ANALYTICAL CHEMISTRY, VOL. 51, NO. 9, AUGUST 1979
quantitation is desired. T h e same complications, no doubt, also exist for all weak acids a n d / o r easilv oxidizable anions, such as sulfite.
LITERATURE CITED (1) H. Small, T . s. Stevens, and W. C. Bauman, Anal. Cbem.,47, 1801 (1975). (2) C. Anderson, Clin. Chem. (Winston-Sabm, N.C.),22, 1424 (1976). (3) F. Smith, Jr., W. Rich, and T. Sidebottom, "Ion Exclusion Coupled to Ion Chromatography: Instrumentation and Application", Second National
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Symposium on Ion Chromatographic Analysis of EnvironmentalPollutants, Research Triangle Park, N.C., October 1978.
William F. Koch Center for Analytical Chemistry National Bureau of Standards Washington, D.C. 20234 RECEIVED for review March 13, 1979. Accepted May 7, 1979.
AIDS FOR ANALYTICAL CHEMISTS Pre-Column Electrochemical Cell for High-Performance Liquid Chromatography Gary W. Schieffer Pharmaceutical Research Division, Norwich-Eaton Pharmaceuticals, Division of Morton-Norwich Products, Inc., Norwich, New York 138 15
Although post-column flow-through electrodes have been used extensively as electrochemical detectors in high-performance liquid chromatography (HPLC) ( I ) , the usefulness of pre-column electrochemical cells apparently has not been explored. Such a cell might provide a means for studying the chromatographic properties of electrochemically generated labile organic compounds or compounds otherwise difficult to obtain or prepare. For example, in HPLC methods requiring the monitoring of the stability of organic compounds (such as active ingredients in pharmaceutical preparations). i t is generally desirable t o test the specificity of t h e method with respect to postulated degradation products before t h e method is qualified for use. However, since it is sometimes difficult, time consuming, and expensive to purchase and/or synthesize all of t h e known or postulated degradation products, a quick, simple method for generating some of the products would be useful. Fortunately, t h e electrochemical oxidation or reduction of organic compounds in many cases yields the postulated degradation products that might be expected to occur during storage through electron transfer with atmospheric oxygen or oxidizing and reducing agents present in the environment (as impurities or matrix constituents). An example is t h e electrochemical oxidation of catecholamines which yields o-quinones and indoles (2,3),products expected to be formed through chemical oxidative degradation ( 4 ) . Thus, electrochemically degrading the analyte in a sample might result in a useful test of the specificity of a chromatographic method for t h e analyte in t h e presence of the degradant. With this in mind, an electrochemical cell was designed and constructed to be inserted between t h e injection port and column of an HPLC apparatus. The major requirements for the cell were a low dead volume to prevent band broadening and a capability for withstanding moderately high pressures (1000-1500 psi) associated with microparticulate packings.
EXPERIMENTAL Cell Design and Construction. A schematic of the cell, which 0003-2700/79/0351-1573$01.0010
is based on an earlier design (51, is shown in Figure 1. The body consisted of two 2-inch (5.1-cm) diameter Plexiglas cylinders both 3/,-inch (1.9-cm) thick, held together with four stainless steel bolts (not shown). The silversilver chloride reference electrode (SSCE) was wound around a Plexiglas post (6.4 mm long and 1.02 cm in diameter) which extended from the upper cylinder into a cavity (2.74 cm in diameter, 8.3 mm deep) in the lower cylinder. Epo-Tek 349 epoxy (Epoxy Technolgy Inc., Watertown, Mass.) was used to seal a 3.07-mm thick glassy carbon disk (Continental Ore Corporation, New York, N.Y.) into the cavity of the lower cylinder, and the SSCE and platinum counter electrode leads into the upper cylinder. Epo-Tek 410E silver-filled conducting epoxy was used to make the electrical contact between the glassy carbon and a copper lead. A 0,025-inch (0.635-mm) diameter channel was drilled through the Plexiglas body with a twist drill bit and continued through the glassy carbon with a stainless steel hypodermic tube attached to an ultrasonic milling machine (Sonipak, Bullen Ultrasonics, Inc., Eaton, Ohio), forming the tubular electrode in the configuration shown in Figure 1. Bolting the two cylinders together exerted pressure on eight cation-exchange membrane washers (0.25 mm thick, 0.63-mm i.d., 2.72-cm o.d., Nafion XR-170, E. I. du Pont de Nemours & Co., Inc., Wilmington, Del.) located in the cavity of the lower cylinder. This provided a leak-free solution bridge between the working electrode and reference and counter electrodes. Cheminert fittings and '/ 16-inch(1.6-mm)Teflon tubing were used for the inlet and outlet. The 0.1 M KC1 inlet and outlet consisted of Tygon tubing press fitted, and sealed with cyclohexanone. Instrumentation. All potentials reported in this paper are given with respect to the SSCE (0.1 M in KCl). Potentials were applied with a Princeton Applied Research Model 364 Polarographic Analyzer, the current output being monitored with a strip chart recorder. A high-performance liquid chromatograph (ALC/GPC 202, Waters Associates, Milford, Mass.) equipped with a U6K injection valve and Model 6000 reciprocating pump (Waters Associates), a variable wavelength detector (Model 770, Schoeffel Instrument Corp., Westwood, N.J.), and a prepacked microparticulate reverse-phase column (p-Bondapak CI8,Waters Associates) was used. D 1979 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 9, AUGUST 1979 KJ/
H
Q, I-.
Z
Lu
CY CY 3 V Y
Q, a 0.0
0.2
0.4
0.8
0.6
1.o
POTENTIAL, V Figure 2. Peak current-potential curve for adrenaline. 0.2 mg/mL adrenaline in 0.01 M H,PO,; 10 pL injected: 1.0 mL/min Flgure I. Pre-column tubular electrode for HPLC. (Cell shown prior to compression by the bolts.) (A) O-ring; (B) cation-exchange membranes; (C) cast epoxy; (D) sample solution inlet; (E) lead to glassy carbon working electrode; (F) 0.1 M KCI inlet; (G) lead to reference electrode (SSCE); (H) sample solution outlet; (J) 0.1 M KCI outlet; (K) lead to platinum counter electrode
The inlet and outlet of the electrochemical cell were connected to the injector and column, respectively, with stainless steel to Teflon tube adapters (STA-1, Bioanalytical Systems, West Lafayette, Ind.). Reagents and Eluent. Adrenaline bitartrate was acquired from Winthrop Laboratories, New York, N.Y., and levodopa equivalent t o USP specifications was used. All other reagents were ACS reagent grade. Solutions of adrenaline and levodopa were made by dissolving the respective compound in 0.1 M phosphoric acid and diluting to 0.01 M with distilled water. The mobile phase was prepared by adjusting a 0.01 M NaH2P04 solution to pH 2.5 with phosphoric acid. This solution was filtered daily through a 0.5-bm Millipore filter and degassed prior to use. The reverse-phase chromatography of catecholamines employing a purely aqueous phosphate buffer eluent has been discussed elsewhere (6). Procedure. Since the electrode geometry allowed less than 1% of the species flowing through at 1 mL/min to be electrochemically transformed, stopped flow was employed to increase the electrochemical yield. The procedure was as follows. The electrode was potentiostated at the desired voltage, the sample injected a t 0.5 mL/min, and the flow stopped as the recorder monitoring the cell current began to respond. After maintaining stopped flow for a time sufficient to yield an easily identifiable peak (usually about 5 min), the pump was started at 0.5 mL/min and the flow rate raised to 1.0 mL/min within 10 s. Injection volumes ranged from 10 to 50 pL. Although smaller injection volumes yielded slightly greater relative electrochemical yields (from 3% for a 50-pL injection to 6 to 10% for a 1-pL injection), much larger absolute amounts oxidized were obtained with the larger injection volumes.
RESULTS AND DISCUSSION Since the hold-up volume is only 10 @L,attachment of the cell t o t h e HPLC system did not noticeably increase peak widths observed with t h e absorbance detector. In addition, no leaks were observed through t h e reference electrode compartment or the Cheminert fittings at the flow rates and pressures employed ( u p t o 1.2 mL/min and 1500 psi). However, after several days use, reflanging of t h e Teflon tubing was generally necessary to prevent the development of leaks. Adrenaline and levodopa were the initial compounds studied to characterize t h e cell since the electrochemistry of catecholamines is well known (2, 3 ) . At p H 3, for example, adrenaline is oxidized t o the o-quinone which readily undergoes ring closure t o form adrenochrome. Other cate-
iL c
485 nm
A 0
B
A
485 nm
5
10
15
MINUTES Figure 3.
Chromatograms of adrenaline and oxidation product: (A,
B) adrenaline, 1, oxidized at 0.9 V, stopped flow fw 5 min; (C)adrenaline oxidized with ferricyanide to adrenochrome, 2. 0.2 mg/mL adrenaline in 0.01 M H,PO,; 50.0 pL injected; 1.0 mL/min
cholamines, on the other hand, show a lesser tendency toward ring closure a t low pHs ( 2 ) . A plot of peak current vs. potential for injections of 0.2 mg/mL adrenaline a t a flow rate of 1.0 mL/min is shown in Figure 2. From the resulting peak current-potential curve, a potential of 0.9 V was chosen for oxidizing adrenaline. From a similar plot, 0.8 V was chosen for levodopa. Chromatograms of adrenaline for which the electrode had been potentiostated a t 0.9 V and t h e absorbance detector monitored a t 280 nm (Figure 3A) and 485 nm (Figure 3B) yielded the parent adrenaline peak and another peak with a retention time of 13.9 min. A chromatogram, monitored a t 485 nm, of a solution of adrenaline t h a t had been oxidized with ferricyanide to a deep red solution of adrenochrome according to the procedure of Mattok and Heacock (7)yielded a peak, shown in Figure 3C, with a retention time t h e same as that observed for the late eluting peak in Figures 3A and 3B. (Aminochromes usually exhibit a broad absorption maximum between 470-490 nm, in addition to strong absorption near 280 nm ( 4 ) . ) Chromatograms of levodopa for which the cell had been potentiostated at 0.8 V and the absorbance detector monitored
ANALYTICAL CHEMISTRY, VOL. 51, NO. 9, AUGUST 1979 1
A
B
C
u 5 0
10
MINUTES Figure 4. Chromatograms of levodopa and oxidization products: (A, B) levodopa, 1, oxidized at 0.8 V, stopped flow for 5 min; (C) levodopa oxidized with ferricyanide to dopachrome, 2. 0.2 mg/mL levodopa in 0.01 M H,P04; 50.0 NL injected; 1.0 mL/min
a t 280 nm yielded a peak (at a retention time of 4.6 min) that eluted just prior to the levodopa peak (Figure 4A). This peak was not evident a t 485 nm (Figure 4B). A chromatogram of a solution of levodopa oxidized to dopachrome with ferricyanide as above yielded a peak (Figure 4C) a t a different retention time than the electrochemically generated peak of Figure 4A.
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Apparently, adrenochrome is the most stable oxidation product of adrenaline in the time scale of the experiment. However, the o-quinone of levodopa does not appear to readily cyclize to form dopachrome a t the low p H of the eluent. This is consistent with the cyclization rate constants for oxidized catecholamines presented by Hawley et al. ( 2 ) . At low pHs, the rate constant for primary catecholamines is much lower than t h a t for adrenaline and isoproterenol, which are secondary amines. Using the present cell and stopped flow procedure, from 3 to 10% of the analyte undergoes electrochemical transformation, making the cell useful primarily for qualitative identification of oxidation products. Increasing the electrochemical yield to 100% might provide an important advantage; namely, the ability to alter the retention or to completely eliminate peaks of electroactive species. By carefully adjusting the potential of the cell, quantitative electrochemical derivatization of potentially interfering components of a chromatogram might be possible, thereby providing an additional variable for controlling selectivity. Such a coulometric cell is presently under design.
ACKNOWLEDGMENT The assistance of W. J. Blaedel and the use of the University of Wisconsin-Madison Chemistry Department machine shop facilities are highly appreciated. LITERATURE CITED (1) Kissinger, Peter T. Anal. Chern. 1977, 49, 445A-456A. (2) Hawiey, M . D.; Tatawawadi, S.V.; Piekarski, S.; Adams, R. N. J. Am. Chem. SOC. 1987, 89,447-50. (3) Sternson, A. W.; Mccreery, R.; Feinberg, B.; Adam. R. N. J . Electronanal. Chern. Interfacial Necfrochem. 1973, 46, 313-21. (4) Heacock, R. A. Chern. Rev. 1959, 59, 181-237. (5) Blaedel, W. J.; Schieffer, G. W. Anal. Chern. 1974, 4 6 , 1564-67. (6) Molnar, Irnre; Horvath, Csaba J . Chromatogf. 1978. 745, 371-81. (7) Manok, G. L.; Heacock, R. A. Can. J . Chem. 1964, 42, 484-85.
RECEIVED for review December 15, 1978. Accepted March 9, 1979.
Determination of Chromium(II1) in the Presence of Large Amounts of Chromium (VI) R. V. Whiteley, Jr. Sandia Laboratories, Albuquerque, New Mexico 87185
A black chromium plating process which was originally designed for preparing decorative coatings is being evaluated for solar applications because it produces a finish with high solar absorptance and low thermal emittance. I t has been shown that the thermal stability of these electroplate finishes is a function of the chromium(II1) content of the black chromium plating bath ( I ) . These plating baths are typically prepared from chromium trioxide and dilute acetic acid solutions. They are rich in chromium(V1) (ca. 150-200 g L-l) and may contain as much as 25 g L-I of iron(II1). The chromium(II1) is produced by reducing some of the chromium(V1) in situ. T h e previously used procedure for a chromium(II1) determination entails a determination first for chromium(V1) and then for total chromium content. The chromium(II1) content is found by difference. Several problems arise from this approach. Since the chromium(II1) content of these baths is usually less than one tenth the chromium(V1) content, the results are based upon subtracting one large value from another large value; the inherent inaccuracies are significant. Furthermore, this determination of total chromium requires the oxidation of the chromium(II1) to chromium(V1) with 0003-2700/79/0351-1575$01 .OO/O
ammonium persulfate which tends to be a rather slow process. An alternative procedure for chromium(II1) delermination which is more rapid and accurate has been developed. Willard and Young (2)have determined chromium(II1) by a potentiometric titration. The chromium(I11) is quickly oxidized to chromium(V1) with an excess of cerium(1V). The excess of cerium(1V) is determined by titrating the solution with a sodium nitrite solution and observing the equivalence point potentiometrically. This method appears to be quite accurate; however, the nitrite-ceric reaction is very slow. This results in a rather laborious titration because it requires several minutes to obtain a fairly stable potential reading after adding titrant. Also, the potential break a t the equivalence point is not particularly well-defined. This makes a plot of the titration curve necessary. Monnier and Zwahlen ( 3 ) have applied the principles of this procedure to a trace analysis for chromium(III), but they achieved better definition in their titration curves by using a platinum-tungsten electrode system. Still, the tungsten electrode does not respond rapidly, and because it is essentially s n “attackable” reference electrode, frequent cleaning is required. The problems associated with the slow nitrite ceric reaction were circumvented in the ‘E 1979 American Chemical Society
