A digital image method of spot tests for determination

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 185 (2017) 310–316

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A digital image method of spot tests for determination of copper in sugar cane spirits Kenia Dias Pessoa a, Willian Toito Suarez a,⁎, Marina Ferreira dos Reis a, Mathews de Oliveira Krambeck Franco a, Renata Pereira Lopes Moreira a, Vagner Bezerra dos Santos b a b

Departamento de Química, Universidade Federal de Viçosa - UFV, Centro de Ciências Exatas e Tecnologia, Viçosa, MG, Brazil Faculdade de Química, Universidade Federal do Pará – UFPA, Instituto de Ciências Exatas e Naturais, Belém, PA, Brazil

a r t i c l e

i n f o

Article history: Received 20 December 2016 Received in revised form 25 May 2017 Accepted 30 May 2017 Available online 31 May 2017 Keywords: Digital image analysis RGB model Sugar cane spirits Copper Spot test

a b s t r a c t In this work the development and validation of analytical methodology for determination of copper in sugarcane spirit samples is carried out. The digital image based (DIB) method was applied along with spot test from the colorimetric reaction employing the RGB color model. For the determination of copper concentration, it was used the cuprizone a bidentate organic reagent - which forms with copper a blue chelate in an alkaline medium. A linear calibration curve over the concentration range from 0.75 to 5.00 mg L−1 (r2 = 0.9988) was obtained and limits of detection and quantification of 0.078 mg L−1 and 0.26 mg L−1 were acquired, respectively. For the accuracy studies, recovery percentages ranged from 98 to 104% were obtained. The comparison of cooper concentration results in sugar cane spirits using the DIB method and Flame Atomic Absorption Spectrometry as reference method showed no significant differences between both methods, which were performed using the paired t-test in 95% of confidence level. Thus, the spot test method associated with DIB allows the use of devices as digital cameras and smartphones to evaluate colorimetric reaction with low waste generation, practicality, quickness, accuracy, precision, high portability and low-cost. © 2017 Elsevier B.V. All rights reserved.

1. Introduction According to Brazilian legislation, sugar cane spirit (cachaça) is the typical and exclusive denomination for distilled spirit made from sugar cane juice produced in Brazil with alcoholic graduation from 38% to 48% by volume at 20 °C obtained by distillation of fermented mash of sugar cane juice with peculiar sensory features [1]. Sugar cane spirit consists primarily of ethanol and water, but also secondary compounds, such as higher alcohols, acids, esters, aldehydes, sugars, among others, which are responsible for characterization and quality of the drink [2]. The process of sugar cane spirit production is usually conducted in distillers made of copper, once it presents a better sensory quality in relation to sugar cane spirit produced in pot stills made of other materials, such as stainless steel, aluminum and porcelain [3]. The use of copper in distillation equipment favors the reduction of acidity as well as the levels of aldehydes and sulfur compounds which impart unwanted flavor and odor in the drink. However, during the distillation process in copper stills or during the time that the still is not in use, occurs the formation of a compound commonly known as verdigris (basic carbonate of copper [CuCO3Cu(OH)2]) [4]. This substance is dissolved by acidified alcoholics vapors contaminating the drink with copper(II), consequently its presence in high concentrations is harmful to human health. ⁎ Corresponding author. E-mail address: [email protected] (W.T. Suarez).

http://dx.doi.org/10.1016/j.saa.2017.05.072 1386-1425/© 2017 Elsevier B.V. All rights reserved.

The most known disease due to the accumulation of copper is called Wilson's disease. It is clinically characterized not only by hepatic and neurological manifestations, but also by copper accumulation in the liver and in the corneas [5,6]. Furthermore, studies have demonstrated that copper is associated with formation of ethyl carbamate in sugar cane spirits, which is a potential carcinogen [7]. Thus, the Brazilian Ministry of Agriculture determines that copper concentrations in sugar cane spirit must not exceed a concentration of 5 mg L−1. In the European Union, the maximum copper concentration permitted is 2 mg L−1 [8,9,10]. Some methods have been described for the determination of copper in alcoholic distillates, like colorimetric [8], spectrophotometry [9,11], diffuse reflectance spectroscopy [12], voltammetry [13,14], ICP-OES (Inductively Coupled Plasma-Optical Emission Spectrometry) [15], total reflection and proton-induced X-ray fluorescence spectrometry [16, 17]. Although, the recommended method for copper determination in Brazilian distilled beverages is the atomic absorption spectrometry (AAS) using the method of standard addition [18]. However, in general, some of these techniques are relatively expensive and, in most cases, require a complex and rigorous pretreatment of the sample [14]. Therefore, the development of analytical methods with high sensitivity, robustness, quickness and low cost of implementation and maintenance becomes a useful alternative for the analysis of analytes and contaminants in the beverages. The analytical methods based on spot test of digital images have become promising alternatives for the determination of several analytes,

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since the spot test procedures are simple, rapid, practical, inexpensive and employ small quantities of chemicals [19–21]. In addition, this method employs moderns and accessible devices, such as cameras, webcams, scanners and mobile phones with built-in cameras, whose resolution and compactability have improved significantly, as well as their costs have reduced in similar proportion [22,23]. By using the spot test coupled to digital image, it is possible to develop in situ analysis at distant locals of difficult access. Moreover, it is not easy to obtain representative samples, to bring them to the laboratory, and to perform analysis, once these are laborious and costly tasks. In addition, handling of samples can present serious analytical errors by contamination and loss of analytes due to changes in physical–chemical and microbiological properties of the samples. These problems can be avoided employing spot tests-digital image based method in situ [22,23]. Li et al. [24] developed an integral optical density (IOD) method combined with the model of response difference of crystallite change (MRDCC) in order to analyze the influence of different sugars on the gelatinization of corn starch. Moreover, Masawat et al. [25] employed an iPhone-based digital image colorimeter (DIC), fabricated as a portable tool for monitoring tetracycline (TC) in bovine milk, to carry out an analysis after pre concentration on C18 SPE (solid phase extraction) sorbent. The presence of sensors in the image capture devices such as CCD (Charge Coupled Device) and CMOS (Complementary Metal Oxide Semiconductor) has allowed the development of analytical methods with high sensitivity, robustness, quickness and low cost of implementation. These sensors are able to convert the intensity of the incident radiation into an analog electrical signal, which is converted to a digital value stored in the form of bits and pixels arranged in a matrix, similar to the RGB model [26]. In the RGB system, three matrices are used to store the information. These matrices are related to the present colors, red (R), green (G) and blue (B). This model is used to establish a relationship between the color components and the concentration of the analyte [21,27]. This correlation cannot be described directly as absorbance, once the measure of image capturing is given by diffuse reflectance [12,28]. However, as the reflected radiation is part of incident radiation that was not absorbed, this radiation could be used for analytical purposes [29,30]. The values relating to responses RGB colorimetric reaction (I) are compared with the values acquired from the R, G, or B obtained from the blank. The equation y = −log(I/I0) supplies the value needed to construct the analytical curve [30–32]. In this work, it is reported the development and validation of analytical methodology for determination of copper concentration in commercial samples of sugar cane spirit. This methodology was based on digital image analysis of spot test involving the reaction between copper ions and the cuprizone that forms a blue chelate, and the images captured by a camera containing a CCD type sensor. The method developed can be applied to in situ determination of copper concentration in sugar cane spirit, which can be important for the quality control, especially for small producers with limited financial resources. Furthermore, the method allows a fast, simple, low cost and low waste generation analysis. 2. Experimental 2.1. Apparatus and Instrumentation The colorimetric reaction for determination of copper(II) in sugar cane spirit was performed directly on a porcelain plaque containing 6 spots. Eppendorf (Germany) automatic pipettes were used to transfer volumes of reagents and/or samples to the porcelain plaque and spot tests were photographed employing a camera Sony Cyber-Shot DSCS730 with resolution of 7.2 Megapixels. For the image capture, it was built a portable simple device with internal lighting system illustrated in Fig. 1(a). This apparatus consists of a

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black plastic box of dimensions 21 × 15 × 7 cm, containing a Unipower Battery 12 V, four LEDs (Light Emitting Diodes) white light BLUEX 20– 100 mA with variable resistors to intensity control, an ON/OFF switch and connectors to couple a 12 V battery charger. To ensure the reproducibility of the acquisition of digital images and minimize the reflection or shadow effects caused by external light, the device was built using four punctual LEDs evenly distributed on top of the equipment. Inside the box it was applied a matte black ink layer to ensure more homogeneous environment. To evaluate the effect of light intensity on the analytical response, it was employed a luximeter Minipa MLM 1011. For the comparative method, the sugar cane spirit samples were analyzed using an Agilent Technologies spectrometer Model 240 FS AA. 2.2. Chemicals and Samples The solutions were prepared with ultrapure water (resistivity N18.0 MΩ cm) obtained from a Millipore Milli-Q system (USA) and all chemical reagents were of analytical grade. The stock solution of Cu(II) 500 mg L− 1 (0.0078 mol L− 1) was prepared by dissolving 0.393 g CuSO4·5H2O in 200 mL of distilled water, after it was stored at room temperature. Cuprizone stock solution (bis(cyclohexanone)oxalildihidrazona) 500 mg L−1 (0.0018 mol L−1) was prepared in a solution of ethanol-distilled water 60% (v/v) and stored at 4 °C. The other solutions were obtained by diluting the stock solution in ethanol-distilled water 60% (v/v). The 0.250 mol L−1 phosphate buffer at pH 8.0 was prepared by dissolving Na2HPO4 and NaH2PO4 in distilled water and stored at room temperature. Stock solutions of concentration 1.0 × 10−4 mol L−1 of the following dyes: Blue Coomassie Brilliant, Tartrazine and Direct Red 80 were prepared by dissolving the respective dye with distilled water, and the other solutions were obtained from dilutions of stock solutions. 2.3. Image Acquisition and Data Analysis The color of an image may be defined as the human perception of the wavelengths combination in the electromagnetic spectrum in the visible light region that reflects on a surface [33]. These combinations were represented by tridimensional arrangement of color such as RGB (Red, Green, Blue) and the channels are correlated with the absorbed selectively certain wavelengths of light [34,35]. The radiation emitted by the white LEDs is composed by a combination of several monochromatic beams that can be represented by RGB tridimensional arrangement. As shown in Fig. 1(b), radiation beams arrive at the solution contained in the porcelain plaque and part of this radiation is absorbed, and further reflected by the solution. This absorption of incident radiation occurs mainly in the complementary color. In fact, if the color blue is displayed by the solution, the channel that has the highest absorption is red [36]. After the absorption of radiation by the solution of the remaining radiation is reflected and reaches at the Bayer filter mosaic, which is a color filter array (CFA) for arranging RGB color filters on a square grid of photosensors. Color is identified by the use of a color filter which allows only one color of light from the visible spectrum into each pixel. In CCD cameras, the level of photons recorded is converted to a proportional electrical signal. This sensor converts the light intensity incident on matrices digital used to store the information. The intensities of the generated color is stored in 256 levels in a scale from 0 to 255 for each primary color (R, G or B) where 0 is pure black (0 for each one of the 3 primary colors) and 255 is pure white (mixture of 255 for each primary colors) [21]. So, for analytical propose, the images acquired from colorimetric reaction are decomposed employing a free software Image J in a system of arrays based on the RGB model. From this, it is possible to obtain analytical responses related to three channels for each spot in the porcelain plaque. For the analytical response − log (I/I0) was used, where I is

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Fig. 1. (a) System built for the image capture (b) it indicates the interaction phenomenon of electromagnetic radiation with a solution at porcelain plaque and how the reflected light reaches the digital camera detector.

the average value of the R, G, or B channel measured by sample or standard solution and I0 is the average value acquired from the analytical blank. Thus, the analytical curve is presented as −log (I/I0) vs. the copper concentration. For this, a proper R G or B channel was used. Other mathematical manipulation could be performed such as the R, G, or B values directly, or its combination as R × G × B generating exponential, polynomial or linear behavior versus concentration. However, −log (I/ I0) is commonly used once it is linearly correlated with the concentration being very employed for colorimetry and spectrophotometry [30, 37,38]. By using this calculation, it is possible to use a reference value (Blank) and to avoid performing the subtraction from the analytical signal, being similar to the Lambert-Beer law for colorimetry. The signal captured from a digital camera is due to reflective, however, this signal is correlated with the absorbed signal, and so, the logarithm signal can be used without loss of performance.

2.5. Copper(II) Determination in Sugar Cane Spirit For the copper determination in sugar cane spirit, it was employed the cuprizone, a bidentate organic reagent, which forms with copper a blue chelate in a slightly alkaline medium [39], as shown in Fig. 2. Initially, studies were conducted to select the concentration of cuprizone that would generate a range of suitable colors for screening analysis type. The concentration of cuprizone was evaluated in four levels using a fixed concentration of Cu(II) (1.25 mg L−1; 0.0019 × 10− 3 mol L− 1): 44 (0.16 × 10−3 mol L−1), 88 (0.32 × 10−3 mol L−1), 132 (0.48 × 10−3 mol L−1) and 176 mg L−1 (0.64 × 10−3 mol L−1) all of them with a fixed volume of 100 μL. Furthermore, to verify the effect of the kinetic of reaction on the analytical response, the reaction was monitored at 1 min intervals for 10 min employing a solution of Cu(II) 1.25 mg L−1 (0.0019 × 10−3 mol L−1) at a volume of 400 μL. The analytical blank was obtained by replacing the Cu(II) solution for distilled water. After reaction optimization, the volumes were

2.4. Optimization of Operating Parameters of Imaging System To evaluate the effect of light intensity on the analytical response it was employed a luximeter and the light intensity incident varied at five levels: 20, 35, 90, 240 and 980 LUX. To evaluate the effect of sample volume on the analytical response, it was varied the volume of the solution of the dye Brilliant Blue 1.0 × 10−5 mol L−1 of 100 to 800 μL with 100 μL of increment on the spot. To verify the RGB channels linear response due to the analyte concentration, analytical curves were constructed employing the dyes Brilliant Blue, Tartrazine and Direct Red 80, whose concentration ranges were tested from 1.00 × 10−6 mol L−1 to 1.00 × 10−4 mol L−1, using the light intensity and the volume determined in earlier studies. The curves were constructed in triplicate, an image for each curve was captured and the coefficient of determination was evaluated. The effect of exchanging cameras on the analytical response was assessed by comparison of two standard curves constructed using Brilliant Blue dye, and the images were captured by a camera Sony Cybershot DSC-W610 with resolution 14.1 Megapixels (MP) and also through camera Sony Cyber-shot DSC-S730 with 7.2 MP. Assays were performed in replicate (n = 7) with only one image being captured for each test. The results were compared using the paired t-test with 95% confidence.

Fig. 2. Scheme of chelate forming reaction between copper (II) ions and cuprizone.

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Table 1 Selectivity study for determination Cu(II) in sugar cane spirits (n = 3). Interferent

Ratio Cu(II):interferent

Relative error (%)

Lead (II)a

1:0.1 1:0.5 1:1 1:1 1:5 1:10 1:1 1:5 1:10 1:0.01 1:0.1 1:0.5 1:1 1:5 1:10 1:1 1:5 1:10 1:1 1:5 1:10

−9.94 −6.46 +6.02 +5.52 −6.73 −12.4 −7.13 −9.85 −10.8 −5.53 −12.1 −10.1 −8.5 −9.3 −15.2 +8.2 +7.4 −4.5 +0.13 −1.79 −1.52

Iron (II)

Iron (III)

Nickel (II)a

Manganese (II)

Aluminum (III) Fig. 3. Intensity values in function of reaction time. Concentration of copper equivalent to 1.5 mg L−1 (n = 3). Zinc (II)

fixed in: 100 μL phosphate buffer pH 9, 400 μL solution of Cu(II) 1.25 mg L−1 or sample and 100 μL of cuprizone. The tests were performed in triplicate, and an image was captured for each test. Effects of possible interferents were evaluated for determining Cu(II) in different proportions of analyte/interferent. The study of the matrix effect was done by comparing the parameters of the analytical curve obtained for standard aqueous solutions of Cu(II) with the parameters of analytical curve using a sugar cane spirit free of copper(II). The mean responses were compared using statistical test F (Snedecor) of homogeneity of variances and through the paired t-test with 95% confidence. Recovery studies were tested in unaged and aged sugar cane spirit samples employing standard addition method using three concentration levels: 0.75; 2.88 and 5.00 mg L− 1. The accuracy of the method was evaluated by comparing the results obtained by digital analysis method with the reference method (Flame Atomic Absorption Spectrophotometry) [40]. The results obtained by both methods were compared using the paired t-test at 95% confidence. The DIB method was applied for determination of Cu(II) in samples of aged and unaged sugar cane spirit collected in supermarkets in the cities of Viçosa and Bom Jesus do Amparo - Minas Gerais/Brazil. No treatment of samples was need before the analysis.

a

Ratio 1:1 showed an intense interference.

3. Results and Discussion 3.1. Imaging System Optimization After preliminary tests, the light intensity was set at 20 LUX due to the greater magnitude of the analytical signal allowing the measurement of lower concentrations of the analyte. To verify the effect on the analytical response, volumes of solutions from 400 to 800 μL were used on the spot, and the intensity of the analytical signal remained nearly constant, with a slight increase until 600 μL. Using 600 μL, an excess of light “brightness regions” appeared only on the edges of the spot, and thus in the center more homogeneous regions were obtained. According to Benedetti et al. [29], the analytical response can be affected by sample volume similar to optical path in spectrophotometry. In fact, the depth of the vessel is correlated with the absorbance and consequently the reflected RGB values. Few microliters of the colorimetric reaction generate poor sensitivity. However, high volumes generate a great amount of residue. Thus, 600 μL was selected for further studies. Calibration curves were built to verify the linearity of concentration range to some solution based on dyes. The linear ranges obtained for the concentration of Brilliant Blue, Direct Red 80 and Tartrazine were: 1.75 × 10−5 to 1.00 × 10−4 mol L−1, 1.00 × 10−6 to 8.35 × 10−5 mol L−1 and 1.00 × 10−6 to 1.00 × 10−4 mol L−1, respectively. The coefficients of determination (R2) were obtained exceeding 0.99, what indicated that the

Table 2 Determination of the concentration of Cu(II) in commercial samples of sugar cane spirits (n = 3).

Fig. 4. Digital image (a) obtained for porcelain plaque containing spot test reactions at different copper(II) concentration levels: (1) 0.0, (2) 0.75, (3) 1.25, (4) 2.50, (5) 3.75, and (6) 5 mg L−1.

Sample Spot test with digital image (mg L−1)

FAAS (mg L−1)

Relative error (%)

ANE1a ANE2a ANE3a ANE4a ANE5a ANE6a ANE7a AE8b AE9b AE10b AE11b

4.19 ± 0.04 1.82 ± 0.01 2.09 ± 0.02 1.02 ± 0.03 bLQ 1.93 ± 0.01 bLD 2.79 ± 0.01 1.97 ± 0.02 3.41 ± 0.03 43.30 ± 0.04

−8.70 −9.77 +6.26 +1.01 – −7.61 – +7.16 −5.41 −8.03 −6.26

a b

3.82 ± 0.35 1.64 ± 0.23 2.22 ± 0.18 1.03 ± 0.11 bLQ 1.78 ± 0.15 bLD 2.99 ± 0.18 1.86 ± 0.34 3.14 ± 0.21 40.60 ± 0.40

ANE = unaged sample. AE = aged sample.

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Table 3 Determination of Cu(II) in sugar cane spirits by several analytical methods. Analytical technique

Beverages

LRa

LODa

LOQa

RSD (%)

Reference

Spectrophotometry Spectrophotometry Spectrophotometry Voltametry Voltametry Flame Atomic Absorption Spectrometry (FAAS) Digital Imaging

Sugar cane spirit Sugar cane spirit Sugar cane spirit Tequila, Vodka, Sugar cane Spirit, Gin Sugar cane spirit Sugar cane spirit Sugar cane spirit

0.18 to 17.0 0.13 to 8.0 0.47 to 10 0.095 to 1.96 0.3 to 5.0 0.10 to 4.0 0.25 to 5.0

0.055 0.02 0.12 0.025 0.11 0.015 0.078

0.18 0.013 0.47 0.082 0.33 0.050 0.26

2.5 1.9 2.6 5.8 1.9 6.9 5.9

[43] [9] [12] [14] [44] [45] Present method

LR = Linear range; LOD = Limit of detection; LOQ = Limit of quantification; RSD = Relative Standard Deviation (medium). a Values expressed in mg L−1.

channels R, G and B linearly respond to these studied concentration range. According to Choodum [34], R, G and B channels absorb wavelengths of light in the range 580–700 nm, 500–580 nm and 400– 500 nm, respectively. Therefore, the spectrums obtained for the dyes were compared with the response obtained by the RGB model showing agreement results and that the Blue Coomassie Brilliant presents response in the R channel, Direct Red 80 in the G channel and Tartrazine on channel B. The effect of the camera resolution was evaluated by comparing standard curves constructed using Brilliant blue dye. The test was done by comparing all analytical responses for each concentration level. The 0.50 value t calculated was lower than the critical t (3.18), indicating that the camera resolution did not influence the analytical methodology. Thus, low cost photographic cameras and smartphones can be used, being very attractive and encouraging for small producers to use them for quality control purposes.

3.2. Copper(II) Determination in Sugar Cane Spirit The colorimetric reaction between Cu(II) and cuprizone is a mixture of colors and therefore, more than one channel can present a satisfactory response to variations in the concentrations of the chelate. Thus, R, G and B channels were monitored in order to determine which of them has greater sensitivity as a function of the copper(II) concentration. The channel R showed a better response, so it was employed for the determination of copper in the carried out studies. In fact, the red color is exactly the complementary color from the reaction blue color. Green and blue channel presented lower sensitivity than red, but the blue channel presented lower sensitivity than expected once the blue radiation is reflected by blue solution. For analyzes of screening type (fast response) involving colorimetric reactions of spot tests, it is essential to find the most stable and color gradient - changing in color levels. In this regard, different concentrations of cuprizone were tested from 44 to 176 mg L−1. The evaluated cuprizone concentrations were always higher than the range of concentration of the tested copper solutions to ensure its maximum complexation. For all evaluated levels, a fixed volume of 100 μL of cuprizone solution was used. For lower concentrations of cuprizone such as 44 and 88 mg L− 1, the color remained almost the same in all spots. In fact, low concentrations of chelant reduce the formation of the product, and so, lower sensitivity. Therefore, the experiments were continued employing the cuprizone solution of 132 mg L−1. For more concentrated cuprizone solution, the analytical signal did not increase significantly. It was evaluated the reaction kinetic of cuprizone and copper ions. The analytical signal increased up to 7 min of reaction as shown in Fig. 3. After this time, there was a decrease of the analytical signal, probably due to the low stability of the generated chelate. After checking the optimal reagent concentrations, volumes used for each solution were set as follows: 400 μL Cu(II), 100 μL of phosphate buffer pH 9 and 100 μL of cuprizone solution. Fig. 4 shows an image of

spot test containing copper concentrations in the range from 0 to 5 mg L−1 after optimized reaction conditions. 3.3. Analytical Features An analytical curve was constructed containing copper concentrations in the range from 0.75 to 5.00 mg L− 1 using the R channel as shown in Fig. 4. The linearity of the developed method was verified by calibration curve analysis using the method named least squares methods (LSM) following the regression equation: − log (I/I0) = −0.0113 + 0.3290 × [Copper(II)], where [Copper(II)], is mg L−1 and r = 0.9999. The limits of detection (LOD) and quantification (LOQ) were calculated using the relations 3 × σ/m and 10 × σ/m, respectively, where σ is the standard deviation of ten times blank analytical and m the slope of the analytical curve. Thus, the LOD and LOQ were 0.078 mg L−1 and 0.26 mg L−1. Some metallic ions that could be present in sugar cane spirit were tested, as described by [41] which consequently may form chelates with cuprizone. Previously, potential interferences showed in Table 1 were analyzed by Flame Atomic Absorption Spectrometry (FAAS) in all sugar cane spirit samples used in this study. The aluminum and zinc were found in some samples, however in concentrations below the quantification limit. The iron ions were found in only one sample at a concentration below the quantification limit. Other metals were not detected in sugar cane spirit samples. Therefore, the metals analyzed were not considered important in determining interfering Cu(II) employing cuprizone as a complexing agent. Recovery studies were established in aged and unaged sugar cane spirits in three concentration levels: 0.75; 2.88 and 5.00 mg L−1. Recovery percentages ranged from 98.3 to 104% were obtained. The results were compared with the values described by [40] that recommends a recovery interval between 80 and 110% to concentration levels of 0.1 mg L−1 to mg L−1. Therefore, all the values obtained from the recovery study were satisfactory. The analytical curves obtained from aqueous solutions and sugar cane spirit in the absence of Cu(II) were compared using F test (Snedecor), and it was found that there are no differences among the variances for the obtained curves. For comparison of means pairs in each concentration level it was used the paired t-test with 95% confidence with n = 3. The 1.19 t value was lower than the t critical (2.77), showing no statistically significant differences in the results by both generated curves. It can be concluded that there were no significant effects of matrices in samples of sugar cane spirit. In addition to the recovery tests, the accuracy was evaluated by comparing the results obtained using the method developed by the results provided by the reference method (Flame Atomic Absorption Spectrophotometry) [41]. Some samples of sugar cane spirit were analyzed using the proposed method and the reference method. The results are shown in Table 2. The 1.29 t value calculated was lower than the critical t (2.30) for n = 3, therefore, no statistically significant differences in the results were generated by both methods at a confidence level of 95% [42]. It can be concluded that the six samples of sugar cane spirit analyzed presented copper(II) concentrations within the limits required by the Ministry of

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Agriculture, Livestock and Supply, except AE11 sample, whose concentration of Cu(II) was approximately eight times higher than allowed by law. In Table 3 some analytical features acquired from DIB were compared to those obtained by other methods reported in the literature for determination of Cu(II) in sugar cane to evaluate its performance. Molecular spectrophotometry, atomic absorption spectrophotometry and voltammetric methods are initially presented, followed by the digital image based method. 4. Conclusions The method developed for the determination of copper(II) in sugar cane spirit presented advantages such as practicality, quickness, low cost, reduced reagent consumption and waste generation. The device employed for image capture is easy to build and it uses low cost components such as LEDs, battery and accessible apparatus as smartphones or digital camera. In addition, it is versatile, robust and portable, very interesting for in situ analysis. The digital image method presented good repeatability and robustness to determine copper, mainly because of the apparatus developed to avoid lack of homogenous with excess of lighting or shadow. Besides, with this method, only one photography is enough to complete the analytical analysis- analytical curve and sample- generating only 600 μL/ spot (miniaturization) at 7 min of analysis. Thus, each analysis could be performed at 35 s. The DIB method presented a good linear range, precision and LOD compared to some spectrophotometric methods as expected, and compatible performance as the reported voltammetric method. Thus, the spot test with digital analysis is ideal to green chemistry purposes. Moreover, this method is a useful analytical tool for laboratory with limited financial resource and for quality control in industries and small producers. Besides, comparing with a work in the literature employing an inductively coupled plasma atomic emission spectroscopy (ICP OES), it showed that all metals tested were found in concentrations lower than the copper concentration in sugar cane spirit samples ratifying the obtained results. Finally, the method can be used for chemical, clinical, medical, forensic, environmental analysis, and as an analytical tool with high potential. Acknowledgment The authors are grateful to Fundação de Amparo à Pesquisa do Estado de Minas Gerais and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Processo No 456543/2014-7). References [1] Ministry of Agriculture, Livestock and Supply, Instrução Normativa n°13, de 30 de junho de 2005. Aprova o Regulamento Técnico para Fixação dos Padrões de Identidade e Qualidade para Aguardente de Cana e para Cachaça. Diário Oficial da União, Brasília, 2005. [2] L. Odello, G.P. Braceschi, F.R.F. Seixas, A.A. da Silva, C.A. Galinaro, D.W. Franco, Avaliação sensorial de cachaça, Quim Nova 32 (2009) 1839–1844, http://dx.doi. org/10.1590/S0100-40422009000700029. [3] R.A. Labanca, M. Beatriz, A. Glória, V. José, P. Gouveia, Determinação dos teores de cobre e grau alcoólico em aguardentes de cana produzidas no estado de Minas Gerais, Quim. Nova 29 (2006) 1110–1113. [4] A.D.J.B. Lima, M.D.G. Cardoso, M.C. Guerreiro, F.A. Pimentel, Emprego do carvão ativado para remoção de cobre em cachaça, Quim Nova 29 (2006) 247–250, http://dx.doi.org/10.1590/S0101-20612008000500014. [5] P.J. Gow, R.A. Smallwood, P.W. Angus, A.L. Smith, A.J. Wall, R.B. Sewell, Diagnosis of Wilson's disease: an experience over three decades, Gut 46 (2000) 415–419, http:// dx.doi.org/10.1136/gut.46.3.415. [6] G. Loudianos, J.D. Gitlin, Wilson's disease, Semin. Liver Dis. 20 (2000) 353–364, http://dx.doi.org/10.1055/s-2000-9389. [7] A.J.B. Lima, M.G. Cardoso, L.G.L. Guimarães, J.M. Lima, D.L. Nelson, Efeito de substâncias empregadas para remoção de cobre sobre o teor de compostos secundários da cachaça, Quim Nova 32 (2009) 845–848, http://dx.doi.org/10. 1590/S0100-40422009000400004.

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