determINatIoN of the reaCtIVe dye Navy Blue Her IN the W ... · JALCA, VOL. 109, 2014 330 determINatIoN of the reaCtIVe dye Navy Blue Her IN the WaSteWaterS of the dyeING ProCeSSeS

JALCA, VOL. 109, 2014

330

determINatIoN of the reaCtIVe dye Navy Blue Her IN the WaSteWaterS of the dyeING ProCeSSeS of

Chrome-taNNed leatherby

l. S. p. SaNtoS,1,2 l. f. CriSpim,2 N. m. C. Silva2* aNd N. S. olivEira1,3

1School of Technology and Management, Polytechnic Institute of Leiria P-2411-901 Leiria, Portugal

2Technological Center for Leather Industries, Apartado 158 – S. Pedro, 2384- 909 Alcanena, Portugal

3Laboratory of Separation and Reaction Engineering (LSRE), Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal

* Corresponding author e-mail: [email protected] Manuscript received April 14, 2014, accepted for publication July 14, 2014.

IntroductIon

Organic synthetic dyes are used in a wide range of industrial applications (e.g., textile, leather, rubber, plastic, food products, pharmaceutical, cosmetic and paper production). Over 10 000 dyes with a total yearly production over 7 x 105 metric tons worldwide are commercially available, and 5-10% of the produced dyes are lost in the industrial effluents2 during synthesis, processing, and use. They are frequently found in wastewaters and are increasingly becoming an environmental problem.1-2

The effluents from dye manufacturing and dye consuming industries exhibit high color, and high chemical and biochemical oxygen demands (COD and BOD).2-3 A very small amount of dye in water is highly visible. The discharge of these effluents into the ecosystems is worrying for both toxicological and aesthetical reasons.2,4 The release of these dye-bearing wastewaters causes damage to the environment as they may significantly affect photosynthetic activity in aquatic life due to reduced light penetration and may also be toxic to some aquatic life due to the presence, in them, of metals, chlorides, etc. Therefore, removal of dyes from aqueous effluents before they are released into the environment is required. All of the various existing technologies that have been used for the treatment of dye-bearing wastewaters have their own limitations, and none were successful in completely removing the color from aqueous industrial effluents.5

In order to monitor removal processes of dyes from wastewaters, i.e. to evaluate the efficiency of those removal processes and to evaluate the residual concentrations of dyes in the treated effluents it is necessary to develop and validate an analytical methodology.

AbstrAct

One of the main environmental problems in the leather industry is the contaminant load and amount of effluent produced during the industrial process. From these effluents we can highlight those resulting from the dyeing processes that contribute to the increase of the COD and BOD of the discharged wastewaters. The aim of the present work is to develop and validate simple, rapid, specific, selective, precise, robust and economical UV-Vis Spectrophotometric method for the estimation of reactive dye (Navy Blue Her) in aqueous solutions and effluents of the dyeing processes of chrome-tanned leather. UV-Vis Spectrophotometric measurement was carried out at a wavelength of maximum absorbance of 610 nm using ultrapure water as the solvent. The developed method was validated with respect to specificity, selectivity, sensitivity, limits of detection and quantification, linearity, precision (repeatabi l ity, intermediate precision) a n d r o b u s t n e s s . T h e c a l i b r a t i o n c u r v e ( Abs = 0.01054 C + 0.00067 ) is linear (r2 = 0.99998) in the concentration range from 3.0 mg/L up to 48.0 mg/L. The limit of detection (LOD) and the limit of quantification (LOQ) are 0.206 mg/L and 0.624 mg/L, respectively. The analysis results and its statistical treatment have proved that this analytical method is specific, selective, precise and robust, and has good repeatability and intermediate precision. Thus the proposed method was approved for all the analyzed parameters, being therefore, properly validated, and can be successfully applied for the estimation of reactive dye (Navy Blue Her) in aqueous solutions and effluents of the dyeing processes of chrome-tanned leather.

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According with Fernandez et al.,1 the removal processes of dyes from effluents can be monitored by determination of analytical indices, and the dye or sub-product concentrations. The most common analytical indices used to monitor removal processes are total organic carbon (TOC), chemical oxygen demand (COD), and absorbance in UV-Visible at the wavelength of maximum absorption (Amax).

1 The value of these three indices is directly related to the dye concentration, so a decrease in any one of them in the process indicates a decrease in the dye concentration.

Several bibliographic references report different analytical methods that have been used for the determination of dyes concentration in different matrices, including UV-Vis Spectrophotometry,6-12 Chromatography11,13-16 Capillary Electrophoresis,17-20 Voltammetry,21-23 and Polarography. Except for UV-Vis Spectrophotometry, these methods require sophisticated instruments and, consequently, have a high operating cost.11 In general, absorption measurements provide useful and reliable results, but they have a limited selectivity in case of multi-components samples.24 This limitation is related to the absorption of several different compounds occurring in the same region of the electromagnetic spectrum resulting in overlapping that inhibits the quantification of individual compounds in the absence of separation process. The direct reading of absorbance is related to contents of analyte only, when overlapping of the spectra of compounds has not occurred or is minimal. This limitation can be overcome by application of the appropriate statistical method in the calibration process to analyze spectra, such as Classical Least Squares (CLS) and Multiple Linear Regression (MLR) or Inverse Least Squares (ILS), Principal Components Regression (PCR), and Partial Least Squares (PLS). And dye spectra can also be used to characterize the dye solution with regard to the two main absorption bands – one in the Visible region and the other in the UV region.1 The disappearance of peaks and the appearance of new peaks in the UV and Visible regions indicate that dyes degrade into low-molecular-weight aromatic compounds.1 According with Sahin et al.,9 the spectrophotometric determinations are preferable due to the possibility of obtaining highly accurate results and good reproducibility for a complex matrix using relatively simple and cheap procedures as compared to other techniques.

In spite of the many studies that report the monitoring of the removal processes of dyes from aqueous solutions and wastewaters with UV-Vis Spectrophotometric measurement at the wavelength of maximum absorption,25-33 not one was found where the UV-Vis Spectrophotometric method was validated. Nor was any validated analytical method found to determine the concentration of the reactive dye used in this study.

Usually, reactive dyes are applied in alkaline medium. However, it is more difficult the use of reactive dyes since they create some constrains in the tanning technicians. It changes

too much the conventional dyeing process. The dyeing with reactive dyes in acid medium is being optimized at the Technological Centre for Leather Industries, CTIC. It was chosen the Navy Blue Her dye for the process optimization, a non-metalized reactive dye that gives shinier colors with high fastness, under specific operating conditions. A part of the optimized dyeing formulation (described in experimental details) was applied to generate the real effluents used in this study.

This work describes the analytical method for determining the concentration of the reactive dye Navy Blue Her in aqueous solutions as well as in effluents of dyeing processes of chrome-tanned leather through UV-Vis Spectrophotometric measurement at the wavelength of maximum absorption, and shows the results that led to its validation. UV-Vis Spectrophotometry was chosen because it provides reliable results using relatively simple and cheap procedures when compared to other analytical methods. The validation of the analytical method was based on the criteria established in the Official Guides (USP, ICH, FDA, EURACHEM,34 USEPA), by determining the parameters: specificity and selectivity, sensitivity, limit of detection (LOD), limit of quantification (LOQ), linearity (homogeneity test of variance and Mandel test), linear and measurement ranges, precision (repeatability, intermediate precision, expanded uncertainty, combined standard uncertainty), solution stability and robustness.34-35

ExpErImEntAl

ApparatusThe absorption spectra and absorbance measurements were carried out with a double-beam UV-Visible Spectrophotometer (model Thermo Electron Corporation Evolution 300 & 600) using a pair of 1.0 cm optical path length quartz cells. An analytical balance (model Mettler AT 200) was used to measure dye mass. A pH meter (model 940 ORION EA) was used to check the pH of the solutions. A centrifuge (model Merlin SPECTRA) was used to accelerate the phase separation. A water purification system (model Purelab ultra ELGA) was used to obtain ultrapure water.

Materials and ReagentsThe Navy Blue Her dye is a monochlorotriazine azo type reactive dye, provided by Technological Centre for Leather Industries, CTIC. It was supplied in confidence and the chemical formula or composition was not provided. Also used were ultrapure water, formic acid (85%), naphthalene sulfonic acid, hydrochloric acid (1 N and 0.1 N) and sodium hydroxide (0.1 N). In this work, the reactive dye and the naphthalene sulfonic acid used were of commercial purity grade and the other chemicals used were of analytical reagent grade. All of them were used without further purification.

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Stock Solution Preparation6.0 g of reactive dye Navy Blue Her was accurately weighted and dissolved in 200 mL of ultrapure water in a 1 L volumetric flask. The solution was shaken, and then the volume was made up to the mark with ultrapure water to give a stock solution containing 6.0 g/L (stock solution I). All the dye solutions used in the described experiments were prepared using the same dye stock solution (stock solution I).

Standard Solutions PreparationThe standard solutions of the reactive dye with a concentration of 0.06 g/L (stock solution II) were prepared by diluting 10.0 mL of the stock solution I of reactive dye Navy Blue Her with ultrapure water in a volumetric flask of 1 L. The pH of the dye solution is a parameter that can influence the behavior of the reactive dye Navy Blue Her in the solution. For that reason, another three dyes solutions, with the same concentration as stock solution II, were prepared and its pH was adjusted to 2.8, 3.6 and 3.9 with diluted formic acid (1:100) at 20ºC.

Preparation of Standard Solutions to Establish the Calibration CurveAppropriate volumes from the standard stock solution II (from 5.0 mL to 80.0 mL) were pipetted out into sixteen different volumetric flasks of 100 mL. The volume was adjusted to the mark with ultrapure water to obtain concentrations of 3.0, 6.0, 9.0, 12.0, 15.0, 18.0, 21.0, 24.0, 27.0, 30.0, 33.0, 36.0, 39.0, 42.0, 45.0 and 48.0 mg/L. The solutions were shaken well.

Preparation of Real Effluent SamplesChrome-tanned leather was treated and dyed at CTIC (Technological Centre for Leather Industries) laboratory. The chrome-tanned leather samples were first washed and neutralized, and then dyed at 30ºC with variable amounts of the reactive dye Navy Blue Her tested according to the following formulations, where proportions are percentage weights relative to shaved weight:

Sample 1: 1000% water and 3% reactive dye Navy Blue Her (run 60 min), and 1% diluted formic acid (1:100) (run 10 min).

Sample 2: 100% water and 4% naphthalene sulfonic acid (run 20 min), 2% naphthalene sulfonic acid and 3% reactive dye Navy Blue Her (run 120 min), 100% water and 1% diluted formic acid (1:100) (run 10 min).



Sample 5: 100% water and 1,5% reactive dye Navy Blue Her (run 60 min), 100% water and 0,5% diluted formic acid (1:100) (run 10 min), 0,5% diluted formic acid (1:100) (run 10 min).

The dyeing baths were carefully collected into cleaned flasks after each dyeing process and were properly labeled as real effluents to subsequent analysis. The naphthalene sulfonic acid was used as dispersing agent, in order to increase the diffusion of the dye along the leather cross-section. It decreases the time needed to obtain a penetrated dyeing.

Description of the Experimental TestsUV-Vis Spectrophotometric MeasurementsThe standard solution of reactive dye Navy Blue Her (stock solution II) was scanned in UV-Vis Spectrophotometer between the 280 nm to 750 nm range using ultrapure water as blank to obtain the dye absorption spectra and to find out the two main absorption bands – one in the Visible region and the other in the UV region.1 The wavelength corresponding to maximum absorbance in the Visible region was found at 610 nm and in the UV region was found at 294 nm (Figure 1). Then the spectra of standard solutions with the same concentration and different pH, and of the real effluents samples were analyzed to verify whether the absorption of several different compounds in the same region of the electromagnetic spectrum (Figure 1) occurred. All the measurements were performed at room temperature. Figure 1 shows the UV-Vis absorbance scans, from 280 nm to 750 nm, for standard solutions of reactive dye Navy Blue Her and samples of real effluents.

Calibration CurveAbsorbance of each standard solution (prepared according to that described in preparation of standard solution to establish the calibration curve) against ultrapure water as blank was measured in triplicate at 610 nm, and the graph of absorbance against concentration was plotted and shown in Figure 2. The regression equation, i.e. the calibration curve and coefficient of determination (r2) were determined (Figure 2) along with the standard deviations of the slope and the intercept (Table II).35 All the measurements were performed at room temperature.

Validation of the Method in StudySpecificity and SelectivityTo evaluate the specificity and selectivity of the analytical method, the standard addition method36-37 was applied in two samples of real effluents of the dyeing processes of chrome-tanned leather (sample 1 and sample 2). The standard addition method was carried out by adding the appropriate volumes from the standard stock solution II (from 5.0 mL to 80.0 mL) into sixteen different volumetric flasks of 100 mL to a known volume of sample of real effluent, and then adjusted to the mark with ultrapure water. Then the same volume of sample

332 WaStEWatErS rEaCtivE dyE dEtErmiNatioN

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of the real effluent was pipetted to a volumetric flask of 100 mL and then adjusted to the mark with ultrapure water. The solutions were shaken well. Absorbance of each solution against ultrapure water as blank was measured in triplicate at 610 nm. All the measurements were performed at room temperature. The graphs of absorbance against concentration were plotted and shown in Figure 3. The regression equations and coefficients of determination were determined (Figure 3). Subsequently, these slopes and coefficients of determination were compared with the obtained calibration curve for the method in study to evaluate its specificity and selectivity.

Linearity and Linear Range Linearity was established through the calibration curve36,38-39 as well as with the homogeneity test of variance and Mandel test. To establish linearity of the method through the calibration curve the response (absorbance) was divided by the respective concentration, these values are named “relative response”. As shown in Figure 4, the “relative response” values were plotted as a function of the concentration, on a log scale. The median line was obtained over the full linear range (Figure 4). By drawing parallel horizontal lines, corresponding to 95% and 105% of the horizontal “relative response” median line (Figure 4), the intersection points can be derived where the method becomes non-linear. In Figure 4 the points that are within these parallel horizontal lines (95% and 105% of the horizontal “relative response” median line) indicate that the respective concentrations of the dye solutions are within the linear range.

To carry out the homogeneity test of variance, ten solutions of the first standard (P1st) and ten solutions of the last standard (P16th) were prepared. In all these solutions, the reactive dye concentration was quantified by measuring the absorbance in triplicate at the wavelength of 610 nm. All the measurements were performed at room temperature. With the obtained data the PG test value was calculated through the Equation 1.35 This PG test value was compared with the Fcritical value (tabulated value of the Snedecor/Fisher’s F-distribution, for n-1 degrees of freedom) to verify if the difference in variance is significant.

Equation 1

where and are the variances associated with the first standard (P1st) and the last standard (P16th), respectively.

The Mandel test was performed with the attained absorbance values of the sixteen standard solutions prepared to establish the calibration curve. With these results the PG test value was calculated through the Equation 2,35 and then this value was compared with the Fcritical value (tabulated value of the

Snedecor/Fisher’s F-distribution, for n-1 degrees of freedom) to verify if the linear regression leads to a better fit of experimental points.

Equation 2

The variances difference (DS2) was calculated through the Equation 3.35 Where Sy/x is the residual standard deviations of the linear calibration curve, Sy

2 is the residual standard deviations of the quadratic calibration curve, and N is the number of standards.

Equation 3

Precision The method precision was determined by repeatability (intra-day precision) and intermediate precision (inter-day precision) studies and is reported as percentage relative standard deviation (% RSD) and coefficient of variation (CV), respectively. Precision was also evaluated through the estimate of the absolute standard deviation (s), average confidence interval and percentage relative standard deviation (% RSD).

The repeatability study of the analytical method was carried out by the same analyst, in the same laboratory, using the same equipment, the same type of reagents and in short intervals of time, by preparing: three solutions of the first standard (P1st); three solutions of the ninth standard (P9th); and three solutions of the sixteenth standard (P16th). Then the respective measurement of absorbance was made in triplicate at the wavelength of 610 nm. All the measurements were performed at room temperature. With the attained values, the parameters needed to evaluate the repeatability of the method were calculated, such as the percentage relative standard deviation (% RSD), the coefficient of variation by Horwitz Trumpet (CV (%)) through the Equation 4 as well as 1 2 and 2 3 of the CV by Horwitz Trumpet.36,39

Equation 4

where C is the mass fraction of analyte expressed as a base 10 potency (to 1 mg/g, C = 10-3 g/g).

Two analysts performed the study of the intermediate precision. Each analyst prepared the sixteen standard solutions and made the respective measuring of the absorbance in triplicate at the wavelength of 610 nm. All the measurements were performed at room temperature. Then, using the obtained values they established the corresponding calibration curves. Subsequently, using the same method and laboratory, each analyst, along a fifteen-day period, prepared ten solutions of the sixth standard (P6th) and made the respective measuring of absorbance in triplicate at the wavelength of 610 nm. All the measurements were performed at room temperature. The


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intermediate precision values were then determined by the simplified method,35 i.e. the relative standard deviation of intermediate precision (Si(T.O.)) through Equation 5 and the coefficient of variation in percentage (CV (%)).

Equation 5

where n is the number of absorbance values, absk is the individual result of absorbance, and abs is the arithmetic average of the individual result of absorbance. In Si(T.O.), the meaning of the abbreviations T and O are different time and different operators (i.e. analysts), respectively.

Stability of Reactive Dye SolutionUsually, the dyes are added to the leather during the dyeing process or sometimes the dye solutions are prepared minutes before it, but there are a few automatic dyeing processes in which the dosage system requires the preparation of the dye solutions in advance. So, normally the dyeing practice doesn’t require a stability study for the dyes solutions.

The validation of an analytical method requires analysis of several parameters, which takes a long period of time, and so the dye stock solution I stayed prepared during a long time at environmental conditions. In order to ensure that the validation of this analytical method is credible, it was secured that the dye solutions were stable for a long period of time.

The stability of the reactive dye solution was evaluated by measuring the absorbance in triplicate at 610 nm, on six different days and along a sixteen-day period, of the same sixteen standard solutions used to construct the calibration curve. All the measurements were performed at room temperature. Six graphs of absorbance against concentration were then plotted, and the regression equations and coefficients of determination were determined. The percentage of relative standard deviation (% RSD) was also estimated.40

RobustnessThe robustness trials were planned and performed according to the Youden test. Three study factors of great interest that can influence the analytical method were chosen, namely, temperature, pH and light.

The robustness test consisted in the preparation of 1 L of the ninth standard solution (P9th), followed by the preparation of four trials in volumetric flasks of 100 mL, using this solution, as described below and shown in Table I:

• the solution of the first trial has a pH value of 5.0 and was placed for 24 h at 22.0ºC;

• the solution of the second trial has a pH value of 4.2 (adjusted with HCl (1N)) and was placed for 24 h at 41.5ºC;

• the solution of the third trial has a pH value of 4.2 (adjusted with HCl (1N)), was placed for 24 h at 22.0ºC and then was placed under UV and Visible light for 48 h;

• the solution of the fourth trial has a pH value of 5.0, was placed for 24 h at 41.5ºC and then was placed under UV and Visible light for 48 h.

The absorbance of each trial was measured in triplicate at 610 nm, and all the measurements were performed at room temperature. With the attained values the needed parameters to evaluate the robustness of the method35 were calculated, such as the effect of each variable (Ri) over the analytical method through the Equation 6, as well as the coefficient of variation (CV) through the Equation 7.

Equation 6

Equation 7

rEsults And dIscussIon

Absorption Spectra of the Dye Solutions and Samples of Real EffluentsAs shown in Figure 1, the absorption spectra of four standard solutions with the same concentration and different pH exhibit two main absorption bands – one in the Visible region (610 nm) and the other in the UV region (294 nm). The two samples of real effluents exhibit the same two peaks of maximum absorption. Nevertheless, naphthalene sulfonic acid also shows a maximum absorption at 294 nm. These results evidenced the absorption of several different compounds (reactive dye Navy Blue Her and naphthalene sulfonic acid) occurring in the same region of the UV region resulting in overlapping that inhibits the quantification of individual compounds. Figure 1 shows that sample 2 has an absorbance value higher than sample 1 at 294 nm because, unlike sample 1, it was generated from a dyeing process in which it was added naphthalene sulfonic acid (as described in the dyeing formulations presented in preparation of real effluent samples). As both organic compounds, reactive dye Navy Blue Her and naphthalene sulfonic acid, have chromophore groups that show maximum adsorption at 294 nm, when they are in the same solution (e.g. sample 2) its absorbance values are added. The maximum absorption wavelength in the Visible region (610 nm) is due to the chromophore group of the reactive dye.12 Figure 1 shows that the overlapping at the wavelength of 610 nm did not occur, and it is possible to quantify the reactive dye Navy Blue Her using direct UV-Vis Spectrophotometric measurements of absorbance at the maximum absorption wavelength of 610 nm.


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Calibration CurveThe calibration curve, shown in Figure 2 and Table II, was constructed in the range of the expected concentrations of 3.0 mg/L up to 48.0 mg/L. This calibration curve shows that the analytical method gives results directly proportional to the concentration of the reactive dye Navy Blue Her in the standard solutions, within a linear range, since the correlation coefficient (r) is greater than 0.9937. Therefore, there is a very strong correlation between absorbance values and reactive dye concentrations, and according to Ribani et al. a r > 0.999 indicates an ideal data adjustment.41

Validation of the Method in StudySpecificity and SelectivityAnalyzing Figure 3 it is evident that the regression equations of the standards with samples and the calibration curve of the standards free of sample are parallel, because they have similar slopes. The method was considered selective,35-37 because there is no interference with other compounds or impurities present in the matrix of the analyzed samples. For that reason, it can ensure that its response (absorbance) at 610 nm is due solely to the chromophore group of the reactive dye Navy Blue Her present in the samples. It was considered specific since it can detect unambiguously this group in the presence of other compounds or impurities that are present in the matrices of the samples, reading the absorbance value at a specific wavelength (610 nm) that identifies the chromophore group.

SensitivityThe sensitivity of the analytical method is the variation of the response (absorbance) in function of the reactive dye concentration, and can be expressed by the slope of the calibration curve. The sensitivity of this analytical method was found to be 0.01054 L/mg (Table II).

Linearity and Linear RangeThe analysis of the linearity of the method through the calibration curve presented in Figure 4 shows that all points used to construct the calibration curve are within the linear range. Figure 4 also shows that there is a greater dispersion at lower concentrations while the dispersion is very good at higher concentrations. The method was considered linear and the linear range was found to be [3.0; 48.0] mg/L. The homogeneity test of variance proved that the PG value (1.655) was lower than the Fcritical (2.423), determined for a 99% confidence level and 29 degrees of freedom. Consequently, the difference of variance was not significant and the work range (3.0 mg/L to 48.0 mg/L) was well adjusted.

The Mandel test showed that the PG value (0.411) was lower than the Fcritical (99.0), given for a 99% confidence level and 2 degrees of freedom. Consequently, the calibration function was considered linear, because the respective non-linear calibration function didn t́ lead to a significant improvement in the adjustment of the points to this function.

Figure 1. Spectra of four standard solutions with different pH, of the naphthalene sulfonic acid, of the formic acid, and of two samples of real effluents obtained in the absorption range between 280 nm and 750 nm.

Figure 2. Calibration curve for the reactive dye Navy Blue Her.

Figure 3. Standard addition method applied on two samples of real effluents of the dyeing processes of chrome-tanned leather.


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be estimated by repeatability, reproducibility and intermediate precision as well as with the estimate of the absolute standard deviation (σ), the average confidence interval and by the percentage relative standard deviation (% RSD).35-36

Precision was evaluated by the estimate of the absolute standard deviation (s), the average confidence interval and by the percentage relative standard deviation (% RSD), and these results are shown in Table II. These values indicate that the analytical method has precision, because the estimate of the standard deviation is low and the% RSD is acceptable (is less than 20%)36, since this method is required to determine the small amounts of reactive dye Navy Blue Her that remain in the effluents of the dyeing processes of chrome-tanned leather.

The uncertainties of each standard used to construct the calibration curve, given by the respective percentage relative standard deviation (% RSD), were small (between 1.3% and 0.1%) and were decreasing with the increasing reactive dye concentration of the standards. Therefore, the calibration curve was constructed with precision, given the low values of the% RSD.34

The repeatability of the analytical method was evaluated by the same analyst, in the same laboratory, using the same equipment and type of reagents, and in short intervals of time. The absorbance values obtained were used to determine the repeatability parameters of the method. The repeatability of the analytical method was confirmed, because all of the standards prepared and analyzed have % RSD values below the corresponding interval between ½ and 2

3 of the CV by Horwitz Trumpet, as shown in Table II. This means that these results show a small dispersion of successive measurements made in the same standards by the same analyst.

Comparing the calibration curves obtained by two analysts (Table II) in the intermediate precision study, it turns out that the curves have similar slopes. The values of the intermediate precision were determined by a simplified method35, using twenty absorbance values. The obtained value for the relative standard deviation of intermediate precision (Si(T.O.)) was found to be 0.00174. The result of the coefficient of variation was found to be 0.912% (Table II). Analyzing these values, it was found that the analytical method has good intermediate precision, because the relative standard deviation on the intermediate precision is small and the coefficient of variation is less than 2%.35 This means that in the same laboratory, this analytical method allows obtaining the same results in spite of the analyst who applies it or when it is applied. Therefore, the data used to construct the calibration curve and to determine the reactive dye concentration in the samples have shown precision, regardless of the analyst who had applied the method.

The combined standard uncertainties were calculated and these values were lower than 0.40% (i.e. were between 0.40%

Limit of Detection (LOD) and Limit of Quantification (LOQ)The limit of detection (LOD) is the lowest amount of an analyte in a sample, which can be detected but not necessarily quantified as an exact value, it was calculated by Equation 8. The limit of quantification (LOQ) is the lowest amount of an analyte in a sample, which can be quantitatively determined, i.e. measured with an acceptable accuracy and precision, it was calculated by Equation 9.42

Equation 8

Equation 9

where Sy/x is the residual standard deviation of the calibration curve and s is its slope.

The LOD and LOQ values were found to be 0.206 mg/L and 0.624 mg/L, respectively. These values are lower than the reactive dye concentration of the first standard (3.0 mg/L).

Measurement RangeThe lower limit of the measurement range was found to be the concentration of the first standard (3.0 mg/L), and its upper limit was found to be 48.0 mg/L, corresponding to the maximum concentration of the standard used to construct the calibration curve with an acceptable uncertainty. [3.0; 48.0] mg/L was found to be the measurement range of this analytic method, where it can be applied with the desired accuracy and precision.

PrecisionThe precision of an analytical method evaluates the results dispersion between independent experiments, repeated over the same sample, similar samples or standards, obtained under specific conditions, around a mean value. This dispersion can

Figure 4. Linear range.


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and 0.27%). Consequently, the standard solutions were prepared with precision. However, it was found that the combined standard uncertainties values were bigger in standards prepared with the smaller volumetric pipettes, namely, with the pipettes of 5.0 mL, 10.0 mL and 15.0 mL. The developed method was considered precise, because the % RSD for repeatability and the CV for intermediate precision were found to be less than 2%.

Stability of Reactive Dye SolutionsThe standard solutions were found stable up to 16 days at room temperature (20.0ºC to 25.0ºC, controlled by an air-conditioning system) because the slopes of the calibration curves obtained in this study are very similar (Table II), i.e. the calibration curves are parallels and the average of percentage relative standard deviation (1.250%) for the trial values determined up to 16 days was less than 2%40, as shown in Table II.

RobustnessThe data resulting from the four trials of the robustness test were used to calculate the effect of each variable (Ri) over the analytical method by Equation 6, as well as to determinate the coefficient of variation (CV) by Equation 7,35 whose values are shown in Table I.

It is considered that the study factors have no effect on the method if the coefficient of variation (CV) of the respective Ri (Rtemperature, RpH and RLight) is less than 2%, and in this case the analytical method shows evidence of robustness.35 Analyzing the results shown in Table I, it was verified that none of the three study factors have influence over the method, since CV values were less than 2%.

The analytical conditions, temperature, pH and light showed no influence in the absorbance. Therefore, temperatures from 22.0ºC to 41.5ºC, pH from 4.2 to 5.0 and for reactive dye

TABLE IValues of the effect of each study factor on the analytical method.

Trails Temperature (24 h) pH Light (UV and Visible) REi

1.º 22.0ºC 5.0 0 h 0.287

2.º 41.5ºC 4.2 0 h 0.285

3.º 22.0ºC 4.2 48 h 0.285

4.º 41.5ºC 5.0 48 h 0.286

Ri 0.002 0.005 0.001 Mean

CV (%) 0.694 1.720 0.467 0.286


solutions exposed to light (UV and Visible) from 0 h to 48 h are suitable conditions where the analytical method can be safely used. Therefore the analytical method could be considered as robust.

Determination of the Reactive Dye Concentration in Samples of Real EffluentsThe performance of the proposed UV-Vis Spectrophotometric method was tested by applying it to the determination of the reactive dye Navy Blue Her in five different samples of real effluents of the dyeing processes of chrome-tanned leather, prepared as described in preparation of real effluent samples.

First, each sample of real effluent was centrifuged at 6000 rpm for 20 minutes. Subsequently, the respective absorbance values of the supernatant solutions were measured in triplicate at 610 nm, according to the proposed method. All the measurements were performed at room temperature. The calibration curve equation (shown in Figure 2 and Table II) was used to determine the reactive dye Navy Blue Her concentration in each sample. Analytical results are shown in Table III. The reactive dye Navy Blue Her concentration of the firsts four samples was higher than the LOQ value, while in the fifth sample was found below the LOD value of the method. This means that the val idated UV-Vis Spectrophotometric method is appropriate to quantify the amount of the reactive dye Navy Blue Her in aqueous solutions as well as in real effluents of the dyeing processes of chrome-tanned leather.

conclusIons

The calibration curve obtained for the wavelength of maximum absorbance (610 nm) is properly adjusted to the work range (3.0 to 48.0 mg/L) and has linearity with a

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TABLE IISummary of validation parameters of UV-Vis Spectrophotometric method.

Parameters Results

λmax 610 nm

Calibration curve equation (Analyst LS) Abs=0.01054 C + 0.00067

Calibration curve equation (Analyst DD) Abs=0.01060 C-0.00017

Coefficient of determination, r2 (Analyst LS and DD) 0.99998

Correlation coefficient, r (Analyst LS and DD) 0.99999

Slope, s (Analyst LS) 0.01054

Intercept, c (Analyst LS) 0.00067

Standard deviation of the slope, σs (Analyst LS) 0.000026

Standard deviation of the intercept, σc (Analyst LS) 0.000525

Sensibility (Analyst LS) 0.01054 L/mg

Linear range (Analyst LS) [3.0; 48.0] mg/L

Sx/y (Analyst LS) 0.00066

LOD (Analyst LS) 0.206 mg/L

LOQ (Analyst LS) 0.624 mg/L

Measurement range (Analyst LS) [3.0; 48.0] mg/L

Precision (Analyst LS)

σ 0.786 mg/L

Average confidence interval [24.99; 26.01] mg/L

% RSD 3.081%

Repeatability (% RSD)(Analyst LS)

P1st 1.516% < ½ CV of Horwitz trumpet (2.404%)

P9th 0.218% < ½ CV of Horwitz trumpet (1.723%)

P16th 0.207% < ½ CV of Horwitz trumpet (1.579%)

Intermediate Precision (Analyst LS)Si(T.O.) 0.00174

CV 0.912%

Stability (Analyst LS)Calibration curve equations

Abs = 0.01054 C + 0.00067 r2=0.99998

Abs = 0.01054 C + 0.00033r2=0.99998

Abs = 0.01057 C + 0.00015r2=0.99992

Abs = 0.01053 C + 0.00108r2=0.99997

Abs = 0.01054 C + 0.00010r2=0.99996

Abs = 0.01042 C - 0.00078r2=0.99983

(% RSD) 1.250%

Robustness (CV) (Analyst LS)

Temperature 0.694%

pH 1.720%

Light 0.467%


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coefficient of determination, r2, of 0.99998. The sensitivity of the method was 0.01054 L/mg. The standard solutions of the reactive dye Navy Blue Her were found to be stable up to 16 days at room temperature (20.0ºC to 25.0ºC). The limit of detection and the limit of quantification are lower than 3.0 mg/L, being respectively 0.206 mg/L and 0.624 mg/L. The application of the standard addition method has shown that the analytical method is specific and selective, because it can detect the reactive dye Navy Blue Her in the presence of other compounds or impurities present in the sample matrix. The analytical method is precise, has good repeatability and excellent intermediate precision. This method has shown robustness for the three study factors (temperature, pH and light) experimentally analyzed. Therefore, the quantification of the reactive dye Navy Blue Her using UV-Vis Spectrophotometric measurement at the wavelength of 610 nm was approved in all the parameters evaluated, and so, the analytical method was properly validated.

No analytical method was found for determination of this reactive dye and the method proposed offers a simple and cheap way for its determination in different samples. The results demonstrated that the proposed methodology is feasible for quantitative analysis of this reactive dye in real samples and could be used for rapid and routine analysis. This method could be also used to quantify the amount of this reactive dye that binds to the substrate (fiber) during the dyeing processes.

The dye studied in this work is classified as reactive, with a lower tendency to bind to textile fibers (in these cases, the quantity of dye lost can reach 50%), and thus are generally used in excess (20%) in mixtures to guarantee a complete and homogeneous dyeing. Therefore, the excess of reactive dye discharged into wastewater could cause considerable environmental harm if these effluents aren’t appropriately treated. The proposed methodology could contribute

economically to the textile and leather industries, through monitoring the exact concentration of reactive dye, reducing losses, minimizing adverse effects on the aquatic environment and supports technic-economics decisions.

AcknowlEdgEmEnts

This work was developed at the Technological Centre for Leather Industries (CTIC), Portugal, in the scope of the Master’s in Energy and Environmental Engineering.

rEfErEncEs

1. Fernandez, C., Larrechi, M. S., & Callao, M. P.; An analytical overview of processes for removing organic dyes from wastewater effluents. Trends in Analytical Chemistry 29 (10), 1202- 1211, 2010.

2. Dawood, S., & Sen, T. K. ;Removal of anionic dye Congo red from aqueous solution by raw pine and acid-treated pine cone powder as adsorbent: Equilibrium, thermodynamic, kinetics, mechanism and process design. Water Research 46 (6), 1933–1946, 2012.

3. Yao, Z., Wang, L., & Qi, J.; Biosorption of methylene blue from aqueous solution using a bioenergy forest waste: Xanthoceras sorbifolia seed coat. Clean 37 (8), 642–648, 2009.

4. Tan, I. A., Hameed, B. H., & Ahmad, A. L.; Equilibrium and kinetic studies on basic dye adsorption by oil palm fibre activated carbon. Chemical Engineering Journal 127 (1-3), 111–119, 2007.

5. Dizge, N., Aydiner, C., Demirbas, E., Kobya, M., & Kara, S.; Adsorption of reactive dyes from aqueous solutions by fly ash: Kinetic and equilibrium studies. Journal of Hazardous Materials 150 (3), 737-746, 2008.


TABLE IIIReactive dye Navy Blue Her concentration in the real effluents samples analyzed.

Sample of real effluents Concentration of the reactive dye Navy Blue Her (mg/L)

1 23.21

2 17.52

3 1.07

4 0.80

5 0.31

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6. Barreto, W. J., Barreto, S. R., Scarminio, I. S., & Inoue, F.; Quantification of Textile Dyes in Industrial Effluent Using UV-Vis Spectrophotometry Combined with Principal Components Regression. Analytical Letters 43 (5), 814-822, 2010.

7. Biparva, P., Ranjbari, E., & Hadjmohammadi, M. R.; Application of dispersive liquid–liquid microextraction and spectrophotometric detection to the rapid determination of rhodamine 6G in industrial effluents. Analytica Chimica Acta 674 (2), 206–210, 2010.

8. Golob, V., & Tusek, L.; VIS absorption spectrophotometry of disperse dyes. Dyes and Pigments 40 (2-3), 211-217, 1999.

9. Sahin, S., Demir, C., & Gucer, S.; Simultaneous UV-Vis spectrophotometric determination of disperse dyes in textile wastewater by partial least squares and principal component regression. Dyes and Pigment 73 (3), 368-376, 2007.

10. Zalacain, A., Ordoudi, S. A., Blázquez, I., Az-Plaza, E. M., Carmona, M., Tsimidou, M. Z., et al.; Screening method for the detection of artificial colours in saffron using derivative UV-Vis spectrometry after precipitation of crocetin. Food Additives and Contaminants 22 (7), 607–615, 2005.

11. Barreto, W. J., Bernardino, N. E., Doi, S. M., & Afonso, R.; Comparison of Chemometric and Chromatographic Methods to obtain Kinetic Parameters for Textile Dyes During a Biodegradation Process. Analytical Letters 45 (12), 1713–1723, 2012.

12. Radi, A., Mostafa, M. R., Hegazy, T. A., & Elshafey, R. M.; Electrochemical Study of Vinylsulphone Azo Dye Reactive Black 5 and Its Determination at a Glassy Carbon Electrode. Journal of Analytical Chemistry 67 (11), 890–894, 2012.

13. Chen, Q. C., Mou, S. F., Hou, X. P., Riviello, J. M., & Ni, Z. M.; Determination of eight synthetic food colorants in drinks by high-performance ion chromatography. Journal of Chromatography A 827 (1), 73–81, 1998.

14. Gennaro, M. C., Gioannini, E., Angelino, S., Aigotti, R., & Giacosa, D.; Identification and determination of red dyes in confectionery by ion-interaction high-performance liquid chromatography. Journal of Chromatography A 767 (1), 87-92, 1997.

15. Minioti, K. S., Sakellariou, C. F., & Thomaidis, N. S.; Determination of 13 synthetic food colorants in water-soluble foods by reversed-phase high-performance liquid chromatography coupled with diode-array detector. Analytica Chimica Acta 583 (1), 103–110, 2007.

16. Pessel, D. H., Couëdor, P., & Verdon, E.; Liquid chromatography–tandem mass spectrometry method for the determination of dye residues in aquaculture products: Development and validation. Journal of Chromatography A 1218 (12), 1632–1645, 2011.

17. Pérez-Urquiza, M., & Beltrán, J. L.; Determination of dyes in foodstuffs by capillary zone electrophoresis. Journal of Chromatography A 898 (2), 271–275, 2000.

18. Giovine, L., & Bocca, A. P.; Determination of synthetic dyes in ice-cream by capillary electrophoresis. Food Control 14 (3), 131-135, 2003.

19. Liu, H., Zhu, T., Zhang, Y., Qi, S., Huang, A., & Sun, Y.; Determination of synthetic colourant food additives by capillary zone electrophoresis. Journal of Chromatography A 718 (2), 448-453, 1995.

20. Pérez-Urquiza, M., Ferrer, R., & Beltrán, J. L.; Determination of sulfonated azo dyes in river water samples by capillary zone electrophoresis. Journal of Chromatography , 883 (1), 277–283, 2000.

21. Zanoni, M. V., Fogg, A. G., Barek, J., & Zima, J.; Electrochemical investigations of reactive dyes; cathodic stripping voltammetric determination of anthraquinone-based chlorotriazine Abstract dyes at a hanging mercury drop electrode. Analytica Chimica Acta, 349 (1), 101-109, 1997.

22. Zanoni, M. V., Sousa, W. R., Lima, J. P., Carneiro, P. A., & Fogg, A. G.; Application of voltammetric technique to the analysis of indanthrene dye in alkaline solution. Dyes and Pigments 68 (1), 19-25, 2006.

23. Zanoni, M. V., Fogg, A. G., Barek, J., & Jiri Zima, J.; Electrochemical investigations of reactive dyes; polarographic determination of anthraquinone-based chlorotriazine dyes. Analytica Chimica Acta 315 (1), 41-54, 1995.

24. Karpinska, J., & Szostak, J.; Determination of chlorprothixene and amitryptyline hydrochlorides by UV-derivative spectrophotometry and UV-solid-phase spectrophotometry. Spectrochimica Acta Part A 61 (5), 975–981, 2005.

25. Rosa, S., Laranjeira, M. C., Riela, H. G., & Fávere, V. T.; Cross-linked quaternary chitosan as an adsorbent for the removal of the reactive dye from aqueous solutions. Journal of Hazardous Materials 155 (1), 253-260, 2008.

26. Santos, S. C., Vilar, V. J., & Boaventura, R. A.; Waste metal hydroxide sludge as adsorbent for a reactive dye. Journal of Hazardous Materials 153 (3), 999-1008, 2008.

27. Daskalaki, V. M., Timotheatou, E. S., Katsaounis, A., & Kalderis, D.; Degradation of Reactive Red 120 using Hydrogen Peroxide in Subcritical Water. Desalination 274 (1), 200-205, 2011.

28. Pereira, M. F., Soares, S. F., Órfão, J. J., & Figueiredo, J. L.; Adsorption of dyes on activated carbons: influence of surface chemical groups. Carbon 41 (4), 811-821, 2003.

29. Furlan, F. R., Silva, L. G., Morgado, A. F., Souza, A. A., & Souza, S. M.; Removal of reactive dyes from aqueous solutions using combined coagulation/flocculation and adsorption on activated carbon. Resources, Conservation and Recycling 54 (5), 283-290, 2010.


JALCA, VOL. 109, 2014

30. Garcia, J. C., Oliveira, J. L., Silva, A. E., Oliveira, C. C., Nozaki, J., & Souza, N. E.; Comparative study of the degradation of real textile effluents by photocatalytic reactions involving UV/TiO2/H2O2 and UV/Fe2+/H2O2 systems. Journal of Hazardous Materials 147 (1), 105-110, 2007.

31. Cristóvão, R. O., Tavares, A. P., Ribeiro, A. S., Loureiro, J. M., Boaventura, R. A., & Macedo, E. A.; Kinetic modelling and simulation of laccase catalyzed degradation of reactive textil dyes. Bioresource Technology 99 (11), 4768-4774, 2008.

32. Maljaei, A., Arami, M., & Mahmoodi, N. M.; Decolorization and aromatic ring degradation of colored textil wastewater using indirect electrochemical oxidation method. Desalination 249 (3), 1074-1078, 2008.

33. Netpradit, S., Thiravetyan, P., & Towprayoon, S.; Application of “waste” metal hydroxide sludge for adsorption of azo reactive dyes. Water Research 37 (4), 763-772, 2003.

34. EURACHEM. The fitness for purpose of analytical methods: A laboratory guide to method validation and related topics, 1998.

35. Relacre - Associação de Laboratórios Acreditados de Portugal; Guia Relacre 13 - Validação de Métodos Internos de Ensaio em Análise Química. Lisboa: Relacre, 2000.

36. Ribani, M., Bottoli, C. B., Collins, C. H., Jardim, I. C., & Melo, L. F.; Validação em Métodos Cromatográficos e Eletroforéticos. Química Nova 27 (5), 771-780, 2004.

37. Brito, N. M., Amarante, O. P., Polese, L., & Ribeiro, M. L.; Validação de Métodos Analíticos: Estratégia e Discussão. Pesticidas: R. Ecotoxicol. e Meio Ambiente, 13 (1), 129-146, 2003.

38. Bressolle, F., Bromet-Petit, M., & Audran, M.; Validation of liquid chromatographic and gas chromatographic methods. Applications to pharmacokinetics. Journal of Chromatography. B, Biomedical and Applications 686 (1), 3-10, 1996.

39. Taverniers, I., Loose, M., & Bockstaele, E. V.; Trends in quality in the analytical laboratory. II. Analytical method validation and quality assurance. Trends in Analytical Chemistry 23 (8), 535-552, 2004.

40. Devkhile, A. B., & Shaikh, K. A.; Method Development and Validation for Third Generation Cephalosporin by UV-Vis Spectrophotometer. International Research Journal of Pharmacy 2 (1), 222-229, 2011.

41. Maluf, D. F., Farago, P. V., Barreira, S. M., Pedroso, C. F., & Pontarolo, R.; Validation of an Analytical Method for Determination of Sibutramine Hydrochloride Monohydrate in Capsules by UV-Vis Spectrophotometry. Latin American Journal of Pharmacy 26 (6), 909-912, 2007.

42. Swamivelmanickam, M., Gomes, A. R., Manavalan, R., Satyanarayana, D., & Reddy, P. G.; Determination and validation of UV Spectrophotometric method for estimation of bicalutamide tablet. International Journal of ChemTech Research 1 (4), 1189-1193, 2009.


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