In the Laboratory
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FIA-Spectrophotometric Method for Determination of Nitrite in Meat Products: An Experiment Exploring Color Reduction of an Azo-Compound José C. Penteado, Lúcio Angnes, and Jorge C. Masini* Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, 05513-970, São Paulo, SP, Brazil; *
[email protected] Paulo C. C. Oliveira Departamento de Química, Universidade Federal de Alagoas–UFAL, Campus A.C. Simões, BR 104/km 97, Tabuleiro dos Martins, 57072-970, Maceió, AL, Brazil
Flow injection analysis (FIA) has been used for many applications in analytical chemistry. Various features, such as high-speed of analysis, great flexibility, excellent reproducibility, and use of discrete samples (1–3) make this method convenient to use. Additional advantages of the method are low cost per analysis, possibility of remote operation, and reduction of waste generation (4, 5). Utilizing flow systems, it is possible to execute, in line, almost all the operations (sampling, separation, dilution, preconcentration, addition, and mixture of reagents, etc.) required in chemical analysis (6, 7). Despite the continuous increase in the number of research articles describing the development of FIA methods, this technique has not been explored extensively during undergraduate analytical chemistry courses (8). FIA can be used to teach many chemical reactions used in classical batch analytical methods. The inherent advantages (speed, automation, small sample volumes, etc.) of the method can be used to motivate the students. FIA experiments exploring spectrophotometric detection are usually preferred for didactic applications owing to the simplicity of the instrumentation and the inherent robustness of this technique (9). In this article we present an experiment involving the reaction of the azo dye 3,7-diamino-2,8-dimethyl-5-phenylphenazinium chloride (usually referred to as safranine O) with nitrite, which decreases the color of the dye. This experiment is a powerful tool to teach many aspects involving optimization, spectrophotometry, and kinetic analysis of real samples. This experiment is appropriate for an instrumental analysis course.
Reagents and Solutions Two stock solutions of safranine O (Sigma-Aldrich) were prepared. One of these solutions was prepared by dissolving 17.0 mg of the dye and 22.4 g of potassium chloride (Merck) with 0.5 mol L᎑1 hydrochloric acid in a 1.00-L volumetric flask. The other solution was prepared similarly, but adding 25.0 mg of safranine O. The standard 1000 µg mL᎑1 nitrite solution was prepared by dissolving 0.1500 g of sodium nitrite (Merck, dried at 100 ⬚C for 4 h, cooled inside a desiccator) in a 100.00-mL volumetric flask. Before diluting to volume, a pellet of sodium hydroxide was added to prevent formation of nitrous acid, which easily decomposes to NO and NO2, and 1 mL of chloroform was also added to inhibit bacterial growth (10, 11). All the working solutions of nitrite were prepared daily by the appropriate dilution of this standard solution with distilled water. Sample Preparation Students were asked to weigh two portions of 5.0 g (on an analytical balance with precision of ±0.1 mg) of commercial sausages. One of these portions was spiked with a known quantity of nitrite to evaluate the extraction effectiveness. Then, both portions were triturated in separate mortars, followed by the addition of 40 mL of distilled water. The mixture was heated at 80 ⬚C for 2 h, with occasional shaking,
S
Experimental
mL/min
Apparatus Spectrophotometric measurements were made using a Hitachi (Model U-1201) double-beam spectrophotometer using a flow cell with 1.000-cm light pathway. The signals were recorded using an Epson printer (Model FX850). A fourchannel peristaltic pump (Ismatec Reglo Model 78016) was used to propel the solutions. The temperature of the reaction coil was maintained constant by a thermostatic bath (Haake Model C1K15, water circulator). Injections of sample or standard in the carrier stream were performed using a Rheodyne 7125 rotary valve.
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H2O 0.6
C P
dye
A
520 nm
W
0.6
D
E
B
Figure 1. FIA manifold for the spectrophotometric determination of nitrite by reaction with safranine O: (A) reagent flasks; (B) peristaltic pump; (C) rotary injection valve; (S) sample or standard nitrite solution; (P) confluence point; (D) thermostatic bath containing the reaction coil; (E) detector; and (W) waste reservoir.
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In the Laboratory
and filtered through 0.45 µm cellulose acetate membrane after reaching the ambient temperature. The filtered extracts were quantitatively transferred to 50.00-mL volumetric flasks, diluted to the mark with distilled water, and immediately analyzed.
also be substituted by inexpensive flow photometers using bicolor LED and phototransistors (13). For optimization, the students were asked to use a 10.0 µg mL᎑1 nitrite solution and perform a set of 16 experiments in which the safranine O concentration, the reaction coil length, the flow rate, the temperature of the reactor, and the sample volume were systematically varied (Table 1). The absorption peak at 520 nm was monitored. The change in the absorption peak, ∆A, was obtained by comparison to the peak height without nitrite present.
Procedure The main parts of the assembly consisted of the reagent flasks, a peristaltic pump, a rotary injection valve, and a spectrophotometric detector (Figure 1). Polyethylene tubes with 0.70 mm i.d. were used to construct the reaction coil. Two channels of the peristaltic pump were used with suitable Tygon pump tubing. One channel carried distilled water to drive the injected sample or standard solutions (nitrite) to the confluence point and then to the detector and waste reservoir. The second channel continuously drove the reagent solution (safranine). At the confluence point the reagent merged with the carrier stream in which sample or standard solutions were injected. The mixture was pumped through the reaction coil immersed in the thermostatic bath maintained at 40 ⬚C, positioned just before the spectrophotometric detector. The waste did not require special treatment before discarding. The system is easy to assemble and if a peristaltic pump is not available, solutions could be driven by gravity instead of mechanically (12). Alternatively, the spectrophotometer could
Hazards The concentrated HCl should be handled with care because it is highly hazardous to skin and eyes. Handling must be done in fume hood and using protective equipment such as eyeglasses and gloves. The concentrated HCl should be transferred to the volumetric flask using a graduated glass pipet fitted with a safety bulb or rubber pipet filler. To handle solid NaOH, eyeglasses and gloves are mandatory because it is also hazardous to eyes and skin. Safranine O may be harmful by inhalation, ingestion, or skin absorption. Additionally, it causes eye and skin irritation and is irritating to mucous membranes and the upper respiratory tract. Chloroform should be handled in a fume hood, avoiding skin contact.
Table 1. Experiments Used for the Factorial Planning of the FIA Parameters Experiment Numbera
Safranine Conc/(µg mL᎑1)
Coil Length/cm
Flow Rate/(mL min᎑1)
Temp/(⬚C)
Sampleb Volume/µL
∆A/103
01
17
200
0.6
30
200
257
02
25
200
0.6
30
100
189
03
17
300
0.6
30
100
210
04
25
300
0.6
30
200
264
05
17
200
1.2
30
100
129
06
25
200
1.2
30
200
201
07
17
300
1.2
30
200
171
08
25
300
1.2
30
100
147
09
17
200
0.6
40
100
216
10
25
200
0.6
40
200
311
11
17
300
0.6
40
200
260
12
25
300
0.6
40
100
227
13
17
200
1.2
40
200
206
14
25
200
1.2
40
100
180
15
17
300
1.2
40
100
158
16
25
300
1.2
40
200
251
17
17
200
0.6
40
100
216
18
17
300
0.6
40
200
261
19
17
200
1.2
40
200
208
20
17
300
1.2
40
100
165
a
Experiments 17 to 20 are replicates of the experiments 9, 11, 13, and 15, respectively. 10.0 µg mL᎑1.
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b
The concentration of nitrite in the sample solution is
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Results and Discussion Safranine O is a reddish-orange-colored dye that belongs to the class of amino compounds. This type of reagent exhibits classical azoic reactions and, in presence of nitrous acid, generates a blue azo dye with low molar absorptivity at 520 nm (Figures 2 and 3). At the wavelength of 520 nm, the starting dye (safranine O) has a high molar absorptivity, while the reaction product does not absorb intensely (Figure 3). The decrease of color intensity of the red azo dye is proportional to the nitrite concentration, allowing anion quantification. Successive absorption spectra, recorded at intervals of 120 s, during the reaction between safranine O and nitrous acid are shown in Figure 3. The first spectrum shows an intense peak at 520 nm, while at 350 nm the signal is practically absent. With the evolution of the reaction, the absorbance at 520 nm decreases and the absorbance at 350 nm increases, as denoted by the arrows in Figure 3. While waiting to record spectra (∼20 minutes), many aspects of the reaction, such as the determination of the kinetic parameters and the choice of best analytical signal, can be discussed.
Optimization of the Reaction Conditions The FIA response depends on the residence time of the sample zone in the flow system and is usually measured at maximum peak height, but being inverted in the present case owing to the color disappearance. There are many parameters that can affect the FIA performance. After preliminary tests, it was observed that the most important variables were: temperature, flow rate, safranine concentration, sample volume, and length of the reaction coil. As previous studies (14) suggested that chloride ions improved the spectrophotometric signal, fixed concentrations of HCl (0.50 mol L᎑1) and KCl (0.30 mol L᎑1) were chosen
H3C
N
CH3
H2N
Nⴙ
NH2
for all experiments. The concentration of the standard sample (NO2−) was fixed in 10.0 µg mL᎑1. To evaluate the most favorable conditions, a series of 2n−1 (n = 5) experiments was planed, based on the Fatorial program (15). For model assessment, a test series consisting of additional four repetitive experiments was used to estimate the variance of the process. The experimental variables, as well as the variation of absorbance observed for each condition studied are contained in Table 1. The experimental data were analyzed by the Fatorial program (15), which assesses the main effects and interactions between different parameters, generating normal and seminormal plots that allow the students to identify the statistically significant parameters and to assess the appropriateness of the approximated models. The semi-normal plot shows that there are four points intensely unaligned. These four points correspond to the parameters: safranine concentration, flow rate, temperature, and sample (nitrite) volume. The statistical treatment shows that the maximum signal is independent of the reaction coil length. To compute the error coefficients of the model, the following equation was applied: V = (X TX )᎑1σ2
where V is the residual quadratic matrix, X TX is the planning matrix, and σ the residual variance associated with the linear regression. From the square root of elements of the main diagonal of V, the model equation can be obtained, ∆A(×103) = 210.6 + 10.7S + 0.05C − 29.8V + 14.6T + 27.7L (2)
where ∆A is the magnitude of the absorbance variation, S is the magnitude of the safranine concentration (µg mL᎑1), C is the magnitude of the coil length (cm), V is the magnitude of the flow rate (mL min᎑1), T is the magnitude of the temperature (⬚C), and L is the magnitude of the sample volume (µL).
+ 2NOⴚ2 + 4Hⴙ
N
CH3
N
0.45
Absorbance
H3C
(1)
0.30
0.15
N N
ⴙ
N
ⴙ
N
ⴙ
+ 4H2O
0.00 600
500
400
300
200
λ / nm Figure 2. Schematic reaction of safranine O with nitrite, which results in loss of the reddish-orange color.
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Figure 3. Absorption spectra as a function of time of a mixture containing 25 µg mL᎑1 safranine O and 10 µg mL᎑1 nitrite. Time interval between scans is 120 s. The arrows denote increasing time.
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Absorbance
Absorbance
0.54
0.53
0.52
0.52
0.51
A
0.50
B
0.51
C 0.49
1000
0
2000
0
1000
2000
3000
Time / s
Time / s Figure 4. Repeatability study for the safranine O–nitrite system. Conditions: 25 µg mL᎑1 of safranine, 4.0 µg mL᎑1 nitrite standard solution, 100-cm coil length, 0.6 mL min᎑1, 40 oC, and sample volume of 200 µL. Relative standard deviation is 2.5%.
Figure 5. FIA readouts for nitrite determination in sausages: (A) for aqueous sausage extract, (B) for a sample spiked before extraction, and (C) for the same sample as A after a spike in the extract.
Despite the reaction coil length being important for optimization of several flow-injection manifolds, the results obtained indicated a low contribution of this parameter to the analytical signal. This fact was explored to discuss the concepts of dispersion and residence time. The possible gain of analytical readout due to the increased residence time obtained with the longer reaction coil is lost by the increased dilution (larger axial dispersion) of the reaction zone when it travels through the longer coil. The best conditions were determined to be: safranine concentration = 25.0 µg mL᎑1, flow rate = 0.6 mL min᎑1, temperature = 40 ⬚C, and sample volume = 200 µL. As the factorial study demonstrated a low contribution of the coil length, some additional experiments using a 100-cm coil were done without significant differences in the results.
To evaluate the dynamic working range, concentrations of nitrite between 1 and 10 µg mL᎑1 were studied. A straight line was obtained for the concentration range between 1.0 and 6.0 µg mL᎑1 (∆A = 126[NO2−] + 0.943, with R2 = 0.996, where ∆A is the variation of absorbance read at the peak maximum).
Real Samples Two sausage samples were analyzed. The accuracy of the method was evaluated spiking a known quantity of nitrite directly in the sausage before extraction, and in a known volume of the extract of the unspiked sausage. In the first case the students evaluated the extraction effectiveness and in the second case, the accuracy of the analytical method. Three solutions were analyzed for each sample: the solution resulting from the unspiked sausage (solution A), the solution from the spiked sausage (solution B), and the solution A spiked with a known quantity of nitrite (solution C). To prepare solution C, 47.00 mL of solution A were spiked with 2.00 or 3.00 mL of a 50.0 µg mL᎑1 NO2− standard solution, adding distilled water, if necessary to bring the volume to 50.00 mL. The absorption signals (in triplicate) for solutions A, B, and C are shown in Figure 5. Quantification of nitrite in these solutions was made by the linear calibration plot for nitrite concentrations between 1.0 and 6.0 µg mL᎑1. Typical recoveries (Table 2) were between 90.4 and 107.2% for the extraction evaluation, and between 101.3 and 104.7% for the analytical method, denoting satisfactory performance of the methodology.
Repeatability and Calibration Curve Using the optimized conditions and a 100-cm reaction coil, the repeatability of the analytical signal was evaluated performing ten repetitive injections of 4.0 µg mL᎑1 nitrite solution. The relative standard deviation of the peak height was 2.5% (n = 10), denoting a good repeatability of the method. A typical readout obtained by the students in the repeatability experiment is presented in Figure 4, which shows the signals obtained for injections performed at intervals of approximately 200 s. This time can be easily shortened to 90 s, leading to a sampling frequency of 40 analyses per hour. The sampling frequency can still be improved by increasing the flow rate after the attainment of the maximum signal.
Table 2. Results of Nitrite Determination in Sausages [N O2−] Spike d
[N O2−]/ (µg mL ᎑1)
[N O2−]/ (µg pe r g᎑1 of s aus age )
5.34
—
2.73
25.6
—
5.49
9.11b
4.09
37.2
107.2
C
5.34
2.0c
4.78
—
104.7
A
5.46
—
2.06
18.9
—
19.8
b
3.53
35.0
090.4
3.0
c
4.99
—
101.1
Saus age Sample
Soln
M as s o f Saus age /g
1
A
1
B
1 2 2 2 a
a
B C
5.05 5.46
Se e t e x t f or de s cipt ion of t he s olut ions .
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b
V alue s in µg g᎑1.
c
Re cov e ry (%)
V alue s in µg mL ᎑1.
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Conclusions The reaction between safranine O and nitrite is rich in details, leading to discussions of many aspects of chemistry. The disappearance of color (instead of the appearance of it) is an unusual situation and relates to many concepts. During reaction time, concepts related to the kinetics of the reaction and some spectroscopic principles can be discussed. Selection of the main variables and data treatment for optimization of various parameters involved in flow-injection experiments are other interesting areas to be examined. In conclusion, the experiment is a useful tool to teach many aspects of modern analytical chemistry. W
Supplemental Material
Instructions and laboratory notes for students are available on the JCE Online. Acknowledgments The authors are grateful to Brazilian foundations (FAPESP, CAPES, and CNPq) for the financial support and fellowships. Literature Cited 1. Ruzicka, J.; Hansen, E. H. Flow Injection Analysis; Willey: New York, 1988.
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2. Karlberg, B.; Pacey, G. E. Flow Injection Analysis. A Pratical Guide; Elsevier: Amsterdam, 1989. 3. Trojanowicz, M. Flow Injection Analysis: Instrumental and Applications; World Scientific: Singapore, 2000. 4. Rocha, J. R. C.; Galhardo, C. X.; Natividade, M. A. E.; Masini, J. C. J. AOAC Int. 2002, 85, 875. 5. Matos, R. C.; Angnes, L.; Araújo, M. C. U.; Saldanha, T. C. B. Analyst 2000, 125, 2011. 6. Sartini, R. P.; Zagatto, E. A. G.; Oliveira, C. C. J. Chem. Educ. 2000, 77, 735. 7. Fang, Z. Flow Injection Separation and Preconcentration; VHC: Weinheim, 1993. 8. Rocha, F. R .P.; Martelli, P. B.; dos Reis, B. F. Quim. Nova 2000, 23, 119. 9. Hansen, E. H.; Ruzicka, J. J. Chem. Educ. 1979, 56, 679. 10. Mousavi, M. F.; Jabbari, A.; Nouroozi, S. Talanta 1998, 45, 1247. 11. Mori, V.; Bertotti, M. Anal. Lett. 1999, 32, 25. 12. Angnes, L.; Richter, E. M.; Augelli, M. A.; Kume, G. H. Anal. Chem. 2000, 72, 5503. 13. Araújo, M. C. U.; Santos, S. R. B.; Silva, E. A.; Veras, G.; Lima, J. L. F. C.; Lapa, R. A. S. Quim. Nova 1997, 20, 137. 14. Kazemzadeh, A.; Ensafi, A. A. Anal. Chim. Acta 2001, 442, 319. 15. Neto, B. B.; Scarminio, I. S.; Bruns, R. W. Planejamento e Optimização de Experimentos; Editora UNICAMP: Campinas, Brazil, 1995.
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