A major drawback of the enzyme electrodes described in the present paper is the relatively slow electrode response. I n particular, if the potentiometric measurement of a solution with a low substrate concentration is made after the potentiometric measurement of a solution with a high substrate concentration, the response time is even longer. To improve the response time of a C02 electrode, a new membrane is urgently needed.
ACKNOWLEDGMENT The authors wish to thank Millard Burroughs for technical help throughout the experiments.
RECEIVEDfor review March 10, 1972. Accepted June 30, 1972. The financial assistance of the National Health Institute (Grant No. G M 17268) and the National Science Foundation (Grant No. G B 12669) is deeply appreciated.
Electrosorption and Separation of Quinones on a Column of Graphite Particles John H. Strohl and Kenneth L. Dunlap Department of Chemistry, West Virginia Uniuersity, Morgantown, W . Va. 26506 I t is possible to adsorb certain compounds on a packed column of graphite particles by applying a potential to the column and flowing a solution of the compound through it. This adsorption can be used as a method of concentrating dilute solutions and separating mixtures of compounds. The concentration of the compounds may then be determined by an appropriate method; UV spectrophotometry of the column effluent and thin-layer chronopotentiometry of the substrate on the column were used as examples. A four-component mixture was successfully separated using the electrosorption technique and analyzed by chronopotentiometry. Several compounds were analyzed at a concentration as low as 5.0 x lO-’M.
A NUMBER OF INVESTIGATIONS of potential dependent adsorption at electrodes have been reported; however, most of these studies have dealt with electrosorption from quiescent solutions. This investigation was undertaken to explore the possibility of observing electrosorption on a column of graphite particles from a solution flowing through the column. This column of graphite particles has a much greater surface area than other electrodes that have been employed in adsorption studies from solution, and thus this electrode could be used to adsorb a proportionally larger amount of material. The study was also initiated to investigate whether it would be possible to utilize this phenomenon as a concentrating technique for the analysis of trace amounts of certain substances, and to study the feasibility of using the column as a means of separating certain mixtures. Several quinones were used to demonstrate the possibilities and limitations of electrosorption of species from a solution flowing through the electrode.
UV flow cell was used as a detector for the column effluent. A Sage syringe pump or a Cole-Partner variable speed Masterflex pump was used to pump liquids through the column a t flow rates ranging from 0.25 to 2 ml/min. The basic design of the electrochemical cell, shown in Figure 1, consists of three concentric electrodes. The reference electrode, F, is a coiled silver wire coated with AgCl and immersed in a 1M KC1 solution, and the counter electrode, E, is a coiled silver wire. The working electrode, G, consists of a 24-cm section of 4-mm i.d. porous Vycor glass tubing packed with 3.10 grams of graphite. In packing the graphite column, a plug of glass wool was inserted in the bottom of the Vycor tubing, and the graphite was packed in the tubing. A Teflon (Du Pont) disk was placed o n top of the column, and then the cell was assembled. A pencil lead, A , was then inserted into the top of the column to make electrical contact with the graphite. With solvent and supporting electrolyte in the graphite column and 1M KC1 outside of the porous Vycor tubing, an ac resistance of 14 ohms was measured between the working and reference electrodes. The solvent used in this study was a l / l , v h mixture of acetonitrile and water. As was observed previously (1, 2), very little adsorption of the compounds occurred with acetonitrile solutions, and it was virtually impossible to wash off adsorbed species when aqueous solutions were used. The supporting electrolyte used in chronopotentiometry was 0.1M KCl. Before the column was used, a potential was applied to the column which was sufficiently negative to reduce the oxygen adsorbed on the graphite and approximately 50 ml of solvent was passed through the column. All solutions were deaerated with nitrogen prior to passage through the column.
EXPERIMENTAL
The quinones used in this study were obtained from Aldrich Chemical Company and Eastman Organic Chemicals. These compounds were recrystallized twice from a waterethanol mixture. The graphite, Asbury Artificial No. 1 , was supplied by Asbury Graphite Mills, Inc. This graphite was extracted with dilute HC1 several times, allowed to dry, and then screened. The size of the graphite used in this study ranged from 105 to 147 microns. The porous Vycor glass tubing No. 7930 was obtained from Corning Glass Works. Burr-Brown operational amplifiers (Model 1506) were used in the construction of a potentiostat, and a Sargent Coulometric Current Source was used for the constant current required in chronopotentiometry. A Bausch and Lomb Spectronic 505 spectrophotometer in conjunction with a 2166
RESULTS AND DISCUSSION
The results of a capacity study of the column are presented below for eleven quinones. A solution of the compound of interest was pumped through the column with a certain potential applied to the column, and the UV absorbance of the column effluent was monitored. In this investigation, the column capacity was defined as that volume of solution which had to be pumped through the column before the compound (1) R. L. Bamberger and J. H. Strohl, ANAL.CHEM., 41,1450 (1969). (2) W. B. Caldwell and J. H. Strohl, Department of Chemistry, West Virginia University, Moryantown, W. Va., unpublished work. 1972.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 13, NOVEMBER 1972
Table I.
Capacities of Compounds Investigated Capacity in Fmoles per gram Compounds studied of graphite" 9,lO-Anthraquinone-1-sulfonic acid Na salt 2.0 Phenanthrenequinone 2.6 Duroquinone 0.26 2,3-Dichloro-5,6-dicyano-l ,Cbenzoquinone 4.7 Acenaphthenequinone 1.1 2,3-Dichloro- 1,Cnaphthoquinone 1.4 1,2-Naphthoquinone-4-sulfonic acid Na salt 0.90 2-Hydroxy-1,4-naphthoquinone 0.16 2-Methyl-1&naphthoquinone 0.39 1,8-Dichloro-9,10-anthraquinone 3.2 1,5-Dichloro-9,10-anthraquinone 5.0 1.O x 10-4Msolutions, 1 ml/min flow rate, and +1.0 V applied.
was observed in the effluent. This capacity could also be related to a certain number of moles of the compound which were adsorbed per gram of graphite since the weight of the graphite (3.10 grams) and the volume and concentration of the solution were known. It was generally found that the capacity of the column increased as more positive potentials were applied to the column until a potential of approximately +1.0 V cs. the Ag/AgCl reference electrode was reached. At more positive potential a decrease in the column capacity of all compounds studied was observed. The cause of this decrease could possibly be due to the preferential adsorption of the solvent a t these potentials or the adsorption of oxygen produced by the electrolytic breakdown of the solvent system. Figure 2 gives plots of the capacity as a function of potential for some of the quinones studied. Of all the compounds studied, only duroquinone showed a general trend of decreasing capacity as more positive potentials were applied to the column. Table I gives the capacity of each compound studied in pmoles/gram of graphite. Additional study indicates that the capacity of the column varies with the concentration of the adsorbate solution pumped through the column and also with the flow rate. Figure 3 gives a plot of capacity as a function of concentration of solutions of 9,10-anthraquinone-l-sulfonicacid sodium salt (AQS). The increase in capacity with increasing concentration as shown in this figure would seem to imply a n equilibrium process for electrosorption. A plot of capacity us. flow rate of a 5.0 X 10-4MAQS solution a t a potential of $1.0 V is given in Figure 4. The capacity decrease with increasing flow rate would seem to indicate an insufficient amount of time for the system to attain equilibrium a t higher flow rates or a n increased stress o n the adsorbed layer preventing a thicker coverage. The following procedure was employed for the chronopotentiometric determination of the quinones adsorbed o n the column. After a chronopotentiogram of the solvent and supporting electrolyte was obtained, a certain volume of the solution was pumped through the column a t a n applied potential where adsorption occurred. The flow was stopped, and cathodic and anodic chronopotentiograms were recorded. The compound was then washed off the column by applying a sufficiently negative potential and pumping solvent through the column. Because of the extremely large surface area of the packed graphite electrode and the subsequent charging current required, the lowest feasible value for the current required in chronopotentiometry was 10 mA for this particular electrode. With lower current values, the potential of back-
Figure 1. Packed graphite electrochemical cell A. B.
C. D. E.
F. G.
H. I.
J. K.
Penal lead Standard joint with silicon stopper Solution inlet Teflon plug with Viton O-rings Counter electrode, Ag wire Reference electrode, Ag wire coated with AgClin lMKCl Working electrode, graphite particles in porous Vycor tubing 50-mm O-ringjoint lMKCl Reference solution inlet Column effluent outlet, to Uv detector
h
J
5 >.
c
0
2a
0
APPLIED POTENTIAL (VOLTS)
s W
J
0
0
525-
*o
0
x
>
t
0
220-
a
a
0
0
-
I
ANALYTICAL CHEMISTRY, VOL. 44, NO. 13, NOVEMBER 1972
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Table 11. Chronopotentiometryof Individual Compounds Adsorbed on the Graphite Column 5.0 X lo-' mole absorbed 1.O X 10-8 mole adsorbed 2.0 X 10-6 mole adsorbed
Compound 7-n Sb 7a Sb Ta 9,lO-Anthraquinone-1-sulfonic acid Na salt 7.8 0.42 15.8 0.47 30.4 7.3 Phenanthrenequinone 0.48 0.52 14.4 28.6 Duroquinone 7.2 0.47 C 14.1 0.48 2,3-Dichloro-5,6-dicyano-l,4benzoquinone 4.0 0.65 7.9 0.57 16.0 2,3-Dichloro-l,4naphthoquinone 7.9 0.50 15.8 31.3 0.48 2-Methyl-1,4-naphthoquinone 8.2 0.42 16.0 0.42 C 1,2-Naphthoquinone-4-sulfonic acid Na salt 6.7 0.48 13.6 0.51 22.5 1,8-Dichloro-9,10-anthra33.0 8.2 0.42 16.3 0.48 quinone 31.8 1,5-Dichloro-9,10-anthraquinone 7.9 0.37 15.7 0.49 Transition time, in seconds, mean of 10 trials, constant current of 10 mA, all solutions were 1.0 X 10-4M. Standard deviation, in seconds. 20 ml of a 1.0 X 10-4M solution of the compound could not be adsorbed due to the small capacity of the compound.
Sb
0.51 0.65 0.55 0.65 0.51 0.57 0.51
Table 111. Chronopotentiometry of Individual Compounds Adsorbed on the Graphite Column Volume of solution pumped Compounds Concentration, M through column, mP Transition time, sec Standard deviation, secb 9,lO-Anthraquinone- I-sulfonic 1.0 x 10-4 5 7.8 0 . 4 2 (10) acid Na salt 2.5 x 10-5 20 7.9 0.35 (10) 5 . 0 x 10-6 100 7.7 0.45 ( 5 ) 5 . 0 x 10-7 1OOOc 7.6 , , (2) 1,8-Dichloro-9.IO-anthraquinone 1.0 x 10-4 5 8.2 0.42 (10) 2 . 5 x 10-6 20 8.2 0.45 (10) 5.0 X 100 8.3 0.47 ( 5 ) 5 . 0 x 10-7 1OOO' 8.0 . . . (2) 5 6.7 0.48 (10) 1,2-Naphthoquinone-4sulfonic 1.0 x 10-4 acid Na salt 2 . 5 x 10-6 20 6.7 . . . (3) 2.5 x 10-6 200 6.4 . , . (3) 5.0 x 10-7 1 m -2 . . . (2) a Potential of +1.0 V and 1 ml/min flow rate unless indicated otherwise. * Numbers in parentheses indicate number of trials. Flow rate of 2 ml/min.
ground chronopotentiograms changed so slowly that it was impossible to interpret a chronopotentiogram. None of the electroanalytical techniques are well suited for this particular electrode because of the extremely large surface area used to provide a large area for adsorption. Thus it was felt that other, more elaborate electroanalytical techniques would not give sufficiently better precision and limit of detection to warrant their use. The results of the chronopotentiometric analysis of samples concentrated by electrosorption are presented in Tables I1 and 111. In Table 11, data are presented for measurements where volumes of 5, 10, and 20 ml of a 1.0 X 1 0 - 4 M ~ ~ l u t i oofneach compound were pumped through the column a t 1 mlimin with a potential of $1.0 V applied to the column. Strohl et af. ( I , 3) have shown that under certain conditions a column of graphite particles can be used as a thin-layer electrode, and transition times in Table I1 appear to be directly proportional to the amount of material present as predicted by thin-layer chronopotentiometry theory. In Table 111, results of the analysis of solutions of different concentrations are presented. 1,8-Dichloro-9,10-anthraquinone (l&DCAQ) and AQS could be analyzed a t a concentration of 2.5 X 10-7M; however, 1,2-naphthoquinone-4-sulfonicacid sodium (3) John H. Strohl and Thomas A. Polutanovich, A n d . Lett., 2,
423 (1969). 2168
salt (NQS) could not be analyzed at 2.5 x lO-7M since it was not quantitatively adsorbed at that concentration. Because of the large charging current of the graphite electrode and slow change in potential, transition times under four seconds could not be determined with any degree of accuracy; so a detection limit of a four-second transition time was set. Ultraviolet spectrophotometry was also used as a method of analyzing the compounds concentrated by electrosorption, and the following procedure was employed. After a certain volume of the solution was pumped through the column a t an applied potential where adsorption occurred, the flow was stopped. The applied potential was made sufficiently negative to desorb the material, and then a certain volume of solvent was pumped through the column. The concentrated sample was collected and analyzed by conventional UV spectrophotometric techniques. Table IV presents the results of a study of this method for the analysis of AQS, 1,8-DCAQ, and 1,5-dichloro-9,10-anthraquinone. In the general case where any type of detection technique could be used after the concentrating electrosorption step, the limit of detection would be dependent upon the sensitivity of the technique and the column capacity at low concentrations. The time required for the concentrating step can be significantly decreased with more sensitive detection techniques. Using chronopotentiometry to analyze a 5.0 X 10-7M, 1,8DCAQ solution which was concentrated by electrosorption,
ANALYTICAL CHEMISTRY, VOL. 44, NO. 13, NOVEMBER 1972
UV Spectrophotometric Analysis of Solutions Concentrated by Electrosorption Average concentration Relative standard Concentration, M Concentrating factor" determined, M* deviation, X b Compounds 9,lO-Anthraquinone-1-sulfonic 1 .o x 10-6 30:lO 0.97 x 10-6 5.4 acid Na salt 5.0 x 10-7 50:lO 5.0X 1 0 - 7 9.2 5.0 X lo-* 500 :1 o c 5.2 X lo-* 12 1,8-Dichloro-9,10-anthraquinone I .o x 10-8 4o:lO 1.0 x 10-6 5.4 5.0 x 10-7 80:lO 4.9 x 10-7 7.9 1,5-Dichloro-9,lO-anthraquinone 1.0x 10-6 45:15 1.0x 10-8 6.1 5.0 x 10-7 90:15 5.1 x 10-7 8.0 a Ratio of volume of sample pumped through the column to volume of the concentrated sample, flow rate 1 ml/min unless stated otherwise. h Average and relative standard deviation were calculated on the basis of ten measurements. Flow rate is 2 ml/min and only 5 trials. Table IV.
_
_
_
_
~
~
~
~~~
Table V.
~~______
Percentage of AQS Remaining on Columna
Volume of solvent pumped through column, ml
Mole initially adsorbed 1.0 x 10-6 2.0 x 10-6 3 .O x 10-6 4.0x 10-6 5.0 X 6.0 x 10-8 40 98 94 II 8 77 67 60 80 85 85 61 57 48 43 1.0 x lO-4M solution of 9,10-anthraquinone-l-sulfonicacid sodium salt (AQS) was used, applied potential of +1.0 V, 1 ml/min flow rate. transition times used to determine percentages.
a concentrating time of 500 minutes was required to pump 1000 ml of solution through the column. When this 5.0 X 10-'M solution of 1,8-DCAQ was concentrated by a factor of five using electrosorption and analyzed by the spectrophotometric method. the concentrating step could be performed in 30 minutes if a flow rate of 2 ml/min were used. Using a fiftyfold concentrating factor, a 5.0 X 10-8M solution of AQS could be analyzed; however, 250 minutes were required for the concentrating step. With additional study, it is hoped that increased flow rates may be employed, thus decreasing the time involved for the concentrating electrosorption step. Electrosorption is also useful for the separation of mixtures. The possibility of separating several compounds depends upon the difyerences in capacity a s a function of applied potential for the particular compounds. A four-component mixture of 1,8-DCAQ, phenanthrenequinone (PQ), AQS, and NQS was successfully separated, and the components remaining o n the column were analyzed by chronopotentiometry. Refer to Figure 2, A-D. At a n applied potential of -0.7 V only 1,8-DCAQ was adsorbed, when the four-component mixture was pumped through the column. When -0.4Vwas applied, only 1,8-DCAQ and PQ were adsorbed as indicated by their transition times. At f0.7 V all components appeared to be quantitatively adsorbed except NQS whose transition time was extremely small. When $1.0 V was applied to the column, all four quinones appeared to be quantitatively adsorbed. Thus, with the proper choice of potential applied to the column, one, two, three, or all of the components can be quantitatively adsorbed o n the column with the unadsorbed components passing through the column. After compounds were electrosorbed o n the column, they were not quantitatively held when solvent was pumped through the column a t an applied potential where they were adsorbed. This desorption process was studied by means of chronopotentiometry of the adsorbed material. Transition times of adsorbed material were compared with transition times of adsorbed material remaining after a certain volume of solvent was passed through the column at a potential where adsorption takes place. Table V gives the results of this study for AQS. This desorption behavior was also studied
FLOW RATE
(ML/MlN)
Figure 4. Capacity as a function of flow rate 5.0 X lO-4M solution of 9,lO-
anthraquinone-1-sulfonic acid Na salt pumped through the column at an applied potential of 1.0 V
+
for NQS, PQ, and 1,8-DCAQ. From this study of the four quinones, a n order of the tendency of the quinones to be desorbed was established: 1,8-DCAQ < AQS < PQ < NQS, where 1,8-DCAQ had the least tendency to be desorbed. In conjunction with this study, competitive adsorption experiments with these four quinones were undertaken. With this study, a certain amount of one of the quinones was adsorbed a t +1.0 V ; then a certain volume of a solution of a second quinone was pumped through the column at +1.0 V, and transition times of the two quinones were compared with expected transition times. The experiment was then repeated, adsorbing a certain amount of the second quinone and then pumping a certain volume of the other quinone through the column. An order of the relative strength of adsorption for these four compounds was obtained: 1,8-DCAQ > AQS > PQ > NQS, where IJ-DCAQ was the most strongly adsorbed compound. This order is consistent with the order of the tendency of the compounds to be desorbed. Electrosorption provides a means of concentrating a number of quinones. This method would be applicable to any sub-
ANALYTICAL CHEMISTRY, VOL. 44, NO. 13, NOVEMBER 1972
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stance that could be appreciably electrosorbed on the packed graphite electrode, and any appropriate method of analyzing the concentrated sample could be employed in conjunction with the concentrating step. The use of electrosorption for the separation of certain mixtures of quinones has also been demonstrated. The feasibility of such a separation depends upon the differences in capacity as a function of potential for the compounds, and thus is applicable to any mixture where components are adsorbed at different potentials. Further work is being conducted on the applicability of these techniques to other series
of compounds and to the use of other electrode materials with the hope of extending the scope of the method. ACKNOWLEDGMENT
The authors acknowledge the help of Rein Valdna, Robert Victor, and Carl Wise in the design and construction of the electrochemical cell. RECEIVED for review March 23, 1972. Accepted July 3, 1972. We are grateful for the support of an NSF Traineeship to K. L. D.
Determination of the Acid or Base Content of Organic Compounds Using Nuclear Magnetic Resonance Spectrometry Yair Deganil and Abraham Patchornik Department of Biophysics, Weizmann Institute of Science, Rehouot, Israel By a prior visual titration of a basic compound with an acidic titrant having a characteristic NMR spectrum, the base content of the titrated compound can be analyzed by NMR spectrometry, in terms of the baseto-titrant proton ratio. Using methanesulfonic acid (MSA) and 2,4,6-trinitrobenzenesulfonic acid (TNBS) as acidic titrants, various basic compounds were thus analyzed. Analogously, various acidic compounds were analyzed by NMR spectrometry following titration with tetramethylammonium hydroxide (TMA). The application of the method for the rapid characterization of basic or acidic reaction products was demonstrated. The method can be extended to other types of stoichiometric titrations, thus broadening the scope of NMR analysis of reactive functional groups.
NUCLEAR MAGNETIC RFSONANCE SPECTROMETRY (NMR) has proved to be a powerful tool for characterizing organic compounds in terms of their different proton types. Organic acids and bases, however, are less easy to characterize as such by N M R spectrometry. The difficulty is especially pronounced in nitrogeneous bases. Tertiary amino groups, lacking a proton, obviously do not appear in the N M R spectrum. In primary and secondary amines, the N-protons appear in a rather broad region. Their bands are affected by proton exchange and by the quadrupole moment of I4N (nuclear spin 1). The latter factor produces the splitting of the N-proton signals into a triplet which is sometimes very broad becoming undetectable. Acidic protons, such as those of carboxylic acids and phenols are more easy to detect, but they may appear in an unfixed shift position depending on the concentration, temperature, and solvent, due to hydrogen bonding. Acidic N-protons of compounds such as cyclic imides cannot be detected at all. In this report we wish to describe a general method for the determination of the relative acid or base content of organic molecules using N M R spectrometry. The method is based on a prior neutralization of the acidic or basic compound with a strong organic base or acid, respectively, which possesses a characteristic NMR spectrum, using an internal visual indicator. By taking the NMR spectrum of the neutral mixture and measuring the 1 Present address, Department of Biological Chemistry, School of Medicine, University of California, Los Angeles, Calif. 90024.
2170
integral ratio of the titrant signal to the rest of the signals, it is possible to determine the content of acidic or basic groups in the compound, in terms of the other protons present in the molecule. This approach thus combines the methods of nonaqueous titration and N M R spectrometry. EXPERIMENTAL
Reagents. Methanesulfonic acid (MSA) and hexadeuterodimethylsulfoxide (DMSO-D6) were obtained from Fluka (puriss). 2,4,6-Trinitrobenzenesulfonic acid (TNBS) was purchased from Aldrich. Tetramethylammonium hydroxide (TMA) was obtained from British Drug Houses as a 25% solution in water. Merck Sharp & Dohme was the source of DzO containing 1 % sodium 2,2-dimethyl-2-silaptane-5-sulfonate (DSS). All compounds analyzed were commercial samples which were used without further purifications. Acidic titrant solutions used were 1 % (v: v) MSA in chloroform and 3 TNBS in acetone-chloroform 2:3 (v: v). For titration of very weak bases, 3% TNBS in acetic acid-acetic anhydride 9 :1 (v: v) was used. The basic titrant used was 1 TMA in isopropanol, prepared by freeze-drying a commercial 25% aqueous solution of the base and dissolving the solid residue in isopropanol. For titration of very weak acids, a similarly prepared 1% TMA in pyridine-methanol (10 :3) was used. Indicators used were methyl red, 0.1 in chloroform; methanil yellow, 0.1 in methanol; methyl violet, 0.2% in acetic acid; thymol blue, 0.1 in isopropanol; and 2,2'dinitrodiphenylamine [prepared according to Eckert and Steiner (I)], 0.2 in pyridine. All solvents used were of analytical grade. Procedure. Five milliliters of about 1 % solution of the acid or the base were titrated with a corresponding titrant to the color change of a suitable indicator (see below). The titrated solution was evaporated to dryness under reduced pressure using a rotary evaporator and the residue was dissolved in 0.5 ml D 2 0 containing 1 % DSS as an internal reference. When the titrant was TNBS, the residue was dissolved in 0.5 ml of DMSO-De, and tetramethylsilane (TMS) was added as internal reference. NMR spectra of the solutions were recorded with a Varian A-60 spectrometer operating (1) A. Eckert and K . Steiner. MoriatsA., 35, 1153 (1914).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 13, NOVEMBER 1972
