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Figure 2. Timing diagram. The purpose of this figure is to show the relative timing of the indicated events. However, the time scales for the various events are not necessarily the same. For example, the clock pulses (IC2) are separated by 8.33 ms whereas the pulses from the exclusive OR output of IC1 occur only every 3-7 s comparable to that reported by Corbusier and Gierst (2) but not as high as has been achieved with some of the previously described devices (3, 5 , 6). However, when the lifetimes of many drops are averaged, the accuracy and precision of the present timer are very high indeed. A precision time mark generator (Tektronix Model 180 A) was used as the input source to test the inherent accuracy and precision of the drop timer. For marks separated by 5 s and counting 8 marks per run, the interval could be measured to within 0.2%. In 20 replicate experiments, a precision of f0.008% was observed. The time mark generator was also used to test the ability of the timer to measure short time intervals. For marks exactly 0.01 s apart, the timer yielded an interval of 0.010006 s after counting 512 marks. Electrocapillary Data. The timer was used to measure the electrocapillary curve for 0.5 M NaC104(pH 4.2) at 25 O C . At least three runs consisting of the measurement of eight drop times were performed at each potential. Even near the potential of zero charge (pzc) the drop time could be measured with a precision better than fO.l% by using the most sensitive current range on the PAR 174. (Triggering of the drop counter requires that the negative edge of the input signal cross +2.6 V. By careful adjustment of the dc offset in the current output of the polarograph, this requirement can usually be satisfied despite the smaller magnitude of currents flowing near the pzc. However, problems can occur under conditions where
c
1
-E/rnV vs SCE Flgure 3. Comparison of electrocapillary data obtained from differential capacitance and drop time measurements. (0)This work; (0)taken from Parsons and Payne ( 7 ) . The two data sets were superimposed at the electrocapillary maximum
the current-time curves contain depressions which cause the input signal to cross the triggering level prematurely.) The data are plotted in Figure 3 along with the surface tension date of Parsons and Payne for aqueous 0.5426 M HC104 a t 25 "C (7). The agreement between the two sets of data is excellent. Thus, the greater convenience of the drop timer described herein has been achieved without sacrificing the quality of the electrocapillary data obtained. Of course, all methods based on drop time measurements share the limitation that adsorption equilibrium must be reached rapidly for the drop time to be a reliable measure of the equilibrium interfacial tension. ACKNOWLEDGMENT 'Charles Klopfenstein provided much valuable advice in the design of the digital circuit. Helpful discussions with Irving Moskovitz are also acknowledged. LITERATURE CITED (1) D. C. Grahame, Chem. Rev., 41, 441 (1947); D. M. Mohilner in "Electroanalytical Chemistry, A Series of Advances", Vol. 1, A. J. Bard, Ed., Marcel Dekker, Inc., New York, 1966. (2) P. Corbusier and L. Gierst, Anal. Chim. Acta, 15, 254 (1956). (3) L. Meites and J. M. Sturtevant, Anal. Chem., 24, 1183 (1952). (4) A. J. Bard and H. B. Herman, Anal. Chem., 37, 317 (1965). (5) B. Nygard, E. Johansson, and J Ologsson, J . Elecfroanal. Chem.. 12, 564 (1966). ( 6 ) G. Papeschi, M. Costa, and S. Bordi, Electrochim. Acta, 15, 2015 (1970). (7) R. Parsons and R. Payne, Z . Phys. Chem. (Frankfurtam Main), 98, 9 (1975).
RECEIVED for review February 28,1977. Accepted April 18, 1977. This work was supported by the National Science Foundation.
Donnan Dialysis Enrichment of Cations James A. Cox" and James E. DiNunzio Department of Chemistry and Biochemistry, Southern Illinois University at Carbondale, Carbondale, Illinois 6290 1
The separation of a sample from a relatively concentrated electrolyte by an ion-exchange membrane results in transfer of ions of the appropriate charge sign from the sample into 1272
ANALYTICAL CHEMISTRY, VOL. 49, NO. 8, JULY 1977
the electrolyte (I, 2). If the volume of the latter solution is smaller, the concentration of the species which originate from the sample may be increased by their transfer into the
electrolyte. This enrichment method, which is termed Donnan dialysis (I),also results in matrix normalization because the electrolyte composition is virtually unchanged as long as the sample is of low ionic strength. The possibility of interference in a subsequent analytical determination is decreased since the membrane tends to exclude high molecular weight neutral compounds and ions of inappropriate charge sign. Analytical determinations in conjunction with preconcentrations by Donnan dialysis have been based upon measurement of the initial rate of transfer of the test species across the ion-exchange membrane and upon the use of a prescribed enrichment time prior to the analytical measurement. Blaedel and Kissel demonstrated that the initial transfer rate into a small volume (about 0.01 mL) of concentrated electrolyte was proportional to the concentration of the test ion in the sample (3). Continuous monitoring of the receiver electrolyte by ion selective electrode potentiometry permitted the rate to be readily measured. Donnan dialysis enrichment for prescribed times was used prior to the voltammetric determination of nitrate ( 4 ) . The quantity of nitrate transferred across the anion-exchange membrane into a 2-mL receiver electrolyte was directly proportional to the initial concentration of nitrate in a sample and the time of contact of the receiver cell with the sample for up to 4 h; thus, definition of a certain enrichment time resulted in linear working curves. The sensitivity was independent of sample size over a wide range. In the present study, a procedure based upon Donnan dialysis for prescribed times was developed for cations. Factors which influence the linearity of the working curves and the precision of the technique were established. EXPERIMENTAL Procedures. The cation-exchange membranes used were type P-1010 and P-4010 (RAI Research Corporation, Hauppauge, Long Island, N.Y.) and Nafion 125 (DuPont). The membrane pretreatment procedure was that described by Blaedel and Kissel (3). The membranes were mounted with equal tension over the 2-cm diameter base of a glass cylinder and attached with O-rings and Teflon tape. From 2.0-5.0 mL of electrolyte were pipetted into the cell. Preconcentration was initiated by placing the membrane face of the cell in contact with a magnetically stirred 200-mL sample. After a prescribed time the cell was removed, and the inner electrolyte was transferred to a sample container for determination by atomic absorption. Apparatus. Controlled stirring was accomplished by continuously monitoring the rotation frequency of a magnetic stirring motor and manually adjusting the unit when necessary. Temperature control was accomplished using a Forma Temp. Jr. Model 2095-2 Refrigerated and Heated Bath and Circulator in conjunction with a jacketed beaker. Unless otherwise stated the temperature was 25 "C. RESULTS AND DISCUSSION Preliminary experiments were performed by preconcentrating a series of Cu(I1) standard solutions in the 10-4-10-6 M range into 0.1 M LiCl. A plot of the Cu concentration in the receiver vs. the original Cu concentration in the sample showed a positive deviation from linearity. The positive deviation from linearity can in part be attributed to the fact that the diffusion coefficient of cations in a cation-exchange membrane increases somewhat with ionic strength (5);however, as shown in Figure 1,the nature of the cations in the sample also influences the transfer rate. In this experiment the initial Cu concentration in the samples was 8.8 x M in all cases, and the ionic strength was varied by addition of MgC12, KC1, A12(S04)3,or Cd(N0J2. The general behavior that the transfer rate of the test species goes through a pronounced maximum as the concentration of a second salt in the sample is varied was also observed when
301 ?
I
P
IO' Figure 1. Effect of various salts on the transfer of Cu(I1) into 0.1 M LiCI. 0, KCI; 0,Cd(N03),; 0,MgCI,; b , A12(S04)3; preconcentration Mx
time, 3 h
cu co
1 0
20
30
cu, v X ' O " Effect of added Mg(I1)on the Cu(I1) working curve. 0, 3.1 M Mg(I1): 0 , 1.3 X M Mg(I1); 0 ,no Mg(I1)added; enrichment time, 30 min; receiver electrolyte, 0.2 M Li2S04;2 X M Co(I1) is used as an internal standard Figure 2.
X
M Mg(I1);D, 1.9 X
potassium was the test ion and either NaCl or MgC1, was added. It is, therefore, unlikely that the behavior in Figure 1was a result of the reported metal ion binding with impurity sites in P-1010 membranes (6). In addition, the Cu determinations after the prescribed enrichment were verified by differential pulse polarography, so the effect is related to the membrane transfer process rather than to the atomic absorption experiment. T o test whether the behavior in Figure 1 was associated with the choice of the membrane, the experiments were repeated with P-4010 and Nafion 125 cation-exchange membranes. The general curve shape was the same. A successful approach to obtaining linear working curves which are not a function of the nature and concentration of other ions in the sample is indicated by Figure 2. In the presence of excess Mg, the working curve becomes linear; however, the slope is a function of Mg concentration. Modification of the enrichment experiment by replacing the 0.2 M Li2S0, receiver electrolyte with 0.2 M MgS04 also results in linear working curves, but now the slope is not a function of added Mg in the sample. Thus the behavior illustrated in Figure 1 can apparently be eliminated by proper selection of the receiver electrolyte. ANALYTICAL CHEMISTRY, VOL. 49, NO. 8, JULY 1977
1273
Table I. Effect of a Cation Added t o the Sample on the Preconcentration of Cu into Various Receiver Electrolytes Enrichment fac-
Receiver 0.2 M MgSO,
Added cation
i'i 10-6 M H+
toru 7.0 7.0 7.2 6.8 7.1
1 x 10-3 M H+ 2X M Ca(I1) 1x M Ca(I1) 6X M Al(II1) 10.1 5 x 10-4 M A ~ ( I I I ) 12.0 0 . 3 M CaCl, ... 5.2 M Mg(I1) 4.1 6x 0.3 M BaCl, ... 4.0 M Mg(I1) 4.6 2x M Al(III), 0.2 M Mg(I1) , . . 10.4 4 x 10-5 M AI(III) 10.3 s x 10-4 M A ~ ( I I I ) 10.4 M Mg(I1) 10.4 1x 0.2 M Li,S04 ... 3.6 M Mg(I1) 6.5 4x
a Receiver Cu concentration after 30 min divided by the original sample concentration, 8.8 x M.
T o test the above hypothesis and to establish guidelines for selection of the receiver electrolyte, the experiments summarized in Table I were performed. Here, various cations of Periodic Groups l A , 2A, and 3A were tested as receivers and as cations added to the Cu samples to be enriched. In the case of Al, a mixture of A1 and Mg was used as high concentrations of the former were subject to hydroxide precipitate formation. Interference with the Cu enrichment generally occurred when the added cation was in a higher group or was of a lower atomic number within the same group than the receiver cation. Within these groups, Al(II1) would therefore be the optimum receiver cation in terms of freedom from interference by cations in the sample. In addition, with the Al(II1) receiver, the presence of Fe(III), Co(II), Ni(II), and Cd(I1) in the sample does not interfere. Comparable studies with Co(I1) and Na as the sample ions yielded the same general results so that the conclusions reached do not apply only to Cu enrichments. Also demonstrated in Table I is that the Al-containing receiver yields the greatest Cu enrichment of the systems tested. The enrichment rate could be further increased by using a higher ionic strength receiver electrolyte as demonstrated by the results in Table 11. Increasing the receiver ionic strength also increases the dilution of the receiver by osmosis. For example, in 1 h the 4 mL of 0.3 M Li2S04is diluted to 4.7 mL. The subsequent quantitative error is eliminated by the use of an internal standard, (or standard addition to the sample can be used) but a partial loss of enrichment will occur. Therefore, the electrolyte used in the further applications was a mixture of 0.2 M MgS04 and 5 X M Alz(S04)3. With high ionic strength solutions on each side, an ionexchange membrane becomes less permselective ( 2 ) . I t therefore follows that with a given receiver electrolyte, there will be an ionic strength above which the transfer rate of a given ion will become a function of sample ionic strength. When A12(S04)3is added to an 8.8 X M Cu(I1) sample, the enrichment factor into the Mg/A1 receiver decreases from 10.4 to 3.5 when the added A1 is increased from 1 X 10-3-2 x 10-3M. When the ionic strength of the sample is varied by addition of NaC1, the enrichment factor is 10 as long as the NaCl concentration is less than 1 X lo-' M, but it decreases to 5 at 2 X lo-' M. In each of the above cases, the ionic strength of the sample is about 0.01 at the point at which it 1274
ANALYTICAL CHEMISTRY, VOL. 49, NO. 8, JULY 1977
Table 11. Effect of Receiver Ionic Strength on the Enrichment of Cu Samples
Receiver 0.1 M 0.2 M 0.3 M 0.1 M 0.2 M 0.3 M
LiCl LiCl LiCl Li,S04 Li,SO, Li,SO,
Ionic strength
Enrichment factora
0.1 0.2 0.3 0.3 0.6 0.9
0.80 2.7 4.3 4.5 8.3 9.4
Receiver Cu concentration after 1 h divided by the original sample concentration, 8.8 X lo-' M; the former concentration is corrected for dilution by osmosis in the high ionic strength samples. begins to influence the transfer rate. Analogous to the previously reported enrichment of nitrate into an anionexchange membrane isolated receiver (4), a t ionic strengths greater than the above limit, the transfer rate of Cu is still directly proportional to the sample concentration; this permits determinations on the basis of the standard addition method. The latter approach is successful as long as the sample ionic strength is less than that of the receiver electrolyte. For example, with 0.1 M and 1 M added NaC1, the enrichment factors into the 0.8 ionic strength receiver are 2 and 0, respectively. For samples with ionic strengths lower than 0.01, a single working curve can be used for quantitative determinations employing Donnan dialysis enrichment. A working curve was obtained by preconcentrating samples in the range 1.4 X 10-7-2.5 X M Cu for 30 min into the Mg/A1 receiver and subsequently determining the Cu concentration in the receiver electrolyte by atomic absorption with a 10-cm air-acetylene flame at 3247.5 A. A linear least squares curve fit with a forced intercept at the origin yielded a slope (enrichment factor) of 9.44 with a standard deviation of 3.8 x lo4. A 5.4 x lo4 M Co(I1) internal standard was used. The sample concentration of Cu which yielded 1% absorption at 3247.5 A was 1.1X M (7 ppb Cu) under these conditions. The previously described working curve utilized an internal standard in order to obtain high precision. The selection of Co(I1) was based upon its having Donnan dialysis behavior similar to that of Cu(I1). This similarity does not account for the linearity of the working curve, however. The primary factors which govern the precision of a Donnan dialysis preconcentration are temperature, membrane area, and stirring rate. The transfer rate across the membrane is directly proportional to temperature over the range 20-50 "C; it increases about 4% per degree in that range when 0.1 M KC1 is the receiver electrolyte and Li is the test cation. The linear relationship, which indicates that the transfer mechanism is not simple diffusion, agrees with the observation of others (7). Increasing the stirring rate from 4 to 7 Hz causes the transfer rate to double. In the absence of an internal standard, the general effect of the above factors is to limit the precision of preconcentration to 8% relative standard deviation based upon a set of 8 identical experiments performed on different days a t ambient temperature, 21-23 "C, with nominal control of stirring. With a thermostated cell and controlled stirring, a 2% relative standard deviation is obtained; without a thermostated cell, but with a stirring control, it is 3%. These values refer to a series of experiments performed with the same cell so that variation in membrane area and thickness is not a factor. If the above enrichment experiments are performed by using eight nominally identical cells in parallel with controlled
temperature and stirring, the relative standard deviation increases to 6%, a value which illustrates the deviation of area and/or thickness of the membranes. By inclusion of the internal standard, the precision is improved to 1.5% relative standard deviation if controlled temperature and stirring are employed and 2.1% if they are not. The fact that the use of the internal standard permits precise use of several cells in parallel without stringent control of temperature and stirring is important. Typical preconcentration times are from 0.5-2 h; therefore, if a batch determination had to be performed by repeated use of the same cell, the technique would not be practical. From the above, it can be concluded that Donnan dialysis provides a precise means of preconcentrating ions. Its primary merits are the freedom from interference by other ions in the sample and potential applicability to all ions of a given charge sign. The major limitations are the influence of ionic strength at values above 0.01 and the possible effect of high concentrations of surfactants. The former would be a problem
if sample digestion were required. The latter was not investigated in this study, but our previous study with anion-exchange membranes, which are considered to be more prone to fouling (8,9),indicated that the transfer rate may not be influenced by their presence ( 4 ) . LITERATURE CITED (1) (2) (3) (4) (5)
(6) (7) (8) (9)
R. M. Wallace, Ind. Eng. Chem., Process Design Dev., 6, 423 (1967). W. J. Blaedel and T. J. Haupert, Anal. Chem., 38, 1305 (1966). W. J. Blaedel and T. R. Kissel, Anal. Chem., 44, 2109 (1972). G. L. Lundquist, G. Washinger, and J. A. Cox, Anal. Chem.,47,319 (1975). T. Ueda, N. Kamo, N. Ishda, and Y. Kobatake, J. fhys. Chem., 76, 2447 (1972). W. J. Blaedel and R. A. Niemann, Anal. Chem., 47, 1455 (1975). H. Kelly, D. Randall, and R. Wallace, Dufont Innovation, 4, 4 (1973). T. R. E. Kressman and F. L. Tye, J. Nectrochem. Soc., 116, 25 (1969). E. W. Lang, E. L. Huffman, and R. E. Lacey, J. Electrochem. SOC.,115, 88c (1968).
RECEIVED for review November 29, 1976. Accepted March 28, 1977. This work was supported in part by the Water Resources Center, University of Illinois, Project A-087-ILL.
Portable Gas Chromatograph for the Acetylene Reduction Assay for Nitrogenase T. Martin Mallard, Clifford S. Mallard, Harry S. Holfeld, and Thomas A. LaRue" National Research Council of Canada, Prairie Regional Laboratory, Saskatoon, Saskatchewan S7N
Crop productivity is often limited by the amount of nitrogen available in the soil. An alternative to fertilizer is the use of legumes which can fix atmospheric nitrogen, particularly if varieties of legumes that fix nitrogen a t higher rates can be developed (I). T o discover these varieties, a convenient method of measuring nitrogen fixing activity is needed. Nitrogenase, the enzyme which reduces nitrogen to ammonia, also reduces acetylene to ethylene. The production of ethylene from acetylene is presumptive evidence for the presence of nitrogenase, and provides an indirect measure of nitrogenase activity. Criswell et al. (2)have reviewed the use and interpretation of this assay. The separation and assay of ethylene are nearly always accomplished by gas chromatography using a flame ionization detector. A color assay based on the selective oxidation of ethylene to formaldehyde has been described (3),but is neither as rapid nor sensitive as the GC assay. Because suitable portable gas chromatographs are usually not available, assay samples from legume test plots must be brought to a laboratory for analysis. There have been very few reports from developing countries on the use of the acetylene reduction system for studying nitrogen fixation, and it is likely that the cost in foreign exchange of obtaining the necessary apparatus limits its use in agricultural research. There is a need for a suitable detecting system which is both inexpensive and portable. The Taguchi Gas Sensor is used in a variety of smoke and gas detectors (4). I t consists of a sintered semiconductor, mainly SnOz, molded around a small filament heater. When the filament is heated in air in the presence of combustible or oxygen-reducing gases, the semiconductor's resistance decreases. The change in sensor conductivity is indicated by an alarm or meter. The sensor responds to hydrogen and combustible organic compounds, but as normally used (4) cannot distinguish between them. We have utilized the Taguchi Gas Sensor as the detector in a gas chromatograph. The design is simple, light, inexpensive, and uses air as a carrier gas. Though other uses should be obvious, the apparatus described here is designed for the separation and estimation of ethylene in the acety-
OW9,Canada
lene-reduction assay for nitrogenase. EXPERIMENTAL Apparatus. The column is 0.03-mm (1/8-inch)0.d. stainless steel tubing, 44 cm long, packed with 22 cm Porapak R and 22 cm Porapak N (Waters Associates, Inc., Milford, Mass.). To make the injector, a Swagelok union tee is slightly machined at the top of one nut acceptor. Two 6-mm silicone rubber septa are held against this by the nut. The other end accepts the column, and the branch accepts the incoming carrier gas (Figure 1). The detector block is machined from brass, and a '/8-inch Swagelok fitting soldered to it (Figure 2). The protective cover is removed from the Taguchi Gas Sensor (Model TGS-812, Figaro Engineering Co., 3-7-3 Higashitoyonaka, Toyonaka City, Osaka 560, Japan; or Southwest Technical Products Corp., 219 W Rhapsody, San Antonio, Texas) and the sensor then pressed into the detector block so that the semiconductor sensor is close to the column exit. The carrier gas flows over the sensor and exits through a hole in the sensor housing. Figure 3 provides the schematic electronic circuit. The change in sensor conductivity is detected by meter deflection. The meter movement is in bridge circuit with two resistors and is balanced in either range with a 50-k linear taper potentiometer. For laboratory use, output to a strip chart recorder or peak area integrator is optional. Procedure. The gas chromatograph is powered by a 12-V power source. The cigarette lighter in an automobile is a convenient source but the polarity of the lighter should be checked first to prevent damage to the instrument. A filtered air supply of about 35 kP should produce a carrier gas flow of about 20 cm3/min. The instrument is turned on after carrier gas is flowing past the sensor. After a 2-3 min warmup, the meter is zeroed at the desired range and a gas sample (0.1-1.0 mL) injected. The operator should note the time using a watch with a second hand. If necessary, the meter needle is re-zeroed just before the ethylene peak is expected to emerge (about 75 s). The maximum needle deflection (peak height) is recorded. The acetylene peak emerges at about 150 s, and the needle can be re-zeroed for another sample approximately 3-5 min after the first injection. The peaks are not symmetrical, and maximum meter deflection is not directly proportional to ethylene concentration. Therefore a calibration is necessary (Figure 4). The sensitivity of the Taguchi Gas Sensor is dependent on the temperature of the semiconductor. The carrier gas removes heat ANALYTICAL CHEMISTRY, VOL. 49, NO. 8, JULY 1977
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