Study of Analytical Parameters of the HPLC Method for

Tartrazine and Sunset Yellow Analysis in Soft Drinks

ELENA DIACU1*, CORNELIA PETRONELA ENE2
1
University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Department of Analytical
Chemistry, 1-7 Polizu, Bucharest, 011061, Romania
2
Institute for Hygiene and Veterinary Public Health, 5 Campul Mosilor Str., 021201, Bucharest, Romania

The study of analytical parameters of the HPLC method for simultaneously determination of Tartrazine and
Sunset Yellow, two of the most widely used synthetic colorants in soft drinks, has been performed. The
results obtained by the specific laboratory over a number of characteristic performance criteria, such as:
linearity of calibration curve, detection limit, quantification limit, recovery, repeatability, reproducibility,
ruggedness and stability are reported. The uncertainty value of the measurand was also determined. The
established performances show the validity of the applied HPLC method for the determination of Tartrazine
and Sunset Yellow in any kind of soft drinks.
Keywords: HPLC method, analytical parameters, Tartrazine, Sunset Yellow, soft drinks

In recent years much attention has been given regarding

the quality of additives which enter into food or drinks
composition. Even the addition of these substances is
permitted by the European Union (Council Directives 94/
34/EC, 1994), they exceed the authorized levels in many
cases. Therefore, in the EU there are regulations controlling
the maximum levels of food additives, including synthetic
pigments in soft drinks [1, 2].
It is well known, that from the two categories of used
food pigments (natural and synthetic), the natural ones
are in the consumer preference, having lower negative
effects on humans than the synthetic ones [3,4].
Nevertheless, the synthetic colorants have increasingly
used in the food processing during the last 20 years,
replacing partially or totally the natural dyes [3-5]. The
reason is that the synthetics dyes, generally, have a lower
price and a higher effectiveness than the natural ones.
Containing normally the azo group and aromatic ring, these
synthetic dyes are known as conferring to the respective
substances harmful properties to people health [2,3].
Therefore, the synthetic dyes have been the subject of
numerous toxicological investigations and they are both
nationally and internationally regulated. The Council
Directive 94/36 has stipulated a reduced number of
permitted synthetic colorants for use in foodstuffs, under
certain conditions and limits in use [1]. Also, in 2007,
Commission Regulation (EC) No 884/2007 have nonauthorized some of food colors which constitute a serious
risk to human health, such as E 128 Red 2G [1, 2].
The pigments, Tartrazine (TAR, trisodium 5-hydroxy-1(4-sulfonatophenyl)-4-(4-sulfonatophenylazo)-H-pyrazole3-carboxylate) and Sunset Yellow (SUN, disodium 6hydroxy-5-(4-sulfonatophenylazo)-2-naphthalenesulfonate) are two synthetic dyes widely used in soft drinks.
TAR and SUN are permitted food colorants by law, the
maximum accepted level by the European Legislation in
food being of 100 ppm for TAR and 50 ppm for SUN, when
individually, and no more than 100 ppm where they are
used in binary mixtures [1]. Therefore, it became of
paramount importance the development of new modern
and powerful analytical techniques able to separate, to

identify and to quantify different food pigments. An

impressive number of determination methods have been
achieved in order to analyze synthetic dyes, separately or
in combination [6-8]. The majority analytical methods for
synthetic colorants are in the range of spectrophotometric
methods and chromatographic ones. Generally,
spectrophotometric methods are time consuming,
because almost always a prior chemical separation
procedure must be involved in order to avoid, the spectral
overlap. In comparison with spectral methods, the liquid
chromatographic ones exceed this inconvenience, offering
the possibility to analyze both pigments with high accuracy
and precision, with a very good detection limit.
The present work describes the study of analytical
parameters of the HPLC method for simultaneously
determination of TAR and SUN in soft drinks by HPLC,
method already published by the authors [8].
Experimental part
All reagents used were of chromatographic purity grade
and all solutions were prepared using bidistilled water. TAR
and SUN were both purchased from SIGMA-Alhrich
(Germany, purity 90%), methanol and acetonitrile were
from Merck (Germany), Chromasolv purity. Monopotassium
phosphate and disodium phosphate were from
SCHARLOW (France).
The chromatographic determinations (described in
details in our previous paper [8]), were carried out using a
high performance liquid chromatograph system Agilent
Series 1100 with diode array detection.
Validation of the method
The validation of the method of TAR and SUN was carried
out on soft drinks samples fortified with respective dyes.
According to the references [9-11] for the validation of the
method, the following specific performance criteria have
been determined using an in house validation procedure:
linearity of calibration curve, detection limit (LOD),
quantification limit (LOQ), recover y, repeatability,
reproducibility, ruggedness, stability and measurement
uncertainty, (U). All these criteria are described bellow.

* email: e_diacu@chim.upb.ro
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1177

Table 1.
EXPERIMENTAL DATA FOR THE DETERMINATION OF LOD AND LOQ

Results and discussions

Calibration curve linearity
The linearity of the method was tested on the basis of
the peak areas of chromatograms for lemon juice samples
fortified with various concentrations of mixed standard
solutions of TAR and SUN (5; 10; 30; 50; 70; 100; 120, 150;
175, and 200 mg/L) in the dynamic concentration range
where it may predict the presence of Tar and SUN in
beverages, respectively 0-200 mg/L. These solutions have
been daily prepared from a stock solution of 1000 mg/L.
The calibration curve was linear for the studied range, with
a good regression coefficient, R=0.99999.
Detection limit (LOD) and quantification limit (LOQ)
In the HPLC technique LOD can be considered as the
concentration where the ratio signal/noise (S/N) is 3/1,
these values being obtained directly using the
chromatographic software. In order to establish the LOD,
four different concentrations of TAR and SUN standard
solutions (1; 0.5; 0.3; 0.1; 0.07 mg/L) were analysed, and
the results for the different concentrations are presented
in table 1 and show a LOD equal with 0.1 mg/L for each
colorant. Consequently, the LOQ is 0.3 mg/L, numerically
equal to three times of LOD.
Recovery %
To determine the recovery of the TAR and SUN, an
unfortified sample of a specific soft dink was analyzed. No
analytical signal was generated in this case, so the colorants
content is considered to be 0 mg/L. Taking into account
the maximum accepted limit for the two colorants in
combination [3, 4], a set of 18 samples of soft drinks were
fortified for each colorant, at three concentration levels, 6
samples for each level, as displayed in table 2, where CD

represents the determined concentration and CI is for the

blank. The recovery percentage is ranged between 92.50101.67% for TAR and 92.14-104.24% for SUN, showing that
the matrix effects are minimum for the determination of
both colorants from soft drinks.
Repeatability, r
Repeatability is the value below which the absolute
difference between single test results obtained under
repeatability conditions (i.e., same sample, operator,
apparatus, laboratory, and short interval of time) may be
expected to lie within a specific probability (typically 95
%) and hence r = 2.8 sr. (sr - standard deviation). As in
the previous experiment is has been used the same HPLC
instrument, operated by the same analyst, in the same
day, the respective values for CD-CI were applied in order to
establish the repeatability. In table 3 there are presented
the obtained values for sr, r and CV (the variation coefficient)
for TAR and SUN.
The obtained values for sr were less than 0.15, showing
a very good repeatability of the analytical data for both
colorants at all three fortification levels.
Reproducibility, R
In order to verify the reproducibility, two sets of
experiments have been performed in two different days,
for each colorant. First set of experiments, collected within
1 day are those presented in table 2. In table 4 are displayed
the analytical data obtained in the same conditions as for
table 2, but in two different days. From the obtained data it
can be seen that for all three fortification levels of TAR and
SUN, the values of sR <2, giving reproducibility to the
proposed method.

Table 2
EXPERIMENTAL DATA FOR THE DETERMINATION OF THE RECOVERY IN TAR AND SUN ANALYSES

Table 3
DETERMINATION OF REPEATABILITY FOR TAR AND SUN ANALYSES

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Table 4
ANALYTICAL DATA OBTAINED IN A DIFFERENT DAY FOR THE DETERMINATION OF REPRODUCIBILITY OF
THE RESULTS. SR= (SR FIRST DAY + SR SECOND DAY)/2 AND THE REPRODUCIBILITY, R= (RFIRST-DAY + RSECOND-DAY)/2

Ruggedness/Stability
The ruggedness of the method was tested, checking if
the requirement sD > sR for HPLC method is executed. Two
soft drink samples were fortified at 50 mg/L concentration
level of TAR and SUN and were analysed using, mainly the
same instrumental operating parameters as indicated in
the method for TAR and SUN determination, but with minor
changes. These consist, in a slightly different pH value for
the sample and the ratio gradient elution. Therefore, the
pH for the soft drink sample was changed at 7.00, in
comparison with the mentioned value of 6.5 of the method
and the gradient elution ratio was A/B=70/30, instead of a
variable gradient used in the method. In both cases, the
analytical peaks are shifted out of accepted limits and the
TAR and SUN cannot be identified and analysed under these
parameters. Accordingly, the method has not ruggedness
under the chosen modifications.

The stability of the method was checked, performing

the control maps (fig 1), where the data in Y-axis represent
the recovery % and the data in the X-axis represent the 20
individual recovery values, the average of the individual
recovery values, the average of the individual recovery
values 2xSD and the average of the individual recovery
values 3xSD.
Measurement uncertainty, U
Taking into account the ISI Gum (1993) [5] and the
EURACHEM guide [7], the following individual uncertainty
components for the total measurement uncertainty, U, in
the analysis of TAR and SUN in soft drinks by HPLC method
should be taken into account: - uncertainty on the
preparation of working standard solution of 100 mg/L; uncertainty on the recovery and on the reproducibility, and
- uncertainty on HPLC instrument. The main components
of uncertainty are detailed in figure 2 and will be treated
hereinafter.

Fig.1. Control-maps of stability for Tartrazine (A) and Sunset Yellow (B) individual recovery value;
the average;
the average 2 x SD; the average 3 x SD

Fig. 2. Main uncertainty sources

and uncertainty budget for
Tartrazine and Sunset Yellow

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Associated uncertainty on working solution preparation,

u(working)
There are two components of uncertainty on the
preparation of the working solution that should be taken
into account: the uncertainty on the preparation of the stock
solutions and is the uncertainty on the preparation of the
working solution itself.
Uncertainty on stock solutions preparation
Two contributions to the uncertainty on stock solutions
preparation have to be taken into account: weighing at
analytical balance and 100 mL volumetric flask.
The purity for bought dyes powders is 90%:

Taking into account the purity of the dyes powders, for

preparing 100 mL of mixed stock solution of 1000 mg/L,
111 mg of TAR and 111 mg of SUN is weighted by analytical
balance. The calibration certificate of Mettler Toledo AT
261 analytical balance has an accuracy for 100 mg 0.01
mg, the uncertainty, u = 0.005 mg, with a coverage factor
k = 2. In order to establish the contribution of analytical
balance to the uncertainty on stock solutions preparation,
10 replicate weighing were performed.
The combined uncertainty of mixed stock solution
preparation, u(mstoc) = 0.295 mg, as the sum of umTAR +
umSUN.
0.17 mg
The uncertainty of a 100 mL volumetric flask Class A on
the uncertainty on stock solutions can have three
influences: the measuring flask, the repeatability and the
temperature effect.
The contribution of the A class 100 mL measuring flask
(Hirschmann Laborgerte GmbH & Co - Germany) to the
volume measurement uncertainty is calculated from the
tolerance 0.1 at 20o C temperature (provided by the
producer), considering a rectangular distribution:

The repeatability uncertainty results from the standard

deviations of 10 replicate fillings and weightings of the 100
mL volumetric flask is: u(Vrepeatability -100mL) = 0.0261 mL.
The contribution to the uncertainty of the flask
calibration temperature (20o C) is assessed taking into
account the temperature variation limits of 4oC and using
the volume expansion coefficient. For the aqueous solution
this coefficient is 2.110-4 and the dilatation coefficient
with the temperature is 100 x 4 x 2.1 x 10-4 = 0.084. The
uncertainty due to variation of the temperature, assuming
a rectangular distribution is u(V Temp. variation 100mL) =
The three uncertainty contributions make the combined
uncertainty for 100 mL volumetric flask:
The quantification of all uncertainty contributions on
stock solutions preparation is given below and the known
formula for the combined uncertainty is applied:
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The combined uncertainty for 1000 mg/L TAR and SUN

stock solution, u(cstock) = 0.003 1000 = 3 mg/L, where c is
the concentration of stock solution.
Uncertainty on working solution preparation
Two contributions to the uncertainty have here to be
taken into account: 10 mL volumetric flask and
micropipette. To prepare 100 mL of working solution of
mixed stock solution 100 mg/L, 1 mL of stock solution
1000 mg/L is diluted in a volumetric flask of 10 mL. In
order to establish the contribution of the measurement of
the volumes on the uncertainty on working solutions similar
procedure and calculus are performed as for the
uncertainty on stock solutions preparation.
The contribution of the A class 10 mL measuring flask
(Hirschmann Laborgerte GmbH & Co - Germany) to the
uncertainty on the volume measurement is calculated from
the tolerance 0.025 at 20o C temperature (provided by
the producer), considering a triangular distribution:

The second contribution to this uncertainty is the

repeatability. According with the Class A 10 mL volumetric
flask certificate, the standard deviation is de 0.006 mL,
taken as uncertainty: u(Vrepeatability-10mL) = 0.006 mL. The
uncertainty of the flask calibration temperature is
calculated from the dilatation coefficient with the
temperature (0.008 =10 x 4 x 2.1 x 10-4), using a rectangular
distribution for the volumetric flask of 10 l mL: u(Vtemp variation
=
All three calculated contributions make the combined
incertitude for the 10 mL volumetric flask uncertainty,
u(Vwork):

To measure 1 mL stock solution a micropipette of 1001000 L is used. There are here two contributions: the
uncertainty due to the micropipette itself and to the
micropipette repeatability. The contribution of the
micropipette (BRAND, Germany) to the uncertainty on 1
mL measurement is assed from the indicated value of the
micropipette certificate, which is 0.0015 mL. Considering
that represent a normal distribution, u(VPipette-100mL) =
0.0015/2 = 0.00075 mL. The repeatability of the
micropipette is obtained from the standard deviation
provided by the producer, considering a rectangular
distribution: u(Vrepeatability - Pipette)=
The sum
of two contributions give the associated uncertainty of the
micropipette:
To calculate the associated uncertainty on working solution
preparation, denoted with u(cworking), the bellow disposal
data (table 5) of all contributions should be taken into
account and the uncertainty formula is applied:

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Table 5
UNCERTAINTY CONTRIBUTIONS IN THE
ASSOCIATED UNCERTAINTY ON WORKING
SOLUTION PREPARATION
Table 6
RSDTAR AND RSDSUN VALUES FOR RECOVERY

fraction of the distribution of values that could reasonably

be attributed to the measurand and represents the
estimated standard deviation of the result [5, 7].
Summarising all the uncertainty components, the
combined uncertainty, u is obtained as follows:
u(cworking) =0.0038 100 = 0.38 mg/L
The uncertainty related to the recovery and to the
reproductibility
The uncertainty related to the recovery was calculated
by the integration of relative standard deviations for TAR
and SUN, RSDTAR and RSDSUN, these being considered as
the uncertainty related to the recovery for each colorant
within 1 day. Table 6 presents the analytical data of standard
deviation (SD) and relative standard deviation (RSD) of
recovery percentage. The associated recovery uncertainty
is the RSDs sum of the values within 1 day, amounting to
0.00312.
The reproducibility was verified by calculating the RSDs
values of six time analyses of a fortified soft drink sample
at 100 mg/L level for TAR and 50 mg/L SUN respectively,
performed between days. Results are reported in table 6.
RSDTAR = 0.00108 and RSDSUN = 0.00164, the associated
uncertainty related to the reproducibility is their sum,
0.00272.
Associated uncertainty for HPLC instrument
Associated uncertainty for HPLC instrument uHPLC is
determined separately for TAR and SUN, being expressed
as the standard deviation: u HPLC_TAR = 0.295 mg/L and
uHPLC_SUN = 0.290 mg/L, uHPLC = 0.2925 mg/L (Calibration
certificate INM no. 05.03-599/ 29.09.2008).
The calculation of the expanded uncertainty, U
Finally, the measurement uncertainty can be expressed
either as the combined standard uncertainty of the
results, denoted with u, or as expanded uncertainty,
symbolised with U. Thus, U defines the interval of TAR and
SUN content that may be expected to encompass a large
REV. CHIM. (Bucharest) 61 Nr. 12 2010

The expanded uncertainty U is calculated using the

simple relationship U = u k, where k is the coverage
factor. Considering a normal distribution of the experimental
data, k=2, U = 1.1226 mg/L.
Conclusions
The evaluation of performance criteria for the HPLC
method validates its goal, which is the application of the
analysis of two of the most used food pigments, Tartrazine
and Sunset Yellow from soft drinks. The described method
is accurate and precise, with a very good reproducibility of
the results (within 1 day RSD N=6,TAR = 0.001304,
RSD N=6,SUN =0.001473 and between days RSD N=6,TAR =
0.000867, RSDN=6,SUN = 0.001816). LOD and LOQ were
established as 100 g/L and 300 g/L for each colorant.
The effect of critical parameters on uncertainty
measurement was investigated, the value of the extended
combined uncertainty being 1.1226 mg/L, which is quite
good for a HPLC method. All these demonstrated that the
HPLC method is appropriate for the determination at g/L
level of Tartrazine and Sunset Yellow in soft drinks samples
and its implementation of this simple and accurate method
for synthetic dyes control will become a very powerful tool
for food safety and for detecting food authenticity.
References
1. MACRAE, R., ROBINSON, R.K., SADLER, M.J., (Eds.), Encyclopaedia
of Food Science, Food Technology and Nutrition, Vol. II, Academic
Press, San Diego, CA (1993)
2. SCHOEFS, B.,Trends in Food Science & Technology, 13, I11, 2002,
p. 361

http://www.revistadechimie.ro

1181

3. GHORPADE, V.M., DESHPANDE, S.S., SALUNKHE, D.K., Food ColorsChapter 4 of Dekker, M., Food Additive Toxicology, Ed. Maga, J.A., and
Tu, A.T., New York, 1995, p. 179
4. *** Council Directive 94/36, European Parliament, Official Journal
of the European Communities, No. L 237/1 and L 237/13, 1994
5. *** Commission Regulation (EC) No 884/2007, European Parliament,
Official Journal of the European Communities, No.L 125, 2007
6. HORWITZ W., LATIMER G. W. Jr.,Official Mthods of Analysis of
AOAC International, 18 Edition, 2005, Revision 3, 2010
7. WROLSTAD, R. E. , ACREE, T. E.,DECKER, E. A. , PENNER, M. H.,
REID, D. S., SCHWARTZ, S. J., SHOEMAKER, C. F., SPORNS,P.,
Handbook of Food Analytical Chemistr y: Pigments, Colorants,
Flavors, Texture, and Bioactive Food Components, J. Wiley & Sons,
New Jersey, 2005.

8. DIACU, E., ENE, C. P., Rev Chim. (Bucharest), 60, no. 8, 2009, p. 745
9.*** ISO GUM, Guide to the Expression of Uncertainty in Measurement,
2nd edn. (1995), with Supplement 1, Evaluation of measurement data,
JCGM 101:2008, Organization for Standardization, Geneva, Switzerland
(2008). ttp://www.bipm.org/en/publications/guides
10. YOUDEN, W. J., STEINER, E.H., Statistical Manual of the AOAC Association of Official Analytical Chemist. AOAC-I, Washington DC
1975
11. *** ISO/IEC 17025:1999. General Requirements for the Competence
of Calibration and Testing Laboratories. ISO, Geneva (1999)
EURACHEM, Quantifying Uncertainty in Analytical Measurement.
Laboratory of the Government Chemist, London ISBN 0- 948926-082 (1995)
Manuscript received: 23.09.2010

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