Digital Image Photometric Analysis of Nickel(II) in Alkaline Solution with Dithizone … · 2020....

Keywords: digital photometry, dithizone reagent, image processing, method validation, Ni(II) analysis, RGB

Digital Image Photometric Analysis of Nickel(II) in Alkaline Solution with Dithizone as Complexing Agent

Using a Mobile Phone Camera

*Corresponding Author: [email protected]

Institute of Chemistry, College of Arts and Sciences University of the Philippines Los Baños, College, Laguna 4031 Philippines

Quantitative analysis of nickel(II) in aqueous alkaline solution, with dithizone as a complexing agent, was done using digital image photometry (DIP) and the results were compared with those using conventional visible spectrophotometry (VS). Digital color photographs of prepared standard and sample solutions of Ni(II) were taken using a fabricated lightbox with a 24-well sample holder and iPhone 5S mobile phone camera. RGB values were determined from the photographs using ImageJ software and plots of Red value, L*, a*, and b* as DIP parameters were constructed in order to calculate Ni(II) concentrations. No significant differences in Ni(II) concentrations were observed at a 95% confidence level based on DIP and VS. Performance criteria were set and various tests were conducted for the limit of detection (LOD), limit of quantification (LOQ), linearity/working range, repeatability, and selectivity. The DIP results satisfied the minimal requirements in terms of method validation criteria according to published guidelines. Among the DIP parameters, b* gave results with the highest linearity and accuracy based on the coefficient of determination (R2), as well as plots of least significant differences (LSDs) and linear regression residuals. The results of the present study show that DIP, which uses a relatively inexpensive mobile phone camera, is a promising alternative to the conventional method using an expensive spectrophotometer.

Philippine Journal of Science149 (3-a): 711-717, October 2020ISSN 0031 - 7683Date Received: 19 Feb 2020

Angelica C. Pagala, Cherry Mae T. Ravidas, Jennifer Q. Pillas, and Ernesto J. del Rosario*

INTRODUCTIONPhotometric methods of analysis have been commonly used to determine the concentration of heavy metals in aqueous solutions, especially industrial effluents and environmental samples. VS is used to measure absorbance or transmittance in combination with a monochromator for selecting a suitable wavelength. Absorbance is the common unit for expressing the attenuation of radiation

because it is a linear function of analyte concentration. However, a spectrophotometer is somewhat expensive and cannot be easily purchased by a laboratory with a limited budget. A relatively cheap digital camera, either stand-alone or part of a mobile phone, is a promising alternative for photometric heavy metal analysis in the presence of a reagent for colored complex formation.

The alternative method of DIP involves taking color images of the solutions using a digital camera followed by image processing based on tristimulus colorimetry.

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The RGB color scheme is commonly used based on the primary colors Red (R), Green (G), and Blue (B) whose values may be calculated using free-access computer software such as ImageJ, SI Color Picker, etc. Color digital still cameras generally use an array of transistors on a charge-coupled device chip with a filter array that allows some detectors for RGB. A digital color image is represented in RGB form with each color component R, G, or B per pixel in the range of 0–255 and conventionally stored using eight bits per color component. Several internet reports have provided valuable information on digital color images, color spaces/models, and image processing (Kelda and Kaur 2014; Skrede 2017).

The DIP method has been previously reported for the quantitative determination of metal ions in aqueous solutions. A study by Suzuki et al. (2006) used a digital still camera to take color photographs [inside a white acrylic box with light-emitting diode (LED) bulb] of a river water sample, the iron content of which was determined based on L*, a*, and b* parameters; a* is the parameter that measures redness-greenness, b* measures yellowness-blueness, and L* measures brightness. Another study by Lahuerta Zamora and co-workers (2010) applied DIP to determine the Ni content of metallic meteorites based on digital images of standard solutions of the Ni dimethyl glyoxime chelate. Several researchers have also used DIP, some with modifications, for quantifying the content of various metals and other inorganic analytes in different samples (Firdaus et al. 2014, 2019; Vedad et al. 2015; Masawat et al. 2016; Barros et al. 2017; Salcedo and Sevilla 2017).

The present study deals with the development of DIP as a photometric method for determining the concentration of Ni(II) in prepared aqueous solutions. Method validation criteria (LOD, LOQ, linearity/working range, repeatability, and selectivity) were applied to the DIP method based on the following parameters: Red value, L*, a*, and b* values.

MATERIALS AND METHODS

Reagents and MaterialsAll chemicals used in this study are reagent grade from Sigma-Aldrich. The reagents used are potassium permanganate (KMnO4), diphenylthiocarbazone (dithizone), ethanol (absolute), nickel sulfate hexahydrate (NiSO4•6H2O), 25% concentrated ammonia solution, cobalt sulfate heptahydrate (CoSO4•7H2O). and anhydrous lead nitrate [Pb(NO3)2].

A Shimadzu UV Mini 1240 Spectrophotometer was used for VS analysis of prepared standard and sample

solutions. Digital photographic data were gathered using an iPhone 5S mobile phone , which was positioned on top of a fabricated lightbox. The latter has an opening that allows images to be taken of solutions inside the lightbox in the absence of stray light. The lightbox was 13 in x 8 in x 7 in made of plywood painted white and lighted by one white-color LED bulb (1.5 W). The sample holder consisted of a 24-well clear plastic plate and was placed at the bottom of the lightbox; this allowed simultaneous analysis of several replicates of different solutions. Figure 1 shows the DIP set-up.

Figure 1. Schematic diagram and photograph of a lightbox with an LED bulb and 24-well sample plate.

Preparation of Standard Ni(II) Solutions Dithizone was dissolved in absolute ethanol in order to prepare the stock Ni(II) solution. For single Ni(II) determination, 1 mL each of 1 ppm, 2 ppm, 3 ppm, 4 ppm, 5 ppm, 6 ppm 7 ppm, and 8 ppm Ni(II) solutions were prepared. Then, 1 mL of dithizone solution was added to each of the solutions. The pH was adjusted to 11–12 by adding 25% concentrated ammonia solution; complex formation of dithizone and Ni(II) is complete at basic condition (Ozturk et al. 2000). The resulting solution was then diluted to 5 mL.

Validation of AccuracyA 50 mM KMnO4 solution was prepared by dissolving 0.3951 g of solid KMnO4 in 50 mL distilled water. The resulting solution was heated for 25 min, filtered through a sintered glass funnel, and used to prepare standard solutions whose absorbance values were determined using VS. The same standards solutions were also subjected to DIP analysis in terms of Red value, L*, a*, and b*; these values were then plotted against concentration. The coefficients of determination (R2) obtained from DIP and VS were compared.

Analysis of Ni(II) Using VS One (1) mL of the prepared Ni(II) solution was mixed with 1 mL of 0.016 M dithizone solution, and the pH of the resulting solution was adjusted to 11 using a 25%

Philippine Journal of ScienceVol. 149 No. 3, October 2020

Pagala et al.: Photometric Analysis of Ni(II) Using a Mobile Phone Camera

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concentrated ammonia solution; this was followed by dilution with distilled water to 5 mL. The absorbance of the sample solution, which was read at the optimal wavelength (λmax) of Ni-dithizone complex (680 nm), was interpolated into the calibration plot in order to determine the Ni(II) concentration.

Gathering of Digital Photometric Data The same standard and prepared sample solutions of Ni(II) were used for both DIP and VS. The mobile phone used for capturing images of the solutions (with the flash turned off) was iPhone 5S with 8 megapixels, f/2.2 aperture, 4.12 mm focal length, and resolution of 1136 x 640 pixels. The photographs were then transferred to a computer and analyzed using ImageJ-Win64 software.

Determination of Analyte Concentration from Red Plot From the RGB data, which were calculated using Image J software, Red values were plotted against concentrations of the standard Ni-dithizone solutions. The Red value was then interpolated from the linear standard plot in order to determine the Ni(II) concentration of the prepared sample solution.

Determination of Analyte Concentration from DIP Plots (Suzuki et al. 2006)The R, G, and B values obtained using ImageJ were linearized and then converted to X, Y, and Z values using the following equations:

(1a)

(1b)

(1c)

The XYZ values were then used to calculate L*, a*, and b* using the equations:

(2a)

(2b)

(2c)

where Xn = 0.98072, Yn = 1.000, and Zn = 1.1823. The analyte concentration was then determined by interpolating the values of L*, a*, and b* of the Ni(II) sample solutions from the standard plot, which was generated from the L*, a*, and b* values of the Ni(II)

standard solutions.

Comparison of VS and DIP Results for Ni(II) Analysis The results obtained from VS were compared with those from DIP based on plots of Red value, L*, a, and b* using t-test at a 95% confidence level. GraphPad Prism 8.4.3 was used to analyze the data responses.

Method Validation (Magnusson and Örnemark 2014).LOD and LOQ (Harris 2010; Huber 2010). Ten blank samples were prepared and the corresponding values of the DIP parameters (Red value, L*, a*, and b*) were obtained. The experimental values at LOD and LOQ were calculated using the conventional method (Harris 2010), as seen in the equations below:

LOD = (3s)/m (3a)LOQ = (10s)/m (3b)

where s is the standard deviation of the blanks and m is the slope of the linear calibration plot.

Linearity/working range. Two sets of working solutions with proper blanks were prepared and subjected to DIP. The corresponding Red value, L*, a*, and b* values were determined and plotted against Ni(II) concentration. The regression statistics and residual plot were also obtained. GraphPad Prism 8.4.3 was used to generate the residual plots.

Precision test for repeatability. The repeatability parameter was assessed using six replicate sample solutions containing Ni(II) and the corresponding analyte concentrations were obtained from the calibration plots of Red value, L*, a*, and b* plots. The standard deviations were then calculated.

Selectivity. The prepared sample solutions containing Ni(II) analyte were analyzed for interferences by Co(II) or Pb(II) in terms of analyte concentration. Selectivity was quantified by comparing the calculated Ni(II) concentrations without interferences and with interferences using the t-test.

RESULTS

Optimization of Set-up for DIPOptimization of the lightbox set-up involved taking images of solutions of KMnO4. The generated R, G, and B values of the standard KMnO4 solutions were plotted and confirmation of Beer’s law was ascertained. Solutions of KMnO4 are reliable and convenient for this purpose. Using DIP, R2 values of 0.9996, 0.9759, and 0.7575 were obtained from R, G, and B plots, respectively (Figure 2).



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The same standard KMnO4 solutions were subjected to VS, for which an R2 value 0.9923 was obtained.

For pairwise comparison between VS and DIP methods, the LSD bars were calculated and plotted in Figure 4. I-beams, which were calculated from the average value of the photometric parameter and its standard (or average) deviation, that do not overlap suggest that the two compared methods are significantly different. On the other hand, overlapping I-beams indicate that results from the two methods do not significantly differ. Figure 4 shows that Ni(II) concentration values calculated from VS do not differ significantly from those obtained using the four DIP parameters because the I-beam for VS overlaps the four I-beams for DIP. However, among the four DIP plots, the Ni(II) concentration values from a* and Red value are significantly different from each other – as well as Ni(II) concentrations from a* and L* – because the I-beams do not overlap.

Table 1. Data on significance testing between VS and DIP plots using t-test.

Plots Ni(II) conc. (ppm)Significantly different from the true value

(4.50 ppm)?

Absorbance (VS) 4.419 ± 0.259 No

Red value 4.627 ± 0.189 No

a* 3.680 ± 0.587 No

b* 4.299 ± 0.058 No

L* 4.401 ± 0.087 No

Figure 2. RGB plots of standard KMnO4 solutions.

Confirmation of Ni(II) Concentration in Prepared Sample Solutions The plot of absorbance against Ni(II) concentration (in the presence of dithizone), when subjected to VS, gave an R2 value of 0.9939. For DIP, plots of b*, L*, a*, and Red values against Ni(II) concentration gave R2 values of 0.9783, 0.9643, 0.9537, and 0.934, respectively – as shown in Figure 3.

Ni(II) concentrations of the prepared sample solutions were then determined by interpolation on the five generated calibration plots based on absorbance, Red value, a*, b*, and L* plots. The Ni(II) concentrations obtained were then compared with the true value (4.50 ppm) using the t-test. As presented in Table 1, the calculated Ni(II) concentration values from the VS plot (absorbance) and from the DIP plots (Red value, a*, b*, L*) were not significantly different from the true value.

Method ValidationMethod description. DIP was the analytical method used to determine the concentration of Ni(II) in prepared sample solutions with dithizone as a complexing reagent. The solution pH was adjusted to pH 12 by the addition of concentrated ammonia solution. Standard solutions containing Ni-dithizone complexes were prepared and their respective RGB values were generated using

Figure 3. Composite calibration plots of absorbance (top), L*, Red value, a*, and b* against Ni(II) concentration.

Figure 4. LSD bars for different methods.



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ImageJ software; this was followed by calculation of corresponding values of Red value, L*, a*, and b*. These DIP parameters were plotted against concentration in order to determine analyte Ni(II) concentration.

LOD and LOQ. The LOD is the lowest analyte concentration that can be detected by the analytical method at a specified level of confidence. On the other hand, the LOQ is the lowest level at which the performance is acceptable for a typical application (Harris 2010). The LOD and LOQ were determined by preparing 10 sample blanks and the corresponding values of Red value, L*, a*, and b* were calculated.

Using Equation 3a, the values of LOD (in ppm) obtained based on Red value, a*, b*, and L* are 0.074, 0.241, 0.177, and 0.075, respectively. On the other hand, the values of LOQ (in ppm) that were calculated using Equation 3b for Red value, a*, b*, and L* are 0.247, 0.804, 0.591, and 0.250, respectively. The same value of LOD, namely 0.075 ppm, was obtained from Red value and L*. Likewise, the same LOQ value of 0.25 ppm was obtained based on these two DIP parameters. These LOD and LOQ values (in ppm) of 0.074 and 0.24 are lower and more acceptable compared to those calculated from a* and b*.

Linearity/working range. The working range is the interval over which the method provides results with an acceptable uncertainty (Magnusson and Örnemark 2014). For the linearity/working range, two sets of nine calibration standards were prepared with 0, 1, 2, 3, 4, 5, 6, 7, and 8 ppm Ni(II) concentration. The respective Red value, L*, a*, and b* values were plotted against the concentration of Ni(II). R2 values for the different DIP parameters can be seen in Figure 3.

Values of simple linear regression residuals for Red value, a*, b*, and L* were calculated and plotted in Figure 5 using GraphPad Prism 8.4.3. Residual plots with a random distribution of data points indicate linearity between the photometric parameter and analyte concentration. This linearity parameter is acceptable in accordance with the performance criteria.

Repeatability. Repeatability is a measure of the variability in results by a single analyst using the same equipment at a short timescale. The results should have minimal deviations in order to be considered as repeatable (Magnusson and Örnemark 2014). For the repeatability parameter, six replicates of the sample solution containing Ni(II) were prepared and the corresponding Ni concentrations were obtained from the calibration curves of Red value, L*, a*, and b* plots. Table 2 shows the calculated Ni(II) concentrations of the prepared sample solutions, as well as their standard deviations.

Table 2. Data on concentration of Ni(II) in prepared sample solutions for assessment of repeatability.

Sample no.Ni(II) concentration (in ppm) using different plots

Red value plot a* plot b* plot L* plot

1 4.912 4.358 4.757 4.645

2 4.794 4.449 4.602 4.447

3 4.886 4.261 4.734 4.636

4 4.819 4.456 4.891 4.446

5 4.759 4.362 4.700 4.405

6 4.703 4.423 4.468 4.328

Standard deviation 0.0783 0.0737 0.144 0.128

Figure 5. Composite plots of linear regression residuals for Red value, b*, a*, and L*.

Selectivity. Selectivity is defined by Magnusson and Örnemark (2014) as “the extent to which the method can be used to determine particular analytes in mixtures or matrices without interferences from other components of similar behavior.” It can be assessed by observing the effect of interfering ions in the detection or quantification of the analyte. In the experiment, this was examined by preparing sample solutions containing only Ni(II), Ni(II) with Co(II), and Ni(II) with Pb(II). Figure 6 is the image for the solutions of Ni(II) with and without interfering Co(II) and Pb(II).

Figure 6. Photograph of interference effects of Co(II) and Pb(II) in Ni(II) solution.



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DISCUSSIONDuring optimization of the lightbox set-up, linear correlation results for the KMnO4 standards gave acceptable R2 values (greater than 0.99) based on absorbance (VS) and Red value (DIP). The results indicated that the experimental set-up for DIP analysis including the mobile phone camera could serve as an analytical colorimeter. Determination of Ni(II) concentration based on linear calibration plots gave comparable results for VS (absorbance) and DIP (Red value, a*, b*, and L* plots); these are summarized in Table 1. No significant differences were observed from the true value (4.50 ppm) of Ni(II) concentration at a 95% confidence level using the one VS and four DIP photometric parameters.

LSD values were calculated based on the results of VS and DIP in order to compare their effectiveness in determining Ni(II) concentration of prepared sample solutions. Figure 4 shows that there are no significant differences between the Ni(II) concentration obtained from the VS and four DIP plots because the VS I-beam overlapped all the other I-beams. However, among the four DIP values, the results for a* showed high standard deviation and a big difference from the results of VS, b*, and L*. The Ni(II) concentration values from Red value and a* are significantly different from each other. The Ni(II) concentrations from a* and L* are significantly different as well. Based on the LSD plot, results from b* and L* showed the best agreement between each other and with VS.

Method validation also involves the determination of LOD and LOQ in order to ensure that the signal reading of the sample is within the proper range. Another parameter obtained was the linearity of the calibration plot where R2 value should be ideally greater than 0.99. Repeatability was also performed to see if the standard deviation falls within an acceptable range. Lastly, the selectivity of the method was validated by adding interferences and testing whether the analyte is still accurately quantified. The same values (in ppm) within experimental uncertainty were obtained for LOD and LOQ of 0.075 and 0.250, respectively based on the Red value and L*. These values of LOD and LOQ are more reasonable compared to much higher values obtained from a* and b*.

One basis for evaluating the linearity of an analytical method is to calculate the values of the R2. Linear plots of VS and DIP parameters versus Ni(II) concentration are shown in Figure 3; corresponding values of R2 for absorbance (VS) and b*, L*, a*, and Red value (DIP) are 0.9939, 0.9783, 0.9643, 0.9537, and 0.934, respectively. Based on these obtained R2 values, it is seen that VS gave results (absorbance) with better linearity than DIP. Furthermore, among the four DIP parameters studied, b* and L* produced plots with the highest linearity. It is interesting to note that for KMnO4 solutions (Figure 2) the values of R2 obtained for absorbance, Red value, and Blue value are 0.9923, 0.9996, and 0.9759, respectively. The results indicate that DIP plots can have higher R2 values than absorbance plots, although the latter have R2 values that are usually greater than 0.99; this explains why VS is generally accepted as accurate and valid as an analytical method.

Residual plots are useful to further test whether the plotted values fall within the linear range. As seen in Figure 5, residual plots for b* and a* showed the most scattered points. This observation supports earlier data, i.e. R2 values in Figure 3 and LSD bars in Figure 4, that b* gave results with the highest linearity among the DIP parameters.

Based on the small standard deviations of Ni(II) concentrations for the prepared sample solutions using Red value, L*, a*, and b* (Table 2), the repeatability parameter for DIP is acceptable in accordance with the performance criteria.

Selectivity test for DIP involved the evaluation of interferences by Co(II) and Pb(II) on Ni(II) determination when these ions are separately added to solutions containing Ni(II). Figure 6 shows that the color of solutions containing interfering ions differs significantly from that of solutions containing only Ni(II). The observed color differences were quantified by comparing the calculated Ni(II) concentration for solutions with and without interferences, as shown in Table 3. The results of the t-test showed that Co(II) and Pb(II) separately exerted significant interference on Ni(II) determination based on either plot of Red value or L*. Results from plots of a* and b* showed, respectively, no significant interference and interference only from Co(II).

Table 3. Effect of interfering ions in the determination of Ni(II) concentration.

SampleNi(II) concentration (in ppm) using different plots

Red value plot a* plot b* plot L* plot

Ni only 4.426 ± 0.064 2.969 ± 0.949 4.640 ± 0.337 4.257 ± 0.238

Ni with Co 6.228 ± 0.089 4.523 ± 0.074 6.667 ± 0.975 6.658 ± 0.147

Ni with Pb 5.691 ± 0.032 5.207 ± 0.311 5.631 ± 0.032 5.534 ± 0.155



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ACKNOWLEDGMENTSThe authors wish to thank Dr. Isagani Padolina, Pascual Pharma Inc., and the University of the Philippines Los Baños Institute of Chemistry’s Analytical Service Laboratory for allowing the use of their laboratory facilities and some chemicals for the study.

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Digital Image Photometric Analysis of Nickel(II) in Alkaline Solution with Dithizone … · 2020....

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