Anal. Chem. 1997, 69, 91-94
Mechanized Sample Workup Interfaced with Flow System in Flow-Reversal Mode for the Determination of Boric Acid in Adulterated Shellfish Encarnacio´n Luque Pe´rez, Angel Rı´os, and Miguel Valca´rcel*
Department of Analytical Chemistry, Faculty of Sciences, University of Co´ rdoba, E-14004 Co´ rdoba, Spain
A mechanical method for the direct determination of boric acid in shellfish is proposed. The sample is weighed, placed in a sample vessel inserted in the flow system, and treated with warm water in order to release the analyte, which is extracted into the aqueous solution. The solution is then continuously filtered to clean the aqueous solution and obtain a clear medium that is processed in the flow system to determine boric acid by spectrophotometrically monitoring its reaction with azomethine H. The determination method is based on the flow-reversal mode and allows the determination of the analyte at the microgramper-milliliter level with a relative standard deviation between (3% and (4% and a throughput of 30 samples h-1. Continuous-flow system in general and flow injection analysis (FIA) in particular have lately aroused much interest for the analysis of a wide variety of water samples, which can be performed with a high degree of automation and simplicity. However, direct analysis of heterogeneous or solid samples by use of automatic systems is confronted with special difficulties. This is why only a few applications of this type have so far been reported. Recently, our research group developed several methods for the direct analysis of complex samples,1 including the determination of nitrate and nitrite in soils by using a hydrodynamic injection probe based on filtration and dialysis,2 soluble sulfate in soils by coupling a filtration probe to a flow system,3 amonium in solid samples,4 trimethylamine in fish using a gas extraction sample device,5 and boron in soils with ultrasonic leaching.6 Boric acid has been used as a preservative in seafood on account of its reducing character and antiseptic power in order to avoid enzymatic darkening, called melanosis, which affects this type of food after fishing. However, its use as an additive has been the subject of bitter disputes. At present, it is forbiden in some countries and allowed in others. In Spain, boric acid has been banned owing to its toxicity;7-9 however, it is occasionally (1) Zhi, Z. L. Ana´lisis Directo de Muestras Complejas mediante Sistemas Automa´ticos de Flujo. Ph.D. Thesis, University of Co´rdoba, Co´rdoba, Spain, 1995. (2) Zhi, Z. L.; Rı´os, A.; Valca´rcel, M. Int. J. Environ. Anal. Chem. 1994, 57, 279-287. (3) Zhi, Z. L.; Rı´os, A.; Valca´rcel, M. Quı´m. Anal. 1994, 13, 121-125. (4) Zhi, Z. L;, Rı´os, A.; Valca´rcel, M. Anal. Chim. Acta 1994, 293, 163-170. (5) Zhi, Z. L.; Rı´os, A.; Valca´rcel, M. Anal. Chem. 1995, 67, 871-877. (6) Chen, D.; La´zaro, F.; Luque de Castro, M. D.; Valca´rcel, M. Anal. Chim. Acta 1989, 226, 221-227. S0003-2700(96)00634-8 CCC: $14.00
© 1996 American Chemical Society
encountered in commercially available seafood. This has created the need for rapid methods for the determination of boric acid in foods in order to facilitate appropriate control of shellfish in the market. An officially recommended method for the determination of boric acid in foods10 (and other matrices11 ) is a manual method that involves sample treatment to extract the analyte, filtration, and manipulation of highly corrosive reagents such as quinalizarin in concentrated mixtures of acetic-sulfuric acids. These steps are time-consuming, hazardous, and the source of substantial errors. In this paper, a new method for the determination of boric acid is proposed. The method is based on flow-reversal methodology,5,12,13 which has proved its utility for automating the preparation of complex samples within the flow manifold where the determination is performed. Solid samples (e.g., prawn, crayfish, shrimp, and small lobster) are treated with warm water leaching. A portion of the leachate is passed through a filtration unit to the analysis system. The boric acid concentration in the solution is finally determined by monitoring its reaction with azomethine H.6,14,15 EXPERIMENTAL SECTION Reagents. A standard stock solution, containing 1000 µg mL-1 boric acid, was prepared by dissolving 0.1 g of the chemical (Merck) in 100 mL of distilled water. Standard working solutions were made by appropriate dilution of the stock. The reagent solution was prepared daily by dissolving 0.5 g of azomethine H (Merck) in about 50 mL of water at 50 °C and diluting to 100 mL. This solution was stored in the dark. The buffer/masking solution was prepared by dissolving 25 g of EDTA (Merck) in water, followed by addition of 68 mL of phosphoric acid (Merck) and 136 mL of ammonia in distilled water to ∼450 mL, pH (7) Multon, J. L. Aditivos y Auxiliares de Fabricacio´n en Industrias Agroalimentarias; Acribia: Zaragoza, 1988; p 661. (8) B.O.E. Spanish Official Bulletin, nu´m 10, de 12 de enero de 1983. (9) Reglamentaciones Te´ cnico-Sanitarias del Sector Alimentario. Tomo I.; AMV Ediciones: Madrid, 1988; p 149. (10) Direccio´n General de Control y Ana´lisis de la Calidad. Ana´lisis de Alimentos. Me´ todos oficiales y Recomendados por el Centro de Investigacio´ n y Control de la Calidad; Ministerio de Sanidad y Consumo. Secretarı´a General para el Consumo: Madrid, 1985; p 1002. (11) Grotheer, E. W. Anal. Chem. 1979, 51, 2402-2403. (12) Rı´os, A.; Luque de Castro, M. D.; Valca´rcel, M. Anal. Chem. 1988, 60, 1540-1545. (13) Zhi, Z. L.; Rı´os, A.; Valca´rcel, M. Anal. Chim. Acta 1996, 318, 187-194. (14) Arruda, M. A. Z.; Zagatto, E. A. G. Anal. Chim. Acta 1987, 199, 137-145. (15) Whitman, D. A.; Christian, G. D.; Ruzicka, J. Analyst 1988, 113, 18211826.
Analytical Chemistry, Vol. 69, No. 1, January 1, 1997 91
Figure 1. Schematic diagram of the manifold used for the determination of boric acid in sellfish. SV, switching valve; R1, reactor 1; R2, reactor 2; filter 1, wool filter; filter 2, cellulose filter.
adjustment to 7.35 after cooling to room temperature, and final dilution to 500 mL with water. All reagents used were analytical-grade chemicals and solutions prepared in distilled water. Apparatus. A Hewlett-Packard 8452A diode array spectrophotometer equipped with a Hellma QS flow cell of 18 µL inner volume and 10 mm light path, controlled by a Hewlett-Packard Vectra ES/12 computer and connected to a HP Think Jet printer, was used. A Commodore 64 computer running BASIC software and a BMC monitor were used to control the reversal cycles of a Gilson Minipuls-3 peristaltic pump. A Rheodyne selection valve and a magnetic stirrer, from SBS, were also used. Manifold and Procedures. The manifold used is depicted in Figure 1. The sample (0.5 g) was weighed into a small sample vessel, and 20 mL of water at 50 °C was added. The vessel was then placed in a water bath at 50 °C, with stirring. After treatment, the aqueous solution containing the analyte was filtered in order to avoid the passage of solid particles in suspension through the flow system, which would decrease the analytical reproducibility. The filtration system used comprised two different filters. The first one was a small piece (∼4 cm length and 2 mm i.d.) of Teflon tubing filled with glass wool and was used to remove the largest solid particles. The second one consisted of a hollow glass tube (∼10 cm length and 5 mm i.d.) placed vertically, with a cellulose filter at the end (fixed with Parafilm) which provided fine filtration. The filter was constructed as follows: a Teflon tube (0.8 mm i.d.), through which the sampled circulated, was placed inside the hollow glass tube, avoiding contact with the cellulose filter of the end. The Teflon tube was fixed inside the glass tube with a piece of Parafilm in such a way that a vacuum was produced 92 Analytical Chemistry, Vol. 69, No. 1, January 1, 1997
in the space between the Parafilm piece and the cellulose filter, facilitating the filtration of the liquid (see Figure 1 for details). The glass tube with the filter, fixed to a cap, was placed in a larger glass tube where the filtered sample was collected. The sample was then pumped through another Teflon tube (0.8 mm i.d.), placed into the largest glass tube, to the spectrophotometer. The peristaltic pump was controlled by a computer running BASIC software that enabled the control of the drum rotation and speed, the start and stop of reversal cycles, and the number of cycles and their duration. The reagent was pumped into the system in the opposite direction of the sample, facilitating contact and reaction between sample and reagent. The interface was then passed as many times as required through the detector, and a multipeak recording was obtained for each sample by monitoring the reaction. The operational sequence was as follows: First, the reagent was introduced into the system and passed by the detection point (leftwards) to zero the instrument. Then, the valve was switched and the reagent driven to waste. Next, the flow system was filled with water and the valve left in the waste position. The sample, weighed and treated in the small vessel placed in the water bath at 50 °C, was pumped through the manifold and filtered by the dual-filter system. Once the manifold was filled with sample, the switching valve was actuated and the reagent introduced into the system in the opposite direction for 15 s (so the interface reached the detection point). Then, the cycle programmer started to control the flow cycles, and the reaction was monitored by the spectrophotometer. When the experiment had finished, the system was flushed with water, thereby being made ready for the next experiment.
a 0.5% azomethine H solution. Because of the strong effect of pH on the reaction,6 a systematic study of the influence of this variable was carried out. The buffer solution was added to the reagent by using an auxiliary peristaltic pump that was actuated separately from the rest of the system for 10 s at a flow rate of 0.6 mL min-1 and then stopped, in order to provide the appropriate reaction pH; however, the reagent was quite unstable and further instabilized when the buffer was added. To overcome this problem, small portions of reagent (5 mL) placed in a small vessel to which the buffer was added were employed instead. The small vessel with reagent was replaced when run out of buffered reagent and the buffer solution added by switching the auxiliary pump for 10 s. This had a twofold purpose: first to avoid reagent decomposition, and second to avoid contamination of the reagent solution. The optimum pH was found to be approximately 7.1, which was adjusted by adding 0.1 mL of buffer to 5 mL of reagent. The wool filter had to be replaced approximately twice a day, in order to avoid clogging. However, the cellulose filter worked longer, being replaced every working day for security reasons. Features of the Proposed Method. Standard solutions containing boric acid at concentrations between 10 and 120 µg mL-1 were used to run the calibration graph for determining the analyte. Depending on the sensitivity and determination rate needed, the number of peaks used from the multipeak recording can vary. In the present work, four peaks were used as a compromise between sensitivity and determination rate. The modulus of the vector for the absorbances from the first to the fourth peak, calculated as follows, was used for calibration purposes: Figure 2. Effect of the most influential variables on the signal provided by the fourth peak in the multipeak recordings: (a) flow rate and (b) reagent concentration.
RESULTS AND DISCUSSION The UV-visible spectrum for the boric acid-azomethine H complex exhibits an absorption maximum at 538 nm, which was used to monitor the reaction and determine boric acid in the food samples. Optimization. One of the critical variables of the system was cycle duration, owing to its strong influence on the analytical information provided by the recordings obtained. Time cycles between 4 and 15 s were tested, and 6 s was found to be the optimal duration. Longer times gave ill-defined peaks, whereas shorter times detracted from sensitivity. The sample flow rate was inversely proportional to the cycle duration; the higher the flow rate, the shorter the cycle time because the interface flowed faster. Low flow rates increased sensitivity and peak definition; however, the analysis time was up to twice as long. Thus, a flow rate of 2 mL min-1 was adopted as a compromise between adequate sensitivity and expeditiousness. Figure 2a shows the effect of the flow rate on the signal. The influence of the reactor (R2) length was also studied. This reactor played a dual role in the system: as a proper reactor, by facilitating mixing and reaction between reagent and analyte; and as a safety device, avoiding contamination of the reagent because of the transfer of analyte to the reagent vessel. A reactor of 100 cm length × 0.7 mm of i.d. was found to be the most suitable as it provides increased sensitivity with minimal dispersion. The reagent concentration was also optimized. As can be seen from Figure 2b, the most sensitive signal was obtained by using
V ) (A12 + A22 + A32 + A42)1/2 where A1, A2, A3, and A4 are the absorbances of the first, second, third, and fourth peaks from the multipeak recording obtained, respectively. Measurements were made at two wavelengths, viz., 538 nm (maximum absorption of the boric acid-azomethine H complex) and 600 nm (minimum absorption of the complex); the difference between the two was used to normalize each response. The figures of merit of the proposed method obtained by using this method of evaluation are listed in Table 1. The calibration graph (run from triplicate measurements for each point) comprised two parts of different slope (sensitivity). The linear range was between 10 and 120 µg mL-1. The precision of the method, expressed as relative standard deviation, for the determination of 50 µg mL-1 boric acid was (3.7% (n ) 11). The detection limits, calculated as the blank signal plus 3 times its standard deviation, were 5.1 and 68.7 µg mL-1 respectively for the two ranges of the calibration graph. The determination rate was obviously dependent on the number of cycles used. To four cycles and including the washing and sample preparation times, the estimated throughput was ∼30 determinations h-1. Interferences. The effect of potential interferences present in shellfish was studied. Table 2 shows the tolerated limits, taken as the largest amount of foreign species yielding a relative error less than (5% of the signal obtained for 50 µg mL-1 boric acid. As can be seen from Table 2, there were no significant interferences from the species studied. The most serious interferences (maximum tolerated ratio of 1:1) were seafood additives,9 such as S2O52-, SO32-, and citric acid, so their concentrations in the samples must be similar to that of boric acid. Anyway, these were Analytical Chemistry, Vol. 69, No. 1, January 1, 1997
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Table 1. Figures of Merit of the Proposed Method for the Determination of Boric Acid
a
equationa
regression coefficient
determination range (µg mL-1 )
throughtput (samples h-1)
detection limitb (mg mL-1)
V ) 0.356 + 0.037C V ) 1.1 + 0.029C
0.9973 0.9993
10-70 70-120
30 30
5.1 68.7
V, modulus of the vector cited in the text; C, concentration of the analyte. b Defined as the blank signal plus 3 times its standard deviation.
Table 2. Tolerated Limits of Interfering Species in the Determination of 50 µg mL-1 Boric Acid by Using the Proposed Method maximum tolerated interferent/boric acid ratio (M:M)
species Na+, K+, NH4+, Cl-, I-, Br-, F-, NO3-, SO42-, C2O42-, benzoatea Mg2+, Al3+ Ba2+, Ca2+, Mg2+, Pb2+ Zn2+ Ni2+, S2O52-,a SO32-,a CO32-,a citric acida a
Table 4. Analysis of Real Samples (Shellfish) by Using Both the Proposed and Official Methods
sample prawn 1
>400:1 50:1 10:1 5:1 1:1
prawn 2
crayfish 1
Used as seafood additive.
Table 3. Analysis of Synthetic Samples of Boric Acid by Using the Proposed Method concn added (µg mL-1)
concn found (µg mL-1)
error (%)
24 36 50 68 88 100 120
24.0 38.6 50.1 67.9 88.8 98.9 120.3
+0.1 +7.2 +0.2 -0.1 +0.9 -1.1 +0.2
negative interferences, which did not give any signal with azomethine H by themselves. On the other hand, Ni(II) is rarely found at such concentrations in shellfish. Analysis of Synthetic and Real Samples. The applicability of the proposed method was checked by analyzing synthetic samples of boric acid. The results obtained are shown in Table 3. As can be seen from the table, the differences between the concentrations found and the those added were in general very small (less than 1%). Finally, the method was applied to real samples, viz., shellfish skins purchased at a local market. Skin samples were removed, ground, and homogenized prior to weighing and analysis. Whole body common prawns were used. Prawns and small lobster specimens were already boiled and salted. To overcome the interference of the matrix effect, the method of the standard addition was used. The results (n ) 3) obtained were compared with those provided by an officially recommended method10 (Table 4). As can be seen from Table 4, they were quite consistent. Thus, the proposed method can be successfully applied to real samples. CONCLUSIONS The proposed method for the determination of boric acid in shellfish based on the joint use of a sample pretreatment unit and
94 Analytical Chemistry, Vol. 69, No. 1, January 1, 1997
crayfish 2
shrimp 1
shrimp 2
small lobster 1
small lobster 2
concn added (µg mL-1) 20 40 60 80 20 40 60 80 20 40 60 80 20 40 60 80 20 40 60 80 20 40 60 80 20 40 60 80 20 40 60 80
concn found (µg mL-1) proposed method official method 18.7 ( 2.9 46.3 ( 1.3 62.2 ( 1.6 75.6 ( 2.3 19.5 ( 1.6 42.5 ( 1.8 56.8 ( 1.7 81.6 ( 2.5 22.9 ( 1.0 40.7 ( 2.1 56.7 ( 4.1 81.4 ( 2.2 21.0 ( 0.9 38.7 ( 0.5 59.6 ( 2.0 80.7 ( 2.5 19.9 ( 1.3 39.2 ( 2.6 61.7 ( 3.8 79.1 ( 2.9 19.0 ( 2.9 42.2 ( 1.9 58.5 ( 2.1 80.3 ( 1.5 18.4 ( 1.4 41.1 ( 1.3 62.5 ( 0.4 78.0 ( 2.5 19.5 ( 1.9 40.3 ( 2.9 61.0 ( 0.5 79.3 ( 0.4
21.0 ( 0.4 40.9 ( 0.4 58.9 ( 0.4 80.1 ( 0.3 20.4 ( 0.3 40.1 ( 0.3 61.0 ( 0.4 79.1 ( 0.5 19.6 ( 0.6 37.9 ( 0.4 61.9 ( 0.6 79.3 ( 0.2 20.0 ( 0.3 43.6 ( 0.4 61.6 ( 0.3 77.0 ( 0.5 18.3 ( 0.6 40.4 ( 0.4 61.2 ( 0.5 79.4 ( 0.4 20.6 ( 0.2 38.4 ( 0.4 61.0 ( 0.3 79.9 ( 0.2 18.1 ( 0.3 40.1 ( 0.1 60.4 ( 0.3 80.1 ( 0.2 19.8 ( 0.5 38.6 ( 0.4 59.4 ( 0.4 81.2 ( 0.6
a computer-controlled flow-reversal injection manifold is proposed. Because sample pretreatment is simplified, the overall determination time is greatly reduced, and some potential sources of error are avoided. The filtration unit used allows clear extracts to be obtained from solid samples to be inserted into a coupled FIA manifold. Compared with the manual method, the proposed method is simpler, faster, and more reliable, as it involves little sample manipulation (pretreatment and filtration are carried out continuously in the flow system). ACKNOWLEDGMENT Financial support provided by the DIGyT (Project No. PB940450) is gratefully acknowledged. Received for review June 26, 1996. Accepted September 19, 1996.X AC960634H X
Abstract published in Advance ACS Abstracts, November 15, 1996.
