- Email: [email protected]
Full automated robotic method for the determination of chloride, nitrite and nitrate in cured meat products
Talanta 46 (1998) 969 – 976
Full automated robotic method for the determination of chloride, nitrite and nitrate in cured meat products A. Velasco-Arjona a, J.A. Garcı´a-Garrido b, R. Quiles-Zafra b, M.D. Luque de Castro a,* a
Department of Analytical Chemistry, Faculty of Sciences, Uni6ersity of Co´rdoba, E-14004 Co´rdoba, Spain b Research Department, Na6idul S.A., E-45500 Torrijos, Toledo, Spain Received 6 June 1997; received in revised form 24 September 1997; accepted 2 October 1997
Abstract A completely automated method to determine the most common parameters in cured meat products is proposed. The approach to full automation is based on the coupling of a robotic station for development of preliminary operations (namely weighing of the sample, grinding, leaching, filtration and transport to the aspiration zone) and a continuous unsegmented manifold for derivatisation and spectrophotometric monitoring of the reaction coloured products. This assembly works in an unattended fashion thus eliminating the bottleneck produced by the determination of these parameters in routine laboratories. The good agreement between results obtained by the proposed method and those from conventional methods for target analytes confirms its excellent performance and usefulness. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Robotic; Chloride–nitrite–nitrate; Meat; Continuous flow
1. Introduction Curing is one of the most widely used technologies in meat manufacturing. The different curing modes (namely dry curing, wet curing, injection curing [1]) are based on the same principle, addition of a mixture of sodium chloride, either nitrite or nitrite plus nitrate salts to the fresh product. Even though the use of these curing agents involve some minor disadvantages, the advantages are so overwhelming [2] that no alternatives are * Corresponding author. Tel.: +34 57 218615; fax: + 34 57 218606; e-mail: [email protected]
available at present. In the case of nitrite and nitrate, some of the most remarkable beneficial aspects of the curing process [3] are as follows; appearance of a red colour due to a reaction between muscular hemoglobin and the nitric oxide generated in nitrite reduction; reaction of nitrite and different compounds such as sugars, alcohols, amines, etc. to yield products which contribute to the typical aroma of meat cured products; antioxidant effect on lipid rancidity; antimicrobial activity, which hinders the growth of pathogenic microorganisms, such as C. Botulinum. The negative effects of curing anions only appear in products with excess amounts of
0039-9140/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0039-9140(97)00349-4
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A. Velasco-Arjona et al. / Talanta 46 (1998) 969–976
these agents [4]. Some of the well-known undesirable effects are; the oxidant action of nitrite on the hemoglobin – Fe(II) complex, which can produce poisonous symptoms; formation of N-nitrosocompounds in the presence of both nitrosamine formers and acid pH; and the toxic action of nitrate, not per se but due to reduction to nitrite. For these reasons, the routine analysis of nitrite and nitrate is common practice in the meat industries in order to keep their levels within a range which ensures the development of the beneficial effects without the toxic aspects. The presence of sodium chloride in the curing process is advantageous, its protective effect hinders or makes it difficult for germ development by decreasing water activity; its conserving action by potentiating the effects of other preservant agents and inhibiting fungi and yeast proliferation, it also increases flavour. High salt concentration in cured products results in consumer rejection. We have developed a fully automated method based on the coupling of a robotic station for sample weighing and pretreatment and a continuous flow system for derivatisation and detection in order to help big meat industries in the development of routine determinations of these three more commonly determined parameters.
2. Experimental section
2.1. Instruments and apparatus The flow injection (FI) manifold was built using a Gilson Minipuls-HP3 peristaltic pump, a Rheodyne 5041 injection valve (adapted for use as switching valve), reduction columns of copperised cadmium of different lengths (1.8 mm i.d.) and a Pye Unicam SP6-500 spectrophotometer furnished with a Hellma 178. 12QS flow-cell and connected to the AD (Analog-Digital) converter of the PEC via its analog output for data acquisition, processing and delivery of the results via a computer program designed in our laboratory, which permits the use of so-called electronic dilution FIA technique [5 – 7]. All tubing used to construct the hydrodynamic system was of PTFE of 0.5 mm i.d.
The robotic station consisted of a Zymate II Plus robot (Zymark, Hopkinton), a System V controller, and the following peripherals.
2.1.1. Master laboratory station (MLS) The proposed method uses two MLSs (MLS1 and MLS2), which consist of three syringes each, intended to dispense liquids in conjunction with the dilute and dissolve unit. Only two of the syringes of MLS1 were used, one of them was connected to a water bath and the other to a filter. Both were also connected to a dispenser; one of the syringe-dispenser assembly was used to dispense water; the other one to dispense filtered solution. 2.1.2. Small-size object all-purpose (SSOAP), big-size object all-purpose (BSOAP) and solid dispenser (SD) hands The SSOAP hand allows the robot to seize centrifuge tubes and objects of similar size. The BSOAP hand allows the robot to seize 250 ml fleakers and objects of similar size. The SD hand (designed by the authors) allows the robot to manipulate solid samples. This hand is installed on the SSOAP hand forming 180° with its fingers and consists of a pair of tweezers which have a basket at their ends (see details in Fig. 1A). 2.1.3. Power and e6ent controller (PEC) The PEC module acts as an interface between the controller, peripherals and robot. 2.1.4. System V controller This module sends orders to the rest of the units, including the robot and controls additional units such as a peristaltic pump, a photometer, switching valves, etc. The RS232 outputs of these units allow them to be operated by the System V via the PEC. 2.1.5. Balance The balance plate for tube weighing was modified as shown in Fig. 1A (closer to the top ofthe balance), thus allowing the fleaker to be manouvred to and from the balance by the robot.
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Fig. 1. Robotic station (A) coupled to a FI manifold (B) for the determination of nitrate, nitrite and chloride in cured meat products. MLS, master laboratory station; SSOAP, small-size object all-purpose hand; BSOAP, big-size object all-purpose hand; SD, solid dispenser hand; PEC, power and event controller; IV, injection valve; SV, selecting valve; RC, redox column; R, reactor; PC, personal computer; D, detector; W, waste; A, carrier solution; b, and c, reagents.
The following units in the flow manifold are also controlled by the robotic station: The peristaltic pump, the HPLC injector (only its valve is employed for automatic injection of the sample into the FI system), the injection valves used as switching valves and the photometer. The controller also makes absorbance readings at short intervals and stores that which is followed by a lower value; that is the maximum absorbance. In addition, an Agimatic-N stirrer, a coffee grinder and a Technicon stainless steel filter were used. The System V controller was interfaced to a Netset 286/400 personal computer.
2.2. Reagents and solutions 2.2.1. Sample pretreatment A 5% di-sodium tetraborate decahydrate aqueous solution and Carrez reagents (a 15% potassium ferrocyanide aqueous solution and a 30% zinc acetate aqueous solution) are used. 2.2.2. Deri6atisation Manual method [8]. Determination of chloride, a 0.1 N AgNO3 and a 10% K2CrO4 aqueous solution. Determination of nitrate and nitrite, 37 g l − 1 CdSO4 aqueous solution, 5% NH4OH aqueous solution and Zambelli reagent
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(52 ml of 1.18 g ml − 1 HCl aqueous solution in a 100 ml volumetric flask is made to volume with distilled water). This solution is transferred to a 200 ml volumetric flask. Sulphanilic acid (1 g) and 1.5 g of phenol are added in this order while heating to 100°C. The solution is cooled down and made to volume with a saturated NH4Cl aqueous solution. FI method. Determination of nitrate and nitrite, a 0.001 M HAcO+1 g l − 1 Na2EDTA solution is used as carrier. Sulphanylamide solution, a volume of 10 ml of HCl containing 0.95 g of sulphanylamide diluted to 250 ml with distilled water. N-1-naphthylethylenediamine solution, 0.36 g of N-1-naphthylethylene-diamine and 2.9 g of sodium chloride is made to volume with distilled water in a 250 ml volumetric flask. Copperisant solution, a 0.1 M Na2EDTA +0.1% CuSO4 solution. Determination of chloride, 0.86 ml of concentrated HNO3 added to an aqueous solution containing 0.086 g of potassium thiocyanate and 0.169 g of HgCl2 · 2H2O and then 12.650 g of Fe(NO3)3 · 9H2O are added. The mixture is made to volume with distilled water in a 250 ml volumetric flask.
2.2.3. Preparation of the copperised minicolumns FI method. A representative amount (ca. 10 g) of cadium granules are rinsed with a 1 N HCl solution and then with distilled water. The metal is placed in a precipitate vessel where 50 ml of a 0.1% CuSO4 solution is added and then stirred until the blue colour of the solution disappears and then rinsed for 10 min with distilled water in order to remove all the semi-colloidal copper formed. The granules of the treated metal are packed in a cylindrical glass column which is coupled to the FI manifold. Manual method. The column is packed with a bed of glass wool and then 20 – 25 cm of cadmium powder. The packed column is successively rinsed with a 0.1 N HCl solution, distilled water and a 5% NH4OH solution. All reagents were supplied by Merck.
weighed (within 1 mg precision) and introduced into a 250 ml Erlenmeyer flask where 5 ml of 5% di-sodium tetraborate decahydrate solution and about 150 ml of distilled water at 60°C is added. The resultant suspension is maintained warm and stirred for 30 min, after which, it is transferred to a 200 ml volumetric flask and 2 ml of each Carrez reagent is added. The suspension is stirred, let cool down and made to volume. Then, it is filtered and the filtrate transferred to a 250 ml Erlenmeyer flask.
2.3.2. Determination of nitrite Between 5 and 10 ml of filtrate is taken and introduced into a 25 ml volumetric flask. Distilled water ca. 20 ml and 1 ml of Zambelli reagent are added and the solution stirred, then stood at room temperature for 10 min. After this, 1 ml of an ammonia concentrated solution is added and made to volume with distilled water. It is let cool down for a further 10 min and the absorbance of the solution is then measured at 436 nm. 2.3.3. Determination of nitrate The filtrate (20 ml) is transferred to a 25 ml Erlenmeyer flask. NH4OH (5 ml) of a 5% solution is added and the mixture is heated until it starts to softly boil it is then passed through the cadmium column. The eluate is collected in a 100 ml volumetric flask. The column is rinsed for 3 min with hot water and the liquid is added to a volumetric flask which is then made up to volume. The nitrite obtained after this treatment is determined in solution by the method previously described. 2.3.4. Determination of chloride The filtrate (10 ml), ca. 100 ml of distilled water and some drops of a 10% K2CrO4 solution are added to a 250 ml Erlenmeyer flask. This solution is titrated with 0.1 N AgNO3 solution until a red colour appears. 2.4. Proposed procedure
2.3. Manual sample pretreatment and determination 2.3.1. Sample pretreatment The meat sample is ground and homogenised. A representative amount (ca. 10 g) of the sample is
2.4.1. Sample pretreatment The robot gets the BSOAP hand, catches a 250 ml fleaker and tares it. The sample is ground using a coffee grinder connected to an AC output (AC2) of the PEC. After this, the robot takes the SSOAP
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hand, adds the sample into the fleaker until the sample weight is between 10 and 12 g. The controller collects the weight-datum, and the robot takes the fleaker out from the balance, sets it under the dilute and dissolve dispenser, adds 5 ml of 5% sodium tetraborate solution inside the fleaker and then places it on the stirrer. It takes the distilled water dispenser, sets it over the fleaker and adds 150 ml of distilled water which is aspirated from a reservoir in a thermostated bath at 60°C. The controller drives the AC output (AC1) of the PEC, the stirrer is turned on and the sample stirred for 30 min maintaining the temperature at 60°C. The robot carries the fleaker from the stirrer and 2 ml of each Carrez reagent is added and then returns it to the stirrer for 20 s. Now, the robot catches the fleaker, places it in the rack and, once the solution is cooled down adds distilled water until the weight is 200 g, homogenises the solution by stirring, takes the filter, introduces it into the solution and acts on the MLS2 syringe, connected to the filter. The syringe is filled with filtered solution and emptied into a clean fleaker. This operation is repeated twice in order to rinse the filter from stuck particles. The filtered solution is aspirated to the continuous manifold. The hydrodynamic system (FI configuration) for the determination of nitrite and nitrate is shown in Fig. 1B. Valve SV lets the sample merge directly with the reagents for nitrite determination (filling position). In the inject position the sample passes through the reduction column before mixing with the reagents. The original nitrite, and nitrite resulting from the reduction of nitrate are thus determined. The concentration of nitrate is calculated by the difference. The continuous manifold for the determination of nitrate is also used for chloride determination. Sulphanylamide and N-1-naphthylethylenediamine solutions are exchanged by distilled water and chloride reagent solution, respectively. The carrier is distilled water in all instances.
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2.4.3. O6erall procedure Once the filtrate has been acquired by the robot, it is aspirated by means of the aspiration probe connected to the injection valve of the flow injection manifold with the aid of the peristaltic pump. First, the nitrite is determined, then the nitrate, in both cases by interpolation of the peak absorbance of the sample in the corresponding calibration line stored in the computer, and finally the chloride is determined using electronic dilution for collection and data treatment. 2.4.4. Computer program for electronic dilution Two main reasons make the use of a computer program necessary for chloride determination. (1) The wide determination range of this analyte (between 3 and 18% w/w) which makes mandatory signal measurement at times longer than the residence time without significant errors. (2) Management of the huge numbers of data obtained in routine analysis of a large series of samples (up to 400 sample day − 1). Fig. 2 shows the flow-chart of the designed program. Signal–time data is collected by the computer at preset intervals from the maximum of the peak. This data is stored either for individual samples or batch samples. In the former case the computer works like a recorder. In the latter case the time at which the absorbance is measured is selected when the data from all the samples have been acquired. Depending on the concentration of the target sample, the time selected can correspond to the maximum or a longer time in the tail of the fiagram where the absorbance is lower due to a higher dispersion. Once the time is selected, the computer provides the absorbance signal, which is imported to a LOTUS chart from which the calibration line corresponding to the selected time is run and the absorbance of the sample interpolated.
3. Results and discussion
2.4.2. Calibration This is accomplished by running a calibration curve with seven standards every week. Three standards are inserted every day in order to correct the slope, if necessary.
3.1. Chemical systems The determination of nitrite is based on the Griess reaction, adopting Shin’s modification [9]
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Fig. 2. Flow-chart of the computer program for electronic dilution.
in order to avoid the manipulation of carcinogenic reagents. The determination of nitrate is based on the same derivatisation reaction after reduction to nitrite. The determination of chloride is based on displacement of thiocyanate in the mercury–thiocyanate complex. Then, the displaced thiocyanate forms a red complex with ferric ion, which is monitored at 480 nm. As the overall process consists of two parts (discontinuous or robotic and continuous or FI), the optimisation of each was accomplished separately.
3.2. Discontinuous or robotic stage: sample pretreatment A computer program was developed to control the different unitary operations (UOs) carried out by the robotic station. The performance of both the program and the different UOs was then checked. In order to obtain a ground sample suitable for handling by the robot (not sticky mass but di-
vided in small particles) the grinding time must be short (about 10 s is sufficient). The solutions were made to weight (as described in Section 2.4) rather than to volume as the robot was a sensorless device.
3.3. Determination step Detailed methods for the determination of nitrite, nitrate and chloride by flow techniques are abundant in the literature. The ruggedness of the method for chloride is guaranteed because of its routine use since 1960 in segmented-flow analysers, and since 1975 in FI. As the aim of this research was focused on overall automation rather than the development of new determination methods, some variables such as reagent concentration, diameter of the tubing system, length of reactors and geometry of the reduction column were not optimised and the values developed by our research team in previous studies [10–12] were used (see Table 1).
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Table 1 Optimum values of variables for the determination of NO− 2 , − NO− 3 and Cl
triplicate was lower than 1.5% in all instances. The sampling frequency was 30 h − 1.
Parameter
Optimum value
− Injection volume (NO− 2 /NO3 ) (ml) − Injection volume (Cl ) (ml) Flow-rate (ml min−1) R1 length (cm) R2 length (cm) Column length (cm) Column diameter (mm)
100 10 1.1 15 200 8.5 3
3.4.2. Chloride determination The characteristics of this system makes it necessary to run only one calibration line. The so called electronic dilution was used in order to adapt the method to the usual wide range of concentrations of this anion in meat (between 3 and 18% (w/w)). The data was acquired and processed using a computer program designed in our laboratory (see flow-chart in Fig. 2). Standards were prepared in a wide range of concentrations. The features of the calibration lines obtained at different times from the maximum of the transient signal are listed in Table 2(B).
3.4. Features of the methods 3.4.1. Nitrite and nitrate determination This method requires running three calibration lines. One for nitrate and two for nitrite (with and without passage of the sample through the reduction minicolumn). A series of solutions were prepared from solutions of both anions within a wide range of concentrations and subjected to sample pretreatment to minimise matrix effects. The data treatment gives the values shown in Table 2(A). Linear ranges between 0.1 – 15 mg l − 1 for nitrite and between 0.5 and 30 mg l − 1 for nitrate were obtained. These ranges are appropriate for the determination of these anions in meat. The RSD obtained for 11 samples of 5 mg l − 1 injected in
3.5. Applications of the method to meat samples The proposed method was validated by applying it to 10 samples of fresh and cured meat from Navidul.The content of the samples was also determined using the manual method. In both cases the concentrations are referred to the filtrated solution. The excellent agreement between both approaches is shown in Table 3, where the precision of the proposed method for each sample, expressed as SD, and that of the manual method, expressed as a reproducibility study during a
Table 2 Features of the methods Anion
Linear range (mg l−1)
Calibration line Slope
Intercept
− (A) Determination of NO− 3 and NO2 −b NO2 0.1–15 NO−c 0.1–30 2 NO− 0.5–30 3
0.178 0.065 0.022
0.012 0.033 0.005
(B) Determination of Cl− t (s)d 0 5–1000 20 6–1000 30 100–2000 400 500–4000
1.473 1.490 0.300 0.079
a
63.5 49.3 34.8 84.8
r
RSD(%)
0.999 0.999 0.999
1.20a 1.42a 1.31a
0.99 0.99 0.99 0.99
0.18 2.77 3.77 3.92
Values calculated from triplicate injection of 11 samples containing 5 mg l−1 of the target analyte. Without passage through the minicolumn. c With passage through the minicolumn. d Time from the maximum b
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Table 3 − − in meat Determination of NO− 2 , NO3 and Cl Samples
Concentration found Manual methoda
Proposed method
1 2 3 4 5 6 7 8 9 10
−1 NO− 2 mg l
−1 NO− 3 mg l
Cl− g l−1
−1 NO− 2 mg l
−1 NO− 3 mg l
Cl− g l−1
3.29 0.2 2.29 0.1 3.4 9 0.2 3.8 9 0.2 3.6 9 0.3 2.7 9 0.2 2.59 0.1 1.9 9 0.1 5.3 9 0.3 3.7 9 0.2
46.29 1.3 37.09 1.2 49.29 0.6 48.59 0.9 41.49 1.2 25.49 1.0 48.89 1.3 44.99 0.8 35.19 1.0 47.19 1.1
3.55 9 0.05 3.35 90.10 3.15 9 0.05 3.20 9 0.05 3.85 90.15 3.35 9 0.10 3.50 9 0.10 2.85 9 0.15 3.50 9 0.05 3.35 9 0.05
3.1 2.3 3.4 2.7 2.6 2.7 3.8 2.7 3.5 3.3
45.4 33.2 42.0 47.7 35.1 23.4 58.9 41.1 28.9 46.7
3.65 3.25 3.05 3.25 3.60 3.35 3.50 2.85 3.50 3.35
a
− − −1 The 1-month reproducibility (n=7) was as follows: RSD (NO− , 2 ) =5.5%; RSD (NO3 ) =3.5%; RSD (Cl ) =0.5% for 1.4 mg l 3.1 mg l−1 and 3.55 g l−1, respectively.
month, demonstrated that the automatic method is more precise than its manual counterpart.
the elimination of human intervention which makes a 24 h working-day feasible.
4. Conclusions
Acknowledgements
A completely automated method for the determination of nitrite, nitrate and chloride in cured meat based on the coupling of discontinuous (robotic)/continuous (flow injection) automated alternatives is proposed. The assembly works in an unattended fashion thus eliminating human intervention with results in a subsequent decrease in the overload created in routine laboratories. The method also allows a reduction in analysis time, as the conventional method involves a number of steps which are deleted in the automated counterpart. This shortening in the time required for the overall process is mainly due to the faster development of the derivatisation/monitoring step, which takes place in non-equilibrium conditions characteristic of FI methods. A decrease in the time required for this step from 20 to 3 min was achieved. The time required for the development of the robotic preliminary operations is similar to that required by the conventional method, the most significant advantage of its implementation being
The authors are grateful to the Spanish Direccio´n General de Investigacio´n Cientı´fica y Te´cnica (DGICyT) for financial support in the form of Grant PB96/0505.
References [1] K. Mo¨hler, El curado, Editorial Acribia, Zaragoza, Spain, 1982. [2] L. To´th, Fleischwirtsch 63 (1993) 208. [3] S.H. Lee, R.G. Cassens, O.R. Fenneman, J. Food Sci. 41 (1976) 969. [4] W. Vo¨sgen, Fleischwirtsch 72 (1992) 1675. [5] J. Ruzicka, Philos. Trans. R. Soc. London 5 (1982) 645. [6] J. Ruzicka, E.H. Hansen, Anal. Chim. Acta 145 (1983) 1. [7] S. Olsen, J. Ruzicka, E.H. Hansen, Anal. Chim. Acta 136 (1982) 101. [8] N.W. Hanson (Ed.), Official, Stantardised and Recommended Methods of Analysis, The Society for Analytical Chemistry, 2nd ed. London, 1973, pp. 155 and 158–160. [9] M.B. Shinn, Ind. Eng. Chem. Anal. Educ. 13 (1941) 33. [10] F. Can˜ete, A. Rı´os, M.D. Luque de Castro, M. Valca´rcel, Analyst 113 (1988) 739. [11] B. Bermu´dez, A. Rı´os, M.D. Luque de Castro, M. Valca´rcel, Talanta 35 (1988) 810. [12] J.S. Cosano, J.L. Calle, J.L. Pinillos, P. Linares, M.D. Luque de Castro, Te´cn. Lab. 147 (1989) 32.
Full automated robotic method for the determination of chloride, nitrite and nitrate in cured meat products A. Velasco-Arjona a, J.A. Garcı´a-Garrido b, R. Quiles-Zafra b, M.D. Luque de Castro a,* a
Department of Analytical Chemistry, Faculty of Sciences, Uni6ersity of Co´rdoba, E-14004 Co´rdoba, Spain b Research Department, Na6idul S.A., E-45500 Torrijos, Toledo, Spain Received 6 June 1997; received in revised form 24 September 1997; accepted 2 October 1997
Abstract A completely automated method to determine the most common parameters in cured meat products is proposed. The approach to full automation is based on the coupling of a robotic station for development of preliminary operations (namely weighing of the sample, grinding, leaching, filtration and transport to the aspiration zone) and a continuous unsegmented manifold for derivatisation and spectrophotometric monitoring of the reaction coloured products. This assembly works in an unattended fashion thus eliminating the bottleneck produced by the determination of these parameters in routine laboratories. The good agreement between results obtained by the proposed method and those from conventional methods for target analytes confirms its excellent performance and usefulness. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Robotic; Chloride–nitrite–nitrate; Meat; Continuous flow
1. Introduction Curing is one of the most widely used technologies in meat manufacturing. The different curing modes (namely dry curing, wet curing, injection curing [1]) are based on the same principle, addition of a mixture of sodium chloride, either nitrite or nitrite plus nitrate salts to the fresh product. Even though the use of these curing agents involve some minor disadvantages, the advantages are so overwhelming [2] that no alternatives are * Corresponding author. Tel.: +34 57 218615; fax: + 34 57 218606; e-mail: [email protected]
available at present. In the case of nitrite and nitrate, some of the most remarkable beneficial aspects of the curing process [3] are as follows; appearance of a red colour due to a reaction between muscular hemoglobin and the nitric oxide generated in nitrite reduction; reaction of nitrite and different compounds such as sugars, alcohols, amines, etc. to yield products which contribute to the typical aroma of meat cured products; antioxidant effect on lipid rancidity; antimicrobial activity, which hinders the growth of pathogenic microorganisms, such as C. Botulinum. The negative effects of curing anions only appear in products with excess amounts of
0039-9140/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0039-9140(97)00349-4
970
A. Velasco-Arjona et al. / Talanta 46 (1998) 969–976
these agents [4]. Some of the well-known undesirable effects are; the oxidant action of nitrite on the hemoglobin – Fe(II) complex, which can produce poisonous symptoms; formation of N-nitrosocompounds in the presence of both nitrosamine formers and acid pH; and the toxic action of nitrate, not per se but due to reduction to nitrite. For these reasons, the routine analysis of nitrite and nitrate is common practice in the meat industries in order to keep their levels within a range which ensures the development of the beneficial effects without the toxic aspects. The presence of sodium chloride in the curing process is advantageous, its protective effect hinders or makes it difficult for germ development by decreasing water activity; its conserving action by potentiating the effects of other preservant agents and inhibiting fungi and yeast proliferation, it also increases flavour. High salt concentration in cured products results in consumer rejection. We have developed a fully automated method based on the coupling of a robotic station for sample weighing and pretreatment and a continuous flow system for derivatisation and detection in order to help big meat industries in the development of routine determinations of these three more commonly determined parameters.
2. Experimental section
2.1. Instruments and apparatus The flow injection (FI) manifold was built using a Gilson Minipuls-HP3 peristaltic pump, a Rheodyne 5041 injection valve (adapted for use as switching valve), reduction columns of copperised cadmium of different lengths (1.8 mm i.d.) and a Pye Unicam SP6-500 spectrophotometer furnished with a Hellma 178. 12QS flow-cell and connected to the AD (Analog-Digital) converter of the PEC via its analog output for data acquisition, processing and delivery of the results via a computer program designed in our laboratory, which permits the use of so-called electronic dilution FIA technique [5 – 7]. All tubing used to construct the hydrodynamic system was of PTFE of 0.5 mm i.d.
The robotic station consisted of a Zymate II Plus robot (Zymark, Hopkinton), a System V controller, and the following peripherals.
2.1.1. Master laboratory station (MLS) The proposed method uses two MLSs (MLS1 and MLS2), which consist of three syringes each, intended to dispense liquids in conjunction with the dilute and dissolve unit. Only two of the syringes of MLS1 were used, one of them was connected to a water bath and the other to a filter. Both were also connected to a dispenser; one of the syringe-dispenser assembly was used to dispense water; the other one to dispense filtered solution. 2.1.2. Small-size object all-purpose (SSOAP), big-size object all-purpose (BSOAP) and solid dispenser (SD) hands The SSOAP hand allows the robot to seize centrifuge tubes and objects of similar size. The BSOAP hand allows the robot to seize 250 ml fleakers and objects of similar size. The SD hand (designed by the authors) allows the robot to manipulate solid samples. This hand is installed on the SSOAP hand forming 180° with its fingers and consists of a pair of tweezers which have a basket at their ends (see details in Fig. 1A). 2.1.3. Power and e6ent controller (PEC) The PEC module acts as an interface between the controller, peripherals and robot. 2.1.4. System V controller This module sends orders to the rest of the units, including the robot and controls additional units such as a peristaltic pump, a photometer, switching valves, etc. The RS232 outputs of these units allow them to be operated by the System V via the PEC. 2.1.5. Balance The balance plate for tube weighing was modified as shown in Fig. 1A (closer to the top ofthe balance), thus allowing the fleaker to be manouvred to and from the balance by the robot.
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Fig. 1. Robotic station (A) coupled to a FI manifold (B) for the determination of nitrate, nitrite and chloride in cured meat products. MLS, master laboratory station; SSOAP, small-size object all-purpose hand; BSOAP, big-size object all-purpose hand; SD, solid dispenser hand; PEC, power and event controller; IV, injection valve; SV, selecting valve; RC, redox column; R, reactor; PC, personal computer; D, detector; W, waste; A, carrier solution; b, and c, reagents.
The following units in the flow manifold are also controlled by the robotic station: The peristaltic pump, the HPLC injector (only its valve is employed for automatic injection of the sample into the FI system), the injection valves used as switching valves and the photometer. The controller also makes absorbance readings at short intervals and stores that which is followed by a lower value; that is the maximum absorbance. In addition, an Agimatic-N stirrer, a coffee grinder and a Technicon stainless steel filter were used. The System V controller was interfaced to a Netset 286/400 personal computer.
2.2. Reagents and solutions 2.2.1. Sample pretreatment A 5% di-sodium tetraborate decahydrate aqueous solution and Carrez reagents (a 15% potassium ferrocyanide aqueous solution and a 30% zinc acetate aqueous solution) are used. 2.2.2. Deri6atisation Manual method [8]. Determination of chloride, a 0.1 N AgNO3 and a 10% K2CrO4 aqueous solution. Determination of nitrate and nitrite, 37 g l − 1 CdSO4 aqueous solution, 5% NH4OH aqueous solution and Zambelli reagent
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(52 ml of 1.18 g ml − 1 HCl aqueous solution in a 100 ml volumetric flask is made to volume with distilled water). This solution is transferred to a 200 ml volumetric flask. Sulphanilic acid (1 g) and 1.5 g of phenol are added in this order while heating to 100°C. The solution is cooled down and made to volume with a saturated NH4Cl aqueous solution. FI method. Determination of nitrate and nitrite, a 0.001 M HAcO+1 g l − 1 Na2EDTA solution is used as carrier. Sulphanylamide solution, a volume of 10 ml of HCl containing 0.95 g of sulphanylamide diluted to 250 ml with distilled water. N-1-naphthylethylenediamine solution, 0.36 g of N-1-naphthylethylene-diamine and 2.9 g of sodium chloride is made to volume with distilled water in a 250 ml volumetric flask. Copperisant solution, a 0.1 M Na2EDTA +0.1% CuSO4 solution. Determination of chloride, 0.86 ml of concentrated HNO3 added to an aqueous solution containing 0.086 g of potassium thiocyanate and 0.169 g of HgCl2 · 2H2O and then 12.650 g of Fe(NO3)3 · 9H2O are added. The mixture is made to volume with distilled water in a 250 ml volumetric flask.
2.2.3. Preparation of the copperised minicolumns FI method. A representative amount (ca. 10 g) of cadium granules are rinsed with a 1 N HCl solution and then with distilled water. The metal is placed in a precipitate vessel where 50 ml of a 0.1% CuSO4 solution is added and then stirred until the blue colour of the solution disappears and then rinsed for 10 min with distilled water in order to remove all the semi-colloidal copper formed. The granules of the treated metal are packed in a cylindrical glass column which is coupled to the FI manifold. Manual method. The column is packed with a bed of glass wool and then 20 – 25 cm of cadmium powder. The packed column is successively rinsed with a 0.1 N HCl solution, distilled water and a 5% NH4OH solution. All reagents were supplied by Merck.
weighed (within 1 mg precision) and introduced into a 250 ml Erlenmeyer flask where 5 ml of 5% di-sodium tetraborate decahydrate solution and about 150 ml of distilled water at 60°C is added. The resultant suspension is maintained warm and stirred for 30 min, after which, it is transferred to a 200 ml volumetric flask and 2 ml of each Carrez reagent is added. The suspension is stirred, let cool down and made to volume. Then, it is filtered and the filtrate transferred to a 250 ml Erlenmeyer flask.
2.3.2. Determination of nitrite Between 5 and 10 ml of filtrate is taken and introduced into a 25 ml volumetric flask. Distilled water ca. 20 ml and 1 ml of Zambelli reagent are added and the solution stirred, then stood at room temperature for 10 min. After this, 1 ml of an ammonia concentrated solution is added and made to volume with distilled water. It is let cool down for a further 10 min and the absorbance of the solution is then measured at 436 nm. 2.3.3. Determination of nitrate The filtrate (20 ml) is transferred to a 25 ml Erlenmeyer flask. NH4OH (5 ml) of a 5% solution is added and the mixture is heated until it starts to softly boil it is then passed through the cadmium column. The eluate is collected in a 100 ml volumetric flask. The column is rinsed for 3 min with hot water and the liquid is added to a volumetric flask which is then made up to volume. The nitrite obtained after this treatment is determined in solution by the method previously described. 2.3.4. Determination of chloride The filtrate (10 ml), ca. 100 ml of distilled water and some drops of a 10% K2CrO4 solution are added to a 250 ml Erlenmeyer flask. This solution is titrated with 0.1 N AgNO3 solution until a red colour appears. 2.4. Proposed procedure
2.3. Manual sample pretreatment and determination 2.3.1. Sample pretreatment The meat sample is ground and homogenised. A representative amount (ca. 10 g) of the sample is
2.4.1. Sample pretreatment The robot gets the BSOAP hand, catches a 250 ml fleaker and tares it. The sample is ground using a coffee grinder connected to an AC output (AC2) of the PEC. After this, the robot takes the SSOAP
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hand, adds the sample into the fleaker until the sample weight is between 10 and 12 g. The controller collects the weight-datum, and the robot takes the fleaker out from the balance, sets it under the dilute and dissolve dispenser, adds 5 ml of 5% sodium tetraborate solution inside the fleaker and then places it on the stirrer. It takes the distilled water dispenser, sets it over the fleaker and adds 150 ml of distilled water which is aspirated from a reservoir in a thermostated bath at 60°C. The controller drives the AC output (AC1) of the PEC, the stirrer is turned on and the sample stirred for 30 min maintaining the temperature at 60°C. The robot carries the fleaker from the stirrer and 2 ml of each Carrez reagent is added and then returns it to the stirrer for 20 s. Now, the robot catches the fleaker, places it in the rack and, once the solution is cooled down adds distilled water until the weight is 200 g, homogenises the solution by stirring, takes the filter, introduces it into the solution and acts on the MLS2 syringe, connected to the filter. The syringe is filled with filtered solution and emptied into a clean fleaker. This operation is repeated twice in order to rinse the filter from stuck particles. The filtered solution is aspirated to the continuous manifold. The hydrodynamic system (FI configuration) for the determination of nitrite and nitrate is shown in Fig. 1B. Valve SV lets the sample merge directly with the reagents for nitrite determination (filling position). In the inject position the sample passes through the reduction column before mixing with the reagents. The original nitrite, and nitrite resulting from the reduction of nitrate are thus determined. The concentration of nitrate is calculated by the difference. The continuous manifold for the determination of nitrate is also used for chloride determination. Sulphanylamide and N-1-naphthylethylenediamine solutions are exchanged by distilled water and chloride reagent solution, respectively. The carrier is distilled water in all instances.
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2.4.3. O6erall procedure Once the filtrate has been acquired by the robot, it is aspirated by means of the aspiration probe connected to the injection valve of the flow injection manifold with the aid of the peristaltic pump. First, the nitrite is determined, then the nitrate, in both cases by interpolation of the peak absorbance of the sample in the corresponding calibration line stored in the computer, and finally the chloride is determined using electronic dilution for collection and data treatment. 2.4.4. Computer program for electronic dilution Two main reasons make the use of a computer program necessary for chloride determination. (1) The wide determination range of this analyte (between 3 and 18% w/w) which makes mandatory signal measurement at times longer than the residence time without significant errors. (2) Management of the huge numbers of data obtained in routine analysis of a large series of samples (up to 400 sample day − 1). Fig. 2 shows the flow-chart of the designed program. Signal–time data is collected by the computer at preset intervals from the maximum of the peak. This data is stored either for individual samples or batch samples. In the former case the computer works like a recorder. In the latter case the time at which the absorbance is measured is selected when the data from all the samples have been acquired. Depending on the concentration of the target sample, the time selected can correspond to the maximum or a longer time in the tail of the fiagram where the absorbance is lower due to a higher dispersion. Once the time is selected, the computer provides the absorbance signal, which is imported to a LOTUS chart from which the calibration line corresponding to the selected time is run and the absorbance of the sample interpolated.
3. Results and discussion
2.4.2. Calibration This is accomplished by running a calibration curve with seven standards every week. Three standards are inserted every day in order to correct the slope, if necessary.
3.1. Chemical systems The determination of nitrite is based on the Griess reaction, adopting Shin’s modification [9]
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Fig. 2. Flow-chart of the computer program for electronic dilution.
in order to avoid the manipulation of carcinogenic reagents. The determination of nitrate is based on the same derivatisation reaction after reduction to nitrite. The determination of chloride is based on displacement of thiocyanate in the mercury–thiocyanate complex. Then, the displaced thiocyanate forms a red complex with ferric ion, which is monitored at 480 nm. As the overall process consists of two parts (discontinuous or robotic and continuous or FI), the optimisation of each was accomplished separately.
3.2. Discontinuous or robotic stage: sample pretreatment A computer program was developed to control the different unitary operations (UOs) carried out by the robotic station. The performance of both the program and the different UOs was then checked. In order to obtain a ground sample suitable for handling by the robot (not sticky mass but di-
vided in small particles) the grinding time must be short (about 10 s is sufficient). The solutions were made to weight (as described in Section 2.4) rather than to volume as the robot was a sensorless device.
3.3. Determination step Detailed methods for the determination of nitrite, nitrate and chloride by flow techniques are abundant in the literature. The ruggedness of the method for chloride is guaranteed because of its routine use since 1960 in segmented-flow analysers, and since 1975 in FI. As the aim of this research was focused on overall automation rather than the development of new determination methods, some variables such as reagent concentration, diameter of the tubing system, length of reactors and geometry of the reduction column were not optimised and the values developed by our research team in previous studies [10–12] were used (see Table 1).
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Table 1 Optimum values of variables for the determination of NO− 2 , − NO− 3 and Cl
triplicate was lower than 1.5% in all instances. The sampling frequency was 30 h − 1.
Parameter
Optimum value
− Injection volume (NO− 2 /NO3 ) (ml) − Injection volume (Cl ) (ml) Flow-rate (ml min−1) R1 length (cm) R2 length (cm) Column length (cm) Column diameter (mm)
100 10 1.1 15 200 8.5 3
3.4.2. Chloride determination The characteristics of this system makes it necessary to run only one calibration line. The so called electronic dilution was used in order to adapt the method to the usual wide range of concentrations of this anion in meat (between 3 and 18% (w/w)). The data was acquired and processed using a computer program designed in our laboratory (see flow-chart in Fig. 2). Standards were prepared in a wide range of concentrations. The features of the calibration lines obtained at different times from the maximum of the transient signal are listed in Table 2(B).
3.4. Features of the methods 3.4.1. Nitrite and nitrate determination This method requires running three calibration lines. One for nitrate and two for nitrite (with and without passage of the sample through the reduction minicolumn). A series of solutions were prepared from solutions of both anions within a wide range of concentrations and subjected to sample pretreatment to minimise matrix effects. The data treatment gives the values shown in Table 2(A). Linear ranges between 0.1 – 15 mg l − 1 for nitrite and between 0.5 and 30 mg l − 1 for nitrate were obtained. These ranges are appropriate for the determination of these anions in meat. The RSD obtained for 11 samples of 5 mg l − 1 injected in
3.5. Applications of the method to meat samples The proposed method was validated by applying it to 10 samples of fresh and cured meat from Navidul.The content of the samples was also determined using the manual method. In both cases the concentrations are referred to the filtrated solution. The excellent agreement between both approaches is shown in Table 3, where the precision of the proposed method for each sample, expressed as SD, and that of the manual method, expressed as a reproducibility study during a
Table 2 Features of the methods Anion
Linear range (mg l−1)
Calibration line Slope
Intercept
− (A) Determination of NO− 3 and NO2 −b NO2 0.1–15 NO−c 0.1–30 2 NO− 0.5–30 3
0.178 0.065 0.022
0.012 0.033 0.005
(B) Determination of Cl− t (s)d 0 5–1000 20 6–1000 30 100–2000 400 500–4000
1.473 1.490 0.300 0.079
a
63.5 49.3 34.8 84.8
r
RSD(%)
0.999 0.999 0.999
1.20a 1.42a 1.31a
0.99 0.99 0.99 0.99
0.18 2.77 3.77 3.92
Values calculated from triplicate injection of 11 samples containing 5 mg l−1 of the target analyte. Without passage through the minicolumn. c With passage through the minicolumn. d Time from the maximum b
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Table 3 − − in meat Determination of NO− 2 , NO3 and Cl Samples
Concentration found Manual methoda
Proposed method
1 2 3 4 5 6 7 8 9 10
−1 NO− 2 mg l
−1 NO− 3 mg l
Cl− g l−1
−1 NO− 2 mg l
−1 NO− 3 mg l
Cl− g l−1
3.29 0.2 2.29 0.1 3.4 9 0.2 3.8 9 0.2 3.6 9 0.3 2.7 9 0.2 2.59 0.1 1.9 9 0.1 5.3 9 0.3 3.7 9 0.2
46.29 1.3 37.09 1.2 49.29 0.6 48.59 0.9 41.49 1.2 25.49 1.0 48.89 1.3 44.99 0.8 35.19 1.0 47.19 1.1
3.55 9 0.05 3.35 90.10 3.15 9 0.05 3.20 9 0.05 3.85 90.15 3.35 9 0.10 3.50 9 0.10 2.85 9 0.15 3.50 9 0.05 3.35 9 0.05
3.1 2.3 3.4 2.7 2.6 2.7 3.8 2.7 3.5 3.3
45.4 33.2 42.0 47.7 35.1 23.4 58.9 41.1 28.9 46.7
3.65 3.25 3.05 3.25 3.60 3.35 3.50 2.85 3.50 3.35
a
− − −1 The 1-month reproducibility (n=7) was as follows: RSD (NO− , 2 ) =5.5%; RSD (NO3 ) =3.5%; RSD (Cl ) =0.5% for 1.4 mg l 3.1 mg l−1 and 3.55 g l−1, respectively.
month, demonstrated that the automatic method is more precise than its manual counterpart.
the elimination of human intervention which makes a 24 h working-day feasible.
4. Conclusions
Acknowledgements
A completely automated method for the determination of nitrite, nitrate and chloride in cured meat based on the coupling of discontinuous (robotic)/continuous (flow injection) automated alternatives is proposed. The assembly works in an unattended fashion thus eliminating human intervention with results in a subsequent decrease in the overload created in routine laboratories. The method also allows a reduction in analysis time, as the conventional method involves a number of steps which are deleted in the automated counterpart. This shortening in the time required for the overall process is mainly due to the faster development of the derivatisation/monitoring step, which takes place in non-equilibrium conditions characteristic of FI methods. A decrease in the time required for this step from 20 to 3 min was achieved. The time required for the development of the robotic preliminary operations is similar to that required by the conventional method, the most significant advantage of its implementation being
The authors are grateful to the Spanish Direccio´n General de Investigacio´n Cientı´fica y Te´cnica (DGICyT) for financial support in the form of Grant PB96/0505.
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