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Multicommutated flow system for the determination of glucose in honey with immobilized glucose oxidase reactor and spectrophotometric detection
Talanta 77 (2009) 1534–1538
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Multicommutated flow system for the determination of glucose in honey with immobilized glucose oxidase reactor and spectrophotometric detection Alexandra Sixto, Moisés Knochen ∗ Universidad de la República, Facultad de Química, Cátedra de Química Analítica. Av. Gral. Flores 2124, 11800 Montevideo, Uruguay
a r t i c l e
i n f o
Article history: Received 7 August 2008 Received in revised form 19 September 2008 Accepted 22 September 2008 Available online 27 September 2008 Keywords: Multicommutated flow system Glucose oxidase Honey Immobilized
a b s t r a c t A new automated method for the determination of glucose in honey is proposed. The method is based on multicommutated flow analysis (MCFA) and employs an immobilized glucose oxidase reactor and spectrophotometric detection at 505 nm of the red quinoneimine formed (Trinder’s method). The calibration curve obeyed a second order equation in the range 0–0.14 g L−1 (h = −2.2199 C2 + 1.3741C + 0.0077, r2 = 0.9991, where h is the peak height (absorbance) and C the concentration in g L−1 ). The method was validated analyzing eight commercial samples, both by the AOAC 954.11 and 977.20 official methods. According to Student’s t-test of mean values, at the confidence level of 95% the results obtained with the proposed method were in agreement with those obtained by the official methods. Precision (sr (%), n = 10) was 3% and the sampling frequency of the system was 20 samples h−1 . © 2008 Elsevier B.V. All rights reserved.
1. Introduction Sugars are the principal constituents of honey. Aside from determining its nutritious and energetic value, they influence some of its important physical characteristics. Glucose is the monosaccharide responsible for the crystallization; honeys with high fructose content and low glucose content are less susceptible to crystallization, which is a phenomenon that decreases the acceptation of this natural product by the consumers. Honey acidity is also strongly related to the glucose content, and the rate fructose/glucose is related to the genuinity, a value below 1 indicating adulteration or the proliferation of osmophiles yeast [1]. The determination of sugars in honey may be achieved by classical procedures, such as those specified in the AOAC standard method [2]; these methods are usually slow and tedious, involving the isolation of the analyte from the matrix. In the aforementioned AOAC method this is carried out by means of classical chromatography involving a column filled with activated carbon and Celite® followed by two different volumetric titrations. The column separation step takes no less than 2 h per sample to which the time necessary for two volumetries should be added. Alternatively glucose may be determined by HPLC with a refractive-index detector [3].
∗ Corresponding author. Tel.: +598 2 9241808. E-mail address: [email protected] (M. Knochen). 0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.09.030
A simpler way to achieve the desired selectivity is employing an enzymatic reaction that is inherently selective. Most common selective procedures for the determination of glucose are based on the catalytic activity of glucose oxidase. Conventional enzymatic methods are costly due to the high consumption of enzymatic reagent, but this disadvantage has been successfully overcome by using immobilized enzymes. This strategy has been widely used in flow injection analysis (FIA) [4,5] where a number of papers have been published describing the use of microcolumns filled with immobilized enzymes as a part of flow systems [6–14]. Various alternatives such as physical adsorption, microencapsulation in membranes, sol–gel entrapment, cross linking or covalent attachment have been explored in the design of suitable flow-through packed-bed, open-tubular reactors or optical/electrochemical biosensors. The advantage of employing a column reactor in comparison to integrated biosensors is the ability of entrapping a much large amount of enzyme. A different flow-based approach is sequential injection analysis (SIA) [15,16]. This technique has been developed to address some drawbacks of FIA, namely the high consumption of reagents and the use of multi-channel manifolds. Methods exploiting this technique for the determination of glucose employing enzymes, have been reported [17,18]. More recently, a different flow technique called multicommutated flow analysis (MCFA) was introduced [19–21]. MCFA shows interesting advantages for automation. It is based on flow systems, where a number of solenoid valves, acting as independent commutators configure a flow network. Solenoid valves are independently
A. Sixto, M. Knochen / Talanta 77 (2009) 1534–1538
actuated under computer control. MCFA allows the implementation of binary sampling [19], a sampling technique where short segments of sample, reagent and carrier are inserted in the flow path. This approach allows modifications of the sample and reagent volumes, as well as extensive modifications of the flow path to be easily carried out under computer control avoiding the need of modifying physically the connections. Multicommutated flow analysis presents some advantages such as a high flexibility and a low investment cost since solenoid valves are inexpensive. This technique has been used successfully in the determination of glucose using enzymes in solution [22]. However, the authors did not found in the literature, previous work dealing with the use of this technique for the analysis of honey. Thus it was considered that the use of multicommutated flow analysis could be useful for the implementation of a novel automated system for the analysis of honey taking advantage of the high speed, flexibility and analytical throughput attained. In this work, a system based on the concept of multicommutated flow analysis was developed for the selective automated determination of glucose in honey. The method was based on work by Trinder [23], exploiting the following reactions: GOD
1535
2.3. Instrumentation and software A four-channel peristaltic pump (Alitea (Stockholm, Sweden) C8/2-XV), fitted with Tygon tubing was used for pumping the carrier, samples and reagents. Commutation was achieved by means of NResearch (West Caldwell, NJ, USA) 161T031 3-way 12 V solenoid valves. These valves were controlled using the individual data bits of the LPT1 parallel port of the computer via a CoolDriveTM driver (NResearch). Reaction coils and connections were made from 0.8 mm (internal diameter) Teflon PFA tubing. A thermostatic water bath set at 37 ◦ C was used to keep the temperature of the enzyme reactor constant throughout the experiments. The software used to control the multicommutated system was compiled in QuickBASIC 4.0 running under MS-DOS 6.0 in a notebook computer. The detection system was composed of a Shimadzu (Kyoto, Japan) UV-240 recording spectrophotometer fitted with an 80 L quartz flow cell (Hellma, Müllheim, Germany) and operated at 505 nm in the time scan mode. Recordings of the signals and peak height measurements were obtained from the graphic recorderprinter of the instrument.
glucose + O2 + H2 O−→gluconic acid + H2 O2 2H2 O2 + 4-aminoantipyrine + 4-hydroxybenzoate POD
−→quinoneimine where GOD stands for glucose oxidase and POD for peroxidase. The quinoneimine formed was detected by its absorption at 505 nm. Glucose oxidase was immobilized in column reactors via Schiff base formation. 2. Experimental 2.1. Reagents Glucose oxidase, Type II-S from Aspergillus niger, 39800 units g−1 solid (Sigma). Peroxidase, Type I, from Horseradish, 113 Purpurogallin units mg−1 solid (Sigma). Aminopropyl-glass beads (170 A, 200–400 mesh, synthesized in the laboratory) were obtained from Professor O. Fatibello-Filho. The following reagents were of analytical reagent grade unless otherwise specified: d-glucose anhydrous (Anedra), dfructose (Sigma, glucose <0.05%), dibasic potassium phosphate (Baker), monobasic potassium phosphate (Fluka), sodium 4hydroxybenzoate (Sigma), 4-aminoantipyrine hydrochloride (Eastman Organic Chemicals), glutardialdehyd 50% solution in water (Sigma–Aldrich), Peroxidase (POD) solution was prepared by accurately weighing 1.0 mg of peroxidase and dissolving in 100 mL of 0.1 mol L−1 phosphate buffer, pH 7. The chromogenic reagent was prepared by dissolving 0.6 g of 4-aminoantipyrine and 9.6 g of sodium 4-hydroxybenzoate in water and diluting with water to 1 L. 2.2. Preparation of the immobilized enzyme Glucose oxidase (GOD) was immobilized onto amino-propyl glass beads using the procedure described by Manera et al. [14]. The reactor was prepared by packing a 40 mm × 2.48 mm internal diameter piece of polytetrafluoroethylene (PTFE) tubing with the immobilized GOD glass beads. Polyethylene frits were used to embed the reactive phase. When not in use the column was stored at 4 ◦ C in 0.1 mol L−1 phosphate buffer (pH 7).
2.4. Multicommutated flow system The multicommutated system (Fig. 1a) consisted of a peristaltic pump (P), four 3-way solenoid valves (V1 –V4 ), a column (C) in a thermostatic bath (TB), a reaction coil (R), and a spectrophotometer used as detector (D). Valve V1 was used for the introduction of the sample, V2 for the carrier (0.1 mol L−1 phosphate buffer, pH 7), V3 for the peroxidase solution and V4 for the mixture of 4-hydroxybenzoate and 4-aminoantipyrine solutions. When not being introduced to the system reagents were recycled to the respective bottles. The operation of the system comprised an analytical cycle, when the same sample (or standard) was repeatedly injected and measured. When the software was run, it requested the control parameters. Afterwards it sent the control commands through the LPT1 port to switch valves V1 –V4 on or off, following the switching pattern depicted in the valves timing diagram in Fig. 1b. At the beginning of the work, a volume of sample mixed with buffer was injected by means of the binary sampling technique [19]. For this purpose valve V1 was switched on and V2 off during a time interval t1 = 1.2 s, and then V1 off and V2 on for t2 = 0.3 s, this routine being repeated for 20 cycles. Thus a nominal volume of 800 L of sample was injected and mixed with buffer. Then a time t3 = 3 s elapsed (V1 and V2 off) and the sample bolus arrived at the column containing the immobilized GOD. The flow was then stopped for t4 = 20 s (V1 off, V2 on) to allow for the following reaction to occur: GOD
glucose + O2 + H2 O−→gluconic acid + H2 O2 Afterwards V2 was turned off for t5 = 0.4 s, then V3 was turned on for t6 = 0.3 s and then V4 turned on for t7 = 0.8 s, this sequence being repeated during 30 cycles. During this sequence, binary sampling was used to mix segments of the reaction product coming out from the column with segments of POD solution and of the chromogenic reagent. Afterwards, V2 , V3 , and V4 were turned off during the signal recording time t8 = 80 s. During the latter time period the carrier buffer solution flowed again through the normal path, the sample plug being displaced into the reaction coil, and the red quinoneimine formed was detected at 505 nm.
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A. Sixto, M. Knochen / Talanta 77 (2009) 1534–1538
Fig. 1. (a) Schematic diagram of the multicommutated flow system. P, peristaltic pump; V1 , V2 , V3 , V4 , 3-way solenoid valves; C, column filled with glucose oxidase immobilized onto controlled pore glass; TB, thermostatic bath; R, reaction coil; D, detection system (spectrophotometer with flow cell); W, waste; 4-HB + 4AAP, chromogenic reagent (solution of 4-hydroxy benzoate and 4-aminoantipyrine); POD, peroxidase solution; Key to valve connections: — connected when valve off, – – – – connected when valve on. (b) Time diagram for activation of solenoid valves. Times in seconds (s). Sample t1
Buffer t2
t until column t3
Stop time t4
Column eluate t5
Peroxidase t6
4HB-4AAP t7
Detection t8
1.2
0.3
3
20
0.4
0.3
0.8
80
After this time, a new analytical cycle was carried out to complete the replicates established in the set-up table. 2.5. Method
Two values of column length were studied, namely 3 and 4 cm. The latter produced higher signals and was chosen. Longer columns however were not tested because the higher mass of immobilized enzyme required would render it unpractical.
Calibration was performed by injecting glucose standard solutions in the range 0–0.14 g L−1 and measuring the peak height (absorbance). Calibration curves were fitted by means of leastsquares regression analysis. An amount of sample (2.5 g) was accurately weighed and dissolved in 20 mL of water under stirring, and finally made up to 25.0 mL. 1.0 mL of this solution was diluted to 250 mL with 0.1 mol L−1 pH 7.0 phosphate buffer. This solution was introduced into the multicommutated system and analyzed (3 injections).
3.2. Validation
3. Results and discussion
3.3. Selectivity
3.1. Influence of operating conditions
In order to assess the potential interference produced by fructose, the following solutions were prepared and measured: a standard solution of fructose (0.1608 g L−1 ), a standard solution of glucose (0.1310 g L−1 ), and a solution containing 0.1608 g L−1 glucose plus 0.1310 g L−1 fructose, i.e. a concentration ratio, for those substances, similar to that present in honey. Each solution was injected 7 times. The signal produced by the fructose standard solution was compared with the blank signal by means of Student’s t-test for mean values. Results of this test (texp = 1.071 < t(0.05, 9.65) = 2.365) do not show evidence of a significant difference between the signal produced by the fructose solution and the blank signal.
The following variables of the flow system were studied: reactor length, column length and stop time at the column. The rest of the variables were fixed based on the results of previous experiments aiming at maximizing the signal. The reaction coil lengths studied were 100, 150 and 400 cm. Considering precision, peak shape and sampling frequency, a reaction coil length of 150 cm was chosen. The influence of stop time at the column was studied between 15 and 30 s; a value of 20 s was chosen as a compromise between precision and maximum signal.
Figures of merit evaluated were linearity, accuracy, precision, detection limit (LD ), quantification limit (LQ ) and sampling frequency. Verification of accuracy was carried out by comparing the results obtained with those obtained by both, the AOAC 954.11 [2] and AOAC 977.20 [3] standard methods. The selectivity of the method was also evaluated.
A. Sixto, M. Knochen / Talanta 77 (2009) 1534–1538
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Table 1 Comparison of the results (percent weight) of the analysis of commercial samples by the proposed multicommutated (MCFA) method and two official AOAC methods. Sample
Results (sr (%))a MCFA (% (w/w))
AOAC 954.11 (% (w/w))
AOAC 977.20 (% (w/w))
AOAC 954.11
AOAC 977.20
1 2 3 4 5 6 7 8
28.5 (0.2) 29.7 (4.5) 28.9 (3.1) 30.1 (0.6) 28.1 (11.4) 28.0 (9.7) 29.0 (11.0) 27.6 (7.1)
27.8 (5.0) 28.9 (3.6) 27.6 (2.1) 29.5 (3.8) 29.5 (5.1) 27.9 (3.6) 27.7 (3.5) 27.8 (6.7)
30.0 (2.7) 29.2 (2.2) 26.9 (2.2) 32.3 (6.4) 25.5 (1.3)
2.3 3.0 4.7 2.0 −4.6 0.1 4.4 −0.7
−5.0 1.7 7.2 −6.7 10.2
a
Relative difference between the MCFA method and the AOAC methods (%)
Figures between brackets are sr (%), percent relative standard deviation (n = 3).
The ratio response to fructose + glucose/response to glucose was compared to the theoretical value of “1” by means of Student’s t-test. The result of this test was |texp | = 1.333 < t(0.05, 6) = 2.447, thus showing no significant interference from fructose under these experimental conditions. 3.4. Linearity, LD , LQ Linearity was evaluated by a 6-point calibration curve in the range 0–0.14 g L−1 of glucose. A second-degree model (h = −2.2199 C2 + 1.3741C + 0.0077, h, peak height (absorbance); C, concentration, g L−1 ) could be fit to the calibration curve (r2 = 0.9991). A slight non-linearity was to be expected given the enzymatic reactions involved. The limit of detection (3, n = 5) was 0.73% and the quantification limit (10, n = 5) was 2.43%, both expressed as percent glucose in honey (w/w). 3.5. Accuracy, precision and sampling frequency Eight commercial samples, representative of the Uruguayan market were obtained, either from stores (six samples, packaged), or from beekeepers (two samples, bulk). All samples were analyzed (three analytical replicates) by the proposed method as well as by the AOAC 954.11 method, and also by the AOAC 977.20 (HPLC) method. Only five of the samples were measured by the latter method. Results are shown in Table 1. Results obtained with the proposed method were compared statistically with those obtained with the official AOAC methods (Table 2). At the confidence level of 95% no evidence was found of a significant difference between the proposed method and the official methods. Table 2 Results of hypothesis testing of the results obtained by the proposed multicommutated (MCFA) method versus two official AOAC methods: Student’s “t”-test of mean values at the confidence level of 95%. Sample
Reference method AOAC 954.11
1 2 3 4 5 6 7 8 a
AOAC 977.20
texp
t(0.05, )
a
texp
t(0.05, )
a
−0.702 −0.607 −1.551 −0.006 0.537 −0.017 −0.571 0.082
2.776 2.776 2.776 4.303 2.776 2.776 2.776 2.776
4 4 4 4.2 4 4 4 4
1.663 −0.460 −2.158 −2.009 −1.383
3.102 2.776 2.571 3.102 2.776
3 4 5 5.1 4
: degrees of freedom.
Also, the relative differences between the results found with the proposed method and those found with the official methods were deemed to be appropriate for the purpose of quality control. The high heterogeneity of the matrix was easily noticeable and reflected in the dispersion of the results when genuine analytical replicates were prepared. Precision was estimated as the standard deviation of the signal produced by a standard solution (0.1267 g L−1 , n = 10). A relative standard deviation sr (%) = 3.0% was found. Furthermore sr (%) was calculated for the analytical process of the determination of commercial samples (three analytical replicates) and is reported in Table 1. Sampling frequency was 20 samples h−1 . 4. Conclusions An automated system based on the use of immobilized glucose oxidase was developed and evaluated for the determination of glucose in honey. The column life was satisfactory with a sensitivity loss of about 10% after the analysis of 50 samples. The method exhibited accuracy, precision and sampling frequency appropriate for the quality control of honey samples. The method based on the use of this system was much faster than the official AOAC method 954.11 based on classical column separation and volumetries, and was also faster than the AOAC 977.20 (HPLC) method which requires about 20 min per run. Acknowledgments The authors thank Universidad de la República, Comisión Sectorial de Investigación Científica (UdelaR-CSIC) for financial support, Professor Orlando Fatibello-Filho (UFSCar, Brazil) for providing the aminopropyl-glass beads, and Alejandra Torre (LATU) for the HPLC determinations. References [1] R. Alves Moreira, C. Bastos de María, Quim. Nova 24 (2001) 516. [2] P. Cunnif, (Ed.), Official Methods of Analysis of AOAC International, 16th ed., Method 954.11, vol. II, AOAC International, Maryland, USA, 1995, p. 22 (Chapter 44). [3] P. Cunnif (Ed.), Official Methods of Analysis of AOAC International, 16th ed., AOAC Method 977.20, vol. II, 1995, p. 25 (Chapter 44). [4] J. Ruzicka, E. Hansen, Flow Injection Analysis, 2nd ed., Wiley, New York, 1989. [5] M. Trojanowicz, Flow Injection Analysis: Instrumentation and Applications, World Scientific, Singapore, 2000. [6] M. Masoom, A. Townshend, Anal. Chim. Acta 166 (1984) 111. [7] P. Linares, M.D. Luque de Castro, M. Valcarcel, Anal. Chim. Acta 202 (1987) 199. [8] C.L.M. Stults, A.P. Wade, S.R. Crouch, Anal. Chim. Acta 192 (1987) 15. [9] M. Kiyoshi, K. Hideaki, M. Hiroaki, O. Yutaka, Anal. Chem. 60 (1988) 147. [10] S.M. Tzouwara-Karayanni, S.R. Crouch, Food Chem. 35 (1990) 109. [11] C. García De María, A. Townshend, Anal. Chim. Acta 261 (1992) 137. [12] W. Xiaojun, M.F. Martin, X. Dan, Analyst 125 (2000) 157. [13] Y. Jiuhong, L. Songqin, J. Huangxian, Biosens. Bioelectron. 19 (2003) 401.
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A. Sixto, M. Knochen / Talanta 77 (2009) 1534–1538 M. Manera, M. Miró, J.M. Estela, V. Cerdá, Anal. Chim. Acta 508 (2004) 23. J. Ruzicka, G.D. Marshall, Anal. Chim. Acta 237 (1990) 329. C.E. Lenehan, N.W. Barnett, S.W. Lewis, Analyst 127 (2002) 997. P. Panoutsou, A. Economou, Talanta 67 (2005) 603. P. Panoutsou, D.G. Themelis, Anal. Chim. Acta 572 (2006) 140. B.F. Reis, M.F. Giné, E.A.G. Zagatto, J.L.F.C. Lima, R.A. Lapa, Anal. Chim. Acta 293 (1994) 129.
[20] F.R.P. Rocha, B.F. Reis, E.A.G. Zagatto, J.L.F.C. Lima, R.A.S. Lapa, J.L.M. Santos, Anal. Chim. Acta 468 (2002) 119. [21] M. Catalá Icardo, J.V. García Mateo, J. Martínez Calatayud, TrAC Trends Anal. Chem. 21 (2002) 366. [22] M. Knochen, M. Pistón, L. Salvarrey, I. Dol, J. Pharm. Biomed. Anal. 37 (2005) 823. [23] P. Trinder, Ann. Clin. Biochem. 6 (1969) 24.
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Multicommutated flow system for the determination of glucose in honey with immobilized glucose oxidase reactor and spectrophotometric detection Alexandra Sixto, Moisés Knochen ∗ Universidad de la República, Facultad de Química, Cátedra de Química Analítica. Av. Gral. Flores 2124, 11800 Montevideo, Uruguay
a r t i c l e
i n f o
Article history: Received 7 August 2008 Received in revised form 19 September 2008 Accepted 22 September 2008 Available online 27 September 2008 Keywords: Multicommutated flow system Glucose oxidase Honey Immobilized
a b s t r a c t A new automated method for the determination of glucose in honey is proposed. The method is based on multicommutated flow analysis (MCFA) and employs an immobilized glucose oxidase reactor and spectrophotometric detection at 505 nm of the red quinoneimine formed (Trinder’s method). The calibration curve obeyed a second order equation in the range 0–0.14 g L−1 (h = −2.2199 C2 + 1.3741C + 0.0077, r2 = 0.9991, where h is the peak height (absorbance) and C the concentration in g L−1 ). The method was validated analyzing eight commercial samples, both by the AOAC 954.11 and 977.20 official methods. According to Student’s t-test of mean values, at the confidence level of 95% the results obtained with the proposed method were in agreement with those obtained by the official methods. Precision (sr (%), n = 10) was 3% and the sampling frequency of the system was 20 samples h−1 . © 2008 Elsevier B.V. All rights reserved.
1. Introduction Sugars are the principal constituents of honey. Aside from determining its nutritious and energetic value, they influence some of its important physical characteristics. Glucose is the monosaccharide responsible for the crystallization; honeys with high fructose content and low glucose content are less susceptible to crystallization, which is a phenomenon that decreases the acceptation of this natural product by the consumers. Honey acidity is also strongly related to the glucose content, and the rate fructose/glucose is related to the genuinity, a value below 1 indicating adulteration or the proliferation of osmophiles yeast [1]. The determination of sugars in honey may be achieved by classical procedures, such as those specified in the AOAC standard method [2]; these methods are usually slow and tedious, involving the isolation of the analyte from the matrix. In the aforementioned AOAC method this is carried out by means of classical chromatography involving a column filled with activated carbon and Celite® followed by two different volumetric titrations. The column separation step takes no less than 2 h per sample to which the time necessary for two volumetries should be added. Alternatively glucose may be determined by HPLC with a refractive-index detector [3].
∗ Corresponding author. Tel.: +598 2 9241808. E-mail address: [email protected] (M. Knochen). 0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.09.030
A simpler way to achieve the desired selectivity is employing an enzymatic reaction that is inherently selective. Most common selective procedures for the determination of glucose are based on the catalytic activity of glucose oxidase. Conventional enzymatic methods are costly due to the high consumption of enzymatic reagent, but this disadvantage has been successfully overcome by using immobilized enzymes. This strategy has been widely used in flow injection analysis (FIA) [4,5] where a number of papers have been published describing the use of microcolumns filled with immobilized enzymes as a part of flow systems [6–14]. Various alternatives such as physical adsorption, microencapsulation in membranes, sol–gel entrapment, cross linking or covalent attachment have been explored in the design of suitable flow-through packed-bed, open-tubular reactors or optical/electrochemical biosensors. The advantage of employing a column reactor in comparison to integrated biosensors is the ability of entrapping a much large amount of enzyme. A different flow-based approach is sequential injection analysis (SIA) [15,16]. This technique has been developed to address some drawbacks of FIA, namely the high consumption of reagents and the use of multi-channel manifolds. Methods exploiting this technique for the determination of glucose employing enzymes, have been reported [17,18]. More recently, a different flow technique called multicommutated flow analysis (MCFA) was introduced [19–21]. MCFA shows interesting advantages for automation. It is based on flow systems, where a number of solenoid valves, acting as independent commutators configure a flow network. Solenoid valves are independently
A. Sixto, M. Knochen / Talanta 77 (2009) 1534–1538
actuated under computer control. MCFA allows the implementation of binary sampling [19], a sampling technique where short segments of sample, reagent and carrier are inserted in the flow path. This approach allows modifications of the sample and reagent volumes, as well as extensive modifications of the flow path to be easily carried out under computer control avoiding the need of modifying physically the connections. Multicommutated flow analysis presents some advantages such as a high flexibility and a low investment cost since solenoid valves are inexpensive. This technique has been used successfully in the determination of glucose using enzymes in solution [22]. However, the authors did not found in the literature, previous work dealing with the use of this technique for the analysis of honey. Thus it was considered that the use of multicommutated flow analysis could be useful for the implementation of a novel automated system for the analysis of honey taking advantage of the high speed, flexibility and analytical throughput attained. In this work, a system based on the concept of multicommutated flow analysis was developed for the selective automated determination of glucose in honey. The method was based on work by Trinder [23], exploiting the following reactions: GOD
1535
2.3. Instrumentation and software A four-channel peristaltic pump (Alitea (Stockholm, Sweden) C8/2-XV), fitted with Tygon tubing was used for pumping the carrier, samples and reagents. Commutation was achieved by means of NResearch (West Caldwell, NJ, USA) 161T031 3-way 12 V solenoid valves. These valves were controlled using the individual data bits of the LPT1 parallel port of the computer via a CoolDriveTM driver (NResearch). Reaction coils and connections were made from 0.8 mm (internal diameter) Teflon PFA tubing. A thermostatic water bath set at 37 ◦ C was used to keep the temperature of the enzyme reactor constant throughout the experiments. The software used to control the multicommutated system was compiled in QuickBASIC 4.0 running under MS-DOS 6.0 in a notebook computer. The detection system was composed of a Shimadzu (Kyoto, Japan) UV-240 recording spectrophotometer fitted with an 80 L quartz flow cell (Hellma, Müllheim, Germany) and operated at 505 nm in the time scan mode. Recordings of the signals and peak height measurements were obtained from the graphic recorderprinter of the instrument.
glucose + O2 + H2 O−→gluconic acid + H2 O2 2H2 O2 + 4-aminoantipyrine + 4-hydroxybenzoate POD
−→quinoneimine where GOD stands for glucose oxidase and POD for peroxidase. The quinoneimine formed was detected by its absorption at 505 nm. Glucose oxidase was immobilized in column reactors via Schiff base formation. 2. Experimental 2.1. Reagents Glucose oxidase, Type II-S from Aspergillus niger, 39800 units g−1 solid (Sigma). Peroxidase, Type I, from Horseradish, 113 Purpurogallin units mg−1 solid (Sigma). Aminopropyl-glass beads (170 A, 200–400 mesh, synthesized in the laboratory) were obtained from Professor O. Fatibello-Filho. The following reagents were of analytical reagent grade unless otherwise specified: d-glucose anhydrous (Anedra), dfructose (Sigma, glucose <0.05%), dibasic potassium phosphate (Baker), monobasic potassium phosphate (Fluka), sodium 4hydroxybenzoate (Sigma), 4-aminoantipyrine hydrochloride (Eastman Organic Chemicals), glutardialdehyd 50% solution in water (Sigma–Aldrich), Peroxidase (POD) solution was prepared by accurately weighing 1.0 mg of peroxidase and dissolving in 100 mL of 0.1 mol L−1 phosphate buffer, pH 7. The chromogenic reagent was prepared by dissolving 0.6 g of 4-aminoantipyrine and 9.6 g of sodium 4-hydroxybenzoate in water and diluting with water to 1 L. 2.2. Preparation of the immobilized enzyme Glucose oxidase (GOD) was immobilized onto amino-propyl glass beads using the procedure described by Manera et al. [14]. The reactor was prepared by packing a 40 mm × 2.48 mm internal diameter piece of polytetrafluoroethylene (PTFE) tubing with the immobilized GOD glass beads. Polyethylene frits were used to embed the reactive phase. When not in use the column was stored at 4 ◦ C in 0.1 mol L−1 phosphate buffer (pH 7).
2.4. Multicommutated flow system The multicommutated system (Fig. 1a) consisted of a peristaltic pump (P), four 3-way solenoid valves (V1 –V4 ), a column (C) in a thermostatic bath (TB), a reaction coil (R), and a spectrophotometer used as detector (D). Valve V1 was used for the introduction of the sample, V2 for the carrier (0.1 mol L−1 phosphate buffer, pH 7), V3 for the peroxidase solution and V4 for the mixture of 4-hydroxybenzoate and 4-aminoantipyrine solutions. When not being introduced to the system reagents were recycled to the respective bottles. The operation of the system comprised an analytical cycle, when the same sample (or standard) was repeatedly injected and measured. When the software was run, it requested the control parameters. Afterwards it sent the control commands through the LPT1 port to switch valves V1 –V4 on or off, following the switching pattern depicted in the valves timing diagram in Fig. 1b. At the beginning of the work, a volume of sample mixed with buffer was injected by means of the binary sampling technique [19]. For this purpose valve V1 was switched on and V2 off during a time interval t1 = 1.2 s, and then V1 off and V2 on for t2 = 0.3 s, this routine being repeated for 20 cycles. Thus a nominal volume of 800 L of sample was injected and mixed with buffer. Then a time t3 = 3 s elapsed (V1 and V2 off) and the sample bolus arrived at the column containing the immobilized GOD. The flow was then stopped for t4 = 20 s (V1 off, V2 on) to allow for the following reaction to occur: GOD
glucose + O2 + H2 O−→gluconic acid + H2 O2 Afterwards V2 was turned off for t5 = 0.4 s, then V3 was turned on for t6 = 0.3 s and then V4 turned on for t7 = 0.8 s, this sequence being repeated during 30 cycles. During this sequence, binary sampling was used to mix segments of the reaction product coming out from the column with segments of POD solution and of the chromogenic reagent. Afterwards, V2 , V3 , and V4 were turned off during the signal recording time t8 = 80 s. During the latter time period the carrier buffer solution flowed again through the normal path, the sample plug being displaced into the reaction coil, and the red quinoneimine formed was detected at 505 nm.
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Fig. 1. (a) Schematic diagram of the multicommutated flow system. P, peristaltic pump; V1 , V2 , V3 , V4 , 3-way solenoid valves; C, column filled with glucose oxidase immobilized onto controlled pore glass; TB, thermostatic bath; R, reaction coil; D, detection system (spectrophotometer with flow cell); W, waste; 4-HB + 4AAP, chromogenic reagent (solution of 4-hydroxy benzoate and 4-aminoantipyrine); POD, peroxidase solution; Key to valve connections: — connected when valve off, – – – – connected when valve on. (b) Time diagram for activation of solenoid valves. Times in seconds (s). Sample t1
Buffer t2
t until column t3
Stop time t4
Column eluate t5
Peroxidase t6
4HB-4AAP t7
Detection t8
1.2
0.3
3
20
0.4
0.3
0.8
80
After this time, a new analytical cycle was carried out to complete the replicates established in the set-up table. 2.5. Method
Two values of column length were studied, namely 3 and 4 cm. The latter produced higher signals and was chosen. Longer columns however were not tested because the higher mass of immobilized enzyme required would render it unpractical.
Calibration was performed by injecting glucose standard solutions in the range 0–0.14 g L−1 and measuring the peak height (absorbance). Calibration curves were fitted by means of leastsquares regression analysis. An amount of sample (2.5 g) was accurately weighed and dissolved in 20 mL of water under stirring, and finally made up to 25.0 mL. 1.0 mL of this solution was diluted to 250 mL with 0.1 mol L−1 pH 7.0 phosphate buffer. This solution was introduced into the multicommutated system and analyzed (3 injections).
3.2. Validation
3. Results and discussion
3.3. Selectivity
3.1. Influence of operating conditions
In order to assess the potential interference produced by fructose, the following solutions were prepared and measured: a standard solution of fructose (0.1608 g L−1 ), a standard solution of glucose (0.1310 g L−1 ), and a solution containing 0.1608 g L−1 glucose plus 0.1310 g L−1 fructose, i.e. a concentration ratio, for those substances, similar to that present in honey. Each solution was injected 7 times. The signal produced by the fructose standard solution was compared with the blank signal by means of Student’s t-test for mean values. Results of this test (texp = 1.071 < t(0.05, 9.65) = 2.365) do not show evidence of a significant difference between the signal produced by the fructose solution and the blank signal.
The following variables of the flow system were studied: reactor length, column length and stop time at the column. The rest of the variables were fixed based on the results of previous experiments aiming at maximizing the signal. The reaction coil lengths studied were 100, 150 and 400 cm. Considering precision, peak shape and sampling frequency, a reaction coil length of 150 cm was chosen. The influence of stop time at the column was studied between 15 and 30 s; a value of 20 s was chosen as a compromise between precision and maximum signal.
Figures of merit evaluated were linearity, accuracy, precision, detection limit (LD ), quantification limit (LQ ) and sampling frequency. Verification of accuracy was carried out by comparing the results obtained with those obtained by both, the AOAC 954.11 [2] and AOAC 977.20 [3] standard methods. The selectivity of the method was also evaluated.
A. Sixto, M. Knochen / Talanta 77 (2009) 1534–1538
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Table 1 Comparison of the results (percent weight) of the analysis of commercial samples by the proposed multicommutated (MCFA) method and two official AOAC methods. Sample
Results (sr (%))a MCFA (% (w/w))
AOAC 954.11 (% (w/w))
AOAC 977.20 (% (w/w))
AOAC 954.11
AOAC 977.20
1 2 3 4 5 6 7 8
28.5 (0.2) 29.7 (4.5) 28.9 (3.1) 30.1 (0.6) 28.1 (11.4) 28.0 (9.7) 29.0 (11.0) 27.6 (7.1)
27.8 (5.0) 28.9 (3.6) 27.6 (2.1) 29.5 (3.8) 29.5 (5.1) 27.9 (3.6) 27.7 (3.5) 27.8 (6.7)
30.0 (2.7) 29.2 (2.2) 26.9 (2.2) 32.3 (6.4) 25.5 (1.3)
2.3 3.0 4.7 2.0 −4.6 0.1 4.4 −0.7
−5.0 1.7 7.2 −6.7 10.2
a
Relative difference between the MCFA method and the AOAC methods (%)
Figures between brackets are sr (%), percent relative standard deviation (n = 3).
The ratio response to fructose + glucose/response to glucose was compared to the theoretical value of “1” by means of Student’s t-test. The result of this test was |texp | = 1.333 < t(0.05, 6) = 2.447, thus showing no significant interference from fructose under these experimental conditions. 3.4. Linearity, LD , LQ Linearity was evaluated by a 6-point calibration curve in the range 0–0.14 g L−1 of glucose. A second-degree model (h = −2.2199 C2 + 1.3741C + 0.0077, h, peak height (absorbance); C, concentration, g L−1 ) could be fit to the calibration curve (r2 = 0.9991). A slight non-linearity was to be expected given the enzymatic reactions involved. The limit of detection (3, n = 5) was 0.73% and the quantification limit (10, n = 5) was 2.43%, both expressed as percent glucose in honey (w/w). 3.5. Accuracy, precision and sampling frequency Eight commercial samples, representative of the Uruguayan market were obtained, either from stores (six samples, packaged), or from beekeepers (two samples, bulk). All samples were analyzed (three analytical replicates) by the proposed method as well as by the AOAC 954.11 method, and also by the AOAC 977.20 (HPLC) method. Only five of the samples were measured by the latter method. Results are shown in Table 1. Results obtained with the proposed method were compared statistically with those obtained with the official AOAC methods (Table 2). At the confidence level of 95% no evidence was found of a significant difference between the proposed method and the official methods. Table 2 Results of hypothesis testing of the results obtained by the proposed multicommutated (MCFA) method versus two official AOAC methods: Student’s “t”-test of mean values at the confidence level of 95%. Sample
Reference method AOAC 954.11
1 2 3 4 5 6 7 8 a
AOAC 977.20
texp
t(0.05, )
a
texp
t(0.05, )
a
−0.702 −0.607 −1.551 −0.006 0.537 −0.017 −0.571 0.082
2.776 2.776 2.776 4.303 2.776 2.776 2.776 2.776
4 4 4 4.2 4 4 4 4
1.663 −0.460 −2.158 −2.009 −1.383
3.102 2.776 2.571 3.102 2.776
3 4 5 5.1 4
: degrees of freedom.
Also, the relative differences between the results found with the proposed method and those found with the official methods were deemed to be appropriate for the purpose of quality control. The high heterogeneity of the matrix was easily noticeable and reflected in the dispersion of the results when genuine analytical replicates were prepared. Precision was estimated as the standard deviation of the signal produced by a standard solution (0.1267 g L−1 , n = 10). A relative standard deviation sr (%) = 3.0% was found. Furthermore sr (%) was calculated for the analytical process of the determination of commercial samples (three analytical replicates) and is reported in Table 1. Sampling frequency was 20 samples h−1 . 4. Conclusions An automated system based on the use of immobilized glucose oxidase was developed and evaluated for the determination of glucose in honey. The column life was satisfactory with a sensitivity loss of about 10% after the analysis of 50 samples. The method exhibited accuracy, precision and sampling frequency appropriate for the quality control of honey samples. The method based on the use of this system was much faster than the official AOAC method 954.11 based on classical column separation and volumetries, and was also faster than the AOAC 977.20 (HPLC) method which requires about 20 min per run. Acknowledgments The authors thank Universidad de la República, Comisión Sectorial de Investigación Científica (UdelaR-CSIC) for financial support, Professor Orlando Fatibello-Filho (UFSCar, Brazil) for providing the aminopropyl-glass beads, and Alejandra Torre (LATU) for the HPLC determinations. References [1] R. Alves Moreira, C. Bastos de María, Quim. Nova 24 (2001) 516. [2] P. Cunnif, (Ed.), Official Methods of Analysis of AOAC International, 16th ed., Method 954.11, vol. II, AOAC International, Maryland, USA, 1995, p. 22 (Chapter 44). [3] P. Cunnif (Ed.), Official Methods of Analysis of AOAC International, 16th ed., AOAC Method 977.20, vol. II, 1995, p. 25 (Chapter 44). [4] J. Ruzicka, E. Hansen, Flow Injection Analysis, 2nd ed., Wiley, New York, 1989. [5] M. Trojanowicz, Flow Injection Analysis: Instrumentation and Applications, World Scientific, Singapore, 2000. [6] M. Masoom, A. Townshend, Anal. Chim. Acta 166 (1984) 111. [7] P. Linares, M.D. Luque de Castro, M. Valcarcel, Anal. Chim. Acta 202 (1987) 199. [8] C.L.M. Stults, A.P. Wade, S.R. Crouch, Anal. Chim. Acta 192 (1987) 15. [9] M. Kiyoshi, K. Hideaki, M. Hiroaki, O. Yutaka, Anal. Chem. 60 (1988) 147. [10] S.M. Tzouwara-Karayanni, S.R. Crouch, Food Chem. 35 (1990) 109. [11] C. García De María, A. Townshend, Anal. Chim. Acta 261 (1992) 137. [12] W. Xiaojun, M.F. Martin, X. Dan, Analyst 125 (2000) 157. [13] Y. Jiuhong, L. Songqin, J. Huangxian, Biosens. Bioelectron. 19 (2003) 401.
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