Table V. Liquid-Junction Potentials between 3.5M Aqueous KCI Bridge and Buffers in Ethanol-Water Solvents a t 25 'C Wt % ethanol in water
(zj-(log 44
-
,n~H)
0.000 0.003 0.086 0.221 0.196 -0.032 -2.91
0.0 16.2 33.2 52.0 73.4 85.4 100.0
logrnYH Er (interpolated) pH units Millivolts 0.00 -0.17 -0.61 - 1.06 -0.93 -0.64 +1.85
0.00 -0.17 -0.52 -0.84 -0.73 -0.67 -1.06
0 -10 -31 -50 -43 -40 -63
Table VI. Standard Potentials of Hydrogen Electrodes in Ethanol-Water Solvents Referred to ,E"(H, H 2 0 ) = 0 (Molal Scale, 25 "C) Wt % ethanol in water ,E"(H, SH), volts 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0
O.Oo0 -0.004 -0.015 -0.030 -0.048 -0.061 -0.067 -0.059 -0.044 -0.030 -0.109
The latter is essentially the liquid-junction potential at the interphase between the nonaqueous buffer and the aqueous KC1 bridge and for ethanol-water solvents, it was found to be a constant characteristic of the medium (41). Combining (41) R. G. Bates, M. Paabo, and R. A. Robinson, 67, 1833 (1963).
J. Phys. Chern.,
the values of log estimated here with the quantities (E, - log determined experimentally in the above study (41), we are able to evaluate the liquid-junction potentials at the boundaries of 3.5M aqueous KC1 and dilute ethanol-water solutions (Table V). Correlation of E M F Series. Values of log m-yH for ethanolwater solvents enable us to calculate the standard potentials of the hydrogen electrode in these solvents on the aqueous emf scale, ,E"(H, SH), from Equation 3. Table VI shows the values of ,E"(H, SH) at even ethanol-water compositions. Any other conventional standard potential of a n electrode reversible to species i in solvent SH, ,E"(i, SH) referred to ,E"(H, SH) = 0, can be converted to its value on the aqueous scale, ,E"(i, SH), via: ,E"(i, SH)
=
,E"(i, SH)
+ ,E"(H, SH)
( 9)
The standard potentials ,E"(i, SH) in any nonaqueous solvent are referred to a single arbitrary zero point-that of the SHE in water [,E"(H, H 2 0 ) = 01. The striking feature of the SHE potentials referred to the aqueous standard state is the fact that for ethanol-water solvents other than anhydrous (or nearly anhydrous) ethanol they do not deviate much from the aqueous value of zero volts. Thus, contrary to general opinion and to our own expectations, the SHE turned out to be a fairly constant reference electrode for ethanol-water mixtures. In anhydrous ethanol, all EO'S exhibit a positive shift by about 0.1 volt, relative to their aqueous values. RECEIVED for review October 23, 1968. Accepted December 27, 1968. Work supported in part by the National Science Foundation under Grant GP-6553. Presented at the Division of Analytical Chemistry, 155th National Meeting, ACS, San Francisco, Calif., April 1968.
Optimization of Parameters for Single Sweep Polarographic Analysis of the Selenium-Diaminobenzidine Complex Donald A. Griffin Department of Agricultural Chemistry, Oregon State Unicersity, Coroallis, Ore. 97331
Determination of the selenium-diaminobenzidine complex by single sweep polarography was investigated. The analytical parameters were studied to find the optimum conditions for analysis. A sweep speed of 100 volts per second and an active derivative of the wave ave maximum sensitivity. Plots of peak current as a unction of sweep speed for the complex (adsorption wave) and for Cd*+ are given for both the normal and the derivative mode. Calibration curves are given for both the normal and derivative modes. A sensitivity of 1 ppb of selenium was obtained using the derivative response. This technique offers equal or greater sensitivity than existing spectrophotofluorometric methods and should be at least as fast. A tentative method for the analysis of selenium in biological samples is given.
P
THENEED for a fast and accurate method for the analysis of selenium at trace levels has been accentuated since it has been found to be an essential trace nutrient in animals. Present procedures for the analysis of selenium are time consuming and/or lack sufficient sensitivity for determining selenium a t concentrations as low as 10 ppb which is normally required 462
ANALYTICAL CHEMISTRY
for nutritional studies. Most analyses are presently done by spectrophotometry or spectrophotofluorometry using colored piazselenol complexes (1-7). These procedures offer a high degree of precision with good sensitivity (to 10 ppb), but are generally time consuming. Neutron activation has not found wide acceptance owing to the special techniques and skills required to measure the short-lived, but most sensitive isotope of selenium (8, 9). Atomic absorption methods lack the (1) K. L. Cheng, ANAL.CHEM., 28, 1738 (1956). (2) G. F. Kirkbright and W. K. Ng, Anal. Chirn. Acta, 35,116 (1966). (3) R. E. Stanton and A. J. McDonald, Analyst, 90, 497 (1965). (4) F. L. Chan, Tuluntu, 11, 1019 (1964).
( 5 ) L. M. Cummins, J. L. Martin, and D. D. Maag, ANAL.CHEM., 37, 430 (1965). ( 6 ) H. H. Taussky, A. Washington, E. Zubillaga and A. J. Milharat, Microchern. J . , 10, 470 (1966). (7) J. H. Watkinson, ANAL.CHEM., 38, 92 (1966). (8) F. J, Conrad and B. T. Kenna, ibid., 39, 1001 (1967). (9) R. C. Dickson and R. H. Tomlinson, I n t . J . Appl. Rudiut. Isotopes, 18, 153 (1967).
to.1
-c.o
-0.1
Starting
Figure 1. Cyclic polarogram of 0.6 ppm solution of selenium (as the complex) in 0.5N HClOl Starting potential-0.1 V, voltage scan -1.0 V, scan speed 100 V/sec, sensitivity 50 @a/div,drop time 4.5 sec, sweep delay 3 sec
required sensitivity (IO) for biochemical studies. Christian, Knoblock, and Purdy (I 1-13) have investigated the conventional polarography of selenium and the diaminobenzidine complex, and have reported a sensitivity of 0.1 pg/ml of selenium. Nangnoit (14) increased sensitivity for the analysis of selenium by IO-fold using single sweep polarography. Conventional polarography has sufficient accuracy and is reasonably fast, but lacks sensitivity in comparison to the spectrophotoiluorometricprocedures. The single sweep polarography of selenium seemed to offer these advantages plus increased sensitivity. The technique of single sweep polarography was applied to the analysis of the diaminobenzidine complex, a significant increase in sensitivity being obtained over and above conventional polarography. The effect of sweep rate on the peak current of the normal and first derivative waves was studied, and calibration curves for the normal and first derivative waves were plotted for several sweep speeds. The corresponding studies were carried out with cadmium for reference purposes. EXPERIMENTAL Instrumentation. A Chemtrix single sweep polarograph was used for this study. The instrument was equipped with a Type 305 dual differential amplifier unit and a Type 205, 3-electrode polarographic time base. The instrument was also equipped with a dual cell-dual DME electrode stand, with provision for mechanical drop synchronization. The time base unit was modified to provide sweep rates from 0.5 to 2000 volts per second. The signal from each channel is amplified by a current amplifier. A variable bucking current is applied at the inputs to allow charging current compensation. The signal from one of the current amplifiers is inverted by a one to one inverting amplifier. The two signals are then summed by a summing amplifier. Either cell may be used in the single cell mode if desired. The signal js then amplified prior to going to the cathode ray tube of the storage oscilloscope. An operational (10) C. S. Rann and A. N. Hambly, Anal. Chirn. Acta, 32,346 (1965). (11) G. D. Christian, E. C. Knoblock and W. C. Purdy, ANAL. CHEM.,37, 425 (1965). (12) G. D. Christian, E. C. Knoblock, and W. C. Purdy, ibid., 35, 1128 (1963). (13) G. D. Christian, E. C. Knoblock, and W. C. Purdy, J . Assoc. Ofic.A g r . Chern., 48, 877 (1965). (14) P. Nangnoit, J . Elecfroanal. Chern., 12, 187 (1966).
-a2 Potential
-a3
-4
-0.5
(volts)
Figure 2. Peak current as a function of the quiescent starting potential for the selenium-diaminobenzidinecomplex (0.1 ppm Se in 0.5N HC10J amplifier derivative circuit with a choice of 3 time constants is available between the summing amplifier and the output amplifier. All work for this investigation was done in the 2-electrode mode against a mercury pool reference electrode. The high concentration acid electrolytes used did not give enough IR drop for the 3-electrode system to be useful. Cells. The cells used were fashioned from the lower 4 cm of 12-mm borosilicate glass test tubes. A platinum wire was sealed into the bottom to make contact with the mercury pool electrode. With this cell a solution volume between 0.20 and 1.5 ml may be conveniently analyzed. The top of the cell was not covered. The cells were not swept with nitrogen because little interference due to dissolved oxygen was observed, with the standard solutions. Oxygen interference was reduced when the sweep speed was increased. The author observed, as have previous workers (13) that the diaminobenzidine complex cannot be bubbled with nitrogen in the presence of mercury as sensitivity is quickly lost. Reagents. The water used for all solutions was singularly distilled. The selenium standard used was made by the method of Watkinson (7). The diaminobenzidine used was reagent grade 3,3'-diaminobenzidine hydrochloride (J. T. Baker) which had been recrystallized once from water. The benzene used was washed with concentrated sulfuric acid and singularly distilled after washing with water and drying. The remainder of the chemicals used were analytical reagent grade. All glassware was soaked overnight in concentrated nitric acid between uses, which decreased the blank to a negligible value. RESULTS AND DISCUSSION Polarographic Behavior Obtained for the Selenium-Diaminobenzidine Complex. A single sweep polarogram of the complex is shown in Figure 1. From this and reference to previous work (13, 15, 16), it was concluded that adsorption waves are obtained because of very strongly adsorbed reactants. The first adsorption wave (larger wave) has two distinct features, a complete absence of diffusion controlled plateau and no reversibility. The second wave on the other hand exhibits some of each of these characteristics. The first adsorption wave was used for all measurements reported in this investigation. The peak current yield varied with the quiescent starting potential used. Figure 2 gives a plot of peak current as a (15) R. H. Wopshall and I. Shain, ANAL.CHEM., 39, 1527 (1967). (16) Ibid., p 1535. VOL. 41, NO. 3, MARCH 1969
463
IOOC
soc
200
IOC
so
20
IC IP BO)
5
Figure 5. Peak readability as affected by scan speed Lower scan 0.2 ppm Se (as complex) 10 V/sec Upper scan 0.02 ppm Se (as complex) 100 V/sec Electrolyte 2 N HCIO,, sensitivity 5 ka/div for both, starting potential -0.1 V, voltage scan -1.0 V, drop time 4.5 sec, scan delay 3 sec
' I
Effect of Sweep Rate on Peak Current of the Normal Wave. Sweep rates from 0.5 to 2000 volts per second were used t o obtain the results shown in Figure 3. The peak current for cadmium ions was a function of the theoretical 0.5 power of the sweep speed. The selenium-diaminobenzidine complex
2
I
P
b
lo
s'o
20
Id0
200
5bO
Sweep Speed W s e c )
Figure 3. Peak current
YS.
sweep speed
1.0 X lO-,M CdCI, in 0.1N HCI 1.2 X 10-6M Se (as complex) in 0.5N HCIO, Drop time 4.5 sec, scan delay 3 sec
= x =
"'sool"1
function of starting potential. The peak width increased with the starting potential, lowering resolution for starting potentials more negative than -0.25 V. Selection of a starting potential was governed by still another effect. The response curves appeared to be linear for voltages between 0 and -0.15 V. At starting potentials more negative than -0.15 V, the sensitivity at the lower concentration levels drops off drastically. The second wave does not appear at starting potentials more negative than the first peak (-0.35 V).
I
1
b
5
Sweep
Figure 6. Peak current waves Figure 4. Polarograms of 0.05 ppm solution of Se (as complex) in 0.5 HCIOl Startingpotential -0.15 V, voltage scan - 1.0V, sensitivity 20 pa/div, scan speeds 10, 100, 1000 V/sec, respectively, from the bottom 464
ANALYTICAL CHEMISTRY
io Speed
YS.
5b
260
so0
sweep speed for first derivative
1 X 10-4M CdCI, in 0.1N HCl Time constant-slope = 0.98 x = 7 msec. o = 15 msec. Time constant-slope = 1.28 =
100
(V/recl
36 msec. Time constant-slope = 1.21
loool
lO0Oj
5001
200.
100’
1
I I
2
5
IO
20
50
100
ZOO
500
Sweep Speed ( V / r r c )
Figure 7. Peak current waves
YS.
sweep speed for first derivative
1.2 X 10-6M Se (as complex) in 0.5N HCIO, x = 7 msec. Time constant-slope = 1.49 15 msec. Time constant-slope = 1.53 O = 36 msec. Time constant-slope = 1.58 Y r
--
gave a linear response with the 0.79 power of the sweep rate. The concentrations were lO-4M for Cd2+ in 0.1M HC1, and 10-6M for Se (as the complex) in 0.5N HC104. These concentrations were selected to allow the greatest possible range of sweep speeds to be used. The precision varied with the sweep speed only slightly for sweep speeds below 1000 V/sec. The optimum precision was at sweep rates from 50 to 300 V/sec. From the above it was found that the optimum sweep speed for the selenium complex is 100 to 500 volts per second, in order to take advantage of the increased peak current and lower noise levels observed. The polarograms in Figure 4 show the increased current resulting from increasing the scan speed with the same solution in the cell. The polarograms shown in Figure 5 show the effect of increasing sweep rate on noise. The ground was removed from an input lead to give a good noise band. The amplifier sensitivity setting was the same for both scans. The upper trace is two scans superimposed. One can see that meaningful measurements are much more easily made at high noise levels (high sensitivity) by using the high sweep speeds. Effect of Sweep Rate on the Peak Current of the First Derivative Response. The solutions used in the previous section were also used to study the effect of sweep rate on the first derivative peak current. The measurements were made using a derivative circuit having a time constant of 7, 15, or 36 msec. The results for cadmium are shown in Figure 6. The reason for the divergent responses is not clear. The theoretical response should have a slope of 1.50; our lower response may
2
5
15 20 50 ppb Sdenium (as complex)
100
200
500
Figure 8. Calibration curves for the selenium-diaminobenzidine complex in 2M HCIOl Peak current YS. ppm selenium (as complex)--derivative circuit time constant 15 ms. Starting potential -0.1 V, voltage scan -1.0 V, drop time 4.5 sec, sweep delay 3.0 sec be explained by a noise filter, giving a nonideal response. The roll off at high frequency is due to the noise filter on the differentiator circuit. Comparable data were obtained for the selenium-diaminobenzidine complex (Figure 7). The response linearity was much better here. The derivative responses were essentially parallel, with peak current increasing by the 1.5 power of the sweep speed. The high frequency filter was again the probable cause of nonlinearity at the higher sweep speeds. In both cases the use of the derivative circuit increased the slope of the response by a factor of 0.70. The advantage of the longer time constants must be weighed against the increased noise incurred. A 20- to 25-msec time constant is the best compromise for these sweep speeds. The active derivative can be used to increase sensitivity as well as to increase resolution and improve base line as seen by comparing Figure 3 with Figures 6 and 7. With the active derivative, the signal is not attenuated at the higher scan speeds as it is for R C derivative circuits. However at the lower scan speeds an R C derivative circuit is preferable because of the lower noise level incurred. Some very preliminary work with a second derivative circuit at these scan speeds has been done and is very promising, as the peak current is again increased considerably and the base line is much flatter than in the first derivative as shown by Perone (17). Calibration Curves. Calibration curves were run for both the complex and cadmium ions at several scan speeds. The results are given in Figures 8 and 9, respectively. The responses (17) S. P. Perone and T. R. Mueller, ibid., 37, 2 (1965). VOL. 41, NO. 3, M A R C H 1969
465
Figure 10. 5 ppb selenium (as complex) first derivative response in 2.OM HCIO,, sensitivity 2 pa/div, other conditions as in Figure 8
i
':xid6
Said6
ib5
2 k 5
Cd
5;16'
16'
2;16~
5ii6'
16'
Concentration (M)
Figure 9. Calibration curves for Cdzt in 0.1N HCI, peak current vs. concentration, starting potential -0.5 V, all other conditions as Figure 8
are linear and the sensitivity can be increased by the use of the derivative circuit. With the use of small cell volumes (0.1 ml) and the derivative circuit, the author has been able t o measure selenium in amounts of 0.05 nanogram with a range of =t15 %. The range with 0.2 nanogram (2 ppb level) samples was less than &52. The limitation on sensitivity was inclined base lines, which presumably could be remedied by the use of ultrapure reagents. The above measurements were obtained using replicate aliquots of a standard solution of the complex. The complexation step is reproducible with a range of less than =t10% at the 2-ppb level. The limit of precision of the tentative method was in the digestion and preparation of the sample. Figure 10 shows the derivative response obtained for a 5-ppb solution in the derivative mode. Method Development. Using the above obtained calibration curves, an attempt has been made to apply the instrumentation to the analysis of biological samples. The method used was essentially that of Christian, Knoblock, and Purdy (13). A 1to 2-gram sample was digested in 15 ml of nitric acid and 5 ml of perchloric acid. To the cooled digest 15 ml of 0.1M EDTA
466
ANALYTICAL CHEMISTRY
and 2 ml of 2.5.44 formic acid were added. The increase in EDTA was required for forage samples. The p H was adjusted to 2.0-3.0 with ammonia, and 2 ml of 3,3'-diaminobenzidine hydrochloride solution containing 10 mg of DAB was added. The complex was allowed t o form for 1 hour. The complex was then extracted by adjusting the pH to 7-8 and extracting with two 10-ml portions of benzene. The complex was then back extracted from the organic layer by extraction with two 1-ml portions of 2 M perchloric acid. This last extraction is very favorable and may be performed with smaller volumes if necessary. No ammonia was added here as it decreased the peak current and was not needed. A portion of the 2 ml of acid solution was added to the cell. Bubbling the sample prior t o putting it into the cell (no mercury present) markedly improved the result obtained. The selenium peak at -0.38 V is read. This procedure has at times shown applicability a t levels at and below 10 ppb in feed and diet samples. The author has experienced considerable unreliability in the method at its present stage of development. The method gives recoveries of about 75 for the entire procedure, with a range of approximately i10 for a single run. Better results were obtained using benzene than with the chlorinated solvents recommended, owing to much less solvent interference and better extraction. The method is presented here so that those interested might be informed.
z z
ACKNOWLEDGMENT
The author thanks Robert Claeys for assistance in the preparation of the manuscript, and Joseph Nelson of Chemtrix, Inc., for his generous assistance with the instrumentation. RECEIVED
26, 1968.
for review September 13, 1968. Accepted December
