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Characterization of a micro spiral flow cell for chemiluminescence detection
Microchemical Journal 75 (2003) 255–264
Characterization of a micro spiral flow cell for chemiluminescence detection Wei Zhang, Neil D. Danielson* Department of Chemistry and Biochemistry, Miami University, Hughes Hall, Oxford, OH 45056, USA Received 30 June 2003; received in revised form 7 August 2003; accepted 13 August 2003
Abstract A 5.5 ml spiral micro-flow cell, mounted in front of a photomultiplier, is made from Teflon capillary (75 cm=100 mm ID) with two inlets for the CL reagent and carrier buffer and a waste outlet. It allows the rapid mixing of CL reagent and analyte and simultaneous detection of the emitted light. Using a flow rate of 25 mlymin for a 0.4 mM luminol-8 mM hemin solution (pH 11.6) and 50 mlymin of carrier buffer (pH 11.6), the slight exponential calibration curve for the flow injection–chemiluminescence (FI–CL) determination of H2 O2 is 2.5–10 mM and the detection limit is 1.5 mM. The detection limit achieved by using a spiral flow cell is 24 times lower than that obtained from a conventional FI system with a low dead volume tee mixer and a 12 ml flow cell in a HPLC fluorometer with the source lamp off. This luminol CL detection method is successfully applied to the enzymatic determination of Llactate by FI. The lactate sample is mixed with polyethylene glycol (PEG)-stabilized lactate oxidase (LO) enzyme and then injected into the buffered (pH 7.5) carrier stream for CL detection of the H2 O2 product. Using the optimal conditions of reaction temperature set to 37.5 8C and flow rates of 45 mlymin for the CL reagent and 60 mlymin for the carrier buffer, the calibration range for lactate is 5–50 mM and the detection limit is 2.9 mM. This method is applied to the determination of L-lactate in beer. 䊚 2003 Elsevier B.V. All rights reserved. Keywords: Spiral micro-flow cell; Flow injection; Luminol chemiluminescence; Hydrogen peroxide; L-lactate; Polyethylene glycol
1. Introduction Chemiluminescence (CL) is the emission of light derived from a chemical reaction when a species in the excited state drops down to the ground state. Besides the often very good sensitivity and selectivity of CL, instrumentally it is a simple technique. Introduction of the sample and *Corresponding author. Tel.: q1-513-529-2872; fax: q1513-529-5715. E-mail address: [email protected] (N.D. Danielson).
CL reagent, often by flow injection (FI) or after liquid chromatography (LC), into a flow cell mounted in front of a photomultiplier tube (PMT) is a common approach. Analytical applications of CL have been reviewed for FI, LC, and immunoassay w1–4x. Biosensors based on immobilized oxidase enzymes for the determination of peroxide by luminol CL are quite common examples w5,6x. Reviews of luminol type CL derivatization reagents for LC and capillary electrophoresis (CE) and CL detectors for CE are recently available w7,8x.
0026-265X/03/$ - see front matter 䊚 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2003.08.003
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The commercial flow cell design for CL detectors is often based on at least two inlet ports, one for the sample and one or two for the CL reagents which connect to a 0.3–0.5 mm i.d.=50–100-cm long Teflon tube arranged in a spiral pattern in front of the PMT. The advantage of the spiral flow cell design is the reaction kinetics for optimum light emission is less dependent on the flow rate of the FI carrier streams or the LC mobile phase flow rate. Because manufacturers of these CL detectors indicate flow cell volumes of 60–120 ml, these detectors are really only suited for conventional analytical LC using flow rates near 1 mlymin and often greater. An application for the determination of peroxide in microalagae by luminol CL chemistry used such a large volume flow cell w9x. Luminol, the Cr(III) catalyst, and the basic pH sample carrier stream were set at 2.5 mly min, 2.5 mlymin and 11 mlymin, respectively, and the injection volume was 400 ml. Peroxide (50 ml sample size) has been determined by FI using immobilized horse radish peroxidase at micro flow carrier flow rates of 0.01–0.1 mlymin with a spiral flow cell w10x. Although the dimensions of the flow cell were 0.96 mm i.d.=6 cm which calculates to a volume of 44 ml, the actual volume was smaller since the cell was packed with immobilized enzyme glass beads. A similar FI method for H2O2 but using immobilized luminol and Co(II) on ion exchange columns involved instrumentally a 200 ml sample loop and a 200 ml spiral flow cell w11x. Subnanomolar levels of H2O2 in seawater were determined by the same luminol reaction using an FI instrument equipped with a 60 ml sample loop and a 660 ml spiral flow cell w12x. A comparison of flow cell designs including the standard quartz flow cell, a helix flow cell, the spiral flow cell, and a novel bundle flow cell was made by FI for the determination of Cr(III) using luminol and H2O2 w13x. The helix, spiral flow cell, and the bundle flow cell all had dimensions of 0.8 mm=50 cm, which calculates to a volume of 251 ml. Injection sizes were 200 ml with a flow rate of 15 mlymin. The sensitivity of the bundle flow cell was found to be 50% better than the spiral flow cell under these experimental conditions. Recently, the FI determination of decylamine using a commercial CL detector equipped with a 120 ml
flow cell with a 12 cm pathlength has been reported w14x. A sample size of 50 ml with a carrier flow rate of 0.75 mlymin was used. Although not commercially available, CL flow cells for microbore and capillary LC have been developed. A study of spiral flow cell tubing dimensions for detection after semi-micro LC using conditions of a 20 ml injection size, 0.1 mly min flow rate, and 1.5 mm i.d.=250 mm reversed phase column has been made w15x. A tubing i.d. of 0.25 mm as compared to 0.5 mm was important to suppress band broadening and the optimum tubing length was found to be 60 cm. The separation of 3-aminoperylene derivatized carboxylic acids was done using a 0.5 ml injection on a 1=250 mm reversed phase column at 0.2 mlymin with peroxyoxalate CL detection in a 100 ml flow cell w16x. Dansylated amino acids were separated by reversed phase LC using similar conditions as just described except a flow rate of 0.6 mlymin was used w17x. A comparison of 5 and 50 ml standard straight flow cells using the peroxyoxalate CL detection chemistry showed because of band broadening, the 50 ml flow cell could be recommended for analytical scale but not microbore LC w18x. A similar straight 0.3 mm i.d. Teflon tube flow celI was investigated for capillary LC w19x. Because the peroxyoxalate CL chemistry has fast kinetics permitting the use of a 200 mlymin combined flow rate for CL reagents as compared to a 10 mlymin mobile phase flow rate, band-broadening effects could be reduced. Peroxyoxalate CL detection for capillary LC was demonstrated using a zero dead volume mixing tee to permit introduction of the CL reagents and then light detection further downstream in a 0.25 mm=4 cm straight PTFE tube w20x. The effective cell volume was estimated to be only 2.1–2.5 ml due to the sheath flow of the CL reagent, which served to reduce extra column band broadening. To the best of our knowledge, a low volume spiral flow cell with tubing less than 0.25 mm i.d. has not been previously reported. Use of small diameter tubing is important since band broadening is dependent on the square of the tubing radius but only proportional to the tubing length. In this paper, a newly designed spiral microflow cell (5.5 ml) using 100 mm i.d. capillary
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Teflon tubing for the rapid mixing of reagents and analyte and simultaneous detection of CL emission was made and characterized using the luminol– H2O2 –hemin CL reaction. The flow rate effects on CL signal intensity and profile were studied during the flow cell characterization. Comparative studies were carried out with a standard FI instrument with a mixing tee and conventional 12 ml flow cell in a HPLC fluorometer with the source lamp off. Polymer stabilized enzyme lactate oxidase (LO) was injected with the sample for the FI–CL determination of L-lactate using the spiral micro flow cell instrument. Factors that affect the CL signal intensity such as flow rate, temperature, activity units (U) of LO and sample stability were also investigated. Finally, this enzymatic FI–CL method was applied to the determination of Llactate in two beer samples. 2. Experimental 2.1. Apparatus and instruments The determination of H2O2 and lactate was carried out with the micro-flow FI–CL instrument diagrammed in Fig. 1a. Two micro-flow pumps (P噛1 and P噛2) with a computer interfaced controller unit composing the Ultra-plus Micro LC system (Micro-Tech Scientific, Mountain View, CA) were used to pump the filtered CL reagent and carrier buffer streams, respectively. Samples of interest (1 ml) were injected with a Rheodyne model 7410 injection valve into the buffer stream. A Model 900 Isotemp refrigerated circulator water bath (Fisher Scientific, Pittsburgh, PA) was used to control the reaction temperature of a mixing coil made from 100 cm=100 mm ID Teflon capillary (Cole–Parmer, Ann Arbor, MI) required for the enzyme-catalyzed oxidation of L-lactate. PEEK tubing of 175 mm i.d.=30 cm (Upchurch Scientific, Oak Harbor, WA) was used to connect the injector to the detector for the H2O2 study or two pieces of 175 mm i.d=40 cm PEEK tubing connected the coil reactor to the injector and detector for the lactate study. The CL signal was continuously measured by a Lumi-Tec photometer (St. John Associates, Inc., Rockville, MD) with a custom-designed 5.5 ml spiral flow cell. The
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design diagram of this flow cell is shown in Fig. 1b. The key part of this flow cell is a spiral 70 cm=100 mm ID Teflon capillary (Cole–Parmer) that is mounted between two disks of diameter 2.5 cm in front of the PMT with two inlets for the mixing of CL reagents and buffer carrier stream carrying the analyte, and a waste outlet. A more detailed description is given in the figure legend. Initially, a nylon capillary 75 mm=80 cm was tried for the spiral flow cell but reliability was poor due to kinking and pressure instability of the tubing. Besides solvent filters, an in-line filter (Upchurch) after the injection valve also protected the reaction coil from particles. Fittings with builtin 0.5 mm PEEK microfilters (Part噛M-560, Upchurch) were used to connect the PEEK tubing to the detector inlets (Fig. 1a). For comparison to the spiral FI–CL detector, a standard FI–CL detector was constructed as follows. The same pump configuration as shown in Fig. 1a was used. No enzyme coil was needed since only the H2O2 –luminol reaction was studied for comparison. The connection between the injector and mixing tee was made by 0.25 mm i.d.=35 cm stainless steel tubing. The CL reagent and carrier buffer with analyte were combined in a low dead volume stainless steel tee (Valco, Houston, TX) before they entered through a 0.30 mm i.d.=30 cm stainless steel tube into the HPLC flow cell for CL measurement. The CL signal intensity was measured by a RF-551 spectrofluorometric detector with a 12 ml quartz square flow cell (Shimadzu, Columbia, MD). The detected emission wavelength was set at 431 nm as recommended w21x and the excitation light source was switched off. All CL signals were recorded by a Chromatopac C-R6A integrator (Shimadzu). 2.2. Reagents and solutions Luminol (3-aminophthahydrazide) was from Aldrich (Milwaukee, WI). Hemin (chloroferriprotoporphyin) from bovine, L-(q)-lactic acid (sodium salt), lactate oxidase (LO) from Pediococcus species (L-0638, 50 units, EC 232-841-6), and polyethylene glycol (PEG) with a molecular weight of 3350 were purchased from Sigma (St. Louis, MO). Hydrogen peroxide (30% solution)
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Fig. 1.
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was obtained from Fisher Scientific. All other chemicals used were of analytical reagent grade. Distilled and doubly deionized water was obtained from the E-Pure deionization system (Barnstead, Dubuque, IA). 2.3. Procedure The CL reagent solution delivered by P噛1 contained 0.4 mM luminol, 8 mM hemin, and 0.1 M sodium phosphate dibasic buffer solution (pH 11.6). The composition of this CL reagent is based on previous work w22x. The carrier buffers delivered by P噛2 to propel the injected analyte were either 0.1 M sodium phosphate diabasic buffer solution (pH 11.6) for the FI–CL determination of H2O2 or 0.1 mM sodium phosphate monobasic buffer solution (pH 7.5) for the determination of lactate. Both buffer solutions were prepared from 0.5 M stock solutions by appropriate dilution with water. H2O2 stock solutions of 20 and 1 mM were prepared fresh daily. Serial dilution of the stock solutions with water provided the required H2O2 standard solutions that were prepared immediately before analysis. For the flow rate optimization work, either a 50 mM H2O2 standard or a 50 mM lactate standard was used. For the calibration curve, each lactate standard solution, made up of 7.5% PEG (vyv), 5 Uyml LOD, lactate and buffer, was prepared in a 1 ml vial immediately before FI–CL analysis. The 1000 mM lactate stock solution was prepared fresh daily. For the H2O2 study, no coil reactor was needed as part of the instrument, however, for the lactate work, the temperature controlled coil reactor set at 37.5 8C was important. In order to study the stability of the lactate samples, a 0.5 ml volume of 100 mM lactate containing 7.5% PEG and 5
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Uyml LO was prepared in each test tube. Those solutions were placed in the ice-bath and stored in the refrigerator before analysis at various times. In order to investigate the stabilization of LO by PEG, a 250 ml volume of 15% PEG aqueous solution was mixed with 50 ml of LO solution with a activity value of 50 Uyml and 150 ml of pH 7.5 phosphate buffer solution in each test tube. Those test tubes were placed in the ice-bath and stored in the refrigerator. Lactate needed for a final concentration of 50 mM was added just prior to analysis. Under the optimal temperature and flow rate conditions, the calibration curve for the determination of L-lactate was obtained. Under the same analytical conditions as before, a 50 ml volume of beer was taken with 50 ml of LO to make each 0.5 ml sample solution with pH 7.5 buffer. The results of L-lactate in beer were calculated from the calibration equation. Both the sample analysis as well as the calibration work should be done using the same enzyme solution to minimize signal response variation from different LO reagent bottles. 3. Results and discussion 3.1. Flowrate studies for the spiral and conventional HPLC flowcells Characterization of the 5.5 ml spiral flow cell flow cell–CL detector was based on injected H2O2 with luminol CL detection using the instrument diagrammed in Fig. 1a but without the reaction coil. The effect of flow rate on the CL signal intensity measured as peak height was studied at both 15 and 25 mlymin for P噛1 over a range of 40, 50, 60, and 70 mlymin for P噛2 (data not shown). The relative standard deviation
Fig. 1. (a) A schematic diagram of the FI–CL instrument for the determination of lactate. The same instrument but without the coil reactor was used for the H2O2 study. P噛1, P噛2smicroflow pumps, PMTsphotomultiplier. Each of the five small rectangles in the solution stream represents a filter. See Section 2.1 for more details. (b) Design diagram for the 5.5 ml spiral flow cell. Top view: spiral flow cell tubing held in place on an Al disk by a clear acrylic retaining disk with two screws w1x, waste outlet w2x also shown. A 5-mm long channel formed in the center of the underside of the acrylic disk is where the inlet tubing w7,8x end and mixing can take place. An outlet from the center of this channel (not shown) is where the tubing to form the spiral shape is connected. Side view: spiral tubing flow cell held between the acrylic disk w3x and the aluminum disk w4x mounted to the bottom Teflon disk w5x all with a diameter of 2.5 cm. Bottom view: waste outlet w6x and inlets w7,8x shown as well as four screws holding the Al and Teflon disks together.
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Fig. 2. Calibration plots for the FI–CL determination of H2O2. (a) 5.5 ml spiral flow cell. (b) 12 ml square flow cell. P噛1 and P噛2 were set at 25 and 50 mlymin for (a) and 55 and 120 mlymin for (b), respectively. See Section 2.3 for more details.
(R.S.D.) of these data range from 3 to 10% for ns6. As the P噛1 flowrate was held constant at either value, the peak became sharper with less time needed to reach the peak height, with the highest peak observed at 50 mlymin. The CL intensity profile with P噛1 at 15 mlymin increased from approximately 15 to a maximum of 17 mV at 50 mlymin and then back down to 14 mV as a function of the P噛2 flowrate. The CL intensity profile with P噛1 at 25 mlymin varied slightly from 19 to a maximum of 21 mV at 50 mlymin and then back to 18 mV as a function of P噛2 flowrate. Using 25 mlymin for P噛1, the baseline peak width decreased from 2.5 to 1.1 min and the peak asymmetry improved by a factor of two as the P噛2 flow rate increased from 40–70 mlymin. Previous work w17x indicated baseline peak widths estimated at 1–2 min for early eluting peaks even at a high flow rate of 0.6 mlymin. The optimal flow rates for P噛1 and P噛2 were 25 mlymin and 50 mlymin, respectively. There was no further optimization of flow rate of CL reagents at P噛1 due to the concern about the problem of pressure buildup inside the Teflon spiral flow cell tubing. Comparative studies were also done on the same CL system as diagrammed in Fig. 1a but without the reaction coil and instead a low volume mixing tee leading to a HPLC detector with a 12 ml square flow cell. Flow rate optimization was studied at
15, 25, and 35 mlymin for P噛1 over a six-point range from 90–140 mlymin for P噛2 (data not shown). The R.S.D. for these data range from 0.5 to 3% for ns4. The improved R.S.D. is likely due to the difference in the stability of the electronic readout of the two detectors. The flow rate responses for P噛2 increased slightly by approximately 4–6 units starting from 11, 17, and 24 mV for the respective increasing P噛1 flowrates before falling off at approximately 130 mlymin. The optimal flow rate of 120 mlymin for P噛2 was found in these gradual profiles for all three P噛1 flowrates. In search of the optimal flow rate of CL reagent for P噛1, the flow rate at P噛2 was set constant 120 mlymin and the flow rate for P噛1 was varied over a seven point range from 15 to 75 mlymin. The CL intensity increased steadily from 14 to 35 mV before leveling off at approximately 50 mlymin. The optimal flow rate for P噛1 was considered to be 55 mlymin. 3.2. Calibration curves and detection limits for H2O2 Under the optimal flow conditions, the relationship between the concentration of each standard H2O2 solution and its CL signal intensity for both the spiral flow cell and the conventional square flow cell was found. For the spiral flow cell (Fig.
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Fig. 3. Flow rate optimization for analysis of lactate. (a) Determination of the optimal flow rate of CL reagent at P噛1 using a 50 mM H2O2 sample. (b) Determination of the optimal flow rate of the carrier buffer at P噛2 using 50 mM lactate with 7.5% PEG and 10 Uyml LO. CL reagent of 0.4 mM luminol, 8 mM hemin, and 0.1 M phosphate buffer (pH 11.6) delivered by P噛1 but the pH 7.5 buffer solution was now the carrier buffer (P噛2). Temperature was set at 38 8C. R.S.D. for these data points ranged from 7 to 18% for ns4.
2a), an exponential profile is noted. The equation of the best fit calibration curve was Ys0.32X 1.41 with a linear regression coefficient r 2s0.9978. A detection limit for H2O2 of 1.5 mM (1.5 pmol for 1 ml sample) was determined from the measurement with three times the signal-to-noise ratio. The reproducibility in terms of relative standard deviation (R.S.D.) was 8.8% (ns5) for a 10 mM H2O2 sample. This absolute detection limit for the spiral flow cell in terms of pmol was also six times lower than that of the similar flow cell made from 6 cm=0.96 mm ID Teflon tubing w10x. For the 12 ml square flow cell (Fig. 2b), a slight exponential profile is again noted from 50 to 1000 mM (ns6). The best fit calibration curve from 50 to 1000 mM was Ys0.0081X 1.16 and r 2s0.9987. The detection limit of H2O2 was 37 mM (37 pmol for 1 ml sample), which was again calculated from three times the standard deviation of the blank measurement. Reproducibility (R.S.D.) was 1.8% (ns7) for a 300 mM H2O2 sample. Although some band broadening in the connecting tubing could have been a factor despite the higher flowrate for the 12 ml fluorescence flow cell FI system, the direct mixing of the CL reagent and H2O2 at the start of the spiral tubing design and the relatively longer residence time in front of the
PMT likely contributed to the lower detection limit and higher sensitivity for the FL–CL determination of H2O2 with the 5.5 ml spiral flow cell. 3.3. L-lactate assay The L-lactate assay was based on the luminol FI–CL measurement of H2O2 that was generated from the oxidation of lactate in the presence of lactate oxidase (LO) using the instrument diagrammed in Fig. 1a. Factors that affected the CL intensity, such as flow rate, temperature, enzyme concentration, and mixing time were considered. Polyethylene glycol (PEG) stabilization of the enzyme activity was also shown. Due to the narrow pH range (pH 6–7.5) for the optimal enzyme activity w23x, the pH 7.5 phosphate buffer replaced the pH 11.6 phosphate as the carrier buffer delivering the injected lactate samples. 3.3.1. Flow rate optimization The flow rate optimization for the lactate assay is shown in Fig. 3. The initial optimization step shown in Fig. 3a was to determine the optimal flow rate of the CL reagent at P噛1 by using 50 mM H2O2 as a sample. As expected, the CL response tended to be higher as the flow rate for
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sity reached 51 mV with 5 Uyml LO. The average CL signal intensity obtained at 1 and 10 Uyml LO was 26 mV and 44 mV, respectively. For the lactate assay, it was decided the optimal reaction temperature was 37.5 8C and the optimal concentration of LOD was 5 Uyml.
Fig. 4. Effect of temperature (8C) on the LO enzyme activity. Optimal flow rates of 45 mlymin and 60 mlymin for P噛1 and P噛2, respectively. Sample: 50 mM lactate in 7.5% PEG with either 1 or 5 Uyml LO. Other conditions as in Fig. 3b. R.S.D. values for six injections were in the range between 7 and 15%.
P噛2 decreased due to a longer residence time for the optimum CL emission intensity. If the flow rate of the CL reagent was below 45 mlymin, any change in flow rate of the carrier buffer at P噛2 had a minimal impact on the CL signal intensity. In the second optimization step using lactate shown in Fig. 3b, the highest CL signal intensity was obtained when the flow rate of carrier buffer was increased to 60 mlymin. This is likely due to mixing in the flow coil was more efficient between the still narrow nondiffused lactate–PEG–LO injection plug and the CL reagent. There was no further attempt to increase the flow rate at P噛2 in case of damage to the Teflon-made flow cell caused by the high pressure buildup inside the flow cell. Therefore, 45 mlymin at P噛1 and 60 mlymin at P噛2 were set as the operational flow rates throughout the remaining lactate studies. 3.3.2. Temperature and enzyme concentration Temperature–CL intensity curves obtained with different LO activities clearly indicated maximum catalytic activity as the reaction temperature reached 37–38 8C (Fig. 4). Surprisingly, strong CL signals were still evident at 60 8C. During the study of the effect of LO concentration on CL intensity with the temperature set at 38 8C, it was observed that the highest average CL signal inten-
3.3.3. Stability of lactate samples The stabilization of lactate dehydrogenase (LDH) by PEG has been used previously in FI and CE studies to ensure reproducibility of the analytical response over a time period of at least 8 h at 30 8C w24,25x. A comparison of the CL signal using lactate samples with and without 7.5% PEG as a function of low temperature storage time is shown in Fig. 5. The variation of CL intensity values was less than 5 mV for the enzyme solution containing PEG while it reached up to 18 mV for the enzyme solution in the absence of PEG. Although the absolute CL response was lower for the first 6 h, PEG did have a positive effect on the stabilization of LO enzyme activity. However, the effect was not as dramatic as that for LDH, which has four subunits. The enzyme LO with 7.5% PEG was mixed directly with the lactate sample solutions prior to FI injection in order to minimize consumption of the expensive enzyme. These samples were kept in an ice-bath stored in the refrigerator. The FI–
Fig. 5. Effect of PEG on the stabilization of LO enzyme activity when PEG–LO samples stored in an ice-bath in the refrigerator. Diamond points–no PEG, R.S.D. 12–20% for ns6–8. Square points – 7.5% PEG. Conditions as in Fig. 3b but 5 Uyml LO and 37.5 8C. R.S.D. 8–16% for ns6–8.
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Fig. 6. Effect of CL signal as a function of storage time for lactate–PEG–LO sample at low temperature. Conditions as in Fig. 3b but 5 Uyml LO and 37.5 8C. R.S.D. for these data were 7–15% for ns6.
CL analyses of those sample solutions were carried out at different times and the results are as shown in Fig. 6. The CL intensity continuously decreased with the mixing time with a slope during the first 2 h of approximately 0.6 mVymin. From 4 to 17 h, the slope was only approximately 0.1 mVymin. The CL signal decay was somewhat surprising considering the low storage temperature. Based on the finding from these experiments, the time spent for lactate, LO, and PEG in solution should be as short as possible. 3.3.4. Calibration, detection limit, and real sample data for L-lactate The relationship between lactate concentration and its CL signal intensity in terms of peak height under optimum analysis conditions is shown in Fig. 7. The calibration range for the lactate assay from 0 to 50 mM was fitted to the equation Ys 0.39Xq6.90 with r 2s0.9923. The detection limit of lactate calculated from three times the standard deviation of the blank measurements was 2.9 mM (2.9 pmol for 1 ml sample). The reproducibility (R.S.D.) was 12.2% (ns4) for 10 mM lactate and 8.8% (ns4) for 25 mM lactate. Compared to other methods for lactate assays, such as the enzymatic FI–CL method based on luminol–H2O2 –horseradish CL reaction with a detection limit of 0.08 mM (8 pmol for 100 ml sample injection) w26x, the FI method based on
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UV detection of NADH generated from the oxidation of lactate catalyzed by lactate dehydrogenase enzyme (LDH) with a detection limit of 0.13 mM (65 pmol for 0.5 ml sample injection) w24x, the FIA method based on photochemical reaction with the detection limit of 18 nM (1.8 pmol for 100 ml injection) w27x, the enzymatic FI–CL method based on the luminol–H2O2 –K3Fe(CN)6 CL reaction with the detection limit of 5 mgyl (44.5 mM) w28x, this FI–CL detection limit of 2.9 pmol for lactate was better or comparative. The proposed method using the external standard calibration method was applied to the determination of L-lactate in two beer samples. The determination of lactate in beer is important since excessive lactate will cause an off-taste and the level of lactate is related to the fermentation process in general for the control of bacterial contamination w29x. For ns5, a sample of George Killian’s Irish Red was found to have 14.5"1.3 mgyl lactate and a sample of Michael Shea’s Black and Tan was analyzed for 8.1"1 mgyl lactate. Both of these values are somewhat low as compared to a previous study for L-lactate in Czech and Italian beers in which levels were on the order of 20–50 mgyl w29x. 4. Conclusions Mixing of the luminol reagent and H2O2 analyte inside the spiral cell with simultaneous detection
Fig. 7. Calibration curve for the FI–CL determination of Llactate using the spiral flow cell. Conditions as in Fig. 3b but 5 Uyml LO and 37.5 8C. R.S.D. for these data ranged from 7.8 to 15.2% for ns4.
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of CL emission resulted in an improvement in sensitivity and detection limit as compared to a tee mixer and a standard fluorescence flow cell. Application to oxidase enzyme chemistry was shown. Although the enzyme (LO) when combined with PEG was quite stable when stored at low temperature, the LO–PEG–substrate (L-lactate) mixture was not for any significant length of time. However, the determination of L-lactate was possible if the LO–PEG–lactate mixture was injected just after preparation. Addition of LO– PEG in the carrier stream should still be cost effective using rapid sample injections spaced approximately 2 min apart. The sample carrier flowrate range of 50–60 mly min for the spiral flowcell FI system is considered more compatible with small microbore HPLC. It is expected this low volume spiral flow cell would minimize band broadening for microbore HPLC applications such as the separation and detection of glucosides w22x. Application to wide bore capillary LC should also be feasible. Acknowledgments We thank Peter St. John of St. John Associates for construction of the spiral flow cell and James J. Bao for helpful discussions. Partial funding for this work was provided by the Procter and Gamble Co and the Miami University Research Advisory Council. References w1x W.R.G. Baeyens, S.G. Schulman, A.C. Calokerinos, Y. Zhao, A.M. Garcia Campana, K. Nakashima, D. De Keukeleire, J. Pharm. Biomed. Anal. 17 (1998) 941. w2x M.G. Sanders, K.N. Andrew, P.J. Worsfold, Anal. Commun. 34 (1997) 13H. w3x A. Roda, P. Pasini, M. Guardigli, M. Baraldini, M. Musiani, Fresenius J. Anal. Chem. 366 (2000) 752. w4x A.M. Garcia Campana, W.R. Baeyens, X.R. Zhang, E. Smet, G. VanDer Weken, K. Nakashima, A.C. Calokerinos, Biomed. Chromatogr. 14 (2000) 166.
w5x K. Hayashi, S. Sasaki, K. Ikebukuro, I. Karube, Anal. Chim. Acta 329 (1996) 127. w6x U. Spohn, F. Preuschoff, G. Blankenstein, D. Janasek, M.R. Kula, A. Hacker, Anal. Chim. Acta 303 (1995) 109. w7x M. Yamaguchi, H. Yoshida, H. Nohta, J. Chromatogr. A950 (2002) 1. w8x Y-M. Liu, J-K. Cheng, J. Chromatogr. A959 (2002) 1. w9x R. Escobar, S. Garcıa-Domınguez, ´ ´ ´ A. Guiraum, O. ´ F.F. de la Rose, Luminescence 15 Montes, F. Galvan, (2000) 131. w10x O. Nozaki, H. Kawamoto, Luminescence 15 (2000) 137. w11x W. Qin, Z. Zhang, B. Lin, S. Liu, Anal. Chim. Acta 372 (1998) 357. w12x J. Yuan, A.M. Shiller, Anal. Chem. 71 (1999) 1975. w13x P. Campins-Falco, L.A. Tortajada-Genaro, F. BoschReig, Talanta 55 (2001) 403. w14x P.J. Fletcher, K.N. Andrew, S. Forbes, P.J. Worsfold, Anal. Chem. 75 (2003) 2618. w15x K. Takezawa, M. Tsunoda, K. Murayama, T. Santa, K. Imai, Analyst 125 (2000) 293. w16x K. Honda, K. Miyaguchi, K. Imai, Anal. Chim. Acta 177 (1985) 111. w17x K. Miyaguchi, K. Honda, T. Toyo’oka, K. Imai, J. Chromatogr. 352 (1986) 255. w18x G.J. de.Jong, N. Lammers, F.J. Spruit, R.W. Frei, U.A.Th. Brinkman, J. Chromatogr. 353 (1986) 249. w19x G.J. de Jong, N. Lammers, F.J. Spruit, C. Derwaele, M. Verzele, Anal. Chem. 59 (1987) 1458. w20x A.J. Weber, M.L. Grayeski, Anal. Chem. 59 (1987) 1452. w21x J. Lee, H.H. Seliger, Photochem. Photobiol. 15 (1972) 567. w22x P.J. Koerner Jr, T.A. Nieman, J. Chromatogr. 449 (1988) 217. w23x F. Mizutani, K. Sasaki, Y. Shimura, Anal. Chim. Acta 55 (1983) 35. w24x J.R. Marsh, N.D. Danielson, Analyst 120 (1995) 1091. w25x J.M. Fujima, N.D. Danielson, J. Cap. Elec. 3 (1996) 281. w26x A. Hemmi, K. Yagiuda, N. Funazaki, S. Ito, Y. Asano, T. Imato, K. Hayashi, I. Karube, Anal. Chim. Acta 316 (1995) 323. w27x T. Perez-Ruiz, ´ ´ ´ J. Martın, ´ C. Martınez-Lozano, V. Tomas, Analyst 124 (1999) 1517. w28x R.W. Min, J. Nielsen, J. Villadsen, Anal. Chim. Acta 312 (1995) 149. w29x S. Girotti, M. Muratori, F. Fini, E.N. Ferri, G. Carrea, M. Koran, P. Rauch, Eur. Food. Res. Technol. 210 (2000) 216.
Characterization of a micro spiral flow cell for chemiluminescence detection Wei Zhang, Neil D. Danielson* Department of Chemistry and Biochemistry, Miami University, Hughes Hall, Oxford, OH 45056, USA Received 30 June 2003; received in revised form 7 August 2003; accepted 13 August 2003
Abstract A 5.5 ml spiral micro-flow cell, mounted in front of a photomultiplier, is made from Teflon capillary (75 cm=100 mm ID) with two inlets for the CL reagent and carrier buffer and a waste outlet. It allows the rapid mixing of CL reagent and analyte and simultaneous detection of the emitted light. Using a flow rate of 25 mlymin for a 0.4 mM luminol-8 mM hemin solution (pH 11.6) and 50 mlymin of carrier buffer (pH 11.6), the slight exponential calibration curve for the flow injection–chemiluminescence (FI–CL) determination of H2 O2 is 2.5–10 mM and the detection limit is 1.5 mM. The detection limit achieved by using a spiral flow cell is 24 times lower than that obtained from a conventional FI system with a low dead volume tee mixer and a 12 ml flow cell in a HPLC fluorometer with the source lamp off. This luminol CL detection method is successfully applied to the enzymatic determination of Llactate by FI. The lactate sample is mixed with polyethylene glycol (PEG)-stabilized lactate oxidase (LO) enzyme and then injected into the buffered (pH 7.5) carrier stream for CL detection of the H2 O2 product. Using the optimal conditions of reaction temperature set to 37.5 8C and flow rates of 45 mlymin for the CL reagent and 60 mlymin for the carrier buffer, the calibration range for lactate is 5–50 mM and the detection limit is 2.9 mM. This method is applied to the determination of L-lactate in beer. 䊚 2003 Elsevier B.V. All rights reserved. Keywords: Spiral micro-flow cell; Flow injection; Luminol chemiluminescence; Hydrogen peroxide; L-lactate; Polyethylene glycol
1. Introduction Chemiluminescence (CL) is the emission of light derived from a chemical reaction when a species in the excited state drops down to the ground state. Besides the often very good sensitivity and selectivity of CL, instrumentally it is a simple technique. Introduction of the sample and *Corresponding author. Tel.: q1-513-529-2872; fax: q1513-529-5715. E-mail address: [email protected] (N.D. Danielson).
CL reagent, often by flow injection (FI) or after liquid chromatography (LC), into a flow cell mounted in front of a photomultiplier tube (PMT) is a common approach. Analytical applications of CL have been reviewed for FI, LC, and immunoassay w1–4x. Biosensors based on immobilized oxidase enzymes for the determination of peroxide by luminol CL are quite common examples w5,6x. Reviews of luminol type CL derivatization reagents for LC and capillary electrophoresis (CE) and CL detectors for CE are recently available w7,8x.
0026-265X/03/$ - see front matter 䊚 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2003.08.003
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The commercial flow cell design for CL detectors is often based on at least two inlet ports, one for the sample and one or two for the CL reagents which connect to a 0.3–0.5 mm i.d.=50–100-cm long Teflon tube arranged in a spiral pattern in front of the PMT. The advantage of the spiral flow cell design is the reaction kinetics for optimum light emission is less dependent on the flow rate of the FI carrier streams or the LC mobile phase flow rate. Because manufacturers of these CL detectors indicate flow cell volumes of 60–120 ml, these detectors are really only suited for conventional analytical LC using flow rates near 1 mlymin and often greater. An application for the determination of peroxide in microalagae by luminol CL chemistry used such a large volume flow cell w9x. Luminol, the Cr(III) catalyst, and the basic pH sample carrier stream were set at 2.5 mly min, 2.5 mlymin and 11 mlymin, respectively, and the injection volume was 400 ml. Peroxide (50 ml sample size) has been determined by FI using immobilized horse radish peroxidase at micro flow carrier flow rates of 0.01–0.1 mlymin with a spiral flow cell w10x. Although the dimensions of the flow cell were 0.96 mm i.d.=6 cm which calculates to a volume of 44 ml, the actual volume was smaller since the cell was packed with immobilized enzyme glass beads. A similar FI method for H2O2 but using immobilized luminol and Co(II) on ion exchange columns involved instrumentally a 200 ml sample loop and a 200 ml spiral flow cell w11x. Subnanomolar levels of H2O2 in seawater were determined by the same luminol reaction using an FI instrument equipped with a 60 ml sample loop and a 660 ml spiral flow cell w12x. A comparison of flow cell designs including the standard quartz flow cell, a helix flow cell, the spiral flow cell, and a novel bundle flow cell was made by FI for the determination of Cr(III) using luminol and H2O2 w13x. The helix, spiral flow cell, and the bundle flow cell all had dimensions of 0.8 mm=50 cm, which calculates to a volume of 251 ml. Injection sizes were 200 ml with a flow rate of 15 mlymin. The sensitivity of the bundle flow cell was found to be 50% better than the spiral flow cell under these experimental conditions. Recently, the FI determination of decylamine using a commercial CL detector equipped with a 120 ml
flow cell with a 12 cm pathlength has been reported w14x. A sample size of 50 ml with a carrier flow rate of 0.75 mlymin was used. Although not commercially available, CL flow cells for microbore and capillary LC have been developed. A study of spiral flow cell tubing dimensions for detection after semi-micro LC using conditions of a 20 ml injection size, 0.1 mly min flow rate, and 1.5 mm i.d.=250 mm reversed phase column has been made w15x. A tubing i.d. of 0.25 mm as compared to 0.5 mm was important to suppress band broadening and the optimum tubing length was found to be 60 cm. The separation of 3-aminoperylene derivatized carboxylic acids was done using a 0.5 ml injection on a 1=250 mm reversed phase column at 0.2 mlymin with peroxyoxalate CL detection in a 100 ml flow cell w16x. Dansylated amino acids were separated by reversed phase LC using similar conditions as just described except a flow rate of 0.6 mlymin was used w17x. A comparison of 5 and 50 ml standard straight flow cells using the peroxyoxalate CL detection chemistry showed because of band broadening, the 50 ml flow cell could be recommended for analytical scale but not microbore LC w18x. A similar straight 0.3 mm i.d. Teflon tube flow celI was investigated for capillary LC w19x. Because the peroxyoxalate CL chemistry has fast kinetics permitting the use of a 200 mlymin combined flow rate for CL reagents as compared to a 10 mlymin mobile phase flow rate, band-broadening effects could be reduced. Peroxyoxalate CL detection for capillary LC was demonstrated using a zero dead volume mixing tee to permit introduction of the CL reagents and then light detection further downstream in a 0.25 mm=4 cm straight PTFE tube w20x. The effective cell volume was estimated to be only 2.1–2.5 ml due to the sheath flow of the CL reagent, which served to reduce extra column band broadening. To the best of our knowledge, a low volume spiral flow cell with tubing less than 0.25 mm i.d. has not been previously reported. Use of small diameter tubing is important since band broadening is dependent on the square of the tubing radius but only proportional to the tubing length. In this paper, a newly designed spiral microflow cell (5.5 ml) using 100 mm i.d. capillary
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Teflon tubing for the rapid mixing of reagents and analyte and simultaneous detection of CL emission was made and characterized using the luminol– H2O2 –hemin CL reaction. The flow rate effects on CL signal intensity and profile were studied during the flow cell characterization. Comparative studies were carried out with a standard FI instrument with a mixing tee and conventional 12 ml flow cell in a HPLC fluorometer with the source lamp off. Polymer stabilized enzyme lactate oxidase (LO) was injected with the sample for the FI–CL determination of L-lactate using the spiral micro flow cell instrument. Factors that affect the CL signal intensity such as flow rate, temperature, activity units (U) of LO and sample stability were also investigated. Finally, this enzymatic FI–CL method was applied to the determination of Llactate in two beer samples. 2. Experimental 2.1. Apparatus and instruments The determination of H2O2 and lactate was carried out with the micro-flow FI–CL instrument diagrammed in Fig. 1a. Two micro-flow pumps (P噛1 and P噛2) with a computer interfaced controller unit composing the Ultra-plus Micro LC system (Micro-Tech Scientific, Mountain View, CA) were used to pump the filtered CL reagent and carrier buffer streams, respectively. Samples of interest (1 ml) were injected with a Rheodyne model 7410 injection valve into the buffer stream. A Model 900 Isotemp refrigerated circulator water bath (Fisher Scientific, Pittsburgh, PA) was used to control the reaction temperature of a mixing coil made from 100 cm=100 mm ID Teflon capillary (Cole–Parmer, Ann Arbor, MI) required for the enzyme-catalyzed oxidation of L-lactate. PEEK tubing of 175 mm i.d.=30 cm (Upchurch Scientific, Oak Harbor, WA) was used to connect the injector to the detector for the H2O2 study or two pieces of 175 mm i.d=40 cm PEEK tubing connected the coil reactor to the injector and detector for the lactate study. The CL signal was continuously measured by a Lumi-Tec photometer (St. John Associates, Inc., Rockville, MD) with a custom-designed 5.5 ml spiral flow cell. The
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design diagram of this flow cell is shown in Fig. 1b. The key part of this flow cell is a spiral 70 cm=100 mm ID Teflon capillary (Cole–Parmer) that is mounted between two disks of diameter 2.5 cm in front of the PMT with two inlets for the mixing of CL reagents and buffer carrier stream carrying the analyte, and a waste outlet. A more detailed description is given in the figure legend. Initially, a nylon capillary 75 mm=80 cm was tried for the spiral flow cell but reliability was poor due to kinking and pressure instability of the tubing. Besides solvent filters, an in-line filter (Upchurch) after the injection valve also protected the reaction coil from particles. Fittings with builtin 0.5 mm PEEK microfilters (Part噛M-560, Upchurch) were used to connect the PEEK tubing to the detector inlets (Fig. 1a). For comparison to the spiral FI–CL detector, a standard FI–CL detector was constructed as follows. The same pump configuration as shown in Fig. 1a was used. No enzyme coil was needed since only the H2O2 –luminol reaction was studied for comparison. The connection between the injector and mixing tee was made by 0.25 mm i.d.=35 cm stainless steel tubing. The CL reagent and carrier buffer with analyte were combined in a low dead volume stainless steel tee (Valco, Houston, TX) before they entered through a 0.30 mm i.d.=30 cm stainless steel tube into the HPLC flow cell for CL measurement. The CL signal intensity was measured by a RF-551 spectrofluorometric detector with a 12 ml quartz square flow cell (Shimadzu, Columbia, MD). The detected emission wavelength was set at 431 nm as recommended w21x and the excitation light source was switched off. All CL signals were recorded by a Chromatopac C-R6A integrator (Shimadzu). 2.2. Reagents and solutions Luminol (3-aminophthahydrazide) was from Aldrich (Milwaukee, WI). Hemin (chloroferriprotoporphyin) from bovine, L-(q)-lactic acid (sodium salt), lactate oxidase (LO) from Pediococcus species (L-0638, 50 units, EC 232-841-6), and polyethylene glycol (PEG) with a molecular weight of 3350 were purchased from Sigma (St. Louis, MO). Hydrogen peroxide (30% solution)
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Fig. 1.
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was obtained from Fisher Scientific. All other chemicals used were of analytical reagent grade. Distilled and doubly deionized water was obtained from the E-Pure deionization system (Barnstead, Dubuque, IA). 2.3. Procedure The CL reagent solution delivered by P噛1 contained 0.4 mM luminol, 8 mM hemin, and 0.1 M sodium phosphate dibasic buffer solution (pH 11.6). The composition of this CL reagent is based on previous work w22x. The carrier buffers delivered by P噛2 to propel the injected analyte were either 0.1 M sodium phosphate diabasic buffer solution (pH 11.6) for the FI–CL determination of H2O2 or 0.1 mM sodium phosphate monobasic buffer solution (pH 7.5) for the determination of lactate. Both buffer solutions were prepared from 0.5 M stock solutions by appropriate dilution with water. H2O2 stock solutions of 20 and 1 mM were prepared fresh daily. Serial dilution of the stock solutions with water provided the required H2O2 standard solutions that were prepared immediately before analysis. For the flow rate optimization work, either a 50 mM H2O2 standard or a 50 mM lactate standard was used. For the calibration curve, each lactate standard solution, made up of 7.5% PEG (vyv), 5 Uyml LOD, lactate and buffer, was prepared in a 1 ml vial immediately before FI–CL analysis. The 1000 mM lactate stock solution was prepared fresh daily. For the H2O2 study, no coil reactor was needed as part of the instrument, however, for the lactate work, the temperature controlled coil reactor set at 37.5 8C was important. In order to study the stability of the lactate samples, a 0.5 ml volume of 100 mM lactate containing 7.5% PEG and 5
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Uyml LO was prepared in each test tube. Those solutions were placed in the ice-bath and stored in the refrigerator before analysis at various times. In order to investigate the stabilization of LO by PEG, a 250 ml volume of 15% PEG aqueous solution was mixed with 50 ml of LO solution with a activity value of 50 Uyml and 150 ml of pH 7.5 phosphate buffer solution in each test tube. Those test tubes were placed in the ice-bath and stored in the refrigerator. Lactate needed for a final concentration of 50 mM was added just prior to analysis. Under the optimal temperature and flow rate conditions, the calibration curve for the determination of L-lactate was obtained. Under the same analytical conditions as before, a 50 ml volume of beer was taken with 50 ml of LO to make each 0.5 ml sample solution with pH 7.5 buffer. The results of L-lactate in beer were calculated from the calibration equation. Both the sample analysis as well as the calibration work should be done using the same enzyme solution to minimize signal response variation from different LO reagent bottles. 3. Results and discussion 3.1. Flowrate studies for the spiral and conventional HPLC flowcells Characterization of the 5.5 ml spiral flow cell flow cell–CL detector was based on injected H2O2 with luminol CL detection using the instrument diagrammed in Fig. 1a but without the reaction coil. The effect of flow rate on the CL signal intensity measured as peak height was studied at both 15 and 25 mlymin for P噛1 over a range of 40, 50, 60, and 70 mlymin for P噛2 (data not shown). The relative standard deviation
Fig. 1. (a) A schematic diagram of the FI–CL instrument for the determination of lactate. The same instrument but without the coil reactor was used for the H2O2 study. P噛1, P噛2smicroflow pumps, PMTsphotomultiplier. Each of the five small rectangles in the solution stream represents a filter. See Section 2.1 for more details. (b) Design diagram for the 5.5 ml spiral flow cell. Top view: spiral flow cell tubing held in place on an Al disk by a clear acrylic retaining disk with two screws w1x, waste outlet w2x also shown. A 5-mm long channel formed in the center of the underside of the acrylic disk is where the inlet tubing w7,8x end and mixing can take place. An outlet from the center of this channel (not shown) is where the tubing to form the spiral shape is connected. Side view: spiral tubing flow cell held between the acrylic disk w3x and the aluminum disk w4x mounted to the bottom Teflon disk w5x all with a diameter of 2.5 cm. Bottom view: waste outlet w6x and inlets w7,8x shown as well as four screws holding the Al and Teflon disks together.
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Fig. 2. Calibration plots for the FI–CL determination of H2O2. (a) 5.5 ml spiral flow cell. (b) 12 ml square flow cell. P噛1 and P噛2 were set at 25 and 50 mlymin for (a) and 55 and 120 mlymin for (b), respectively. See Section 2.3 for more details.
(R.S.D.) of these data range from 3 to 10% for ns6. As the P噛1 flowrate was held constant at either value, the peak became sharper with less time needed to reach the peak height, with the highest peak observed at 50 mlymin. The CL intensity profile with P噛1 at 15 mlymin increased from approximately 15 to a maximum of 17 mV at 50 mlymin and then back down to 14 mV as a function of the P噛2 flowrate. The CL intensity profile with P噛1 at 25 mlymin varied slightly from 19 to a maximum of 21 mV at 50 mlymin and then back to 18 mV as a function of P噛2 flowrate. Using 25 mlymin for P噛1, the baseline peak width decreased from 2.5 to 1.1 min and the peak asymmetry improved by a factor of two as the P噛2 flow rate increased from 40–70 mlymin. Previous work w17x indicated baseline peak widths estimated at 1–2 min for early eluting peaks even at a high flow rate of 0.6 mlymin. The optimal flow rates for P噛1 and P噛2 were 25 mlymin and 50 mlymin, respectively. There was no further optimization of flow rate of CL reagents at P噛1 due to the concern about the problem of pressure buildup inside the Teflon spiral flow cell tubing. Comparative studies were also done on the same CL system as diagrammed in Fig. 1a but without the reaction coil and instead a low volume mixing tee leading to a HPLC detector with a 12 ml square flow cell. Flow rate optimization was studied at
15, 25, and 35 mlymin for P噛1 over a six-point range from 90–140 mlymin for P噛2 (data not shown). The R.S.D. for these data range from 0.5 to 3% for ns4. The improved R.S.D. is likely due to the difference in the stability of the electronic readout of the two detectors. The flow rate responses for P噛2 increased slightly by approximately 4–6 units starting from 11, 17, and 24 mV for the respective increasing P噛1 flowrates before falling off at approximately 130 mlymin. The optimal flow rate of 120 mlymin for P噛2 was found in these gradual profiles for all three P噛1 flowrates. In search of the optimal flow rate of CL reagent for P噛1, the flow rate at P噛2 was set constant 120 mlymin and the flow rate for P噛1 was varied over a seven point range from 15 to 75 mlymin. The CL intensity increased steadily from 14 to 35 mV before leveling off at approximately 50 mlymin. The optimal flow rate for P噛1 was considered to be 55 mlymin. 3.2. Calibration curves and detection limits for H2O2 Under the optimal flow conditions, the relationship between the concentration of each standard H2O2 solution and its CL signal intensity for both the spiral flow cell and the conventional square flow cell was found. For the spiral flow cell (Fig.
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Fig. 3. Flow rate optimization for analysis of lactate. (a) Determination of the optimal flow rate of CL reagent at P噛1 using a 50 mM H2O2 sample. (b) Determination of the optimal flow rate of the carrier buffer at P噛2 using 50 mM lactate with 7.5% PEG and 10 Uyml LO. CL reagent of 0.4 mM luminol, 8 mM hemin, and 0.1 M phosphate buffer (pH 11.6) delivered by P噛1 but the pH 7.5 buffer solution was now the carrier buffer (P噛2). Temperature was set at 38 8C. R.S.D. for these data points ranged from 7 to 18% for ns4.
2a), an exponential profile is noted. The equation of the best fit calibration curve was Ys0.32X 1.41 with a linear regression coefficient r 2s0.9978. A detection limit for H2O2 of 1.5 mM (1.5 pmol for 1 ml sample) was determined from the measurement with three times the signal-to-noise ratio. The reproducibility in terms of relative standard deviation (R.S.D.) was 8.8% (ns5) for a 10 mM H2O2 sample. This absolute detection limit for the spiral flow cell in terms of pmol was also six times lower than that of the similar flow cell made from 6 cm=0.96 mm ID Teflon tubing w10x. For the 12 ml square flow cell (Fig. 2b), a slight exponential profile is again noted from 50 to 1000 mM (ns6). The best fit calibration curve from 50 to 1000 mM was Ys0.0081X 1.16 and r 2s0.9987. The detection limit of H2O2 was 37 mM (37 pmol for 1 ml sample), which was again calculated from three times the standard deviation of the blank measurement. Reproducibility (R.S.D.) was 1.8% (ns7) for a 300 mM H2O2 sample. Although some band broadening in the connecting tubing could have been a factor despite the higher flowrate for the 12 ml fluorescence flow cell FI system, the direct mixing of the CL reagent and H2O2 at the start of the spiral tubing design and the relatively longer residence time in front of the
PMT likely contributed to the lower detection limit and higher sensitivity for the FL–CL determination of H2O2 with the 5.5 ml spiral flow cell. 3.3. L-lactate assay The L-lactate assay was based on the luminol FI–CL measurement of H2O2 that was generated from the oxidation of lactate in the presence of lactate oxidase (LO) using the instrument diagrammed in Fig. 1a. Factors that affected the CL intensity, such as flow rate, temperature, enzyme concentration, and mixing time were considered. Polyethylene glycol (PEG) stabilization of the enzyme activity was also shown. Due to the narrow pH range (pH 6–7.5) for the optimal enzyme activity w23x, the pH 7.5 phosphate buffer replaced the pH 11.6 phosphate as the carrier buffer delivering the injected lactate samples. 3.3.1. Flow rate optimization The flow rate optimization for the lactate assay is shown in Fig. 3. The initial optimization step shown in Fig. 3a was to determine the optimal flow rate of the CL reagent at P噛1 by using 50 mM H2O2 as a sample. As expected, the CL response tended to be higher as the flow rate for
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sity reached 51 mV with 5 Uyml LO. The average CL signal intensity obtained at 1 and 10 Uyml LO was 26 mV and 44 mV, respectively. For the lactate assay, it was decided the optimal reaction temperature was 37.5 8C and the optimal concentration of LOD was 5 Uyml.
Fig. 4. Effect of temperature (8C) on the LO enzyme activity. Optimal flow rates of 45 mlymin and 60 mlymin for P噛1 and P噛2, respectively. Sample: 50 mM lactate in 7.5% PEG with either 1 or 5 Uyml LO. Other conditions as in Fig. 3b. R.S.D. values for six injections were in the range between 7 and 15%.
P噛2 decreased due to a longer residence time for the optimum CL emission intensity. If the flow rate of the CL reagent was below 45 mlymin, any change in flow rate of the carrier buffer at P噛2 had a minimal impact on the CL signal intensity. In the second optimization step using lactate shown in Fig. 3b, the highest CL signal intensity was obtained when the flow rate of carrier buffer was increased to 60 mlymin. This is likely due to mixing in the flow coil was more efficient between the still narrow nondiffused lactate–PEG–LO injection plug and the CL reagent. There was no further attempt to increase the flow rate at P噛2 in case of damage to the Teflon-made flow cell caused by the high pressure buildup inside the flow cell. Therefore, 45 mlymin at P噛1 and 60 mlymin at P噛2 were set as the operational flow rates throughout the remaining lactate studies. 3.3.2. Temperature and enzyme concentration Temperature–CL intensity curves obtained with different LO activities clearly indicated maximum catalytic activity as the reaction temperature reached 37–38 8C (Fig. 4). Surprisingly, strong CL signals were still evident at 60 8C. During the study of the effect of LO concentration on CL intensity with the temperature set at 38 8C, it was observed that the highest average CL signal inten-
3.3.3. Stability of lactate samples The stabilization of lactate dehydrogenase (LDH) by PEG has been used previously in FI and CE studies to ensure reproducibility of the analytical response over a time period of at least 8 h at 30 8C w24,25x. A comparison of the CL signal using lactate samples with and without 7.5% PEG as a function of low temperature storage time is shown in Fig. 5. The variation of CL intensity values was less than 5 mV for the enzyme solution containing PEG while it reached up to 18 mV for the enzyme solution in the absence of PEG. Although the absolute CL response was lower for the first 6 h, PEG did have a positive effect on the stabilization of LO enzyme activity. However, the effect was not as dramatic as that for LDH, which has four subunits. The enzyme LO with 7.5% PEG was mixed directly with the lactate sample solutions prior to FI injection in order to minimize consumption of the expensive enzyme. These samples were kept in an ice-bath stored in the refrigerator. The FI–
Fig. 5. Effect of PEG on the stabilization of LO enzyme activity when PEG–LO samples stored in an ice-bath in the refrigerator. Diamond points–no PEG, R.S.D. 12–20% for ns6–8. Square points – 7.5% PEG. Conditions as in Fig. 3b but 5 Uyml LO and 37.5 8C. R.S.D. 8–16% for ns6–8.
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Fig. 6. Effect of CL signal as a function of storage time for lactate–PEG–LO sample at low temperature. Conditions as in Fig. 3b but 5 Uyml LO and 37.5 8C. R.S.D. for these data were 7–15% for ns6.
CL analyses of those sample solutions were carried out at different times and the results are as shown in Fig. 6. The CL intensity continuously decreased with the mixing time with a slope during the first 2 h of approximately 0.6 mVymin. From 4 to 17 h, the slope was only approximately 0.1 mVymin. The CL signal decay was somewhat surprising considering the low storage temperature. Based on the finding from these experiments, the time spent for lactate, LO, and PEG in solution should be as short as possible. 3.3.4. Calibration, detection limit, and real sample data for L-lactate The relationship between lactate concentration and its CL signal intensity in terms of peak height under optimum analysis conditions is shown in Fig. 7. The calibration range for the lactate assay from 0 to 50 mM was fitted to the equation Ys 0.39Xq6.90 with r 2s0.9923. The detection limit of lactate calculated from three times the standard deviation of the blank measurements was 2.9 mM (2.9 pmol for 1 ml sample). The reproducibility (R.S.D.) was 12.2% (ns4) for 10 mM lactate and 8.8% (ns4) for 25 mM lactate. Compared to other methods for lactate assays, such as the enzymatic FI–CL method based on luminol–H2O2 –horseradish CL reaction with a detection limit of 0.08 mM (8 pmol for 100 ml sample injection) w26x, the FI method based on
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UV detection of NADH generated from the oxidation of lactate catalyzed by lactate dehydrogenase enzyme (LDH) with a detection limit of 0.13 mM (65 pmol for 0.5 ml sample injection) w24x, the FIA method based on photochemical reaction with the detection limit of 18 nM (1.8 pmol for 100 ml injection) w27x, the enzymatic FI–CL method based on the luminol–H2O2 –K3Fe(CN)6 CL reaction with the detection limit of 5 mgyl (44.5 mM) w28x, this FI–CL detection limit of 2.9 pmol for lactate was better or comparative. The proposed method using the external standard calibration method was applied to the determination of L-lactate in two beer samples. The determination of lactate in beer is important since excessive lactate will cause an off-taste and the level of lactate is related to the fermentation process in general for the control of bacterial contamination w29x. For ns5, a sample of George Killian’s Irish Red was found to have 14.5"1.3 mgyl lactate and a sample of Michael Shea’s Black and Tan was analyzed for 8.1"1 mgyl lactate. Both of these values are somewhat low as compared to a previous study for L-lactate in Czech and Italian beers in which levels were on the order of 20–50 mgyl w29x. 4. Conclusions Mixing of the luminol reagent and H2O2 analyte inside the spiral cell with simultaneous detection
Fig. 7. Calibration curve for the FI–CL determination of Llactate using the spiral flow cell. Conditions as in Fig. 3b but 5 Uyml LO and 37.5 8C. R.S.D. for these data ranged from 7.8 to 15.2% for ns4.
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of CL emission resulted in an improvement in sensitivity and detection limit as compared to a tee mixer and a standard fluorescence flow cell. Application to oxidase enzyme chemistry was shown. Although the enzyme (LO) when combined with PEG was quite stable when stored at low temperature, the LO–PEG–substrate (L-lactate) mixture was not for any significant length of time. However, the determination of L-lactate was possible if the LO–PEG–lactate mixture was injected just after preparation. Addition of LO– PEG in the carrier stream should still be cost effective using rapid sample injections spaced approximately 2 min apart. The sample carrier flowrate range of 50–60 mly min for the spiral flowcell FI system is considered more compatible with small microbore HPLC. It is expected this low volume spiral flow cell would minimize band broadening for microbore HPLC applications such as the separation and detection of glucosides w22x. Application to wide bore capillary LC should also be feasible. Acknowledgments We thank Peter St. John of St. John Associates for construction of the spiral flow cell and James J. Bao for helpful discussions. Partial funding for this work was provided by the Procter and Gamble Co and the Miami University Research Advisory Council. References w1x W.R.G. Baeyens, S.G. Schulman, A.C. Calokerinos, Y. Zhao, A.M. Garcia Campana, K. Nakashima, D. De Keukeleire, J. Pharm. Biomed. Anal. 17 (1998) 941. w2x M.G. Sanders, K.N. Andrew, P.J. Worsfold, Anal. Commun. 34 (1997) 13H. w3x A. Roda, P. Pasini, M. Guardigli, M. Baraldini, M. Musiani, Fresenius J. Anal. Chem. 366 (2000) 752. w4x A.M. Garcia Campana, W.R. Baeyens, X.R. Zhang, E. Smet, G. VanDer Weken, K. Nakashima, A.C. Calokerinos, Biomed. Chromatogr. 14 (2000) 166.
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