Detection limits and Selectivity in Electrochemical detectors are in direct contact with the solution under scrutiny. Electrodes can be used to drive chemical changes that are detected by another sensor, or electrodes can be used to detect the products of chemical changes wrought by other processes. The close chemical relationship between the electrode and the solution allows better analytical systems to be developed and provides challenging questions in the understanding of figures of merit.
Stephen 0. Webe JotwrT. Llmg Oeparrment of Chemir
University of Pittsburgh Pmsburgh, PA 15260
109-27W/88/0360-903A/SO 1 .SO/O ) IQRR American Chemical Societv
ANALYTICAL CHEMISTRY,
nal and noise generation and signal-to noise ratio (SiN), the improvement o qualitative information content, ana control of selectivity of the detector. In each area there are many opportunities to increase our knowledge; we will describe some relevant research. We hope to demonstrate that there is much to learn and that the electrochemical approach can be made more versatile and powerful than it is today. Predictingsignals and nolse The foundation of any technique for quantitative analysis is the relationship between the measured quantity and the amount of analyte in the sample. The simplest electrochemical detectors operate at constant potential (constant oxidizing or reducing power). When exposed to a flowing stream of solution, the electrode yields a current that is the sum of a background current and any current caused by the presence of electroactive species. Because one measures current at these electrodes, they are called amperometric. Equations relating the current to the concentration of the analyte are dependent on manv factors such as fluid flow rate, U,ita kinematic viscosity, v , the solute’s diffusion coefficient, D, and the cell’s dimensions (1).The kinematic viscosity is the ratio of the fluid‘s viscosity to density. Examples of electrochemical detectors include the thinlayer cell, the tubular electrode, the impinging flow electrode with larg, spacer, and the impinging flow elec trode with small spacer. Equations for the current generated in these and other electrochemical detectors have recently been tabulated (2,3). One can appreciate the signal-generatingmechanism by considering the example equation for the thin-layer detector (Figure 1) operating at low efficiency (high flow rates or with small electrodes). The sensitivity is the current (i) per concentration ( C ) :
sensitivity = i/C = 1.467nFU”3(DA/b)2’3 (1) In this equation, nF is the number of mulomhs of electrons per mole of analyte, A is the electrode area, and b is the height or thickness of the channel. This equation is appropriate when the applied potential is sufficient to keep the concentration of analyte at the surface equal to zero. Several general observations can be made from this equation. The current is time independent. The equation applies to a steady-state cell in which one unit of analyte flows in per second and a fraction, f, equal to il nFCU, is electrolyzed. The complementary fraction, 1% flows to waste. The sensitivity increases (although the efficiency decreases) as the flow rate of the mobile phase increases. S04A
-
ANALYTICAL CHEMISTRY, VOL. 60,
fusion is increased. (Consequently, electrochemical detectors in supercritical fluid chromatography should be quite sensitive.) When the electrode area, A, is inaeased, the sensitivity also increases, hut the increase in sensitivity is not proportional to the increase in electrode area. This can be understood by imagining that the electrode is made up of a series of adjacent strips running across the direction of flow. The current at the second strip is not as great as that at the first strip because of the depletion of material from electrolysis at the first electrode. Thus an incremental addition of electrode results in a less than proportional increase in signal. Finally, the thickness of the channel, b, can be decreased to increase current. The gradient of fluid velocity at the surface is greater with a lower b. This increases the rate of the replenishment of the solution near the electrode. For cells of typical dimensions and working under typical conditions, the efficiencyofthe cell,f , is on the order of 0.01. One should be careful not to interpret the term “low efficiency” in a pejorative sense. The flame ionization detector in gas chromatography is a lowefficiency detector, but it is nonetheless important and powerful. The discussion above, in its most general terms, suffices for an understanding of any low-efficiencydetector. 1) are High-efficiency detectors (f somewhat different. Electrodes of high efficiency have heen called coulometric. This is a misnomer; one measures current with these cells. They got the name coulometric because in the ideal case, where f = 1, the analyte is completely consumed by an electrode process, as in a coulometric titration. A preferable name is “high-efficiency cell.” Signal production in them has been described in some detail (4). In these cells (Figure Z), which are often made
-
Figure 1. Examples of restricted-flow geometry for low-efficiencycells. In sach example. laminar flow camles wlutionr pas1 me OIemode w h a e elecwoiysir occurs. (a1Flow I.(b) llow out. (c) wrking elmode. (dl spacer. IAdapled hom Relerence 1.1
In these detectors, molecules are oxidized or reduced after diffusing from a point in the fluid to the electrode surface. The rate at which this process occurs depends on the local concentration gradient (through Fick’s laws). In the thin-layer cell, if the cell is well designed, the flow past the electrode is laminar. In laminar flow, the fluid moves parallel to the surface of the electrode; consequently, there is no bulk motion of analyte toward the electrode surface. The effect of the flow on the current is indirect. By replenishing solution that has been depleted of analyte by fresh analyte-containing solution, the flow increases the concentration gradient and thus the flux of analyte to the electrode. Whenever the fluid’s viscosity is absent from the sensitivity equation, as above, there is no transport of solute toward or away from the electrode surface by bulk fluid motion alone. Of course, if the diffusion coefficient, D,is increased, the flux caused by difNO. 15, AUGUST 1. 1988
Figure 2. High-efficiency cells. Increasing the electmde surtace area and the length of time that me solution Is exposed m me elechode both Increase elticiewy. (a) Flow in. (b) llow (ut, (0) wwklng alacbode. (d) spacer. (Adapted horn Reference 1 .)
4 WAYS TO MAKE SAMPLE PREP F--T... E...A -
-
1 I...
1 -
FLORISIL@PR NOW AVAILABLE IN EASY-TO-USE, PREPACKAGED COLUMNS FOR PESTICIDE ANALYSIS
Florisil PR - specifically designed for pesticide analysis. IS now available in 100 mq or 500 ma easy-to-use, Large 10 ml reservoir capacity, laboratory robot compatible. Convenient - eliminates packing your own tubes. Special grade Florisil made specifically for the analysis of pesticide residue. CIRCLE 3 ON READER SERVICE CARD
a
# A
F
CIRCLE 5 ON READER SERVICE CARD
High efficiency extraction columns are disposable, fast and reliable for extracting drugs of abuse from urine for TLC screening. The hydrophilic packing eliminates emulsions and time-consuming manual separations for more consistent results. Available pre-buffered to pH 9.0 or pH 4.5. Also: Chem Elut'* - High purity columns for GC, HPLC, UV-Vis or RIA. Extract 0.3 to 300 ml sampIes.
I
CIRCLE 4 ON READER SERVICE CARD
N E W C%/OH SINGLEPHASE COLUMN FEATURES BOTH POLAR ANDrNON-POLAR INTERACTIONS
HL 1 BL-..SPE SAMPLES. WITH MEGA BOND ELUT"
Large volume extractions and clean-up can be challenging, especially if they are extremely "dirty". New Mega Bond Elut columns handle bulk sample extraction with ease and accuracy. Up to 50 ml reservoir. Ideal for extracting herbicides, pesticides, proteins, peptides and decomposition products of reactants. High capacity. Available in 1, 2, 5, and 10 gram columns. Available in C18, C8, CN, NH, SI. and Florisil'. Specify Mega Bond Elut - the only large scale sample extractio columns from your SPE source.
EXTRACTION COLUMNS FOR DRUG SCREENING
'23
These liahtlv loaded C18 columnsfeature enhanced selectivity that results from hydrophobic and polar interactions. Large 10 ml reservoir capacity means you can handle sample extractions with ease and accuracy. Use CWOH Bond Elut LRC'* columns for: Convenient, single-column separation of Vitamin D metabolites. Easy extraction of nitrogen-containingdrug metabolites. CIRCLE 6 ON READER SERVICE CARD
Analytichem International
:
AEubsldlawalVarlan Assoclafes
24201 Frampton Avenue, Harbor City, CA 90710 USA (800) 421-2825 FAX. (213) 539-4270
The Source for Tomorrow's SPE Technoloau.
linearly with an increase in area: s
= 2s,
(3,
If the noises are completely uncorrelated, as they would be if the noise came from independent electrochemical processes occurring at each electrode, then p would he zero, and one would have s
Figure 3. Simplified version of the detector as an electrical device with generic c
rent and voltage noise. The vwlable banen represenla me euxllwy-refsrsnmslsctmde s w m and pomtbsm. The interfaoh1 lmpedanca 16 4.tho vdt%e m b la %, me cwentmla¶1s b, ard me valueofttu, fembsck naistsnoe 111
e.
of a porous material such as reticulated carbon, the current from a small increment of the electrode decreases exponentially along the electrode. This is simply because of the decrease in the bulk concentration of the material being electrolyzed. As material is electrolyzed, the rate of electrolysis decreases because the diffusive flux, driven by the difference between the bulk and surface concentrations of analyte, decreases. Although the understanding of signal production in both low- and highefficiencycells is fairly good for simple Faradaic reactions, noise is less well undershd. Theoretical analysis (5) shows that noise will decrease as one decreases the electrode area to a point, and then it will he constant. Area-dependent noise includes contributions from the fluctuations in the working electrode-reference electrode potential difference and environmental influences on the interfacial impedance. For example, the operational amplifier used to make the current-to-voltage conversion has an input voltage noise; the reference electrode and its liquid junction can also generate voltage noise. To see how this affects the measurement, think of adding a noise voltage to the voltage being applied in the potentiostat (Figure 3). It will generate a current, e,,/.zi, with a corresponding output voltage from the current-tovoltage converter equal to -Rp,,/zi. The interfacial impedance, zi,is halved when the electrode area is doubled; thus, this noise is proportional to the electrode area. Fluctuations in the interfacial impedance caused by fluctuations in temperature or concentration of some adsorbate will lead to a fluctuation in the measured current that is also proportional to the electrode area (or more precisely, capacitance). Electrical engineers always assume that capacitances are noiseless, and any noise they generate is put into a circuit as an equivalent voltage or current noise. However, we have allowed the interfacial capacitance to have a “noise” because it is a 906A
physically reasonable assertion that the interfacial capacitance fluctuates in an analytical system. The input current noise in the current-to-voltage converter amplifier is the constant contribution. The current noise, in, adds to the signal and yields a constant voltage noise, -i&, in the output of the current-to-voltage converter. This noise becomes more important when Rr is large, as it needs to be with s m a l l electrodes. This simple picture demonstrates why the consideration of electrode area is important to the S i N question. The limiting noise at electrodes is mostly instrumental and environmental (6).at least in our hands. (Certainly there are ways to study fundamental noise procesaes of particular electrochemical interest [q, but conditions that reveal this noise do not usually hold in the detector case.) Consequently, a parallel array of electrodes feeding current into a single amplifier has a noise that is proportional to electrode area (as long as the total area is large enough so that the constant noise, in, is not dominant). However, if the noise source were the random nature of the fundamental Faradaic procesa itself, then the noise at each electrode in the array would be independent. The noise in this case would increase as the square root of the area. This can be explained by the following statistical argument. Consider the simple case of two electrodes of equal area and sensitivity. They are connected in parallel. The variance (2)of the current signal from the pair is (8) 02
=
.:+ 4 +
2U10?p12
(2)
The standard deviation of the signal from each electrode is identified by the subscript, and p12 is the correlation coefficient between the two noises. When the source of noise is voltage noise in the current-to-voltage converter (as it often is), then both electrodes feel the Same perturbation and the correlation coefficient hetween them is 1. Then, because 01 and u2 are of equal magnitude, one finds that the noise increases
ANALYTICAL CHEMISTRY. VOL. BO, NO. 15, AUGUST 1, 1988
= @a1
(4)
Data indicating the linearity of the standard deviation of the current signal with electrode area for carhon fiber electrodes are shown in Figure 4. The noise is proportional to the electrode capacitanceor area (forthe same material). There is an important inference to be taken from the demonstration of this dependence: Improved instrumentation can improve the theoretical lower limit of detection in electrochemical detectors. Although large electrodes give the highest signal, there is an electrode area that yields a maximum S/N.This is illustrated in Figure 5, which is actually a simplification of a more complex (and interesting) set of relationships. Three curves for “signal” are shown. In the fmt, the contribution to the total current from molecules diffusing beside the electrode is small. If the shape of the electrode is changed so that its perimeter-to-area ratio is increased (curves 2 and 3), then the contribution from edge diffusion is increased. This will result in an increase in the current density (currenthea) at the electrode and an increase in the sensitivity. If one assumes that the electrode capacitance is independent of the shape, then the S/N increases as the perimeter-to-area ratio increases (9). As a practical matter. the flux induced by the flow is quite large, and the effect just described would he difficult to perceive experimentally unless the characteristic dimensions of the electrode (aredperimeter) were on the order of micrometers (for typical fluid velocities). It is also beneficial to break the electrode up into pieces separated by an insulator (as in the Kel-Graf electrode). This works because of “depletion layer recharge” (IO,II), or hydrodynamically assisted diffusional relaxation of the concentration perturbation established by the electrolysis. The insulating space between electrodes allows diffusion to fd in the region of solution that has contributed its analyte to signal production at the electrode surface. This is similar to pulse amperometry but is carried out spatially instead of temporally. The optimum area also depends on the frequencyof the measurement being made; the signal and the noise do not have the Same Fourier spectra (9). Often, the offending noise in an analysis is very low frequency or drift. In this case, a differential procedure can
work to one’s advantage. Two working electrodes are used to accomplish a sip nificant immunity to drift. This is only one of many ways in which multiple electrodes can be used to one’s advantage (12).If the noise in environmental, then two identical electrodes at the same potential in the same cell will be exposed to the same perturbations. The resulting noises are correlated. From the propagation of errors (8). one finds that the noise in the difference of the currents generated at the two electrodes does not contain components that are correlated, or common to both electrodes. For example, when the temperature changes, the reference electrode and liquid junction potentials change. These potential changes induce a double-layer charging that is seen as a drift in the background currents of the two electrodes. The current resulting from this charging-discharging process is removed by the subtraction procedure. One might guess that the signal would be eliminated as well. Recall the explanation given above for the relationship between signal and area. The same reasoning applies to this case. The second electrode, beiig shielded by the first, only gives about 4096 of the signal of the fvst electrode (13).Taking the difference in the signals from the fvst and second electrodes yields 6 W of the original signal with a large reduction in drift for a net increase in the S/N (14). Because of a detailed knowledge of the physics of detectors, quantitative predictions of signals for simple geometries can he made. Progress is beiig made in understanding the sources of noise. It is clear that instrumental improvements will reduce noise, and signal can be increased by taking advantage of the influence of geometry on mass transfer. Much more is possible by taking advantage of chemistry, as the following interesting example shows. Recently electrochemical transistors were developed by Wrighton and coworkers (15). In these devices, charge injection by analyte into a poorly conducting polymer film yields a signal. The polymer fhcovers two adjacent electrodes separated by an insulating space. When analyte injects charge into the film connecting the two electrodes, the polymer becomes conducting and an increase in the current through the polymer film (between the two electrodes) is observed. The current between the electrodes is larger than the current drawn from the analyte, so there is an amplification. The ramifications of this are twofold The gain in sensitivity may lead to lower detection limits, and simple electron transfer at an electrode surface is not the only means by which an electroactive analyte can be induced to yield a signal.
Figure 4. Root mean square noise as a function of electrodt in this expBrimBnt. lO-gm dkmster carbon libsr eisctmdes were mnnected in parallelto yield mie b bode areas 01 wiou6 maglibudes. The el&& area was estimated hwn he quaaiateadyatate voltemmeby (2 mV/s) of fsnocene carboxylate. he pen circies are tor me bend 0.140 HZ wim an LW p+ tentioatat (modifid m have a 0.05 s time -tam). The cIQsBd squares are for the 0.1-40 HZ Lmnd using a Kelthley 427 picoammeler as me ansnt-twoltspe conyBrt(K. me v o w mise in me lamrr insbw menl was lovnd in tha same band to be 1.1 X 10-e V. On the haiuntai axis, 1 wit = 3.3 X 10- cm2.
I
Fkun 5. Slanal, noise, and sianal-tc-noise ratio.
rabie I. Detection limits in voltammetry in TIOW cells” Sweep type Staircase Staircase
Staircase Square wave Coulostatic Potential scan amperometric
Detedlon llmlt
System
Solid electrodeloxidation Solid electrodeloxidation
Solid electrodeloxidation Mercurylreduction Solid electrode Dual solid electrode
5 X lo-’ M 1 X IO-* M 1 x t0-7 M 5 x to-7 M -4 X lo-’ M 10-7 M
Reference no. 20
I
t8,t9 21 16.17
22,23 24,25
*These limits are intended as a guide, and mly order 01 magnitude significance should bt nferred. All data are fw the 1 ~ 1 8 rate 8 ~ range 01 0.5-1 .O V I S . In some cases, the injected volumt lad to be guessed. Cmcenlralionr are given as injected concentratlons.
Improved qalltatlve i n f m t i i Under ordinary conditions, the electrochemical detector gives no qualitative information. I t is expected that chromatography will provide the qualitative information, and the electrochemical detector is used for quantitation. Voltammetry adds a dimension of qualitative information to the analytical process. Voltammetric (potential scanning) detectors, in conjunction with chromatographic separation, can increase the information content derived from the analytical process over that ohtained in conventional liquid chromatography with electrochemical detection (LCEC). Being able to plot a chromatovoltammogram, a 3-D plot with current vs. time and potential, gives one a second “separation” (1619). Having such a data array allows one to quantitate the components of a multicomponent peak (if the components have unequal redox potentials) or to identify a compound by its redox potential. Methods development also is made easier with these detectors. With a chromatovoltammogram in hand, one can easily choose the optimum potential for an amperometric detector in LCEC. However, these advantages do not come without a cost. The disadvantage of voltammetric detection is that poorer detection limits accompany the gain in selectivity. Of course, often detection limit is not the real issue and it can be given up for the sake of the enhanced selectivity of voltammetric detection. Many researchers are concentrating on improving the detection limit of voltammetric detection methods, and much progress has been made in improving classical methods and in inventing new ones. The primary obstacle that keeps voltammetric detection from approaching the low detection limits of amperometric detection is the high background current caused by double-layer charging and unknown Faradaic processes induced by the changing poten908A
tial. There are several ways to minimize this effect. Background subtraction (20) can he used. It decreases noise at very low frequencies, or drift. It also can decrease bit noise. If one is acquiring data by computer, and the background current is a significant portion of the measured current, then the dynamic range available for the signal current is diminished. When the range available is small enough, the discrete nature of the signal becomes evident. Subtraction of the background before analog-to-digital conversion is required for this problem. Background subtraction following analog-to-digital conversion is helpful in revealing the wanted signal when the dynamic range is sufficient for the task. The application of voltammetry in detectors for flowing streams requires rapid scanning capability. One needs to obtain voltammograms several times during a single chromatographic peak. One is naturally led to small electrodes because of the lower time conntant that they display. One must be aware, however, that the low time constant displayed hy microelectrodes is principally caused by the divergent flux lines for migration of the supporting electrolyte. If one is to use this effect, the physical boundaries of the flow cell must allow the divergence of the flux lines to occur. If they do not, then the resistance in solution will he greater than if they did, and the time constant will increase accordingly. There are several ways to change the potential at an electrode surface. The methods that have been used include staircase voltammetry, in which a discrete approximation to a ramp is used; square wave voltammetry, in which a square wave is superimposed on a staircase: and coulostatic potentiometry, in which the leakage of a charge pulse through the Faradaic impedance is observed. Smircase voltammetry has yielded good results at solid electrodes in flowinjection analysis and chromatography at around I VIS sweep rate. Represen-
ANALYTICAL CHEMISTRY, VOL. 60. NO. 15, AUGLIST 1. 1988
tative data can be seen in Table I. These data should be taken to mean that one can perform reasonable quantitative analysis a t the micromolar level with discrimination along the potential axis. There is no agreed-upon method to use for reporting the detection limit in these cases. One gets a different answer from making measurements of signal and noise on individual voltammograms and from making measurements of signal and noise on a reconstructed “constant potential” chromatogram (20).Furthermore, one gets less noise by averaging several background scans and several voltammograms. The detection limit should be defined within the context of the task one has set out to accomplish using the voltammetry. Square wave voltammetry is well suited to mercury b e c a m the relaxation of the transient induced by the pulses is reproducible. Table I shows the detection limits are similar to those seen with the staircase approach. Coulostatic detectors also have been used as potential scanning detectors. Charge pulses are used to change the electrode-solution potential. The approaches of Last (22) and Nieman (23) are slightly different, and they yield similar results (see Table I). The detection limits are poorer than those for potentiostatic techniques. A combination of voltammetric and amperometric detection has been used recently. Trubey and Nieman (24) combined rapid-scanning coulostatic detection with amperometric detection, whereas Lunte e t al. (25) used staircase voltammetry with amperometric detection. The advantage obtained is the combination of selectivity of potential scanning with the low detection limits of amperometry. The concept in the two papers is similar; the upstream electrode changes the solution concentration of the analyte as the potential on the electrode is scanned. The prcduct of the upstream scanned electrode can be detected a t constant potential in complete analogy to rotating ring disk experiments. Also, if the product of the upstream electrolysis is not electroactive, the decrease in the electrolysis of the analyte can be seen a t the constant potential downstream electrode when the potential at the upstream electrode is right for the electrolysis of the analyte. The key is that one electrode causes concentration changes as a function of potential while another, without the background current induced by altering the potential, detects the events. Both research groups needed to correct for the time delay caused by the separation of the electrodes in the flow direction. Detection limits are better for the staircase approach and are apparently limited by instrument design flaws, not noise. As Table I shows, de-
I
I
Figure 8. Fourier transform of the noise in cyclic voltammetry with a 2.1-mm-long 10-pm carbon fiber electrode and using a I-Hz sine wave as a perturbation.
Fbure 7. Fourier Vansfam of a cyclic voltammogram of 160 pM Vis(2,2'-bipyridine)iron (11) in aqueous phosphate buffer (PH 7).
Noiw 16 bund by removing an average Sean horn a %$I 01 24 wnliguws WP cycle (0-1-0-1-0 V) scans. Fwier lransbnning each a m , and summing me powers of each. The square mot 01 me resuii is plotled. Note low pow= at hequencies abovt, me tundanmntal.
The voiiammogam resulted horn lhe application of a 1-nr sins w m and is
tection limits are competitive. We have recently begun investigations of noise in voltammetry. The problem resulted from a consideration of the bandwidth required to obtain a voltammogram. For a triangle wave potential, one needs to acquire data up to a frequency about 35 times that of the fundamental frequency. One then pays a severe cost in noise by expanding the handwidth that much. Such high frequencies are needed because the current response to a potential change is nonlinear. The stimulation of the interface at one frequency will lead to signal power at higher frequencies. Thus, the triangle wave, which consists of the fundamental and odd harmonics, induces power at and above each of the Fourier components of the perturbing wave. As a result, the power in the signal overlaps the power in the background. It would be beneficial if one could arrange it so that the portion of the Fourier spectrum containing signal power did not contain background power. This suggests the use of a sine wave perturbation, One can obtain voltammograms with a lower handwidth using a sine wave perturbation. Furthermore, if one is willing to do the necessary signal processing, one can make measurements in the frequency domain in regions of the spectrum where there is virtually no background. Figures 6 and 7 are Fourier transforms of the noise and signal, respectively, in 24 cyclic scans (0-14-1-0 volts) using a sine wave at 1.0 Hz. There is considerable signal power in the few Hz region but the noise power is low in the same region. This is reflected in Figure 8,where the S/Nis shown as a
kckckgound-subwacled.
function of the upper and lower cutoff frequencies of a bandpass filter. Figure 9 shows that useful voltammetric information exists in a limited frequency band. The nonlinearity of the voltammetric response becomes an advantage. One can benefit from this harmonic distortion if one perturbs with a sine wave. The same distortion exists with the triangle wave perturbation, but it is difficult to extract the signal in the frequency domain. In recent studies (26),we found that noise in the voltammetric experiment, when applying the potential with an
analog source, increases linearly with electrode area. Polished ellipsoids created from 10-am fibers embedded in epoxy resin and glass showed a higher slope than those seen for cylinders composed of the same kind of fiber. This is consistent with the expected difference in their capacitances. The noise also increases with scan rate because of the increase in bandwidth as the scan rate increases. We have also found that the baseline reproducibility is superior when one applies the potential with a digital-to-analog converter compared with the application of the
-
Figure 8. Sianal-to-noise ratio as a function of bandwidth for Ficlures 6 and 7. band is plnted horn back lo horn the high and is plotled I& to right me peak me-
The low end Ot
spordstoadstectlon limnolabout2X 10-'M.
ANALYTICAL CHEMISTRY, VOL. 60, NO. 15, AUGVST 1. 1988
-
SOSA
potential with even the best available sine wave source (0.01% rms noise in the sine wave put out hy the generator). Background subtraction is more effective with digital-to-analog converterbased potential application, but the noise in the digital case is not uniformly lower than in the analog case. The current state of affairs is easily summarized. Detection limits for oxidations a t solid electrodes using M voltammetry are in the 1-5 X range (tens of picomoles) for sweep rates in the 1V f srange. Potential resolution, except in the coulostatic case, is less than RTfnR thus, it is not instrument limited. For reductions a t mercury with square wave voltammetry, detection h i t s are about an order of magnitude poorer. If one is in the fortunate position of doing flow analysis a t very low flow rates, then the efficiency of the detectors improves a t no cost in noise. In the case of detection in microbore chromatography, the detection limit is improved (18,19).Experiments designed to allow understanding of the noise-background problem in voltammetric detection in flowing streams are underway in our laboratory. What information is needed from the analysis? Does one need to detect trace amounts of a substance in a relatively simple mixture where low detection limits are essential, or is it a complex mixture where low detection limits are not necessary but selectivity is important? The information needed and the
character of the sample should determine how the analysis is done. Future electrochemical detectors can be visualized as being capable of both amperometric and voltammetric detection, thereby allowing the experimentalist to make a choice depending upon what the application requires.
control over Selectivity One may be faced with a particularly complex mixture, in which case one wants a highly selective detector to minimize the number of interfering compounds. The electrochemical detector, through the control of potential, is reasonably selective. Can it be made more so? Yes. By using modified electrodes, one can improve the selectivity of the detector. One may also look longingly at the impressive detection limits and low cost of the detector and wish that its sphere of applicability could be increased. Actually, because all molecules are oxidizable and reducible, in principle one ought to he able to use the electrochemical detector as a general detector, applicable to many more analytes than it is now. The fault in this argument is that one of the molecules to which the detector will become sensitive is the solvent. One could increase the electrochemical driving force to make the detector “see” more compounds, but it would see a lot more solvent than solute and thus be useless. The key to expanding the sphere of ap-
Figure 9. Inverse Fourier transform of the data in Figure 7 in the 2.5-10-Hz band srnwlhd filter (shown lor relerenat (. , , .). ReC(Bngu1Brliner (-);
#IO*
.
- - -). The ffiginal (background subtracted) vOltarnmOgarnis
ANALYTICAL CHEMISTRY, VOL. 60, NO. 15, AUGUST 1. 1988
plicability of the detector is a controlled increase in solute reactivity. These two pursuits-increasing detector selectivity and increasing detector reactivity-are inherently chemical. We will briefly examine some approaches in the final section of this article. Increased selectivity. Size-exclusion selectivity can be built into an electrochemical detector. By using cellulose acetate modifications of electrode surfaces, one can restrict the diffusion of analyte molecules and therefore create a device with molecular size-dependent sensitivity (27-30).An electrode surface covered with a dense film of cellulose acetate excludes virtually all electroactive species except hydrogen peroxide (27,28). This can be used in a large number of biological assays that rely on oxidase enzymes. For example, Wilson’s group has developed a rapid and sensitive immunoassay using a glucose oxidase label (31). The use of a cellulose acetate film on the electrode in the electrochemical detector obviates the electrochemical interferences from the remainder of the molecules in the system. Wang (29,301, recognizing the limitations of a peroxide-selective detector for general use, hydrolyzed the dense films with base to obtain response to larger molecules such as uric acid and NADH. The LCEC chromatogram from human urine is greatly simplified by using the modification. Also, the dense cellulose acetate film dramatically decreases the deleterious effects of proteins on the electrode response to analyte. In some of our work on developing immunoassays and looking ahead to biosensors, we are anxious to take advantage of the size-exclusion selectivity provided by the dense cellulose acetate films. However, we find the molecular weight cutoff of the base-hydrolyzed films to be too low (about 6 6 0 0 daltons). We would like to increase the molecular weight cutoff to allow the passage of larger molecular weight molecules such as peptide hormones. At the same time, we wish to reject offending species from the matrix. How high can we push the molecular weight limit and still minimize biological interferences? How can the molecular weight cutoff be extended? In experiments using ultrafiltration on solutions of electroactive species and various biological media (32),we have determined that a molecular weight cutoff of 5000 daltons protects the electrode, and a cutoff of 30,000 daltons does not. How then can we increase the cutoff up to a few thousand daltons? Preliminary results in this laboratory (3335) indicate that the molecular weight range can be extended considerably by using the phase-inversion
membrane process on electrode surfaces. The modifications consist of a cellulose acetate skin that is supported by a low-density reticulated layer that lies on the electrode. The outer skin provides the permselectivity. Because of the way in which the membranes are formed, their character is considerably different from the more easily prepared dense films. Heineman’s group is exploring y-irradiation-induced polymer coatings (36). One of the desired results of the work is permselectivity. Although the results in both labs are recent, it is evident at this stage that, whereas the polymer-coated electrodes are more rugged, the phase-inversion modified electrodes are more selective and respond more rapidly. The development of such barriers will be useful for both electrode protection and selectivity. Furthermore, the use of more than one selectivity-enhancing scheme should lower detection limits in real samples. Besides multiple layers on the electrode surface (30),one might combine these barriers with voltammetry or selective chemistry to achieve superior selectivity. Expanding the sphere of applicability. The key to expanding the range of compounds for which electrochemical detection is appropriate is a controlled increase in the analyte reactivity. Increasing the electrochemical driving force increases analyte reactivity indiscriminately with concomitant loss in selectivity and detection limit. Some of the interesting and relatively unexplored areas for achieving a controlled increase in solute reactivity are photoelectrochemistry, adsorption and heterogeneous atom-transfer reactions, and chemically modified electrodes. Photoelectrochemistry. Excited states are better oxidizing and reducing agents than the corresponding groundstate species. Consequently, by using light correctly, one should be able to selectively increase the reactivity of a certain class of analytes. It is unlikely that an excited molecule would yield a net transfer of electrons with a metal or carbon electrode. Energy transfer quenching of the excited state is a far more probable event. To use the light to one’s advantage, one must arrange for some chemistry to occur that will conserve the photon energy as electrochemical energy. One way of conserving the energy is to cause the photosensitive analyte to react in the excited state with a molecule in solution. Then, in principle, one could detect either of the two species involved in the reaction. Griller’s work (37,38)demonstrates the ability to detect species that donate a hydrogen atom to the t-butylperoxy radical (generated photochemically). The radical from the parent compound can be oxi-
dized or reduced. Many neutral radicals have modest potentials for oxidation, so this procedure should be of Value in detection work. Some ketones photodissociate into radical species. This has been used by Griller as well to study radical electrochemistry (37,38). Krull’s group has used this to great advantage in detection work (39);nanogram (2 X lo-’ M) detection limits were found for benzaldehyde. What is more important is the clear evidence for the selective response of the detector caused by the presence of the light. Our efforts (40, 41) have revolved around a well-studied molecule, R ~ ( b p y ) 3 ~(bpy + = 2,2’-bipyridine). We have used this molecule to develop an understanding of the capabilities and limitations of the photochemical approach to detection. In this technique, the analyte species, Ru(bpy)S2+, is photooxidized, and a purposely added electron transfer quencher is reduced. The Ru(bpy)g3+is then cathodically reduced. It acts as a photosensitive mediator. We have shown detection limits of 1 X M in a flowing stream for this molecule. The challenge is to apply this chemistry to analytes of interest. Currently, this would require labeling of the analyte in a precolumn reaction. Using this model system, we have found that photocurrent potential curves have a much higher (-lo2) S / N if one avoids directly striking the electrode surface with light (42).Thus, enveloping a fiber electrode in a column of laser light is more effective than blasting the electrode surface with a perpendicular beam. Krull’s group (43) has also used a technique that detects stable products of photolysis. A long-time, high-flux exposure to photons is followed by amperometric detection of the products. This has been effectively applied to organohalides. Surface chemistry. The chemistry of the platinum surface is complex. Johnson and co-workers (44) have recently extended the applicability of electrochemical detection to molecules containing N, 0, or S by taking advantage of Pt surface chemistry. The application of a positive potential pulse to a Pt electrode leads to the oxidation of the surface. A negative pulse causes reductive dissolution of the surface oxides leading to a clean Pt surface. Molecules in solution with an affinity for platinum have an opportunity to adsorb, and they now can do so on the clean surface. Taking the potential to some value that might oxidize an adsorbate then detects it. The real power of the technique is in the controlled selectivity that is gained by controlling the various potentials. Many adsorbates are not oxidized at modest potentials, and they block the electrode from oxide formation. This
leads to a decrease in signal in the presence of the adsorbate. Pulse-to-pulse reproducibility is good, so the background is well behaved. Other chemical routes exist that lead to the possibility of further selectivity in the time domain. Thus one may see a negative peak, no peak, or a positive peak from an analyte that can be desorbed by oxide formation, depending on the time after the application of the final pulse that the current measurement is made and the chemical nature of the adsorbate. There are metals that form chemically reactive oxides on their surfaces. By appropriate control of solution conditions, mainly pH, and electrode potential, it is possible to create a surface that can be used to carry out specific reactions. For example, Johnson’s group has explored a variety of materials for use in oxygen atom transfer reactions (45).By doping PbOz with Bi, they prepared a surface that had much improved kinetics for the oxidation of Mn2+.The nickel oxide surface is catalytic for carbohydrate oxidations and can be used for their determination in chromatography and flow-injection analysis (46,47).A basic solution is required, so addition of base may be needed if one is using chromatography in a neutral or acidic eluent. The use of metal electrodes that have catalytic properties will open up new classes of analytes for detection. A difficulty will be that the background current arising from the natural corrosion reaction that generates the catalysts will yield significant background currents. Chemically modified electrodes. Such electrodes have recently begun to play a more important role in detection. We have already pointed out some applications of the modified electrode strategy in altering the molecular weight selectivity of the detector and in creating an electrochemical transistor. The catalytic properties of modified electrodes can be used to increase the range of applicability of the sensor. Engstrom (48, 49) showed dramatic and reproducible changes in redox potential and wave shape following oxidative and reductive treatment of glassy carbon electrodes. The electrochemical pretreatment has been successfully applied to the determination of hydraM detection zines with lo-’ to 2 X limits. The overpotential for the oxidation of the hydrazines is lowered by as much as 1 V because of the electrochemical treatment. Similarly, after receiving a pulse of photons from a nitrogen laser, the glassy carbon surface shows improved voltammetry for the champion of slow kinetics and irreproducibility, ascorbate ion (50) (among others). The improvement disappears during a period of about an hour. Repetitive pulsing and measurement of
ANALYTICAL CHEMISTRY, VOL. 60, NO. 15, AUGUST 1, 1988
9llA
current after a pulse should stabilize t h e improved response. In neither case is the mechanism of the improved performance well understood. R a t h e r t h a n altering t h e n a t u r a l electroactive surface-solvent interface, one can modify t h e surface with “foreign molecules,” as is well known. One example of this is t h e cobalt phthalocyanine modification t h a t has been shown to be effective in catalyzing a number of important reactions for detection (51, 52). A requirement of the electrode modification, when accomplished with molecules t h a t are not native to t h e surface or the solvent, is t h a t t h e modification be long lived. T h e friction from the flowing fluid in t h e detector and the chemistry of t h e environment will certainly conspire to limit the usefulness of most modified electrodes. Self-regenerating surfaces, protected surfaces, or thick overlayer-type modifications (such as ruthenium purple [ 5 3 ] )would seem t o be t h e best.
Conclusion T h e development of electrochemical detectors parallels t h e development of t h e science of electrochemistry. T h e marvelous thing about research in this area is that electrochemistry is chemistry. Microstructural and chemical alterations of electrode surfaces can be made elegantly and precisely because
of the natural, and controllable, reactivity of t h e surface. More powerful detection techniques will be one result of this. T h e processes that occur a t a n electrode can be influenced by light, giving a new channel of selectivity to detection. Finally, it is worth noting that studies of noise have all demonstrated t h a t t h e limiting factors in the lower limit of detection are the presence of Faradaic current from components of t h e system t h a t are of no analytical interest, and instrumental noise. T h e fundamental limit has not been approached with a n electrochemical detector. More selectivity and better sample preparation will improve t h e chemical environment problem, and perhaps some clever approaches t o t h e instrumentation will solve t h e latter problem.
References (1) Weber, S. G.; Purdy, W. C. Ind. Eng. Chem. Prod. Res. Dev. 1981,20,593. (2) Weber, S. G. In Detectors for Liquid Chromatography; Yeung, E. S., Ed.; Wiley Interscience: New York, 1986; pp. 229-91. (3) Stulik, K.; Pachkovh, N. Electroanalytical Measurements in Flowing Liquids; Ellis Harwood, Ltd.: Chichester. England. 1987, pp. 60-1. (4) Sioda, R. E.; Keating, K. B. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1982; Vol. 12, pp. 1-52. (5) Morgan, D. M.; Weber, S. G. Anal. I
,
Chem. 1984,56,2560-67. (6) Berger, T. Abstracts of Papers, Pitts-
burgh Conference, Atlantic City, NJ; 1986;Abstract 1059. (7) Bezegh, A.; Janata, J. Anal. Chem. 1987, 59,494-A. (8) Box, G. E.; Hunter, W. G.; Hunter, J. S. Statistics for Ermrimenters: Wilev: New York, 1978; App: 3. (9) Weber, S. G. Presented at the International Electroanalytical Symposium, Chicago, IL, 1985; paper 27. (10) Fosdick, L. E.; Anderson, J. L. Anal. Chem. 1986,58,2481. (11) Cope, D. K.; Tallman, D. E. J . Electroanal. Chem. 1986,205,101. (12) Roston, D. A.; Shoup, R. E.; Kissinger, P. T. Anal. Chem. 1982,54,1417 A. (13) Matsuda, H. J . Electroanal. Chem. 1968,16, 153. (14) Lunte, C. E.; Shoup, R. E.; Kissinger, P. T. Anal. Chem. 1985,57,1541. (15) Chao, S.; Wrighton, M. S. J . Am. Chem. SOC.1987,109,2197. (16) O’Dea, J.; Osteryoung, J. Anal. Chem. 1980,52,2215. (17) Reardon. P.A.: O’Brien. C.E.: Sturrock, P. E. Anal. h i m . Acta 19sh, 162, 175. (18) White, J. G.; Jorgenson, J. W. Anal. Chem. 1986,58,2992. (19) White, J. G.; St. Claire, 111, R. L.; Jorgenson, J. W. Anal. Chem. 1986,58, 293. (20) Caudill, W. L.; Ewing, A. G.; Jones, S.; Wightman, R. M. Anal. Chem. 1983, 55, I
“
1877. (21) Gunasingham, H.; Tay, B. T.; Ang,
K. P. Anal. Chem. 1987,59,262.
(22) Last,T. A. Anal. Chem. 1983,55,1509.
(23) Barnes, A. C.; Nieman. T.A. Anal. Chem. 1983.55.2309. (24) Trubey, ’R. D:; Nieman, T. A. Anal. Chem. 1986,58,2549.
Biotechnology - and Materials Photon Activation Analysis Christian Segebade, Hans-Peter Weise, and George John Lutz 1988. viii
+ 705 pages. Illustrations.
17 x 24 cm. ISBN 0-89925-305-9. Hardcover $193.00 This book provides an overview of the application of photonuclear reactions to activation analysis. It is intended to serve as both a source of general information on photon activation analysis and as a practical reference manual to accompany the analyst’s laboratory work. Emphasis is placed upon experimentally obtained analytical, qualitative, and quantitative data. The results of the authors’ laboratory work, as well as a large amount of literature data, are evaluated and presented as completely as possible. An elementary theoretical introduction is provided to assist those without a special knowledge of photonuclear physics.
From the Contents Activation Analysis - the general principle Photonuclear reactions Activating radiation sources Photon spectrometers * Properties and yields of radionuclides produced through photonuclear reactions Analytical application Bibliography Subject Index orwe iubicci
IO (hanee
fids. See how chemkts. through their ability to-manipulatechemical structures,will significantly influence our national economic vitality. learn the history behind presentday biotechnology and see where advances are predicted. look at the progress being made in the new recombinant DNA technology and materiais produced in high
technology. Fully illustrated.this beautiful book begins with a discussion of biotechnology 0 What Is Biotechnolcg? 0 Biotechnology:A New Marriage of Chemistry and Bioiogy 0 Synthetic Tools for Molecular Biology 0 Molecular Basis for Drug Design Taming the Chemistry of Proteins 0 Protein Engineering. Then advances in the chemistry of materiais are featured 0 Chemical Research in Materials Science 0 Simulation of Atoms and Molecules 0 Materials for Advanced Electronic Devices Chemistry of Materials for Energy Production,Conversion, and Storage 0 High-StrengthComposite Materials. Biotechnoiog and Materials Science expiores the dramatic role chemistry will play in shaping our future.Scientist and nonscientist alike who are interested in chemistry at its most exciting will find this book both informative and enjoyable. Mary 1. Good. Edtor, Aiiied-Signai Corporabon 144 pages (1988) Clothbound 144 oaaes 119881 PaDerbound
Walter de Gruyter, Inc. 200 Sah Mill River Road Hahthorne, N Y 10532 CIRCLE 170 ON READER SERVICE CARD
912A
ISBN 0-8412-1472-7 LC 88-14544
US 8. Canada $24.95 Export $29.95
ANALYTICAL CHEMISTRY, VOL. 60, NO. 15, AUGUST 1 1988
iSBN 0-8412-1473-5 LC 88.14544
(25) Lunte, C. E.; Ridgway, T. H.; Heineman, W. R. Anal. Chem. 1981.59.761. (26) Long! J.T.; ,Weber, S. G., submitted for publication m Anal. Chem. (27b,pwman, D. P. US. Patent 3979274, I9I".
(28) Sittampalam, G.; Wilson, G. S. Anol.
Chem. 1983,55,1608. (29) Wang, J.; Hutchins, L.D. Anal. Chem. 1985,57,1536. (30) Wang, J.; Tuzhi, P. Anal. Chem. 1986,
J. M. Clin. Chem. ( Winston-Salem) 1983. 29,1665. (41) Elbicki, J. M.; Morgan, D. M.; Weber, S. G. Anal. Chem. 1985,57,1746. (42) Berry, W. F.; Weber, S. G. J. Electroanal. Chem. 1986,208,77. (43)Selavka. C. M.; Jim, K-S.; Krull, I. S.; Sheih. P.; Yu, W.; Wolf, M. Anal. Chem. l988,60,250. (44) Austin, D. S.;Polta, J. A,; Polta, T. 2.; Tang, A.D.-C.; Cahelka, T.D.; Johnson, D. C. J. Electroanal. Chem. 1984, 168,
.
907 I_.
(45) Yeo, I. H.; Johnson, D. C. J. Eleetroehem. Soe. 1987.134.1973. (46)Yuan, C. J.; Huber, C. 0. Anal. Chem. 1985,57,180. (47)Reim, R. E.; Van Effen, R. M. Anal. Chem. 1986,58,3203. (48)Engstrom, R. C.; Strasser, V. A. Anal. Chem. 1984,56,136. (49) Engstrom, R. C. Anal. Chem. 1982,54, min (50) Poon, M.; McCreery, R. L. Anal. Chem. 1987.59,1615. (51) Santos, L.M.; Baldwin, R.P. Anal. Chem. 1987,59,1766. (52)Halbert, M. K.; Baldwin, R. P. A n d . Chim. Acto 1986,187.89. (53) Con, J. A,; Kulesza, P. J. Anal. Chem. 1984.56,1021. The authors thank the National Institutes of Health for supporting the research that was described in this report. Without this assistance we could not have carried out the work.
"
. .
r . . . : .i+ . . . . ' ."@. . ... . . .* . i .f...;. . :. ,,,., ..l',.;. I
. .
': _ .
. . .
!
Stephen G. Weber ( l e f t ) a, n associateprof~ssorof chemistry and affiliate of the Center for Biotechnology and HioengineerinR at the University of Pittsburgh, received a Ph.D. degree i n 1979 f r o m McCill University. Under the direction of William C. Purdy, he was involved i n t h e development o f a homogeneous electrochemical immunoassay. His research interests are t h e use of excited-state processes i n detection work, the SIN problem in electrochemistry, electrostatics in chromatography, and t h e understanding of stochastic processes i n chromatograp h y and electrochemistry. H e has gained a n appreciation o f t h e m a n y similarities between electrochemistry and chromatography through his work i n both areas. J o h n T. Long received a E.S. degree in chemistry f r o m Geneva College (Beaver Falls, PA) in 1981 and will graduate from t h e Uniuersity o f Pittsburgh with a Ph.D. in chemistry in August 1988.His research interests include electrochemistry and chromatogiaphy, with emphasis in electrochemical detection and t h e application of microelectrodes. Long is a member of t h e American Chemical Society, t h e Society f o r Electroanalytical Chemistry, t h e Society f o r Analytical Chemists of Pittsburgh, t h e Spectroscopy Society of Pittsburgh, and t h e X i Chapter of Phi Lambda Upsilon.
WHEATON Manufacturers Since 1888
1000 N. Tenth Street Millville. NJ 08332,USA In USA, Call Toll-Free: 1-800/225-1437, Ext. 2768 TWX: 510-687-7580. FAX: 1-609/825-1131 CIRCLE 172 ON READER SERVICE CARD
ANALYTICAL CHEMISTRY, V M . 60. NO. 15. AUGUST 1, 1988
913,.
