Note: In lieu of an abstract, this is the article's first page. Click to increase image ... The first decade of flow injection analysis: from serial a...
0 downloads 0 Views 656KB Size
Anal. Chem. 1983, 55, 497-501 (18) Bender, M. L.; Turnguest, 8. W. J . A m . Chem. SOC. 1957, 7 9 , 1889- 1893. (17) Sllien, L. G.: Martell, A. E. "Stability Constants of Metal-Ion Complexes"; The Chismlcal Society: London 1964. (18) Ackerman, M. J. 6.; Ackerman, J. J. H. J . fhys. Chem. 1980, 8 4 , 3151-3157.


(19) Wang, S.M. Ph.D. Dissertation, Kent State University, Kent, OH, 1981. (20) Jakobsen. H. J.; Ellis, P. D. J . fhys. Chem. 1981, 8 5 , 3367-3369.

for review September 309 lg8'. Resubmitted 23, 1982. Accepted November 8, 1982.

Nonsegmented Rapid-Flow Analysis with UltravioIetNisible Spectrophotometric Determination for Short Sampling Times Peter W. Alexander" and Amlius Thallb Department of Analytical Chemistry, University of New South Wales, P.O. Box 7, Kensington, New South Wales, Australia 2033

The development of a nonsegmented, rapid-flow system wlth short sampling times In shown to allow hlgh-speed sampling with a UV-vislble spectrophotometric detector. For methyl orange as a model color indlcator, a sampling rate of 360 samples h-' is obtained experimentally with a 3-s sampllng time followed by a 7 a flush In a narrow bore (0.5 mm), short pathlength (800 mm) flow. The sampllng is performed manually with a precision of 0.85% relatlve standard devlatlon (RSD) and negligible carry-over, wlth a dwell time of 8 s. Determination of chloride by a fast chemical reactlon Is used as an example to compare rapld-flow analysis wlth flow-injectlon and bubble-gating techniques.

Studies of theoretical factors (1)and sample peak formation ( 2 ) in air-segmented continuous flow analyzers have been reported. It was concluded (2) that operation with short sampling times with sharp sample peak formation is inferior t o steady-state operation, particularly when computer acquisition of the sample data is required. It is clear, however, from the work of Evenson e t al. (2),that increased sampling speed can be achieved bj7 operation of continuous-flow systems under non-steady-state conditions. Increased sampling sipeeds have instead been obtained by bubble-gating techniques (3-5) and by the development of various flow-injection analysis (FIA) systems (6-10). FIA operates under non-steady-state conditions and gives sharp sample peak readout, a ~reviewed i by several authors (6-10). A comparison of the segmented-flow and FIA systems has been described recently ( 5 ) . In this study, we report the results obtained by using a nonsegmented flow analysis system designed to maintain rapid-flow velocity through the system. With the improved hydraulic control and more sensitive UV-vis spectrometric detectors now available, we show that a rapid-flow analysis system can be operated a t very short sampling times to give precise data with no carry-over between samples, without the need for either air-segmented flow or flow injection of the sample. With the short sampling times, sampling rates as high as 360 samples h-' are demonstrated with a peak readout equivalent t o the flow-injection technique b u t without the need for a loop injector. In previous rapid-flow studies (11-14) we showed that both air-segmented and nonsegmented flow can be used to give very rapid sample analysis with ion selective electrodes (12),polarography (11, 13),and plasma emission spectrometry (14) as detectors in flow systems. In this work, however, we have miniaturized the complete flow system with a UV-vis spec-

trometric detector and show that nonsegmented flow can be used for fast sample-throughput with manual sampling provided rapid-flow velocity and short pathlength flow systems are used with fast chemical reactions.

EXPERIMENTAL SECTION Reagents and Solutions. Methyl orange (May & Baker) was used as an acid-base indicator for flow-analysis experiments. A stock solution of methyl orange (50 mg L-') was prepared in distilled water. Sample solutions in the concentration range 1-6 mg L-l were then prepared by serial dilution of the stock solution. The acid reagent was AnalaR hydrochloric acid (Ajax Ltd.) prepared in 0.1 M concentration in aqueous solution. The reagent for chloride determination was prepared according to a published procedure (9) by dissolving and mixing mercuric thiocyanate (0.626g), ferric nitrate (30.3 g), and concentrated nitric acid (4.72g) in water, adding methanol (150 mL), and diluting to a final volume of 1 L. The standard chloride solutions were prepared from a stock solution of sodium chloride containing 1.648 g in 1 L of distilled water. Chloride standards in the range 0.1-10.0 mg L-' were prepared by serial dilution in distilled water. Instrumentation. A Desaga Type 131-900 peristaltic pump with six channels equipped with a speed controller was used with a Pye-Unicam (SP-400)spectrophotometer, fitted with a flowthrough cuvette of 10 mm pathlength and 20 pL volume (Zeiss). The spectrophotometer was coupled to a Linear strip chart recorder. All pump tubing and transmission tubing was of PVC (Elkay). Procedure. Sample solutions of methyl orange were pumped into the acid reagent stream, as shown in Figure 1A. The tubing for the sample line was 0.5 mm internal diameter and the mixing point was a zero dead-volume T-piece constructed from stainless steel with 0.5 mm bore. After mixing of the sample and reagent, the solution was pumped through uncoiled tubing (0.5 mm bore) to the optical flow cuvette in the spectrophotometer set at 510 nm and then to waste. A debubbler constructed of glass with 1.0 mm bore was inserted in the flow path just prior to the cuvette, in order to remove intersample bubbles. The line lengths of each flow segment are shown in Figure lA, giving a total flow path of 80 cm for the sample solution. The experimental procedure consisted of initially pumping the flush solution (water) through the system continuously until a steady base line absorbance reading was established on the chart recorder. Samples were then aspirated into the system by manual sampling with timing measured using a stopclock, followed by a flush solution also timed accurately between each sample. The absorbance readout was continuously recorded at the fixed wavelength of 510 nm. The only air bubbles introduced into the system occurred between each sample and the following flush solution. The bubbles were removed by use of the debubbler. Flow rates were controlled by use of the pump speed controller, using fixed-bore pump tubes of 0.63 mm for the sample line and 1.02 mm for the reagent for all experiments. The maximum flow rates possible using the pump tubes in this system are shown in

0003-2700/83/0355-0497$01.50/00 1983 American Chemical Societv



' min _i


I 1 u

Figure 1. Schematic diagrams of the continuous-flow system showing flow rates (mL min-') for sample and reagent, and line lengths for each flow segment: R, reagent; S,sample; F, flush; D, debubbler (0.2 mL min-'); W, waste; DL, diluent; C, mixing coil (50 cm); (A) manifold for methyl orange; (B) manifold for chloride determination.

Figure 1A. With the debubbler operating at 0.2 mL min-', the total flow rate through the optical cuvette was 4.2 mL min-' at maximum pump speed. Flow rates were varied in the range 1.C-4.2 mL min-l through the cuvette. The procedure for chloride determination was the same as above except that the manifold was changed as shown in Figure 1B. The reagent used was mercuric thiocyanate mixed with ferric nitrate in a single solution,as described above. The diluent, water, was pumped into the reagent stream in order to dilute the reagent by a factor of 1.2/3.0. A mixing coil of 50 cm length and 0.5 mm bore was inserted in the flow path to facilitate mixing of the chloride sample with the reagent. Sampling was again performed manually with accurate timing by using in this case 2 s for the sampling and 8 s for the flushing solution.

RESULTS AND DISCUSSION As an example of fast color-forming reaction in a flowing stream, methyl orange was used as an acid-base indicator. The dye was prepared in aqueous sample soutions in the concentration range, 1.0-6.0mg L-l, and aspirated into an acid reagent stream (0.1 M HCl), giving a color change from yellow to pink. Absorbance of the solution was monitored a t 510 nm. Flow System. The flow system was designed on the basis of the principles (6-10) required to minimize dispersion of the sample in the flowing stream, i.e., short reaction pathlength (80 cm) and narrow bore flow tubing (0.5 mm). The system was basically a nonsegmented flow system, as shown in Figure 1,where each methyl orange sample was aspirated into the acid reagent stream for 3 s, followed by a flush solution for 7 s, using a Teflon sampling probe 22 cm in length. However, the introduction of an air bubble between each sample and wash solution was unavoidable with this sampling technique, and hence a debubbler was required just prior to the flow cell. Since only two bubbles were introduced for each sample, the debubbler flow rate could be kept very low (0.2 mL min-l), so that very little solution is lost through the debubbler (-5% of the total flow). The disadvantage of this approach is that the inclusion of a debubbler has the effect of increasing the dispersion in comparison to a completely nonsegmented flow system. Despite this problem, however, very fast sampling rates were achieved. Response Curves. The absorbance changes recorded after the indicator samples were aspirated into the reagent stream are shown in Figure 2. The samples were aspirated for 3 s in triplicate with a water flush for 7 s between each sample. The absorbance readings in Figure 2 indicated a sharp peak for each sample with significant tailing almost back to the base line absorbance between samples with the above timing sequence, equivalent to a sampling rate of 360 samples h-l. The readout in fact is exactly the same shape as observed in previously reported flow-injection studies (6-10). The shape of the readout can be attributed to the very short sampling time used. The steady-state recording for the dye



II I 'L i Flgure 2. Absorbance changes recorded for methyl orange samples (concentrations shown) aspirated into the flow system with 3 s sampling and 7 s flush. Steady-state response (SS) is shown for the sample at 6 mg L-'.




Flgure 3. Carry-over (a) and precision (b) of replicate determinations

of methyl orange (concentrations shown) with a 3 s sampling time and 7 s wash.

sample with 6.0 mg L-' concentration is shown in Figure 2 for comparison with the fast sampling results. I t is obvious that the sharp sample peaks observed are approximately 90% of the steady-state absorbance value. A sampling time of 6.6 s was required to reach 98% of the steady-state absorbance. During the wash period, the sample tailed into the wash due to dispersion and gave a response curve the inverse of the rise curve. Precision and Carry-Over. The effects of sample carry-over and sampling time on precision and accuracy are shown in Figure 3. The peak height reproducibility depends on several experimental factors but the most important is the sampling time. With the 3-9 manual sampling used here, a precision of 0.85% RSD for peak height replicate measurements from Figure 3 was found for n = 6 determinations. The carry-over or cross-contamination between consecutive samples is determined by the wash time. Sufficient wash time between samples must be allowed for the dispersed tail to approach the base line closely, as required in FIA. The results observed here, as given in Figure 3, showed that carry-over from a high to low sample was negligible for a wash time of






TIM? 151

Figure 4. Steady-stateabsorbance peaks plotted at various flow rates: (a) 4.2, (b) 3.2, (c) 2.2, (d) 1.2 mL min-', for methyl orange (6.0 rng L-9.


Table 11. Effect of Flow Rate on Sample Peak Widths at a Fixed Sampling Time of 2 s

Table I. Effect of Flow Rate on Steady-State Peak Widths flow rate, mL min-l

rise timea

fall timea

4.2 3.2 2.8 2.3 2.2 1.2 1.0

6.6 8 .O 9.8 11.1 10.7 13.3 17.8

7.9 9.4 11.0 13.0 15.0 25 28.5





peak width,

Figure 5. Effect of flow rate on sample peaks with 2 s sampling time and the followlng flow rates: (a) 4.2, (b) 3.2, (c) 2.8, (d) 2.3, (e) 1.9, (f) 1.4 mL min-', for methyl orange (6.0 mg L-I).


calcd sampling rate, h-l

14.5 17.4 20.8 24.1 25.7 38.3 46.3

248 207 173 149 140 94 78

flow rate, mL min-' 4.2 3.2 2.8 2.3 1.9 1.4

sampling peak rate, dwell sample reagent width samples time, v01,~ vol? ( A t 9 8 ,s ) h-' s PL PL 7.3 8.8 10.0 11.2 13.6 16.0

493 409 360 321 265 225

8 9 10 11 13 17

40 32 26 22 18 14

390 374 351 328 326 299

a Times to reach 98%(of the steady-state and base line absorbance values,

a Volumes required for each analysis calculated from the flow rates and sampling rates.

7 s. The total sampling/wash cycle of 10 s therefore allowed an overall sampling ratle of 360 samples h-l. The precision of this method is dependent on the reproducibility of the residence of the sample in the system from the time of aspiration to peak maximum. The method precision therefore depends on maintaining a constant flow rate and the accurate timing of sampling and flush. In contrast, a valve switching procedure is commonly used in FIA (19) without the need for the accurate timing necessary in this rapid-flow procedure, but still requiring a precisely controlled flow rate. The precision of 0.85% RSD obtained reflects the reproducibility of the residence time and compares favorably with FIA precision (9). Effect of Flow Rate on Response Times at Steady State. The flow rate of the solution stream through the optical cuvette was found to harve a marked effect on the time required for the absorbance to reach steady state. Figure 4 shows the absorbance plotted against time during continuous sampling taken from fast recordings of the absorbance changes approaching the steady-stata value. With the bore and pathlength fixed, increased flow rate and hence flow velocity through the cuvette c a u ~ e dincreased speed of approach to the steady-state absorbance up to 4.2 mL min-l. The results from Figure 4 are summarized in Table I showing the peak widthti for base line resolution a t various flow rates, together with the calculated change in sampling rate. The peak widths (Atb) were found to be inversely related to the flow rate ( q ) , and SI log-log plot was found to be linear with a slope of -0.85 and intercept of 1.74 at log q = 0 with a correlation coefficient of 0.9950. Effect of Flow Rate on Response at Short Sampling Times. Sample peaks were recorded at various flow rates after sampling for a short time, such that the steady-state absor-

bance value was not reached. Figure 5 shows the effect of peak broadening and increased dispersion at the slow flow rates, together with the increased dwell time in the system. The resulting peak widths for base line resolution (98%) are summarized in Table 11,together with the effect on calculated sampling rate. The results in Table I1 were obtained in such a way that the sample volume changed with flow rate when the sampling time was kept constant at 2 s. The peak width was therefore related to both flow rate and sampling volume aspirated into the reagent stream, unlike the results a t steady state where sampling volume was constant. It is clear, however, from Figure 5 that the effect of changing sample flow rate is very similar to the effect of increased pathlength or dwell time in a flow-injection system described by Ruzicka and Hansen (9). Increased dispersion occurs due to the increased dwell time and smaller volumes taken, and hence sampling rate is slowed down at the slower flow rates in our system. The irregularity of the response curves at low pumping rates is clearly shown, but by use of rapid flow, dispersion is limited, and improved sensitivity and reproducibility of the response curve are obtained. Sample and Reagent Consumption. The maximum pump rates for aspiration of the sample, flush, and reagent into the flow system are given in Figure 1. Calculations based on the pumping rates and sampling and wash times were used to obtain the sample and acid reagent volumes consumed for each sample analyzed. The results for a 3 s/7 s sample-flush cycle show low consumption of sample (60 pL) and reagent (507 pL) for each sample test. The overall dwell time of the sample in the system was measured to be 8 s for a total pathlength of 80 cm, allowing almost immediate readout of results.




10 0

1c.o Cl-ImgC'l

20 2 0


Flgure 6. Effect of sampling time on sample peaks at a fixed flow rate of 4.2 mL min-': (a) 0.5, (b) 1.0, (c) 2.0, (d) 4.0 s, for a methyl orange sample at 6.0 mg L-' concentration.

Table 111. Effect of Sampling Time on Peak Formation at a Fixed Flow Rate samppeak ling samp- widtha rate, ling (at,, , samples time, s s) h-' 0.5

1.o 2 .o 3.0

4.0 5.0

4.8 5.9 7.5 9.5 11.2 12.9

750 610 480 379 321 279

%SS 32.5

49.4 15.3 90.6 94.8 96.0

w Flgure 7. Determinaiion of chloride at a sampling rate of 360 samples h-' with the flow manifold shown in Figure lB, using manual sampling with 2 s sampling time and 8 s flush time.

Table IV. Comparison of Performance of Various Flow-Analysis Methods for Determination of Chloride

sample reagent vol,b vol,b pL pL 10

sampling rate, samples h" sample volume, p L reagent volume, pL dwell time, s ref

256 315

20 40 60 80 100

rapid flow

400 507 597 688

Baseline-to-baseline peak width at a fixed flow rate of Calculated from sampling rates and flow rates.

flow injec- bubble tion gating




40 200

30 370


8 9

4.8 272 500 5, 17

this work


4.2 mL min-I through the optical cuvette.




Table I1 shows further calculations for 2 s samplings, based on the results in Figure 5, for the changes obtained in sample and reagent consumption, dwell time, and sampling rate after decreasing the overall flow rate. The sampling rate was again clearly improved by increasing the flow rate, but at the expense of higher sample and reagent consumption. Effect of Sampling Time. In addition to the improved sampling rate obtained by increasing the rate of flow of sample transport through the system, it is also obvious from Figure 4 that sampling rate can be improved by decreasing the sampling times, so that the absorbance never reaches the steady-state value. The effect of decreasing sampling time was experimentally measured and the results are shown in Figure 6. The peak height decreased at very short sampling times down to 0.5 s, and the peak width also decreased. It can be concluded therefore that the combination of rapid flow with short sampling times allows rapid sampling rates to be obtained. The peak-width data are summarized in Table 111,together with percent steady state obtained at each sample time and the sample volume used. With the flow rates given in Figure 1, the sampling rates, sample size, and reagent consumption can be calculated for each sampling time, as shown in Table 111. The loss in sensitivity at short sampling times is much the same as occurs in FIA when small sample volumes are injected. But the sampling speed is greatly improved. From Figure 6 and Table 111, speeds up to 750 samples h-l with 10 pL sample size are predicted. This result agrees fairly well with predictions described by Snyder (5) on the basis of dispersion theory in air-segmented systems, while Tijssen (15)has pre-

dicted 900 samples h-l in nonsegmented flow with 0.2 mm bore tubing. Determination of Chloride. As an example of a fast chemical reaction in the flowing stream, chloride samples were analyzed by the rapid-flow method using the reaction of chloride with mercuric thiocyanate and ferric nitrate (16). The flow manifold shown in Figure 1B was used for the chloride method, where the total reagent flow rate for diluent and reagent (3.0 mL mi&) and the sample line flow rate (1.2 mL mi&) were as close as possible to the methyl orange study. For sampling and wash times of 2 s and 8 s, respectively, and after mixing in a 50-cm coil, sharp sample peaks were again obtained, as shown in Figure 7 . For chloride concentrations in the range 2-10 mg L-I, peak heights increased linearly with concentration, and the peak width time was the same as observed for methyl orange (Figure 2). Although the dwell time of 10 s was slightly longer because of the 50-cm coil in the system, precision of 0.7% RSD was obtained for 10 replicates of the chloride sample at 10 mg L-I. The detection limit for chloride determination was found to be 0.017 mg L-l, obtained after scale expansion of the chart recorder by a factor of 10. In any flow system such as this one, the detection limit depends on a number of factors including the molar absorptivity of the colored reaction product, the noise level of the base line on maximum scale expansion, and the extent of dispersion of the sample in the stream. The detection limit found here was calculated from twice the base line noise level (0.000 24 absorbance units), in agreement with the reported values (16) of 0.015 to 0.055 mg L-l obtained by conventional manual procedures. Comparison w i t h O t h e r Flow Methods. The results given here for determination of chloride allow a direct comparison with FIG and bubble-gating methods, as summarized


in Table IV. The FIA method for chloride reported by Ruzicka and Hansen operates with a low flow rate of 0.8 mL min-I, but at a low sampling rate of 130 samples h-I, while the bubble-gating technique (5,17) uses still lower flow rates in the range 0.1-0.5 mL m i d at a sampling rate of 150 samples h-l. Table IV shows the sampling rates, the required sample and reagent consumption per test, and the sample dwell time in each system. Although the bubble-gating method has the advantage of very low sample consumption, the rapid flow method shows faster response with lower reagent consumption and a shorter dwell time in the system. A more recent development (18) in bubble gating has given improved sampling rates of up to 360 samples h-l for nitrite determinations after miniaturization of the system. The possibility of using short sampling times to further improve the bubble-gating sampling rate is obvious when the present results are compared to the bubble-gated results of Patton et al. (18),but again with the requirement of more complex detector design than used in this study. The approach to sampling taken in this study therefore offers an alternative to FIA and bubble gating for fast sampling in a continuous-flow system. Because dispersion is the critical factor which limits sampling rate, it is of interest to compare the response patterns observed in this study of the sample aspiration technique to the theoretically expected response for dispersion in a nonsegmented stream. Dispersion Characteristics. Despite the fact that two air bubbles occur in this system, dispersion takes place from the sample into the waqh preceding and following each sample. The system differs from FIA because of the two bubbles, but in practice, we found that the flow results conform to the diffusion-convection equation described by Vanderslice et al. (19).

The dependence of peak widths (A&, s) on experimental parameters was shown to be (19)


Atb = 35.4a2f

9)OA4 L


where q is the flow rate (mL min-I), D is the diffusion coefficient (cm2s-l), a is the tube radius (cm), L is the pathlength (cm), and f is a constant dependent on the sensitivity of the detector instrumentation. Hence, the time dispersion is proportional to the square of the radius and to (L/q)0,64. In comparison, the bore (0.5 mm) and pathlength (80 cm) in our system were as low as practicable in order to interconnect the instrument modules and without causing excessive pressure build up, and the maximum flow rate obtainable was 4.4 mL min-'. From the results given in Tables I and 11, the peak widths measured to give 98% base line resolution were found to give a linear log-log relationship with flow rate, and the slopes were not significantly different for the two cases of steady-state peaks and the short sampling peaks in Tables I and 11. The slope found to be -0.85 indicates that the peak width is proportional to q-a,85 for the constant bore and pathlength used in this flow system. The flow rate exponent is therefore larger than found by Vanderslice et al. (19),as predicted by solution of the convection-diffusion equation. This difference can be attributed to the deformations caused by the method of sample introduction, the use of debubbler,


and the variations in bore through the pump, transmission tubing, debubbler, and flow cuvette. However, the change in value of the exponent when compared to the ideal case of bolus injection into a nonsegmented carrier stream is small enough to be insignificant in terms of an advantage in sampling speed and sample and reagent consumption. Automatic Sampling. Our results in this study therefore indicate that basically there is no difference between the air-segmented and flow-injection techniques when rapid flow velocity is maintained. The method of sample aspiration used in the least complex method for automated sampling, and can be used to advantage, in principle, to obtain extremely high sampling rates in flow analysis. In practice, the sampling rate of 360 samples h-l achieved with the experimental system given here is in reasonable agreement with the calculated rate in Table 111, but sampling rate is experimentally limited by the timing possible with the manual sampling/flush procedure employed. Obviously, to maintain the required manual short timing for analysis of many samples is not possible in practice. However, the advantage of the short timing technique is the considerably simplified approach to automated sampling over the loop injection (9,lO)and bubble-gating techniques ( 3 , 4 ) . Automatic samplers with the timing sequence established in this study seem to be practicably feasible, for example, with a lower limit of 1 s for sampling and wash times. Hence, it is reasonable to conclude the procedure described here could be easily automated without the added complexity of the above techniques, provided that short pathlength systems with rapid-flow velocity can be utilized. Many applications of RFA may be possible, similar to those developed for the FIA technique (6-10).

LITERATURE CITED Thiers, R. E.; Cole, R. R.; Kirsch, W. J. Clln. Chem. (Winston-Salem, N . C . ) 1987. 73. 451-467. Evenson, M: A.; Hicks, G. P.;Thiers, R. E. Clin. Chem. (Wlnston-Sa/em, N . C . ) 1970, 76,606-611. Habig, R. L.; Schlein, 8. W.; Waiters, L.; Thiers, R. E. Clin. Chem. (Wlnston-Salem, N . C . ) 1989, 75, 1045-1055. Neeley, W. E.; Wardlaw, S. C.; Sing, H. C. Clln. Chem. (Wlnston-Sa/em, N . C . ) 1974, 20,424-427. Snyder, L. R. Anal. Chlm. Acta 1980, 7 1 4 , 3-18. Betteridge, D. Anal. Chem. 1978, 50, 832A-846A. Ranger, C. 6. Anal. Chem. 1981, 53, 20A-32A. Mattola, H. A. Anal. Chem. 1981, 53, 1312A-1316A. Ruricka, J.; Hansen, E. H. "Flow-Injection Analysis"; Wiley: New York, 1981. Stewart, K. K. Talanta 1981, 28, 789-797. Alexander, P. W.; Shah, M. H. Talanta 1979, 26, 97-102. Alexander, P. W.; Seegopaul, P. Anal. Chem. 1980, 52, 2403-2406. Alexander, P. W.; Marpaung, H. Talanta 1982, 29, 213-217. Alexander, P. W.; Finlayson, R. J.; Smythe, L. E., Thaiib, A. Analyst (London) 1982, 107, 1335-1342. Tijssen, R. Anal. Chlm. Acta 1980, 7 7 4 , 7 1-89. Florence, T. M.; Farrar, Y. J. Anal. Chlm. Acta 1971, 54, 373-377. Morgenstern, S.;Rush, R.; Lehman, D. I n "Advances in Automated Analysis, Technicon International Congress 1972"; Mediad Inc.: Tarrytown, NY, 1973; pp 27-31. Patton, C. J.; Rabb, M.; Crouch, S. R. Anal. Chem. 1982, 54, 1113-1118. Vanderslice, J. T.; Stewart, K. K.; Rosenfeld. A. G.; Higgs, D. J. Talanta 1981, 28, 11-18.

RECEIVED for review August 23,1982. Accepted November 23,1982. We gratefully acknowledge the award of a research fellowship to A.T. by the Australian Development Assistance Bureau.