL
Signal drift 30 mV/min
Max. waiting time 32 s
I(pol) 1 μA
EP criterion 30 mV
EP recognition greatest

Fig. 2: Titration curve of the bi-voltametric ascorbic acid

Calculation determination with DPIP as titrant

Titer References
βAA × VA • G.Pongracz
f=
cDPIP × VEP1 × MAA Neue potentiometrische Bestimmungsmethoden für
Ascorbinsäure und dessen Verbindungen
f: Titer of the DPIP solution Fresenius Z. Anal. Chem. 253, 1971, 271 – 274
βAA: Concentration of the standard solution in g/L
VA: Added volume of ascorbic acid solution in mL
cDPIP: Concentration of titrant in mol/L
VEP1: Titrant consumption until the first equivalence
MAA: Molar mass of ascorbic acid in g/mol
(176.12 g/mol)

Sample
VEP1 × cDPIP × f × 1000 × MAA
βAA =
VA × d

βAA: Mass concentration of ascorbic acid (AA) in

mg/L
VEP1: Titrant consumption until the first equivalence
point in mL
cDPIP: Concentration of titrant in mol/L
f: Correction factor («titer») without unit
1000: Conversion factor
MAA: Molar mass of ascorbic acid in g/mol
(176.12 g/mol)
VA: Aliquot volume in mL
d: Dilution factor

VS + VEx
d=
VS

VS: Sample volume in mL

VEx: Volume of extraction solution in mL

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 Application Bulletin 98/5 e
Determination of ascorbic acid (vitamin C) and its compounds

Photometric determination Sample preparation

• Juices without pulp can be analyzed directly.
Instruments
• For tablets and other vitamin preparations, a diluted
• Titrator with MET mode solution in degased dist. water is prepared. Thereof an
• 10 mL burette aliquot is used for the titrimetric determination.
• Stirrer • Foods, juices with pulp, stimulants, and animal feeds
are extracted using the appropriate procedures.
Colored substances can remain in the sample, because
Electrode they do not interfere with the determination of ascorbic
Optrode (520 nm) 6.1115.000 acid.

Reagents Analysis

• 2,6-Dichlorophenol-indophenol, DPIP For both titer and sample a blank determination has to be
carried out. If the sample is directly measured (without
• Sodium hydrogen carbonate
extraction) the same blank value can be used for titer and
• Oxalic acid dihydrate sample.

Blank
Solutions For the titer the following blank determination is performed:
Titrant c(DPIP) ≈ 0.001 mol/L 30 mL oxalic acid solution are titrated with c(DPIP) ≈
Approx. 330 mg DPIP is dissolved 0.001 mol/L until after the equivalence point using the
in about 250 mL hot dist. water Optrode at 520 nm.
(50 – 60 °C) containing 100 mg If the sample is extracted, an aliquot of the extraction solution
NaHCO3, transferred into a is pipetted into a titration beaker, 30 mL oxalic acid solution
1000 mL volumetric flask and is added and followed by the titration with c(DPIP) ≈
made up to the mark with dist. 0.001 mol/L until after the equivalence point using the
H2O. Optrode at 520 nm.
The solution can be stored up to
one week in a brown glass bottle Titer
in a refrigerator. 1 to 3 mL standard solution is pipetted into the titration vessel
The titer of this solution has to be and 30 mL oxalic acid solution is added. The solution is then
determined daily. titrated with c(DPIP) ≈ 0.001 mol/L until after the equivalence
point using the Optrode at 520 nm.
Oxalic acid solution w(H2C2O4) = 2%
28 g oxalic acid dihydrate is Sample
weighed into a 1 L volumetric The sample or an aliquot of the extracted sample solution
flask, dissolved in dist. H2O. The (containing at least 0.2 mg ascorbic acid) is added into the
flask is then filled up to the mark titration beaker, 30 mL oxalic acid solution is added and the
with dist. H2O. solution is then titrated with c(DPIP) ≈ 0.001 mol/L until after
the equivalence point using the Optrode at 520 nm.

Standard
Ascorbic acid β(ascorbic acid) = 0.5 g/L Parameters
standard 50.0 mg dried ascorbic acid is Mode MET U
weighed into a 100 mL volumetric Stirring rate 6
flask and made up to the mark
Start volume 0.5 mL
with the oxalic acid solution.
Pause 30 s
This solution has to be freshly
prepared. Signal drift 30 mV/min
Max. waiting time 32 s
Volume increment 0.05 mL

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 Application Bulletin 98/5 e
Determination of ascorbic acid (vitamin C) and its compounds

EP criterion 15 mV Example determination

EP recognition greatest

Calculation

Titer
βAA × VA
f=
cDPIP × (VEP1 - Blanktiter ) × MAA

f: Titer of the DPIP solution

βAA: Concentration of the standard solution in g/L
VA: Added volume of ascorbic acid solution in mL
cDPIP: Concentration of titrant in mol/L
VEP1: Titrant consumption until the first equivalence
Blanktiter: Volume of the blank consumption for the titer
determination in mL
MAA: Molar mass of ascorbic acid in g/mol
(176.12 g/mol) Fig. 3: Titration curve of the photometric determination of
ascorbic acid in blood orange juice

Sample
(VEP1 - Blanksample ) × cDPIP × f × 1000 × MAA Comments
βAA =
VA × d • Samples containing iron, tin or copper salts as well as
sulfite, thiosulfate or sulfur dioxide cannot be analyzed
βAA: Mass concentration of ascorbic acid (AA) in using this method.
mg/L • Instead of 2,6-dichlorophenol indophenol, other
VEP1: Titrant consumption until the first equivalence oxidizing titrants can be used as well, e.g. iodine,
point in mL
iodide/iodate, chloramine-T, Fe(III). Here apart from
Blanksample: Volume of the blank consumption for the
sample determination in mL ascorbic acid other oxidizable compounds present in
the sample will also be determined.
cDPIP: Concentration of titrant in g/L
f: Correction factor («titer») without unit • Juices with pulp should be extracted because the pulp
1000: Conversion factor disturbs the measurement signal leading to large signal
MAA: Molar mass of ascorbic acid in g/mol drifts and long titration times.
(176.12 g/mol)
VS: Aliquot volume in mL
References
d: Dilution factor
• ISO 6557/2
VS + VEx Fruits, vegetables and derived products –
d=
VS Determination of ascorbic acid content – Part 2: routine
methods
VS: Sample volume in mL • Schweizerisches Lebensmittelbuch – Method1560.1
VEx: Volume of extraction solution in mL Determination of ascorbic acid in food stuff and
cosmetics, titrimetric
• L. Erdey, G. Svehla
Ascorbinometric Titrations, Akademiai Kiado, Budapest,
1973

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 Application Bulletin 98/5 e
Determination of ascorbic acid (vitamin C) and its compounds

Polarographic determination Sample preparation

• Drinks, fruits and vegetable juices can be analyzed
Instruments directly
• VA instrument • For tablets and other vitamin preparations, a diluted
capable of operating a mercury electrode and solution in degased ultrapure water is first prepared, an
supporting DP mode aliquot of which is then used for the determination.
• Foods, stimulants and animal feeds are extracted using
Electrode appropriate procedures.

WE Multi-Mode Electrode pro 6.1246.120

Mercury drop capillary 6.1226.030 Analysis
AE Separate Pt rod electrode 6.0343.000 Measuring solution
RE Ag/AgCl reference electrode 6.0728.020
10 mL ultrapure water
c(KCl) = 3 mol/L
1 mL acetate buffer pH 4.6
Electrolyte vessel 6.1245.010
filled with c(KCl) = 3 mol/L 0.5 mL sample

10 mL ultrapure water, 1 mL acetate buffer pH = 4.6 and

Reagents
0.5 mL sample are pipetted into a polarographic vessel. After
• Sodium acetate anhydrous, CH3COONa, for analysis,
degasing the measuring solution with nitrogen for 300 s a DP
CAS 127-09-3
polarogram is recorded using the parameters given below.
or
The concentration of ascorbic acid is quantified by two
Sodium acetate trihydrate, CH3COONa · 3H2O, for
analysis, CAS 6131-90-4 additions of ascorbic acid standard solution β(ascorbic acid)
= 1 g/L.
• Acetic acid, w(CH3COOH) = 100%, for analysis, CAS
64-19-7
• L-ascorbic acid (vitamin C), C6H8O6, for analysis, CAS Parameters
50-81-7 Determination
• Ultrapure water, resistivity >18 MΩ·cm (25 °C), type I No. of additions 2
grade (ASTM D1193) No. of replications 2
Voltammetric
Solutions Electrode DME
Acetate buffer 8.2 g sodium acetate anhydrous Measuring mode DP – Differential Pulse
pH = 4.6 or 13.61 g sodium acetate Stirring speed 2000 min-1
trihydrate is dissolved in ultrapure Hydrodynamic measurement No
water. 6 mL acetic acid is added
Sweep
and the solution is made up to
Equilibration time 10 s
100 mL with ultrapure water.
Start potential -0.05 V
End potential 0.2 V
Standard
Pulse amplitude 0.05 V
Ascorbic acid β(Ascorbic acid) = 1 g/L
Pulse time 0.04 s
standard solution 50 mg L-ascorbic acid is dissolved
Potential step 0.06 V
in degased ultrapure water and
made up to 50 mL. This solution Potential step time 0.6 s
has to be freshly prepared every Sweep rate 0.01 V/s
day. Substance + calibration
Calibration Standard addition
Name Vitamin C

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 Application Bulletin 98/5 e
Determination of ascorbic acid (vitamin C) and its compounds

Peak potential 0.1 V keep the standard in the dark. The standard solution
Tolerance 0.05 V should only be used on the day of preparation.

Baseline Linear • Larger quantities of chloride ions (e.g. in sauerkraut)

Automatic interfere with the polarographic determination of
ascorbic acid. They are removed from the sample
solution by precipitation with silver nitrate and
Example determination subsequent filtration.

Vitamin C References
1.00u
• D. Amin
Application of differential pulse polarography to the
800n assay of ascorbic acid
Microchem. J., 28, 1983, 174 – 179.
600n • S. Kozar, A. Bujak, J. Eder-Trifunovic, G. Kniewald
I (A)

Determination of L-ascorbic acid in fresh and processed

fruit and vegetables by differential pulse polarography
400n
Fresenius Z. Anal. Chem., 329, 1988, 760 – 763.

200n
Author

0
Competence Center Titration
0 0.10 0.20 Competence Center Voltammetry
U (V)
Metrohm International Headquarters
Vitamin C
c = 243.506 mg/L
+/- 0.999 mg/L (0.41%)

800n

600n
I (A)

400n

200n

-0.011
0
-10.0m -5.00m 0 5.00m 10.0m 15.0m

c (g/L)
Fig. 4: Polarogram and calibration curve of a determination of
ascorbic acid in orange juice.

Comments
• Ascorbic acid is sensitive against oxygen and light.
Therefore it is recommended to degas the deionized
water for the preparation of the standard solution and

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