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Mercury thread electrode in a flow cell
ANALYTICA CHIMICA ACTA ELSEVIER
Analytica
Chimica Acta 332 (1996) 165-171
Mercury thread electrode in a flow cell H.G. Jayaratna”, Bioanalytical Received
C.S. Bruntlett,
P.T. Kissinger
Systems Inc., West Lafayette, IN 47906, USA
12 March 1996; revised 24 April 1996; accepted 29 April 1996
Abstract A mercury thread electrode modified with hydrophilic dialysis membrane was first introduced in 1994. Incorporation of this electrode in a flow stream is now introduced. The flow of solution and mercury are conveniently achieved using two syringe pumps. The cell can be used with a sample loop of a desired volume; hence, it allows flow injection applications.
Characterization of the flow cell was performed considering various experimental parameters using lead(I1) as the analyte. Calibration curves for the lead(I1) exhibit good linearity. Keywords: Stripping voltammetry; Flow methods; Mercury; Dialysis membrane
1. Introduction The electrochemical community greatly appreciates the benefits of mercury as an electrode material. The high overvoltage for hydrogen ion reduction, easily renewable surface and the large liquid range of mercury are responsible for its widespread use. It is an ideal choice for the determination of metal ions in aqueous and other media. Mercury electrodes are also being used as detector electrodes in liquid chromatography for the determination of organic compounds such as sulfides, thiols, peroxides, and nitro-organics. Since the description of the first polarographic apparatus by Heyrovsky in 1921, there have been various forms of mercury electrodes. These include the Dropping Mercury Electrode (DME), Hanging Mercury Drop Electrode (HMDE), Streaming Mer-
* Corresponding 1102.
author. Tel.: +l 317463 4527; fax: +l 317 497
0003-2670/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved PII SOOO3-2670(96)00229-2
cury Electrode (SME), Mercury Film Electrode (MFE), Static Mercury Drop Electrode (SMDE), and Controlled Growth Mercury Electrode (CGME) [ 1,2]. A disadvantage of mercury drop electrodes is that it is difficult to recycle the used mercury collected in the analytical solution. In addition, mercury electrodes exposed to complex samples such as blood, urine, etc. are easily fouled. Polymer modified MFEs have been useful for electrochemical studies in complex samples. The use of polymer (for example, Nafion) over a mercury film electrode and their application in trace metal determination have been investigated [3-51. Development of a flow cell using a mercury electrode is essential for an automated electrochemical analyzer, especially for trace metal determinations. Placing a mercury drop electrode in a flow cell has been attempted by many workers with limited success [5-71 The difficulties with the mercury drop in a flow cell are: the instability of the drop and the
166
H.G. Jayaratna et al./Analytica
noise introduced by the vibrations of the drop due to the flow of solution. Recently we introduced a new form of polymer modified mercury electrode titled, Mercury Thread Electrode (MTE) [8] for determining trace metals in biological samples. This electrode is constructed by enclosing a mercury column within a hydrophilic dialysis membrane tube of -150 urn diameter. The dialysis membrane prevents proteins and other large molecules present in biological samples from fouling the electrode surface while allowing intimate interaction with small molecules and elements. This paper describes the incorporation of the MTE in a flow cell arrangement and explores some of its properties.
2. Experimental 2.1. Reagents and solutions All chemicals were of analytical grade and were used without further purification. Cyclic Voltammetry (CV) experiments employed 0.1 mM lead(I1) in 0.1 M HCl. Lead solutions used in Anodic Stripping Voltammetry (ASV) were prepared by diluting 1OOOppm lead(I1) standards (Certified Atomic Absorption Standards, Fisher Scientific Company, Fair Lawn, NJ) with 0.1 M HCl background solution. Deionized water was used throughout this work for rinsing and solution preparation purposes. All glassware was stored in 1.OM HNOs acid bath and rinsed with water before use.
Chimica Acta 332 (1996) 165-I 71
MTE. Quantitative data analysis (calibration plot or standard addition plot) was performed with the BAS 1OOW electrochemical software. Syringe pumps (BAS Bee, BAS, West Lafayette, IN) were used for pumping analyte solution and mercury through the MTE. 2.3. Flow Cell Design One of the important features of the MTE design is that it can easily be made into a flow cell as shown in Fig. 1. This design utilizes a hydrophilic dialysis membrane tube transversely inserted through a Tygon tube. The Tygon tube used as the solution carrier had an internal diameter of -1.5 mm and an outer diameter of -3 mm. The internal diameter of this tube essentially defined the length of the MTE. A length of -2 cm of this tube defined a cell volume of -40 ul. The total length of the membrane tube used was approximately 8 mm which left about 2.5 mm lengths on either side of the Tygon tube. These ends were inserted into two pieces of polyacrylonitrile (PAN)
2.2. Instrumentation The instrument used for potential control in all the experiments was the BAS lOOB/W Electrochemical Workstation (BAS, West Lafayette, IN) which included a computer equipped with an Intel 486DX (33 MHz) microprocessor and a LaserJet IIIP printer. The Osteryoung Square Wave Stripping Voltammetry (OSWSV) and Differential Pulse Stripping Voltammetry (DPSV) techniques available with this instrument were chosen as the ASV modes. Other techniques such as, Osteryoung Square Wave Voltammetty (OSWV) and Linear Scan Voltammetry (LSV) were also used. CGME in the static drop electrode mode was used for comparison work with
OUTLET
(NJX
,
Fig. 1. Flow cell design for the MTE.
H.G. Jayaratna et al./Analytica
tube of internal diameter -1 mm with variable length and sealed with epoxy. These were used as mercury inlet and outlet. The outlet tube was connected to a vial to contain the used mercury. A syringe with a stainless steel needle that fit snugly into the inlet tube was chosen for delivering the mercury by means of a syringe pump (Bee pump, BAS). The same needle served as the working electrode contact. Any discontinuity in mercury was easily observed through the transparent PAN tubes. The Tygon tube ends were sealed on to the cell block with epoxy in order to prevent leaks. The ability to observe the electrode and the solution inside the cell is advantageous. In the case of blood, the sample was observed making contact with the MTE and timing for subsequent experiments was determined. Inlet and outlet ports were also made of plastic (PEEK) and attached to the central cell block with screws as shown (Fig. 1). The Teflon gaskets allowed leak-proof connection of inlet and outlet ports to the cell block and provided ionic communication between electrodes. The inlet port was a 1 cm thick PEEK disk with a plastic fingertight ferrule (Teflon or PEEK). A small diameter plastic tube of length -5 cm (0.65 mm o.d. and a dead volume of 1.2 ul/ 10cm) was used as the solution inlet tube. The same type of fingertight fittings were used to connect this tube to an injection valve (Rheodyne, model 7125) which allowed the use of a sample loop for convenient sample handling. A BAS Bee pump with a 5 ml syringe was connected to the inlet port for the delivery of solution. The outlet port was a similar diameter PEEK disk with a stainless steel tube of internal diameter of N 1 mm and a silver disk of diameter - 1 mm epoxied as shown in Fig. 1. The stainless steel tube served as the outlet tube as well as the auxiliary electrode. The silver disk was coated with AgCl to serve as the reference electrode for the electrochemical cell. This reference had an operational life time of about one day at low pH (< 1) values and a potential about 90mV negative of regular Ag/AgCl reference. Recoating of the reference with AgCl was conveniently done by polishing the electrode and placing a drop of AgCl coating solution (CF 2200, UniJet Reference Solution, BAS). Regenerated cellulose hollow fiber dialysis membranes of 13 000 and 18 000 molecular weight cut off
Chimica Acta 332 (1996) 165-l 71
167
(MWCO), were purchased from Spectrum Medical Industries (Houston, TX). Nephross Allegro H.F., a third type of membrane of -20000 MWCO (Boxter, Netherlands), was also used. Internal diameter and the wall thickness of these membranes were specified as 200 and 8 urn, respectively. The membranes were stored at room temperature before use in the MTE fabrication.
3. Results and discussion The initial focus of this MTE research is trace metal determination. The work described here shows preliminary data using the flow cell design with aqueous lead(I1) samples. The sample was delivered to the cell directly from a syringe or through a Rheodyne valve with a sample loop of desired volume. The syringe pumps used for both mercury and solution allowed flow rates in the range from l600 pl min-’ with a 5 ml syringe. One of the most important advantages of the MTE is the ability to renew the electrode surface by advancing the mercury inside the membrane tube. A plastic syringe (5 ml) connected to a BAS Bee pump was used to move mercury through the membrane tube. Cyclic and stripping voltammetry were used to evaluate performance. 3.1. Cyclic voltammetry Cyclic voltammetry of 0.1 mM lead(I1) in 0.1 M HCl was performed with the flow cell arrangement which consisted of MTE with 18 000 MWCO membrane. The solution was pumped at -25 ul min-’ with the syringe pump. Fig. 2 shows the cyclic voltammetry between -200 and -600 mV with a scan rate of 100 mV s-l. Voltammogram A was recorded with stationary mercury, whereas the voltammogram B with moving mercury at a rate of about 8 ul min-‘. In A, the cyclic voltammogram has peak potential difference (A,??,) of 33 mV which indicates nearly ideal situation with fast electron transfer at the electrode for the redox couple of Pb(II)/Pb. During the forward scan lead(I1) is reduced and plated into the mercury thread. In the reverse direction, plated lead is oxidized and diffused into the flow stream to be swept away. The mercury thread
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lnit E (mV) = -200 High E (mV) = -200 Low E (mV) = -600 lnit P/N = N V (mV/s) =I00 Sweep Segments = 2 Slmpl Int (mV) =I Quiet T (s) = 2 (AA’) = 1 E-6
Chimica Acta 332 (1996) 165-171
the tube sealing procedure used in the fabrication procedure is satisfactory. Second, it is absolutely necessary to maintain a stationary mercury column during stripping analysis or the application will result in erroneous data. The cell design discussed in this paper occasionally showed loss of signal due to selfadvancement of mercury. Improvements to the cell design are currently being made to avoid this problem. 3.2. Renewal of Mercury
Sens t -0.20
I
I -0.30
I
I -0.40
I
I -0.50
/ -0.60
Potential/V
Fig. 2. Cyclic voltammetry of 0.1x10~3moll~’ lead(I1) in 0.12 mol-’ HCl. (A) Stationary mercury; (B) Moving mercury.
with the reduced metal, therefore, needs to be kept stationary during the oxidative scan of cyclic and stripping voltammetry. The volume of the mercury thread exposed to the solution was about 2.7 nl (-370ng of mercury). With the given flow rate of 8 plmin-‘, this only takes 200ms. to pass the solution region of 1.5 mm length. Hence, the slightest movement of mercury could result in loss of analytical signal. The effect of flowing mercury on CV is shown by the scan B. Voltammogram B does not show an oxidation peak because the flowing mercury moves the section of the mercury thread with the plated metal out of the solution area well before it reaches the oxidizing potential for lead. But, the continuously moving mercury shows a reduction peak identical to that of stationary system. Since all the other parameters remain the same, the peak heights could have been changed in this situation only if the electrode area changed due to fluctuation in mercury pressure and expansion of the membrane during the flow. Therefore, it can be assumed that the electrode area does not change due to the flow of mercury. Two aspects that can be identified from the cyclic voltammetry data provided here are quite important for the use of the MTE. First, the solution is in contact with only the mercury column in the membrane portion within the Tygon tube and there is no flow of solution out into the bulk of mercury in the inlet and outlet tubes. This, in turn, indicates that
The response from stripping voltammetry was investigated to determine if the mercury advancement before each experiment has any effect on the magnitude of the signal. Lead solutions of 50 and 1OOppb concentration in a background electrolyte of 0.1 M HCl were used for this purpose. The flow cell was assembled with syringes containing these solutions. Analyses were performed with DPSV with a deposition time of 2min. Two sets of 5 stripping voltammograms were recorded with and without mercury advancement for each concentration with 2min of plating time. The average peak currents observed for these two concentrations were in the range of 50 and 80nA with an RSD less than ~2%. Therefore, it appears that frequent advancement of mercury is not necessary in clean water samples. However, in real samples, the need will be determined by the contaminants and MWCO of the membrane. 3.3. Effect of solution Jlow The response at the MTE is mainly governed by the rate of diffusion of the analyte through the membrane. Can the mass transport to the electrode be enhanced by increasing the flow rate of the solution? In order to answer this question an experiment was performed with membranes of different MWCO; namely, 13 000 and 18 000. A solution of lead (0.1 mM) in 0.1 M HCl was employed and the response at different flow rates was measured using square wave voltammetry. The data collected are shown in Fig. 3. Each point is the average of five readings that had an RSD < 1%. Fluctuation of average data over the entire range of flow rates was also N 1% for both membranes. Therefore, the
H.G. Jayaratna et al./Analytica
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30
60
Chimica Acta 332 (1996) 165-l 71
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Flow Rate (pL / min) Fig. 3. Solution flow rate effects on OSWV response at MTEs with two different MWCO: (0) 13 000; (0) 18000. Solution=O. 1 x 10~3moll~’ lead(l1) in O.l2mollHCl, Init. E=-2OOmV, Final E=-700mV, S.W. Amp.=25, Freq.=60Hz, Step E=4 mV, Quite time=2 s, Sensitivity=1 x 10m6 AV-‘.
I
response at the electrode is not dependent on the flow rate and the mass transfer limiting factor appears to be the rate of diffusion of the analyte in the membrane, at least for the MWCOs used here. There is a greater response at higher MWCO due to the higher rate of diffusion. Membranes with much larger pore size may show some dependence on the flow rate. Membranes of MWCO up to 20000 (Nephross) did not show flow rate dependence. This is very favorable for the application of the MTE, because different flow rates can be utilized to alter the residence time of the sample at the electrode. Hence, the plating time in ASV experiments can be adjusted without affecting the magnitude of the response, especially, with small amounts of sample. Continued investigation is necessary for better use of this strategy. 3.4. MTE for J~OWinjection analysis In order to test the ability of the MTE to detect a sample portion injected into the flow stream reproducibility was explored next. An MTE of -20000 MWCO was connected to a Rheodyne valve with a 100 pl sample loop and then 0.1 mM lead(I1) in 0.1 M HCl were injected. The detection of this sample was achieved by stepping the potential of the electrode to -700 mV and monitoring the response with time. Fig. 4 illustrates the detection of two 100~1 portions of lead solution at the MTE.
0
I
200
I
400
I
1 600
Time I set Fig. 4. Time base response observed at MTE with a membrane of 20 000 MWCO for 100 pl portions of sample injected through a Rheodyne valve with a 100 11 loop. Electrode potential=-700mV.
The particular membrane used here receives equal quantities of analyte reproducibly. The samples were injected at 120 and 350s. from the start of the potential step experiment. The solution flow rate was -60 ~1 min-‘. A response time (time from injection to the half peak height) of -25 s. was measured and it includes the time for the sample front to come in contact with and diffuse through the membrane before detection. The same cell arrangement and parameters were used with a gold wire electrode in place of MTE and performed the experiment with 0.1 mM ferrocene solution. The data obtained had a similar appearance with a response time difference of -2.8 s. This difference represents the time for diffusion of the analyte through the membrane in the case with MTE. Based on a membrane wall thickness of 6.5 pm, a diffusion coefficient for lead(I1) in the membrane was calculated to be -7.5 x lop8 cm2 s-‘. This value closely resembles 5.4 x 5 x lop8 cm2 s--l reported in ref. [8]. The slight difference between these two values probably represents the difference in MWCO used.
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H.G. Jayaratna et al./Analytica
3.5. Linearity of stripping response Common mercury electrodes, mainly MDE and MFE, have been shown to give linear response over wide concentration ranges in stripping analysis. Saturation of the mercury phase by the plated metal could contribute to non-linear behavior of the MFEs results [9,10]. This is determined by the length of the plating time and the concentration of the analyte in the solution. Especially with MFEs, non-linearity can also result from the loss of integrity of mercury due to the formation of amalgam with the substrate metal and the low stability when plated on to the carbon surface. Another factor that determines linearity, especially with MDE, is the diffusion of the plated metal out into the bulk of the mercury in the capillary as the plating time is lengthened [ 101 Mercury thread electrode, on the other hand, does not suffer from the loss of integrity due to amalgam formation because no substrate metal is used. The stability of mercury electrode in a flow stream or a stirred solution is not in question because of inclusion in a membrane tube. But, non-linearity due to the saturation effects and the loss of analyte into the bulk mercury by diffusion could be anticipated. The linearity studies in the O-100 and 100-400 ppb lead(I1) ranges were performed separately. The MTE was fabricated with a regenerated cellulose membrane of -20000 MWCO. The background electrolyte employed was 0.12 M HCl. The DPSV was used as the electrochemical technique in the potential range of -800 to -2OOmV. A set of stripping voltammograms at five concentrations were obtained for the latter range with a plating time of 2 min. Peak currents were in low uA range. Five data points with RSD <2% at each concentration were collected and the plot of peak current vs. concentration showed excellent linearity with correlation coefficient (R) of 0.999. Other relevant fitting parameters were as follow: slope=0.96 uA ppbb’, intercept=-0.24 PA. A similar curve in the lower concentration range resulted an R value of 0.986 with a slope of 0.0042 pAppbb’ and an intercept of 0.053 PA. Five data points at each concentration were collected with a plating time of 60s. The magnitude of the peak currents recorded was in tenths of uA range. A lower plating time was used in order to see the noise level, detection limit and the feasibility of shorter analysis
Chimica Acta 332 (1996) 165-171
time. The noise level observed at 1Oppb concentration was ~5%. Based on 3 times the noise, a detection limit less than 2 ppb was estimated. The use of longer plating times will improve the precision and lower the detection limit. 3.6. Effect of plating time Non-linearity could result from the saturation effects. In order to evaluate the extent of this effect in MTE, stripping experiments were carried out with different plating times. The concentration of lead(I1) was fixed at 5Oppb in 0.1 M HCl. The stripping experiment was performed in square wave voltammetry mode. The plating time was varied from 1 to 5 min in steps of one minute collecting 3 data points at each. A plot of peak current vs. plating time showed good linearity with an R value of 0.996. Therefore, saturation does not occur with the highest plating time employed here. According to the linearity data discussed above, the MTE has excellent linearity with 2 min plating in the 100400 ppb lead(I1) range and saturation has not come into play. The problem of saturation, if it exists, can be avoided by lowering the plating time. For the concentrations 1OOppb or above, a plating time of 1 min or less would be more than adequate. During the analysis of actual blood or other biological samples for trace metals, higher plating times might be necessary if the diffusion of the analyte through the membrane is hindered in the presence of large proteins. 3.7. Application
to biological samples
The behavior of the membrane in biological samples was tested with blood samples received from the Blood Lead Proficiency Testing Program conducted by the US Department of Health and Human Services. In order to release bound lead [ 111, 6 M HCl (20 ~1) was added to whole blood (100 ~1) followed by dilution with water (880~1) to bring the acid concentration to 0.12 M. Final dilution of blood was by a factor of 10. This method resulted in a precipitate upon addition of 6M acid to the sample. The mixture, after stirring, was centrifuged to remove the precipitate. The clear solution was then injected into the solution stream (0.12 M HCl) with the help of
H.G. Jayaratna et al./Analytica Chimica Acta 332 (1996) 165-171
1
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response is independent of the flow rate, but it is limited by the rate of diffusion of the analyte through the membrane as discussed in the ref. [8]. The MWCO used will partially govern this situation. The disposal of used electrode material is made quite convenient because the mercury line was designed to be a completely closed system. The user would be able to advance the used mercury into a waste container which can then be recirculated when necessary without any direct physical contact. The cost of plastic tubing system is minimal and hence it is disposable. Some attention is required to make sure no mercury is retained in any of the tubes before discarding.
Potential / V Fig. 5. DPSV curves obtained for blood containing -65Oppb lead(I1). Scan Rate=20 mV s-‘, Pulse Amp.=50 mV, Sample Width=17 ms, Pulse Period=200 ms, Pulse Width=50 ms, Quite Time=10 s, Flow rate=25 ~1 min-‘, Plating time=4 min.
100 1.11sample loop. The determination of lead was achieved by DPSV. Stripping voltammograms from three consecutive runs are shown in Fig. 5. Relative standard deviation of the three runs shown here is less than 1% with an excellent S/N ratio. Therefore, prevention of electrode fouling appears to be effective due to the presence of cellulose membrane. The actual whole blood volume being used in the cell for each run is approximately 10 ul and contained -650ppb. The concentration seen by the electrode is 65ppb because of the dilution. Further evaluation of the performance of MTE in blood samples and quantitation of lead is due publication elsewhere [12].
Acknowledgements This work was financially supported by National Institute of Health (NIH) under the SBIR grant #l R43 ES0699 1-O.
References 111 PT. Kissinger and W.R. Heineman, Laboratory
121 131 141 151 [61 r71
4. Conclusion A flow cell design for the MTE is introduced and several aspects of it are investigated. The current design is simple and can easily be connected to a conventional liquid chromatography sampling valve. The performance of the membrane in a flow stream appears promising. The amount of mercury used is minimal (~200 mg/lOO runs). The electrochemical
PI [91
[lOI [ill [W
Techniques in Electroanalytical Chemistry, 2nd edn., Marcel Dekker, New York, 1995. Z. Kowalski, K.H. Wong, R.A. Osteryoung and J. Osteryoung, Anal. Chem., 59 (1987) 2216. B. Hoyer and T.M. Florence, Anal. Chem., 59 (1987) 2839. J.C. Vidal, R.B. Vinao and J.R. Castillo, Electroanalysis, 4 (1992) 653. W.W. Kubiak and Z. Kowalski, Talanta, 41 (1994) 1319. A. Trojanek, F. Opekar and K. Holub, J. Electroanal. Chem., 251 (1988) 41. H.B. Hane Kamp, Polarographic Continuous-Flow Detection, Rodopi, Amsterdam, 198 1. H.G. Jayaratna, Anal. Chem., 66 (1994) 2985. H.G. Jayaratna, Stripping/Plating Analysis at Carbon and Metallic Interdigitated Electrodes, Ph.D. Thesis, The Ohio State University, 1993. J. Wang, Stripping Analysis: Principles, Instrumentation and Application, VCH, Deerlield beach, FL, 1985. B.J. Feldman, J.D. Osterloh, B.H. Hata and A. D‘Alessandro, Anal. Chem., 66 (1994) 1983. H.G. Jayaratna, C.S. Bruntlett and PT. Kissinger, Clin. Chem., in preparation.
Analytica
Chimica Acta 332 (1996) 165-171
Mercury thread electrode in a flow cell H.G. Jayaratna”, Bioanalytical Received
C.S. Bruntlett,
P.T. Kissinger
Systems Inc., West Lafayette, IN 47906, USA
12 March 1996; revised 24 April 1996; accepted 29 April 1996
Abstract A mercury thread electrode modified with hydrophilic dialysis membrane was first introduced in 1994. Incorporation of this electrode in a flow stream is now introduced. The flow of solution and mercury are conveniently achieved using two syringe pumps. The cell can be used with a sample loop of a desired volume; hence, it allows flow injection applications.
Characterization of the flow cell was performed considering various experimental parameters using lead(I1) as the analyte. Calibration curves for the lead(I1) exhibit good linearity. Keywords: Stripping voltammetry; Flow methods; Mercury; Dialysis membrane
1. Introduction The electrochemical community greatly appreciates the benefits of mercury as an electrode material. The high overvoltage for hydrogen ion reduction, easily renewable surface and the large liquid range of mercury are responsible for its widespread use. It is an ideal choice for the determination of metal ions in aqueous and other media. Mercury electrodes are also being used as detector electrodes in liquid chromatography for the determination of organic compounds such as sulfides, thiols, peroxides, and nitro-organics. Since the description of the first polarographic apparatus by Heyrovsky in 1921, there have been various forms of mercury electrodes. These include the Dropping Mercury Electrode (DME), Hanging Mercury Drop Electrode (HMDE), Streaming Mer-
* Corresponding 1102.
author. Tel.: +l 317463 4527; fax: +l 317 497
0003-2670/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved PII SOOO3-2670(96)00229-2
cury Electrode (SME), Mercury Film Electrode (MFE), Static Mercury Drop Electrode (SMDE), and Controlled Growth Mercury Electrode (CGME) [ 1,2]. A disadvantage of mercury drop electrodes is that it is difficult to recycle the used mercury collected in the analytical solution. In addition, mercury electrodes exposed to complex samples such as blood, urine, etc. are easily fouled. Polymer modified MFEs have been useful for electrochemical studies in complex samples. The use of polymer (for example, Nafion) over a mercury film electrode and their application in trace metal determination have been investigated [3-51. Development of a flow cell using a mercury electrode is essential for an automated electrochemical analyzer, especially for trace metal determinations. Placing a mercury drop electrode in a flow cell has been attempted by many workers with limited success [5-71 The difficulties with the mercury drop in a flow cell are: the instability of the drop and the
166
H.G. Jayaratna et al./Analytica
noise introduced by the vibrations of the drop due to the flow of solution. Recently we introduced a new form of polymer modified mercury electrode titled, Mercury Thread Electrode (MTE) [8] for determining trace metals in biological samples. This electrode is constructed by enclosing a mercury column within a hydrophilic dialysis membrane tube of -150 urn diameter. The dialysis membrane prevents proteins and other large molecules present in biological samples from fouling the electrode surface while allowing intimate interaction with small molecules and elements. This paper describes the incorporation of the MTE in a flow cell arrangement and explores some of its properties.
2. Experimental 2.1. Reagents and solutions All chemicals were of analytical grade and were used without further purification. Cyclic Voltammetry (CV) experiments employed 0.1 mM lead(I1) in 0.1 M HCl. Lead solutions used in Anodic Stripping Voltammetry (ASV) were prepared by diluting 1OOOppm lead(I1) standards (Certified Atomic Absorption Standards, Fisher Scientific Company, Fair Lawn, NJ) with 0.1 M HCl background solution. Deionized water was used throughout this work for rinsing and solution preparation purposes. All glassware was stored in 1.OM HNOs acid bath and rinsed with water before use.
Chimica Acta 332 (1996) 165-I 71
MTE. Quantitative data analysis (calibration plot or standard addition plot) was performed with the BAS 1OOW electrochemical software. Syringe pumps (BAS Bee, BAS, West Lafayette, IN) were used for pumping analyte solution and mercury through the MTE. 2.3. Flow Cell Design One of the important features of the MTE design is that it can easily be made into a flow cell as shown in Fig. 1. This design utilizes a hydrophilic dialysis membrane tube transversely inserted through a Tygon tube. The Tygon tube used as the solution carrier had an internal diameter of -1.5 mm and an outer diameter of -3 mm. The internal diameter of this tube essentially defined the length of the MTE. A length of -2 cm of this tube defined a cell volume of -40 ul. The total length of the membrane tube used was approximately 8 mm which left about 2.5 mm lengths on either side of the Tygon tube. These ends were inserted into two pieces of polyacrylonitrile (PAN)
2.2. Instrumentation The instrument used for potential control in all the experiments was the BAS lOOB/W Electrochemical Workstation (BAS, West Lafayette, IN) which included a computer equipped with an Intel 486DX (33 MHz) microprocessor and a LaserJet IIIP printer. The Osteryoung Square Wave Stripping Voltammetry (OSWSV) and Differential Pulse Stripping Voltammetry (DPSV) techniques available with this instrument were chosen as the ASV modes. Other techniques such as, Osteryoung Square Wave Voltammetty (OSWV) and Linear Scan Voltammetry (LSV) were also used. CGME in the static drop electrode mode was used for comparison work with
OUTLET
(NJX
,
Fig. 1. Flow cell design for the MTE.
H.G. Jayaratna et al./Analytica
tube of internal diameter -1 mm with variable length and sealed with epoxy. These were used as mercury inlet and outlet. The outlet tube was connected to a vial to contain the used mercury. A syringe with a stainless steel needle that fit snugly into the inlet tube was chosen for delivering the mercury by means of a syringe pump (Bee pump, BAS). The same needle served as the working electrode contact. Any discontinuity in mercury was easily observed through the transparent PAN tubes. The Tygon tube ends were sealed on to the cell block with epoxy in order to prevent leaks. The ability to observe the electrode and the solution inside the cell is advantageous. In the case of blood, the sample was observed making contact with the MTE and timing for subsequent experiments was determined. Inlet and outlet ports were also made of plastic (PEEK) and attached to the central cell block with screws as shown (Fig. 1). The Teflon gaskets allowed leak-proof connection of inlet and outlet ports to the cell block and provided ionic communication between electrodes. The inlet port was a 1 cm thick PEEK disk with a plastic fingertight ferrule (Teflon or PEEK). A small diameter plastic tube of length -5 cm (0.65 mm o.d. and a dead volume of 1.2 ul/ 10cm) was used as the solution inlet tube. The same type of fingertight fittings were used to connect this tube to an injection valve (Rheodyne, model 7125) which allowed the use of a sample loop for convenient sample handling. A BAS Bee pump with a 5 ml syringe was connected to the inlet port for the delivery of solution. The outlet port was a similar diameter PEEK disk with a stainless steel tube of internal diameter of N 1 mm and a silver disk of diameter - 1 mm epoxied as shown in Fig. 1. The stainless steel tube served as the outlet tube as well as the auxiliary electrode. The silver disk was coated with AgCl to serve as the reference electrode for the electrochemical cell. This reference had an operational life time of about one day at low pH (< 1) values and a potential about 90mV negative of regular Ag/AgCl reference. Recoating of the reference with AgCl was conveniently done by polishing the electrode and placing a drop of AgCl coating solution (CF 2200, UniJet Reference Solution, BAS). Regenerated cellulose hollow fiber dialysis membranes of 13 000 and 18 000 molecular weight cut off
Chimica Acta 332 (1996) 165-l 71
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(MWCO), were purchased from Spectrum Medical Industries (Houston, TX). Nephross Allegro H.F., a third type of membrane of -20000 MWCO (Boxter, Netherlands), was also used. Internal diameter and the wall thickness of these membranes were specified as 200 and 8 urn, respectively. The membranes were stored at room temperature before use in the MTE fabrication.
3. Results and discussion The initial focus of this MTE research is trace metal determination. The work described here shows preliminary data using the flow cell design with aqueous lead(I1) samples. The sample was delivered to the cell directly from a syringe or through a Rheodyne valve with a sample loop of desired volume. The syringe pumps used for both mercury and solution allowed flow rates in the range from l600 pl min-’ with a 5 ml syringe. One of the most important advantages of the MTE is the ability to renew the electrode surface by advancing the mercury inside the membrane tube. A plastic syringe (5 ml) connected to a BAS Bee pump was used to move mercury through the membrane tube. Cyclic and stripping voltammetry were used to evaluate performance. 3.1. Cyclic voltammetry Cyclic voltammetry of 0.1 mM lead(I1) in 0.1 M HCl was performed with the flow cell arrangement which consisted of MTE with 18 000 MWCO membrane. The solution was pumped at -25 ul min-’ with the syringe pump. Fig. 2 shows the cyclic voltammetry between -200 and -600 mV with a scan rate of 100 mV s-l. Voltammogram A was recorded with stationary mercury, whereas the voltammogram B with moving mercury at a rate of about 8 ul min-‘. In A, the cyclic voltammogram has peak potential difference (A,??,) of 33 mV which indicates nearly ideal situation with fast electron transfer at the electrode for the redox couple of Pb(II)/Pb. During the forward scan lead(I1) is reduced and plated into the mercury thread. In the reverse direction, plated lead is oxidized and diffused into the flow stream to be swept away. The mercury thread
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lnit E (mV) = -200 High E (mV) = -200 Low E (mV) = -600 lnit P/N = N V (mV/s) =I00 Sweep Segments = 2 Slmpl Int (mV) =I Quiet T (s) = 2 (AA’) = 1 E-6
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the tube sealing procedure used in the fabrication procedure is satisfactory. Second, it is absolutely necessary to maintain a stationary mercury column during stripping analysis or the application will result in erroneous data. The cell design discussed in this paper occasionally showed loss of signal due to selfadvancement of mercury. Improvements to the cell design are currently being made to avoid this problem. 3.2. Renewal of Mercury
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Fig. 2. Cyclic voltammetry of 0.1x10~3moll~’ lead(I1) in 0.12 mol-’ HCl. (A) Stationary mercury; (B) Moving mercury.
with the reduced metal, therefore, needs to be kept stationary during the oxidative scan of cyclic and stripping voltammetry. The volume of the mercury thread exposed to the solution was about 2.7 nl (-370ng of mercury). With the given flow rate of 8 plmin-‘, this only takes 200ms. to pass the solution region of 1.5 mm length. Hence, the slightest movement of mercury could result in loss of analytical signal. The effect of flowing mercury on CV is shown by the scan B. Voltammogram B does not show an oxidation peak because the flowing mercury moves the section of the mercury thread with the plated metal out of the solution area well before it reaches the oxidizing potential for lead. But, the continuously moving mercury shows a reduction peak identical to that of stationary system. Since all the other parameters remain the same, the peak heights could have been changed in this situation only if the electrode area changed due to fluctuation in mercury pressure and expansion of the membrane during the flow. Therefore, it can be assumed that the electrode area does not change due to the flow of mercury. Two aspects that can be identified from the cyclic voltammetry data provided here are quite important for the use of the MTE. First, the solution is in contact with only the mercury column in the membrane portion within the Tygon tube and there is no flow of solution out into the bulk of mercury in the inlet and outlet tubes. This, in turn, indicates that
The response from stripping voltammetry was investigated to determine if the mercury advancement before each experiment has any effect on the magnitude of the signal. Lead solutions of 50 and 1OOppb concentration in a background electrolyte of 0.1 M HCl were used for this purpose. The flow cell was assembled with syringes containing these solutions. Analyses were performed with DPSV with a deposition time of 2min. Two sets of 5 stripping voltammograms were recorded with and without mercury advancement for each concentration with 2min of plating time. The average peak currents observed for these two concentrations were in the range of 50 and 80nA with an RSD less than ~2%. Therefore, it appears that frequent advancement of mercury is not necessary in clean water samples. However, in real samples, the need will be determined by the contaminants and MWCO of the membrane. 3.3. Effect of solution Jlow The response at the MTE is mainly governed by the rate of diffusion of the analyte through the membrane. Can the mass transport to the electrode be enhanced by increasing the flow rate of the solution? In order to answer this question an experiment was performed with membranes of different MWCO; namely, 13 000 and 18 000. A solution of lead (0.1 mM) in 0.1 M HCl was employed and the response at different flow rates was measured using square wave voltammetry. The data collected are shown in Fig. 3. Each point is the average of five readings that had an RSD < 1%. Fluctuation of average data over the entire range of flow rates was also N 1% for both membranes. Therefore, the
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Flow Rate (pL / min) Fig. 3. Solution flow rate effects on OSWV response at MTEs with two different MWCO: (0) 13 000; (0) 18000. Solution=O. 1 x 10~3moll~’ lead(l1) in O.l2mollHCl, Init. E=-2OOmV, Final E=-700mV, S.W. Amp.=25, Freq.=60Hz, Step E=4 mV, Quite time=2 s, Sensitivity=1 x 10m6 AV-‘.
I
response at the electrode is not dependent on the flow rate and the mass transfer limiting factor appears to be the rate of diffusion of the analyte in the membrane, at least for the MWCOs used here. There is a greater response at higher MWCO due to the higher rate of diffusion. Membranes with much larger pore size may show some dependence on the flow rate. Membranes of MWCO up to 20000 (Nephross) did not show flow rate dependence. This is very favorable for the application of the MTE, because different flow rates can be utilized to alter the residence time of the sample at the electrode. Hence, the plating time in ASV experiments can be adjusted without affecting the magnitude of the response, especially, with small amounts of sample. Continued investigation is necessary for better use of this strategy. 3.4. MTE for J~OWinjection analysis In order to test the ability of the MTE to detect a sample portion injected into the flow stream reproducibility was explored next. An MTE of -20000 MWCO was connected to a Rheodyne valve with a 100 pl sample loop and then 0.1 mM lead(I1) in 0.1 M HCl were injected. The detection of this sample was achieved by stepping the potential of the electrode to -700 mV and monitoring the response with time. Fig. 4 illustrates the detection of two 100~1 portions of lead solution at the MTE.
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Time I set Fig. 4. Time base response observed at MTE with a membrane of 20 000 MWCO for 100 pl portions of sample injected through a Rheodyne valve with a 100 11 loop. Electrode potential=-700mV.
The particular membrane used here receives equal quantities of analyte reproducibly. The samples were injected at 120 and 350s. from the start of the potential step experiment. The solution flow rate was -60 ~1 min-‘. A response time (time from injection to the half peak height) of -25 s. was measured and it includes the time for the sample front to come in contact with and diffuse through the membrane before detection. The same cell arrangement and parameters were used with a gold wire electrode in place of MTE and performed the experiment with 0.1 mM ferrocene solution. The data obtained had a similar appearance with a response time difference of -2.8 s. This difference represents the time for diffusion of the analyte through the membrane in the case with MTE. Based on a membrane wall thickness of 6.5 pm, a diffusion coefficient for lead(I1) in the membrane was calculated to be -7.5 x lop8 cm2 s-‘. This value closely resembles 5.4 x 5 x lop8 cm2 s--l reported in ref. [8]. The slight difference between these two values probably represents the difference in MWCO used.
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3.5. Linearity of stripping response Common mercury electrodes, mainly MDE and MFE, have been shown to give linear response over wide concentration ranges in stripping analysis. Saturation of the mercury phase by the plated metal could contribute to non-linear behavior of the MFEs results [9,10]. This is determined by the length of the plating time and the concentration of the analyte in the solution. Especially with MFEs, non-linearity can also result from the loss of integrity of mercury due to the formation of amalgam with the substrate metal and the low stability when plated on to the carbon surface. Another factor that determines linearity, especially with MDE, is the diffusion of the plated metal out into the bulk of the mercury in the capillary as the plating time is lengthened [ 101 Mercury thread electrode, on the other hand, does not suffer from the loss of integrity due to amalgam formation because no substrate metal is used. The stability of mercury electrode in a flow stream or a stirred solution is not in question because of inclusion in a membrane tube. But, non-linearity due to the saturation effects and the loss of analyte into the bulk mercury by diffusion could be anticipated. The linearity studies in the O-100 and 100-400 ppb lead(I1) ranges were performed separately. The MTE was fabricated with a regenerated cellulose membrane of -20000 MWCO. The background electrolyte employed was 0.12 M HCl. The DPSV was used as the electrochemical technique in the potential range of -800 to -2OOmV. A set of stripping voltammograms at five concentrations were obtained for the latter range with a plating time of 2 min. Peak currents were in low uA range. Five data points with RSD <2% at each concentration were collected and the plot of peak current vs. concentration showed excellent linearity with correlation coefficient (R) of 0.999. Other relevant fitting parameters were as follow: slope=0.96 uA ppbb’, intercept=-0.24 PA. A similar curve in the lower concentration range resulted an R value of 0.986 with a slope of 0.0042 pAppbb’ and an intercept of 0.053 PA. Five data points at each concentration were collected with a plating time of 60s. The magnitude of the peak currents recorded was in tenths of uA range. A lower plating time was used in order to see the noise level, detection limit and the feasibility of shorter analysis
Chimica Acta 332 (1996) 165-171
time. The noise level observed at 1Oppb concentration was ~5%. Based on 3 times the noise, a detection limit less than 2 ppb was estimated. The use of longer plating times will improve the precision and lower the detection limit. 3.6. Effect of plating time Non-linearity could result from the saturation effects. In order to evaluate the extent of this effect in MTE, stripping experiments were carried out with different plating times. The concentration of lead(I1) was fixed at 5Oppb in 0.1 M HCl. The stripping experiment was performed in square wave voltammetry mode. The plating time was varied from 1 to 5 min in steps of one minute collecting 3 data points at each. A plot of peak current vs. plating time showed good linearity with an R value of 0.996. Therefore, saturation does not occur with the highest plating time employed here. According to the linearity data discussed above, the MTE has excellent linearity with 2 min plating in the 100400 ppb lead(I1) range and saturation has not come into play. The problem of saturation, if it exists, can be avoided by lowering the plating time. For the concentrations 1OOppb or above, a plating time of 1 min or less would be more than adequate. During the analysis of actual blood or other biological samples for trace metals, higher plating times might be necessary if the diffusion of the analyte through the membrane is hindered in the presence of large proteins. 3.7. Application
to biological samples
The behavior of the membrane in biological samples was tested with blood samples received from the Blood Lead Proficiency Testing Program conducted by the US Department of Health and Human Services. In order to release bound lead [ 111, 6 M HCl (20 ~1) was added to whole blood (100 ~1) followed by dilution with water (880~1) to bring the acid concentration to 0.12 M. Final dilution of blood was by a factor of 10. This method resulted in a precipitate upon addition of 6M acid to the sample. The mixture, after stirring, was centrifuged to remove the precipitate. The clear solution was then injected into the solution stream (0.12 M HCl) with the help of
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response is independent of the flow rate, but it is limited by the rate of diffusion of the analyte through the membrane as discussed in the ref. [8]. The MWCO used will partially govern this situation. The disposal of used electrode material is made quite convenient because the mercury line was designed to be a completely closed system. The user would be able to advance the used mercury into a waste container which can then be recirculated when necessary without any direct physical contact. The cost of plastic tubing system is minimal and hence it is disposable. Some attention is required to make sure no mercury is retained in any of the tubes before discarding.
Potential / V Fig. 5. DPSV curves obtained for blood containing -65Oppb lead(I1). Scan Rate=20 mV s-‘, Pulse Amp.=50 mV, Sample Width=17 ms, Pulse Period=200 ms, Pulse Width=50 ms, Quite Time=10 s, Flow rate=25 ~1 min-‘, Plating time=4 min.
100 1.11sample loop. The determination of lead was achieved by DPSV. Stripping voltammograms from three consecutive runs are shown in Fig. 5. Relative standard deviation of the three runs shown here is less than 1% with an excellent S/N ratio. Therefore, prevention of electrode fouling appears to be effective due to the presence of cellulose membrane. The actual whole blood volume being used in the cell for each run is approximately 10 ul and contained -650ppb. The concentration seen by the electrode is 65ppb because of the dilution. Further evaluation of the performance of MTE in blood samples and quantitation of lead is due publication elsewhere [12].
Acknowledgements This work was financially supported by National Institute of Health (NIH) under the SBIR grant #l R43 ES0699 1-O.
References 111 PT. Kissinger and W.R. Heineman, Laboratory
121 131 141 151 [61 r71
4. Conclusion A flow cell design for the MTE is introduced and several aspects of it are investigated. The current design is simple and can easily be connected to a conventional liquid chromatography sampling valve. The performance of the membrane in a flow stream appears promising. The amount of mercury used is minimal (~200 mg/lOO runs). The electrochemical
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Techniques in Electroanalytical Chemistry, 2nd edn., Marcel Dekker, New York, 1995. Z. Kowalski, K.H. Wong, R.A. Osteryoung and J. Osteryoung, Anal. Chem., 59 (1987) 2216. B. Hoyer and T.M. Florence, Anal. Chem., 59 (1987) 2839. J.C. Vidal, R.B. Vinao and J.R. Castillo, Electroanalysis, 4 (1992) 653. W.W. Kubiak and Z. Kowalski, Talanta, 41 (1994) 1319. A. Trojanek, F. Opekar and K. Holub, J. Electroanal. Chem., 251 (1988) 41. H.B. Hane Kamp, Polarographic Continuous-Flow Detection, Rodopi, Amsterdam, 198 1. H.G. Jayaratna, Anal. Chem., 66 (1994) 2985. H.G. Jayaratna, Stripping/Plating Analysis at Carbon and Metallic Interdigitated Electrodes, Ph.D. Thesis, The Ohio State University, 1993. J. Wang, Stripping Analysis: Principles, Instrumentation and Application, VCH, Deerlield beach, FL, 1985. B.J. Feldman, J.D. Osterloh, B.H. Hata and A. D‘Alessandro, Anal. Chem., 66 (1994) 1983. H.G. Jayaratna, C.S. Bruntlett and PT. Kissinger, Clin. Chem., in preparation.