Fuel 212 (2018) 236–239

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Fuel
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Full Length Article

Determination of ethanol in gasoline by high-performance liquid MARK

chromatography
⁎
Lorena Morine Avilaa, Amanda Pereira Franco dos Santosb, ,
Danielle Ignácio Mançano de Mattosb, Cristiane Gimenes de Souzab, Débora França de Andradec,
Luiz Antonio d'Avilab
a
Instituto de Química, Universidade Federal Fluminense, Niterói, Rio de Janeiro 24020-141, Rio de Janeiro, Brazil
b
Engineering Program in Chemical and Biochemical Processes, Escola de Química, Universidade Federal do Rio de Janeiro, 21941-909 Rio de Janeiro, Brazil
c
Instituto de Química, Universidade Federal do Rio de Janeiro, Cidade Universitária, 21941-909 Rio de Janeiro, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: This study employed non-aqueous reversed-phase high-performance liquid chromatography (HPLC) with re-
Gasoline fractive-index detection and methanol as a mobile phase to quantify the ethanol content in gasoline. The ad-
Ethanol vantages of HPLC are its good separation versatility, high resolution, relatively short analysis time, and auto-
HPLC mation. Standard samples were prepared to obtain a standard curve and partial validation. Accuracy, precision,
linearity, selectivity for methanol (adulterant), and measurement uncertainty were investigated. In the partial
validation analysis, the accuracy of the proposed method was identified as its main advantage over the reference
method.

1. Introduction
When fuel is adulterated, it is usually done by adding a lower cost
product in order to obtain illicit financial gains from its retail. The il-
legal addition of excess ethanol to gasoline is arguably the easiest and
most common form of adulteration, since ethanol is already a compo-
nent of the gasoline/ethanol blend used in Brazil (27% ± 1% by vo-
lume) and is cheaper than gasoline. According to the national bulletin
of the Fuel Quality Monitoring Program run by the Brazilian fuel reg-
ulatory agency (ANP), the ethanol content of gasoline is the biggest
cause of noncompliance found in samples of gasoline [1,2].
The reference method for determining ethanol content, described in
ANP resolution N°40 of October 25, 2013, is detailed in Brazilian
standard NBR 13992/2015, issued by the Brazilian technical standards
association (Associação Brasileira de Normas Técnicas, ABNT) [3,4]. It
is a quick, simple, practical method that can be done in the field to
check gasoline purity. However, because of these very features, its
measurement uncertainty is 1 vol%, and it has the added limitation of
quantifying other water-soluble alcohols like methanol, impairing its
accuracy.
The adulteration of ethanol by methanol has been verified in Brazil
by the ANP [5–8], and this has motivated the development of tests to
determine methanol content in ethanol [9,10].
The American Society for Testing and Materials (ASTM) has its own
standard, D5501, published in 2012 and reapproved in 2016, which
describes a standard method using gas chromatography (GC) for the
determination of the ethanol (20 mass% or over) and methanol
(0.01 mass%–0.6 mass%) content of fuels [3,11]. This method is not so
easy to implement, as it is done using long columns (100 m and 150 m),
which, while they do exist, are harder to find on the market and very
expensive. In the case of the 100 m column, the initial temperature of
the column is supposed to be 15 °C, but this is unfeasible in much of
Brazil as it is lower than ambient temperature, meaning that a cooling
stage would first have to be employed. Further, the specific density of
all the samples must be calculated to correct the results obtained [11].
In Brazil, ABNT set up a working group at its Ethanol Fuel Study
Commission to create a reference method for quantifying ethanol and
methanol in gasoline and ethanol fuel. The chosen method was GC, but
after much research no reference method for quantifying ethanol in
gasoline was produced, since the compounds in gasoline are eluted in
the same time as ethanol [12].
There are different methods described in the literature for analyzing
oxygenates in gasoline for quality control and regulatory purposes, like
Fourier-transform near-infrared spectroscopy [13] with partial least
square (PLS) calibration [14,15]; Raman spectroscopy [16]; synchro-
nous fluorescence spectroscopy with principal component regression or

⁎
Corresponding author at: Centro de Tecnologia, Bloco K02, LABCOM, Cidade Universitária, Rio de Janeiro 21941-909, Brazil.
E-mail addresses: morineavila@gmail.com (L.M. Avila), apfranco@eq.ufrj.br (A.P.F. dos Santos), danielleignacio@eq.ufrj.br (D.I.M. de Mattos),
cristianegimenes@yahoo.com.br (C.G. de Souza), debora.franca.andrade@gmail.com (D.F. de Andrade), davila@eq.ufrj.br (L.A. d'Avila).

http://dx.doi.org/10.1016/j.fuel.2017.10.039
Received 21 February 2017; Received in revised form 22 September 2017; Accepted 6 October 2017
0016-2361/ © 2017 Elsevier Ltd. All rights reserved.
L.M. Avila et al. Fuel 212 (2018) 236–239

PLS calibration models [17]; a combination of excitation-emission
matrix fluorescence spectroscopy with multiway partial least square
regression (N-PLS) and unfolded PLS [17]; batch injection analysis with
detection by multiple-pulse amperometry [18]; Terahertz spectroscopy
[19]; proton nuclear magnetic resonance spectroscopy (1H NMR) [20];
cyclic voltammetry and multivariate calibration [21,22]; and custo-
mized mobile near-infrared spectrometry [23].
ASTM D5599/2010 describes a procedure for determining oxyge-
nate content in gasoline by GC, but only for the 0.1–20% by mass range,
which is not enough for the Brazilian fuel market [24].
ASTM D4815/2013 describes a GC method for determining methyl
tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), diisopropyl ether
(DIPE), tert-amyl alcohol, and C1–C4 alcohols in gasoline. However, this
method is employed to determine alcohol levels of 0.2 mass% to
12 mass%, ruling out its use for ethanol-based fuels and making it un-
feasible for testing Brazilian gasolines [25].
In this context, this study proposes the use of high-performance li-
quid chromatography (HPLC) with refractive index detection to de-
velop a quick method for quantifying the ethanol content in gasoline. It
is hoped that this method may replace the existing reference test for
laboratory analyses and the monitoring and inspection of gasoline sold
in Brazil, whose drawbacks are its minimum uncertainty of ± 1% (by
volume) and the fact that it is not accurate enough for determining
ethanol levels in gasoline if it is adulterated with methanol.
Calibrated glassware (10.00 mL volumetric flask, 1.00 mL volu-
metric pipette, and 10–100 µL variable volume automatic micropipette)
was also used in the preparation of this sample.
2.2. Analysis of the samples by high-performance liquid chromatography
(HPLC)
All the samples were analyzed using non-aqueous reversed-phase
high-performance liquid chromatography (HPLC) with a Dionex
UltiMate™ 3000 quaternary pump (Thermo Scientific, Massachusetts,
USA), a Shodex RI-101 refractive index detector (ECOM, Czech
Republic), and an UltiMate™ WPS-3000 autosampler (Thermo
Scientific) with a 100 µL sample loop. An Acclaim™ column (Thermo
Scientific) measuring 250 mm long and 4.6 mm internal diameter was
used with an octadecylsilane phase with 5 µm particle size and 120 Å
pore diameter. The mobile phase was 100% pre-filtered HPLC-grade
methanol (Tedia Brazil, Rio de Janeiro, Brazil). Total analysis time was
15 min at 40 °C (temperature of column oven), with a constant flow rate
of 1 mL/min and 10 µL injection volume. The samples were injected in
quadruplicate to evaluate repeatability. The chromatograms were
analyzed using Chromeleon 6.80 SR11 software (Thermo Scientific)
with manual integration.
2.3. Partial validation of proposed method

In order to use objective evidence to ascertain whether the proposed

2. Materials and methods
2.1. Preparation of standard samples of gasoline with ethanol
Eighteen standard samples of gasoline containing different quan-
tities of anhydrous ethanol fuel, ranging from 19 to 40% (by volume),
were prepared. The gasoline was supplied by Companhia Brasileira de
Petróleo Ipiranga and the ANP inspection department, and support was
provided by the Fuel and Petroleum Products Laboratory at the School
of Chemistry, Federal University of Rio de Janeiro (LABCOM/EQ/
UFRJ). All the glassware used in preparing the samples (1.00, 2.00,
5.00, and 10.00 mL volumetric pipettes; 10–100 µL variable volume
automatic micropipette; and 50.00 mL volumetric flask) were cali-
brated in advance. Six of the 18 samples were used to plot an analytical
curve and 12 were used to verify the analytical curve and to partially
validate the proposed method, as shown in Table 1.
Aside from the 18 standard samples, a sample of gasoline containing
12.5 vol% ethanol and 12.5 vol% methanol was prepared to verify ac-
curacy when ethanol was adulterated with methanol and to demon-
strate its selectivity for ethanol.
Table 1
Standard samples of gasoline containing different proportions of anhydrous ethanol fuel.
Standard Samples for Analytical Curve Standard Samples for Verification
method produces reliable results that are fit for purpose – i.e. whether it
meets the requirements and standards for the specific proposed use –
we assessed its linearity, selectivity for methanol (adulterant), accu-
racy, precision, and measurement uncertainty [26–30].
Linearity was checked by the coefficient of determination (R2) of the
straight line. Values over 0.99 indicated the linear working range,
where the angular coefficient of the straight line could be considered
constant [26].
The selectivity of the HPLC method for methanol was checked by
injecting a sample of pure gasoline (matrix) and a standard sample
containing 12.5 vol% ethanol, 12.5 vol% methanol, and 75 vol% gaso-
line, since no certified reference material exists for such a determina-
tion.
In order to assess the accuracy of the verification samples, we used
Eq. (1) to ascertain relative error (Erel) [26]:
xlab−x v
Erel = ∗100
xv (1)
where:
Erel is relative error (%);
Xlab is the single value obtained experimentally or the mean of the
laboratory results;
Xv is the value accepted as being true.

Sample Code Nominal Ethanol Value Sample Code Nominal Ethanol Value In this study, the relative error of the 12 verification samples
(% volume)* (% volume)* (V1–V12) was calculated, assuming a 95% confidence interval for the
A1 20.00 V1 19.00
statistical treatment of the results obtained. As such, the maximum
A2 22.00 V2 20.00 admissible error was set at 5%.
A3 24.00 V3 21.00 The repeatability limit (r) calculated by Eq. (2) and the total am-
A4 26.00 V4 23.00 plitude of the measurements (difference between the highest and lowest
A5 28.00 V5 23.00
value from a dataset) were used to evaluate the precision of the pro-
A6 30.00 V6 25.00
V7 26.00 posed method [27].
V8 27.00 r = 2.8∗Sr (2)
V9 27.00
V10 31.00 where:
V11 32.00
V12 40.00
r is the repeatability limit;
* The nominal values were corrected using the data from the glassware calibration Sr is the standard deviation of repeatability, meaning the standard
certificates (see Table 2, in Results). deviation of the results for each concentration.

237
L.M. Avila et al.
Finally, the expanded uncertainty of measurement of the HPLC
method described here, for a 95% confidence interval, was calculated
according to the Guide to the Expression of Uncertainty in
Measurement (ISO GUM) [30]. It considered the following sources of
uncertainty: the calibration of the pipettes and flasks used to prepare
each standard sample; the purity of the ethanol used; the straight-line
equation obtained; the preparation of the control sample; and the re-
peatability of the measurements. For each source of uncertainty, we
ascertained what type of uncertainty was involved and what method
was best suited to determining and calculating it. We carried out type A
and type B evaluations of uncertainty and closely observed the con-
tributions made by more than one source of uncertainty so that no
calculations were duplicated. Electronic spreadsheets were used for the
calculations of combined uncertainty, effective number of degrees of
freedom, coverage factor, and expanded uncertainty. The spreadsheet
showing the uncertainty of measurement calculations for this HPLC
method is included in the Supplementary material.
3. Results and discussion
3.1. Analysis of samples by high-performance liquid chromatography
(HPLC)
All the samples analyzed by HPLC had the same chromatographic
profile, as shown in Fig. 1. The anhydrous ethanol fuel was eluted in
around 3 min, unlike the constituent parts of the gasoline, whose re-
tention times ranged from 4 min to 10 min.
3.2. Partial validation of proposed method
To plot the analytical curve, the six standard samples of gasoline
containing anhydrous ethanol fuel (nominal values: 20 vol%, 22 vol%,
24 vol%, 26 vol%, 28 vol%, and 30 vol%) were analyzed by HPLC in
quadruplicate, generating a dispersion graph in Excel of ethanol con-
centration (% volume) versus anhydrous ethanol fuel peak area
(µRIU*min), as shown in Fig. 2. As the coefficient of determination
(R2 = 0.9910) indicated a linear correlation, the equation of the ana-
lytical curve (y = 2.1608 x + 17.978) was used to quantify the ethanol
concentrations determined by HPLC in the 12 verification samples for
the partial validation of the proposed method.
Table 2 shows the results of the HPLC analyses of the anhydrous
ethanol fuel content in the 12 verification samples performed in
quadruplicate. The results in Table 2 show that the method was valid
with regard to accuracy, since all the relative errors obtained in the 12
verification samples were lower than 5%, the level set as the maximum
admissible error (95% confidence interval).
The results also show that the total amplitude of the measurements
was lower than the repeatability limit in all the verification samples
analyzed, which indicates the method was valid in terms of repeat-
ability.
Fig. 3 shows the HPLC chromatogram of the standard sample
1200
Ethanol
1000
800
Height (μRIU)
600
400
200
0
0 2 4 6 8 10 12 14
-200
Time (min)
Fig. 1. HPLC chromatogram of a standard sample containing 28% (vol) ethanol.
Fuel 212 (2018) 236–239
Analy cal Curve
85
y = 2.1608x + 17.978
R² = 0.991
80
area (μRIU*min)
75
70
65
60
19 21 23 25 27 29 31
ethanol on entra on (% volume)
Fig. 2. Analytical curve showing ethanol concentration (% volume) against area
(µRIU*min).
containing 12.5 vol% ethanol and 12.5 vol% methanol in gasoline A,
highlighting the ethanol peak after 3 min retention time, set against the
HPLC chromatogram of pure gasoline (without ethanol). From Fig. 3, it
is clear that there is no interference (superimposition) of the ethanol
and gasoline peaks at this retention time to prevent the quantification
of the anhydrous ethanol fuel content in the blend. The chromatogram
of the gasoline matrix (filled line) does not peak at the same retention
time as the ethanol (dotted line). In addition, methanol cannot be de-
tected if methanol is the mobile phase, showing that the HPLC method
proposed is selective for ethanol.
The quantity of anhydrous ethanol fuel determined for this sample
using the reference method (ABNT NBR 13992/2008) was 25 vol%,
because both methanol and ethanol are water-soluble. However, the
proposed HPLC method found the ethanol content to be 11.97 vol
% ± 0.43 vol%. In other words, it was not affected by the presence of
an interferent, which puts it at an advantage over the reference method
currently used in Brazil.
The expanded uncertainty of measurement of the HPLC method for
determining the level of ethanol in the gasoline based on the standard
samples used to plot the analytical curve was calculated at 0.56 vol%
(Fig. 4S, supplementary material). The uncertainty of measurement in
HPLC is a quantitative indication of the quality of the result obtained,
and is therefore a tool that makes the analytical results and thus the
analyses conducted using HPLC more reliable.
In making the calculation, the contributions of the following sources
of uncertainty were considered: the calibration of the pipettes and
flasks used to prepare each standard sample; the purity of the ethanol
used; the straight-line equation obtained; the preparation of the control
sample; and the repeatability of the measurements. The spreadsheet
showing the uncertainty of measurement calculations for this HPLC
method is included in the Supplementary material (Fig. 4S).
4. Conclusion
The proposed high-performance liquid chromatography method
proved effective for quantifying the anhydrous ethanol fuel content of
gasoline for its speed, simplicity, accuracy, and precision, demonstrated
by the statistical tools used in its partial validation.
However, its greatest advantage is the accuracy it was found to
have, as it suffered no interference from the other constituent parts of
the matrix (gasoline) or the interferent (methanol). This is one of the
drawbacks of the traditional method and the main reason why this
research was done.
16
We conclude that the HPLC method described in this study is pro-
mising for the accurate determination of the ethanol content of gaso-
line. The expanded uncertainty of this method (0.56 vol%) is also lower
than that of the current reference test (1.00 vol%).
238
L.M. Avila et al. Fuel 212 (2018) 236–239

Table 2
Anhydrous ethanol fuel content in the verification samples, determined by HPLC.

Sample Nominal Ethanol Value (% Anhydrous Ethanol Fuel Content (% volume)

volume)*
Mean of Quadruplicate Measurements (% Standard Absolute Error Relative Error Repeatability Limit Amplitude
volume) Deviation (%)

V1 19.01 19.76 0.04 0.75 3.95 0.12 0.10

V2 19.99 19.51 0.20 0.48 2.41 0.55 0.42
V3 20.99 21.84 0.96 0.84 4.02 2.70 2.01
V4 22.98 23.57 0.85 0.60 2.61 2.38 1.75
V5 22.95 23.33 0.09 0.37 1.63 0.25 0.22
V6 24.99 25.45 0.18 0.46 1.84 0.50 0.43
V7 25.96 26.38 0.34 0.42 1.63 0.95 0.71
V8 26.96 27.28 0.50 0.32 1.20 1.41 1.18
V9 26.95 27.61 0.11 0.66 2.46 0.30 0.22
V10 31.04 30.08 0.50 0.96 3.08 1.41 1.11
V11 32.06 30.88 0.31 1.18 3.68 0.88 0.76
V12 39.99 38.55 1.17 1.44 3.60 3.27 2.69

* Corrected nominal values.

1000 Metanol em Etanol Combustível para o Monitoramento da Qualidade de

Combustíveis [Colorimetric Kit for Detection of Methanol in Ethanol Fuel for
900 Monitoring Fuel Quality]. Quim Nova 2015;38:280–4. http://dx.doi.org/10.5935/
800 12,5% EtOH + 12,5% MeOH 0100-4042.20140305.
[11] ASTM D5501/2016. Standard Test Method for Determination of Ethanol and
700
Height (μRIU)

Pure Gasoline Methanol Content in Fuels Containing Greater than 20% Ethanol by Gas
600 Chromatography. 2012. (Reapproved in 2016).
[12] NBR 16041. Etanol Combustível - Determinação dos Teores de Metanol e Etanol por
500 Cromatografia Gasosa [Ethanol Fuel - Determination of Methanol and Ethanol
400 Content by Gas Chromatography]. 2015.
[13] Corsetti S, Zehentbauer FM, McGloin D, Kiefer J. Characterization of gasoline/
300 ethanol blends by infrared and excess infrared spectroscopy. Fuel
200 2015;141:136–42.
[14] Fernandes HL, Raimundo Jr. IM, Pasquini C, Rohwedder JJR. Simultaneous de-
100 termination of methanol and ethanol in gasoline using NIR spectroscopy: effect of
gasoline composition. Talanta 2008;75:804–10.
0
[15] Conklin Jr. A, Goldcamp MJ, Barrett J. Determination of ethanol in gasoline by
2.8 2.9 3.0 3.1 3.2 FTIR spectroscopy. J Chem Educ 2014;91:889–91.
Time (min) [16] Fortunato FM, Vieira AL, Gomes Neto JA, Donati GL, Jones BT. Expanding the
potentialities of standard dilution analysis: determination of the ethanol in gasoline
Fig. 3. HPLC chromatograms of a standard sample containing 12.5 vol% ethanol and by Raman spectroscopy. Microchem J 2017;133:76–80.
12.5 vol% methanol and a sample of pure gasoline. [17] Kumar K, Mishra AK. Quantification of ethanol in ethanol-petrol and biodiesel in
biodiesel-diesel blends using fluorescence spectroscopy and multivariate methods. J
Fluoresc 2012;22:339–47.
Acknowledgements [18] Pereira PF, Marra MC, Munoz RAA, Richter EM. Fast batch injection analysis system
for on-site determination of ethanol in gasohol and fuel ethanol. Talanta
2012;90:99–102.
The authors wish to thank Companhia Brasileira de Petróleo [19] Arik E, Altan H, Esenturk O. Dielectric properties of ethanol and gasoline mixtures
Ipiranga and ANP for the gasoline supplied. by terahertz spectroscopy and an effective method for determination of ethanol
content of gasoline. J Phys Chem 2014;118:3081–9.
[20] Turanov A, Khitrin AK. Proton NMR characterization of gasoline-ethanol blends.
Appendix A. Supplementary data Fuel 2014;137:335–8.
[21] Pereira PF, Souza RMF, Munoz RAA, Richter EM. Simultaneous determination of
ethanol and methanol in fuel ethanol using cyclic voltammetry. Fuel
Supplementary data associated with this article can be found, in the 2013;103:725–9. http://dx.doi.org/10.1016/j.fuel.2012.07.034.
online version, at http://dx.doi.org/10.1016/j.fuel.2017.10.039. [22] Abreu REL, Paz JEM, Silva AC, Pontes MJC, Lemos SG. Ethanol fuel adulteration
with methanol assessed by cyclic voltammetry and multivariate calibration. Fuel
2015;156:20–5.
References [23] Lutz OMD, Bonn GK, Rode BM, Hucka CW. Reproducible quantification of ethanol
in gasoline via a customized mobile near-infrared spectrometer. Anal Chim Acta
2014;826:61–8.
[1] Portaria MAPA N°75, March 5, 2015. Brazil, 2015. [24] ASTM D5599/2010. Test Method for Determination of Oxygenates in Gasoline by
[2] http://www.anp.gov.br, (accessed in February 2017). Gas Chromatography and Oxygen Selective Flame Ionization Detection. 2010.
[3] ANP Resolution N°40, October 25, 2013. Brazil, 2013. [25] ASTM D4815/2013. Standard Test Method for Determination of MTBE, ETBE,
[4] NBR 13992/2015. Gasolina Automotiva - Determinação do Teor de Etanol Anidro TAME, DIPE, Tertiary-Amyl Alcohol and C1 to C4 Alcohols in Gasoline by Gas
Combustível [Automotive Gasoline - Determination of Anhydrous Ethanol Fuel Chromatography. 2013.
Content]. 2015. [26] Lopes PA. Validação de Métodos de Ensaios para Laboratórios de Ensaios de
[5] http://economia.estadao.com.br/noticias/geral,anp-descobre-nova-adulteracao-do- Calibração [Validation of Test Methods for Calibration Laboratories]: Internal
alcool-hidratado,4518e, (accessed in February 2017). publication of method validation course. Rio de Janeiro, Brazil: Rede de Tecnologia;
[6] http://oglobo.globo.com/economia/petroleo-e-energia/anp-encontra-metanol-em- 2008.
alcool-hidratado-para-veiculos-20467071, (accessed in February 2017). [27] Rego ECP. Validação de Métodos de Ensaios Químicos [Validation of Chemical
[7] http://blogs.oglobo.globo.com/ancelmo/post/anp-encontra-16-milhoes-de-litros- Testing Methods]: Internal publication of method validation course. Rio de Janeiro,
de-combustivel-adulterado-em-tanques-de-shell-ipiranga-e-br.html, (accessed in Brazil: Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMETRO); 2013.
February 2017). [28] Ribeiro FAL, Ferreira MMC, Morano SC, Silva LR, Schneider RP. Planilha de
[8] http://odia.ig.com.br/rio-de-janeiro/2016-11-16/anp-interdita-postos-da-ilha-que- Validação: Uma Nova Ferramenta para Estimar Figuras de Mérito na Validação de
vendiam-alcool-adulterado.html, (accessed in February 2017). Métodos Analíticos Univariados [Validation Spreadsheet: a new tool for estimating
[9] E.C.S. Oliveira, A. Cunha Neto, R.C. Silva, E.V.R. Castro, V. Lacerda Jr., W. Romão, figures of merit for the validation of univariate methods.]. Química Nova
R.G. Pereira, T. Sten, Determinação de Adulteração de Etanol Combustível com 2008;31:164–71. http://dx.doi.org/10.1590/S0100-40422008000100029.
Metanol por Ressonância Magnética Nuclear Associada a Ferramentas [29] http://www.inmetro.gov.br/Sidoq/Arquivos/Cgcre/DOQ/DOQ-Cgcre-8_04.pdf,
Quimiométricas [Determination of Adulteration of Ethanol Fuel with Methanol by (accessed in February 2017).
Nuclear Magnetic Resonance Associated with Chemometric Tools], in: 52nd [30] http://www.inmetro.gov.br/inovacao/publicacoes/gum_final.pdf, (accessed in
Brazilian Congress of Chemistry, Recife, Brazil, 2012. Available at: http://www. March 2015).
abq.org.br/cbq/2012/trabalhos/9/845-14325.html.
[10] Guilherme BCM, Mateus AM, Paulo AZS. Kit Colorimétrico para Detecção de

239