468

Amperometric Flow Injection Analysis of Organic Thiols and Proteins Achmad Hidayat,' D. Brynn Hibbert*+ and Peter W. AEexunder+'

+ Department of Analytical Chemistry, The University of New South Wales, Sydney, Australia 2052 ++Department of Physical Sciences, University of' Tasmiania, PO Box 1214, Launceston, Tasmania, 7250

Received: January 2, 1995 Final version: March 3, 1995

Abstract The amperometric determination of thiols by injection into a mercury carrier stream is shown to be a sensitive method when using a tungsten wire as the working electrode in a three-electrode flow cell. Hg2+ gives sharp peaks when injected into phosphate buffer at pH 3.1 at an operating voltage of -0.24V (against a Ag/AgCl reference electrode). Hg2+ was determined in the range 0.43 to 25.0pM with linear response. Injection of cysteine into the mercury carrier causes a decrease in the cathodic current, and sharp flow injection peaks are observed with a peak width of approximately 60s. Optimization of various parameters was performed, including buffer effects, mercury concentration, injection volume, and the effects of various cations and anions on the response were determined. Linear calibrations in the range 0.25pM to 100pM for cysteine and 1.3 to 1000 ng for the other thiols glutathione, thiourea, albumin, and DNA were found. The responses to cysteine and thiourea were most sensitive, and interference from silver and iodate was observed at concentrations in excess of the thiols. The method may be used as a rapid means of detecting thiols in various samples and as an HPLC detection method.

Keywords: Thiols, Flow injection analysis, Cysteine

1. Introduction

Electrode materials such as graphite, glassy carbon, and the conventional dropping mercury electrode (DME), various mercury electrodes, amalgamated or mercury-coated metallic electrodes, and mercury pool electrodes have been used for detecting metals and organic thiols and proteins [l-71. Graphite and glassy carbon are now commonly used in HPLC detection methods, but are subject to poisoning, and ordinary carbon electrodes have a high overpotential for electrooxidation of these organic species [ 1, 21. Consequently, their electrochemical quantitation following liquid chromato- graphy (LC) is usually performed at a mercury or a mercury amalgam [8, 91 electrode at which the mercury sulfide species formed can be oxidized at comparatively low potentials. However, methods based on mercury electrodes are too inconvenient for flow analysis detection methods. The over- potential for the oxidation of thiols may be decreased by modification of carbon and graphite [2, lo]. Recently, Cox and Gray [2] applied a glassy carbon electrode coated with ruthenium cyanide for the detection of insulin, cysteine and glutathione with detection limits below the pM level. How- ever, the preparation of these chemically modified electrodes (CMEs) is often tedious and experience with electrochemical techniques is needed.

Hou and Wang [lo] used a Prussian blue-modified glassy carbon electrode, but this electrode was not stable in flowing systems when the applied potential was more positive than 0.9 V. They coated the electrode with Nafion to improve the stability and reproducibility in the flow stream.

An amperometric sensor constructed with a working metallic tungsten electrode vs. a reference saturated calomel electrode (SCE) and a counter platinum electrode in a static, 20mL electrochemical cell has been reported for detecting thiols and proteins [ll]. It is shown that good detection limits (in the range 0.05-0.2 mgmL-') and sensitivity are achieved when organic thiols are injected into mercury (11) reagent solution buffered in 0.005 M sodium acetate at pH 4.7, at an operating voltage of -0.20 V. The principle of this method is to measure the excess of mercury (11) ions after complex reaction with organic thiols (RSH) to form mercury complexes (RS)2Hg

[13,14]. Complexation reduces the Hg2+ activity in the solution, and thus reduces the cathodic current of the ion.

The use of flow injection with amperometric detection has several advantages over a conventional static system. Construc- tion of flow-through cells is more simple, and due to the smaller size better performance can be obtained, for example, simple operation, rapid sampling rate, smaller reagent and sample consumption, good precision and sensitivity, and possible application for HPLC.

The aim of this study is to determine the performance of the detection of thiols in flow injection analysis. The experimental parameters, such as working potentials, flow rate, volume of injection, pH, and concentration of buffer and reagent solution are investigated, and some factors which caused poor repro- ducibility are identified and eliminated. A variety of organic thiols, particularly those of biological significance, were tested. Finally the sensor was utilized for detecting a mixture of organic thiols after separation with HPLC.

2. Experimental

2.1. Apparatus

A PAR-I 74 polarograph was used together with an X - Y and Omniscribe recorder. A single line flow injection manifold was used, consisting of a Gilson-Minipuls-3 peristaltic pump, injection port, transmission tubing of 0.5 mm inside diameter and the flow detection system. The flow detection system was a perspex flow through cell, described previously by Alexander et al. (121, with a widened (4.0 mm) compartment for the silver- silver chloride reference electrode. This compartment was filled with hot liquid agar (20% w./v. in 1% potassium chloride solution). The working electrode was a tungsten wire of l.Omm in diameter and platinum wire was used as an auxiliary electrode. The electrodes were made from wires of 99% purity obtained from Johnson Matthey, UK. The silver- silver chloride reference electrode was prepared by electrolysis at a silver wire electrode (1.0mm diameter) in 1% potassium chloride solution by application of a potential of +0.6V for 5 min.

Electrounulysis 1996,8, No. 5 0 VCH Verlugsgesellschaft mbH, 0-69469 Weinheim, 1996 1040-0397/96/0505-468 $ 10.00+.25/0

Amperometric FIA of Organic Thiols and Proteins 469

2.2. Chromatography

A liquid chromatograph with electrochemical detector utiliz- ing post column reaction with Hg2+ consisted of two recipro- cating dual-piston pumps, one for delivering mobile phase at mostly 0.3 mL min-' and the other for the post column reaction (1 .O mLmin-I). Isocratic separation was performed on a commer- cial 5 mm octadecylsilyl-modified silica (C18) column (250 x 4.6mm I.D., Zorbax ODs, DuPont). The chromatograms were recorded on an Omniscribe chart recorder and interfaced to an Apple IIe computer. Further data manipulation was performed using a Macintosh computer.

2.3. Reagents

Phosphate buffer (0.1 M NaH2P04 and 0.1 M Na2HP04) were prepared from analytical grade reagents and deionized distilled water. A stock solution of Hg2+ was prepared by dissolving the appropriate amount of HgC12 in the buffer solution. The stock solutions were made up to the appropriate concentration and pH as required. L-Cysteine hydrochloride hydrate and gluthatione (reduced form) were purchased from BDH. m-penicillamine, 3-mercaptopropanoic acid, albumin (bovine), phenyl-thiourea and deoxy-ribonucleic acid (DNA) were obtained from Sigma Chemicals Co. These standard solutions were prepared daily.

12.5 pM

10 pM

~

7.5 pM

5.0 pM

2.5 pM 1111 2.4. Procedures

The current at tungsten for the reduction of 10 pM Hg2+ was determined by injecting 1OpL of the ion into the flow system (0.1 M phosphate or 0.1 M acetate buffer of pH 3.1) at potentials from -0.13V to -0.30V and flow rate of lSmLmin-'. The potential giving the highest current, -0.240 V (vs. silver-silver chloride), was used as the operating potential for further experiments. The effects of concentration and pH of buffer solution and concentration of Hg2+ and the injection volume were studied by varying the respective parameters. The effect of flow-rate was investigated by varying the pump setting with single tubing of 2 mm internal diameter. The peak height and width for the reduction of Hg2+ were measured.

Experiments that studied the analysis of cysteine and other thiols were performed by injecting 10 pL of a thiol standard into a carrier stream, without deaeration, containing Hg2+. Each measurement was performed in triplicate and the conditions were optimized as described above. To determine the stability of the electrodes and working range of cysteine, cysteine standards between 2.5 and 12.5 pM were recorded each day for four weeks.

Interference effects were studied by injecting 1OpL of solutions containing 7.5 pM cysteine and 100 pM cations of nitrate salts, or anions of potassium salts, into phosphate buffer containing 20 pM Hg2+. The current changes were compared with the current change due to injecting 1OpL of 7.5pM cysteine solution alone. The ions tested were Ag', Br-, Co2+, Cu2+, Fe2+, Fe3+, 107, Mn2+, Ni2+, NO;, NO;, Pb2+, SCN-, SO:-, Sr2+, and Zn2+. These ions were prepared by dissolving the appropriate salts in distilled water to make up solution of 0.2 pM. One mL of each solution was mixed with 1 .O mL of 200 pM buffer solution containing 7.5 pM cysteine. The mixture was injected into the system, and detected by the tungsten electrode.

HPLC separation of a mixture of thiols was detected using the cell described above and post column reaction with a carrier stream containing 20 pM Hg2+.

2 min

Fig. 1. Typical peaks for the injection of Hg2+ standards in 0.1 M phosphate buffer of pH 3.1, at a tungsten working electrode (-0.240 V vs. Ag/AgC1).

3. Results and Discussion 3.1. Response to Hg2'

Typical flow injection peaks for the reduction of Hg2+ standard solutions are shown in Figure 1. The standards were recorded by injecting 1OpL of the solutions into 0.1 M phosphate buffer of pH 3.1, at potential -0.240V and flow rate of 1 .5m~min- ' .

The changes in current produced varied linearly with con- centration of Hg"+ in the range from 2.5pM to 12.5pM Hg2+, with a correlation coefficient of 0.999 and slope of 0.53pA (pM Hg2+)-'. The precision obtained when 12.5pM Hg2+ was successively injected into phosphate buffer nine times was 1.8% (RSD). A detection limit (3gblank) and sensitivity of 0.43 pM Hg2+ and 32.1 nA (pM Hg2+)-' mm-2 respectively, were obtained in this flow system. This value is better than previous experiments in static systems which had a detection limit of 2.0 pM Hg2+ and sensitivity of 9.6 nA (mM Hg2+)-' mm-2 [l 11. The effects of improved transport are clearly seen in the FIA results.

3.2. Response to Cysteine

When cysteine was injected into phosphate buffer containing 15pM Hg2+ the current was reduced significantly as cysteine reacted with Hg2+ to form the complex compound of mercury cysteinate. The negative peak rapidly returned to the base line current as the sensor detected the following fresh Hg2+ solution. Typical FI peaks and calibration graphs for the injection of cysteine standards are shown in Figures 2 and 3 respectively.

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470 A . Hidayat et al.

5 min -

Fig. 2. Typical peaks for the injection of cysteine standards in 0.1 M phosphate buffer of pH3.1 containing 15,uM Hg2+, at a tungsten working electrode (-0.24 V vs. Ag/AgCl).

The response of tungsten electrode to Hg2+ at different flow rates is given in Table 1.

The changes in current varied linearly with cysteine concen- tration with a correlation coefficient of 0.994 and slope of 0.22pA (pM cysteine)-' in the range from 1.0pM to 100pM. Above 100 pM the current curves as the added cysteine saturates the mercury. This behavior is to be expected in a system in which the mechanism involves complex formation. It may be inferred that the maximum change in current for 300 pM Hg2+ is about 33p.A. An additional factor that is observed at high cysteine concentration is adsorption of uncomplexed cysteine on the electrode leading to severe peak tailing. The sensitivity in the flow system was 13.3nA (pM cysteine)-'rnmp2 which was

/I" 10 - b

0 200 400 Cysteine /pM

600

Fig. 3. Graph of peak height against cysteine concentration for the injection of 10 mL of 20pM cysteine into a 0. I R.1 phosphate buffer of pH 3.1 containing 0 100pM Hg2+, 0 200pM Hg2'~, W 300pM HgZC,at a tungsten working electrode (-0.24OV vs. Ag/AgCl). Insert is the linear part at low cysteine concentration after injection into 100pM Hg''.

Elcctrounalysis 1996,8, No. 5

higher than previous experiments in a static system [sensitivi- ty = 5.8 nA (pM cysteine)-l mrn-*], and therefore the detection limit is also better in the flow system (0.25 pM compared with 0.4 pM cysteine), again due to the improved transport in a flow system. Sampling rates of 60 samples per hour are obtainable with this flow system, with precisions of less than 2% (RSD).

We note that the peak width is some 2.5 times greater in the analysis of cysteine than in the calibration of mercury (Table 1). Kolthoff [ 151 found irregularities in polarographic anodic wave when using alkaline buffer due to the formation of a film of HgRS in the surface of the electrode which caused leveling off of the current. The linearity of the FIA calibration was better than in the static system when the mercury cysteinate film tended to form on the cathode in the more alkaline conditions of that experiment.

3.3. Parameters Affecting the Sensitivity of Cysteine Determination

Increasing the concentration of Hg2+ increases the sensitivity of the method. The slope of the cysteine standard increased from 87 nA pM-' to 283 nA pM-I when the concentration of the H$+ in the carrier solution was increased from 7.5 pM to 25 pM.

Increasing the buffer concentration from I mM to 100mM increased the peak current and slope of the standard cysteine cali- bration plot (1 5 pM Hg2') from 18 nA pM-' to 220 nA pM-'. This is a significant increase and may reflect the interaction of Hg2+ with phosphate, or simply a reduction in resistance of the solu- tion. For values less than pH4.8, decreasing the pH of the buffer solution increased the change in current, therefore increas- ing the sensitivity of the method. A pH of 3.1 was chosen as opti- mum. The electrode itself did not respond to the buffer at pH 3.1.

Both peak current of Hg2+, and the current change due to injection of cysteine (Table 1) fell as the flow-rate was increased from 1.5 to 5.6mLmin-'. This reduction is due to the dilution effect caused by increasing the flow rate. However, increasing the flow rate also increased the rate of analysis, due to a decrease in the peak width.

Amperometric FIA of Organic Thiols and Proteins 47 1

Table 1. Effect of flow rate on peak current and peak width of injected Hg" (1OpL of 20pM) and of cysteine (10pL of 20pM) into Hg2' (20 PM).

Flowrate Peak current [PA] Peakwidth Is] [ m ~ m i n - ' ]

Hg2+ Cysteine Hg2' Cysteine

1.5 2.0 2.9 25 60 3.2 1.8 2.6 20 50 4.5 1.7 2.4 18 40 5.6 1.6 2.2 14 35 ____ ~ ~ _ _ _ _ _ _ _ _ _ _ _ _ _ _

The peak height increased linearly with injection volume to 20 mL of 20 pM cysteine into 20 pM Hg2+ after which it levelled out.

3.4. Interferences

Within the concentration ranges studied, only Ag+ and 10; significantly interfered with the Hg2+ reduction current. Silver ions increased the cysteine peaks by 7.2%, whereas 10; reduced cysteine peak by 8%. Further study confirmed that the maximum concentrations of ions which did not affect the cysteine response were 1 pM for Ag' and IO;, 10 pM for Cu2+ and Fe3', and 5pM for SO",. Silver ions will be reduced at -0.240V [E" (Ag+/Ag)= t0.58V vs. Ag/AgCI] and so will augment the reduction current. The observed increased change in current may arise from interaction between the thiol and silver ion. Iodate may also be reduced (10; + 6H' + 6e + I-+ 3H20, E" = 0.68V vs. Ag/AgCl at pH 3.1) giving the expected smaller cysteine peak.

This finding supports the previous studies by Alexander et al. [Il l , Sugawara et al. [16], Csejka et al. [17], and Forsman [18]. The high formation constant of mercury cysteinate ensures that when mercury ions predominate in the solution this complex is formed.

3.5. Calibration of Cysteine and Other Thiol Compounds

Calibration data for cysteine, thiourea, glutathione, DNA and albumin are given in Table 2. The results confirmed the previous study in the static system 1111, that organic compounds containing thiol groups undergo complex formation with Hg2+ and reduce the reduction current of Hg2+ with different sensitivities. Table 2 shows that cysteine was the most sensitive among the compounds studied.

This is similar to results from CSV reported by Forsman [3] which is selective toward the sulfhydryl-containing amino acids in a mixture of amino acids. Injection of 1 .O pM solutions of L-leucine, m-alanine, DL-histidine, L-aspargine, L-arginine, L-methionine glycine, lycine, aspartic acid, and glutamic acid caused no detectable current change. As a result of this

Table 2. Working concentration ranges and calibration data for various thiols.

Compound Range [pgmL-'] Slope [ P A pg-' mL] Correlation coejicient

Cysteine 0.13-1.50 I 303 0.983 Thiourea 0.50-2.25 0.585 0.997 Glutathione 1.50-25.0 0.083 0.995 DNA 6.25-100.0 0.010 0.984 . Albumin 6.25-100.0 0.008 0.98 1

.~ .. ~- ~~~~

5 1 0 1 5 2 0 2 5 3 0 3 5

Timelmin

Fig. 4. HPLC of 20 mL of 1: cysteine (75 ng), 2: oL-homocysteine (50 ng), 3: reduced glutathione (75 ng), 4: m-penicillamine (75 ngj, 5: 3- mercaptopropanonic acid (50 ng) and 6: thiourea (Song). Details of column and post column reaction are given in Table 3.

Table 3. Retention time ( k ' ) and concentration of selected organic thiols after reverse phase separation by HPLC. Column - CIS; mobile phase: 0.05 M 3-chloroacetate/l% methanol, pH 2.3; flowrate: 0.3 mL min-'; postcolumn reaction: 0.05 M phosphate buffer, pH 2.3 containing 15 pM HgZt; flowrate: 1 .OmLmin-' ~ . .. . .- ~~~ -.

Thiol k ' Amount injected Current Iminl Ingl [PA1

Cysteine 0.8 15 oL-homocysteine I .5 50 Reduced glutathione 2.0 75 o~-penicillamine 2.9 50 3-mercaptopropanoic acid 4.6 50 Thiourea 6.5 15

1.4 0.7 0.6 0.7 0.1 0.1

selectivity for the analysis of sulphydryl compounds by HPLC it is only necessary that these compounds are separated from each other and not necessarily from the other amino acids and peptides in the sample.

The detection limit obtained in this flow system is better than in a static system and appears promising when compared ,with amperometric methods using chemically modified carbon-based electrodes [16, 191. The advantage of the tungsten electrode, as most metallic electrodes, is that it is easy to prepare with good stability. The long-term operational stability showed 80-9O0/0 of the initial sensitivity over a 3-month period. This lifetime is superior to that of a CME which degrades because of the gradual leaching of the CME surface. In fact, some decrease in response has been observed previously even for conventional carbon paste electrodes in LCEC upon long-term exposure to a binary mobile phase containing a small fraction of organic components. Surface contamination also causes problems with a HMDE or mercury pool electrode. Florence [7] reported that the baseline did not return to a normal position after seven cleaning scans following the analysis of cysteine.

3.6. Response of Sensor to Thiols after Separation by HPLC

A mixture of cysteine, DL-homocysteine, reduced glutathione, DL-penicillaniine, 3-mercaptopropanonic acid and thiourea were reversed-phase separated with the mobile phase reported by Shea and MacCrehan [20]. Figure 4 demonstrates that the thiols were base line separated with acceptable retention time. Table 3 gives retention times and sensitivities and shows

Elecrroanalvsis 1996, 8, No. 5

412 A . Hidayat et al.

that a tungsten detector.

wire is a promising alternative as an HPLC 131 U. Forsman, Anal. Chim. Acta 1984,100, 141. [4] P.W. Alexander, M.H. Shah, Talanta, 1979,26,91. [5] P.W. Alexander, U. Akapongkul, And. Chim. Acta 1983, 148, 103. [6] R. Eggli, R. Asper, Anal. Chim. Acta 1978, 101, 253. 171 T.M. Florence. J. Electroanal. Chem.. 1979. 97. 237

4. Acknowledgement i8j R.F. Bergstrom, D.R. Kay, J.G. Wagner, J.Chromatogr., 1981,222,445. [9] L.A. Allison, R.E. Shoup, Anal. Chem,, 1983,55, 8.

[lo] W. Hou, E. Wang, J. Electroanal. Chem., 1991, 316, 155 [ l l ] P.W. Alexander, A. Hidayat, D.B. Hibbert, Electroanalysis 1995, 7,290. 1121 P.W. Alexander, P.R. Hadad, M. Trojanowicz, Anal. Chim. Acta 1985, The authors are to the Indonesian Agency for

Agricultural Research and Development for a fellowhip in 171, 151. support of A. Hidayat.

5. References

[l] M.K. Halbert, R.P. Baldwin, Anal. Chem., 1995, 57, 591 [2] J.A. Cox, T.J. Gray. Anal. Chem., 1989, 61, 2462.

[I31 J. S . Fritz, T. A. Palmer, Anal. Chem., 1961, 33, 98 [I41 E.P. Serjeant, in Chemical Analysis, Vol. 69, Potentiometry and

Potentiometric Titrations, Wiley Sons, New York, 1984. [I51 I. M. Kolthoff, C. Barnum, Anal. Chim. Acta 1940, 62, 3061. [16] K. Sugawara, S. Tanaka, M.Taga, J . Electroanal.Chem., 1991,316, 305. [I71 D.A. Csejka, S.T. Nakos, E.W. DuBord, Anal. Chem. 1975, 47,322. [18] U. Forsman, J. Electroanal. Chem., 1981, 122, 215. [I91 A. Cox, T. J. Gray, in Contemporary Electroanalytical Chemistry (Ed:

[20] D. Shea, W.A. MacCrehan, Anal. Chem., 1988,60, 1449. A. Ivaska), Plenum Press, New York, 1990, p. 267-281.