line at a time ( 1 3 ) . Here a measurable readout must be obtained within, say, 30 s after injection, otherwise a reasonable sampling rate of about 90 samples/h cannot be maintained. On the other hand, extremely short, yet precisely reproducible reaction times, which cannot be maintained in air segmented streams, can be usefully employed in enhancing the selectivity by exploiting differences in reaction rates (e.g., inorganic phosphate in serum ( 9 ) ,or albumin by bromcresol green). From the viewpoint of routine determination of many samples, the lack of automated sampling devices is indeed a drawback, yet it is an engineering problem which has a feasible solution ( 2 1 ) ,especially in connection with a rotary valve system. I t is a t present difficult to estimate the future impact of the flow injection technique on practical analyses, although the method already has found its way into routine laboratories in Sweden, Brazil, and Canada. I t is believed, however, that this new approach will confirm the inherent advantages of nonsegmented continuous flow systems and demonstrate their great potential in analytical research and routine work. These systems are simple to construct and operate and readily allow new principles to be employed in chemical analysis. The most recent example of this development is the series of works of‘ Dutt and Mottola (22) where repetitive determinations with reagent regeneration result in a nearly “zero reagent consumption” technique. Also very fast, automated titrations, which are impossible to execute in an air segmented system have recently been developed ( 1 2 ) . It is indeed natural that “the new concepts are replacing the old ones, for [the analytical chemistry, as any other] science is not a museum of finished constructions“ (23). Unsegmented flow systems have been known for many years. What has kept them from coming into general use has been the limitation of our knowledge as to how to employ the dispersion patterns in open narrow tubes for analytical purposes. By combining the feat of sample injection with controlled dispersion and reproducible timing, a new method of continuous flow analysis has been developed. Thus, at last a breakthrough has been made which is not only an alternative to the AutoAnalyzer system but offers new possibilities in fast reaction analyses.
Sir: Scientific comment on the letter from Ruzicka et al. (I) is very difficult, as most of the conclusions in it are drawn from unpublished work that is either in press or in preparation. The promised article on the theory of unsegmented flow should be of particular interest. Readers of this journal were recently given a summary of the theory of band spreading in segmented continuous flow analysis (2),and some references to more detailed theoretical treatments can be found there. In their Letter, Ruzika et al. have set up a straw man for their arguments by making subtle changes in what I wrote in my recent paper ( 3 ) . Compare their statement, “This leads him to the conclusion that the minimum conceivable Re for the unsegmented streams can be as low as 150 ...”, to the actual words used: “The data in Table I suggest that the Reynolds number for a successful unsegmented analytical system can be as low as 150.” My sentence was carefully worded to stay within the bounds of the information that was then available in the literature. I made no reference to minimum conceiuable values of Re. Much depends on what is considered to be a “successful” system. Of the many performance factors that are important in any analytical system, much of the emphasis in the early publications on unsegmented continuous flow has been on high sampling rates. The new data in the Letter from Ruzicka et al. show quite clearly how the maximum sampling rate falls off with decreasing Re. The following values are listed in their
LITERATURE C I T E D M. Margoshes, Anal. Chem., 49, 17 1977). L. J. Skeggs, Am. J . Clln. Pathol., 28, 311 (1957). D. A. Lane and J. A. Sirs, J . Phys. E : Sci. Instrum., 7, 51 (1974). J. Ruzicka and E. H. Hansen, Anal. Chim. Acta. 78, 145 (1975). (5) J. Ruzicka and J. W. B. Stewart, Anal. Chlm. Acta, 79, 79 (1975). (6) J. W. B. Stewart, J. Ruzicka, H. Bergamin Filho, and E. A. Zagatto, Anal. Chim. Acta, 81, 371 (1976). (7) J. Ruzicka, J. W. B. Stewart, and A. E. Zagatto, Anal. Chim. Acta, 81, 387 (1976). ( 8 ) J. W. B. Stewart and J. Ruzicka, Anal. Chim. Acta, 82, 137 (1976). (9) E. H. Hansen and J. Ruzicka, Anal. Chlm. Acta, 87, 353 (1976). (IO) J. Ruzicka, E. H. Hansen, and E. A. Zagatto, Anal. Chim. Acta, 88, 1 (1977). (1 I ) E. H. Hansen, J. Ruzicka,and B. Rietz, Anal. Chim. Acta, 89 241 (1977). (12) J. Ruzicka, E. H. Hansen, and H. Mosbaek, Anal. Chim. Acta. 92, 219 (1977). (13) J. Ruzicka and E. H. Hansen, Anal. Chim. Acta, (in preparation). (14) G. Taylor Proc. R . Soc.,(London), Ser. A , 219, 186 (1953). (15) R. Aris, Proc. R . Soc., (London). Ser. A , 235, 67 (1956). (16) 0. Levenspiel, Ind. Eng. Chem., 50, 343 (1958). (17) G. Taylor, Proc. R . SOC. (London), Ser. A , 225, 473 (1954). (18) G. Taylor, Proc. R . Soc.(London), Ser. A , 223, 446 (1954). (19) Industrial Method No. 94-70W/B, “Orthophosphate in Water and Wastewater“, Revised Jan. 1976, Technicon 1ndusWk.l Systems, Tanytown, N.Y. 10591. (20) Technicon Method No. SE 4-0005 FD4 ‘Thloride”, April 1974, Technicon Instruments Corporation, Tarrytown, N Y. 10591, (21) K.K . Stewart, G. P. Beecher. and P. E. Hare, Anal. Biochem., 70, 167 ( 1976). (22) V. V . S. Eswara Dutt and H. A. Mottoia, Anal. Chem., 49, 776 (1977). (23) J. Bronowski. “The Ascent of Man“, 2nd ed..Little, Brown and Co., Boston, Mass., 1976, p 20. (1) (2) (3) (4)
Jaromir Ruzicka* Elo H a r a l d H a n s e n H a n s Mosbaek Francisco J o s e K r u g ’ Chemistry Department A The Technical University of Denmark Building 207 2800 Lyngby, Denmark ‘Permanent address, CERA. C.P. 96, 13400 Piracicaba, S.P. Brazil
RECEIL-ED for review February 2 2 , 1977. Accepted July 18, 1977. Our gratitude is being expressed to the Danish International Development Agency for providing a scholarship for one of the authors (F.J.K.).
Table I for three unsegmented flow phosphate methods:
Re
Sampling rate (samples/h)
440 360 34
420 225 100
In today’s clinical chemistry, the sampling rate and the sample consumption are both important, the former because of the large work load in many clinical chemistry laboratories and the latter because of the increasing variety and frequency of analyses being done on patient sera. The instrument designer cannot afford to improve one of these parameters at the expense of the other, nor can he afford to ignore many other parameters that characterize a suitable analytical instrument and method. It is therefore instructive to compare the recently described method for determination of chloride and inorganic phosphate in serum by unsegmented continuous flow analysis with the results from an advanced, but well established, instrument for segmented continuous flow analysis, Technicon’s SMAC ( 4 ) . Both analytical systems employ similar reagent compositions. SMAC performs 20 analyses in parallel a t a rate of 150 serum samples per hour. The amount of serum consumed per test is 4.6 p L for chloride and 7.9 pL for inorganic phosphate. In the unsegmented flow method that was A N A L Y T I C A L CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
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developed for serum analysis ( 5 ) ,alternative flow systems require 60-1L samples at a rate of 125 per hour or 30-pL samples at 80 per hour. For phosphate in serum ( 5 ) ,the sample size is 200 pL and the rate is 130 per hour. An alternative method for inorganic phosphate consumes a 100 pL sample a t 50 per hour, and the results from that method are unsatisfactory. In the words of Hansen and Ruzicka (5,p 361), “However, even when the long dialysis unit and pumping speeds as low as 0.8-1 mL min-’ were used with a measuring cell with an optical path length of 20 mm, the highest absorbance signals recorded were only of the order of 0.1 absorbance unit.” Unsegmented flow systems have been known for many years, particularly as reaction systems following ion-exchange chromatography columns. The limitations of unsegmented flow have kept these systems from coming into general use. T h e renewed interest in unsegmented continuous flow, principally by the Copenhagen group, may eventually develop
facts to invalidate the conclusions I drew in my article. I leave it to the readers to decide if the information published to date has had this effect.
LITERATURE CITED
-
J. Ruzicka. E. H. Hansen. H. Mosbaek. and F. J. Krua. Anal. Chem.. 40. preceding paper L R. Snyder, J. Levine, R Stoy, and A. Conetta, Anal. Chem.,48, 942A (19761 - -, M. Margoshes, Anal. Chem., 49, 17 (1977). J Isreeli and W. Smythe, in “Advances in Automated Analysis”, Mediad, Inc.. Tarrytown, N. Y.. 1973, Vol. 1, D 13; also 5 following in the - .DaDers . same volume. E. H. Hansen and J. Ruzicka, Anal. Chim. Acta, 87, 353 (1976). \
Marvin Margoshes Technicon Instruments Corporation, Tarrytown, New York 10591 RECEIVED for review April 12, 1977. Accepted July 18, 1977.
Stabilizing the Manganese Tetramethylenedithiocarbamate/Methyl Isobutyl Ketone Extract Sir: Tweeten and Knoeck ( I ) recently studied the chelation of six trace metals by sodium diethyldithiocarbamate (NaDEDTC) and their extraction into isoamyl alcohol for analysis by atomic absorption spectrophotometry. They obtained poor precision and extraction efficiency and concluded: “Based on these experiments, the method of solvent extraction was considered unacceptable for use for multiple metal analysis of a natural water system. Copper is the only trace metal which can be quantitatively determined by this method.” These poor results are rather surprising since many other investigators have used dithiocarbamate chelating agents quite successfully with a variety of extracting solvents. I have also experienced similar precision problems though when extracting Mn with ammonium tetramethylenedithiocarbamate (ATMDTC, commonly but incorrectly known as ammonium pyrrolidine dithiocarbamate) into methyl isobutyl ketone (MIBK). One advantage of ATMDTC over other dithiocarbamates is its much slower decomposition rate in acidic solution (2-10). There is also some evidence that its extracts may be more stable (10, 11). The reproducibility problem I’ve experienced has been traced to the instability of the extracts. Previous investigators have also reported that the Mn dithiocarbamate extracts exhibit a rapid decrease in absorbance (11, 14-19). Other metal dithiocarbamates are more stable. Brooks et al. (12) reported that iron and nickel complexes were stable for 3 h, lead and zinc for 5 h, and cobalt and copper for 24 h when extracted from seawater with ATMDTC into MIBK. Kremling and Peterson (13),also using the ATMDTC/MIBK system, obtained similar results for iron and copper when extracted from seawater. Shendrikar et al. (14) found the ATMDTC extracts of zinc, cadmium, and lead to be stable for less than 1 day. It is apparent that the finite time stability of the dithiocarbamate extracts is a factor that must be taken into account when designing extraction procedures. For most dithiocarbamate extractable metals, it is inadvisable to use a lengthy extraction procedure (several hours or overnight) because of this instability problem. This may be the reason that Tweeten and Knoeck ( I ) experienced difficulties in their study. I t is unfortunate that the long-term storage of dithiocarbamate extracts is not possible. If they were more stable, 1862
ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
then extractions of water samples could be performed at the sampling sites. This would considerably reduce the volume and weight of samples returned to the laboratory for analysis. There have been few attempts to improve the stability of the dithiocarbamate extracts. Yanagisawa et al. (11)found the Mn-ATMDTC/MIBK extract to be more stable than the Mn-NaDEDTC/MIBK extract. They also observed that the ATMDTC and NaDEDTC extracts of Mn were more stable (90 min) when extracted into alcohol and ester solvents. Tweeten and Knoeck ( I ) noted that metal-DEDTC standards in isoamyl alcohol could be used over a 2-day period with reproducible absorption values. Jenne and Ball (19) increased the stability of the Mn-ATMDTC/MIBK distilled water extract from 6 h to at least 3 days by the addition of 20% (v/v) acetone. Olsen and Sommerfeld (20) circumvented the problem by the lengthy process of evaporating the MIBK from the extract and redissolving the residue in 1:l acet0ne:O.l N HC1. Of the many metals extractable by dithiocarbamates, manganese seems to exhibit the worst stability characteristics. I have briefly investigated the Mn-ATMDTC/MIBK system with the objective of improving the stability of the extracts. Hopefully a method for stabilizing the Mn extracts will also be applicable to other dithiocarbamate extractable metals.
EXPERIMENTAL Apparatus. All analyses were performed with a Jarrell-Ash single beam atomic absorption spectrophotometer equipped with a Perkin-Elmer nebulizer and single-slot burner. Reagents. ATMDTC from Fisher Scientific Company was used throughout, and all other chemicals were reagent grade. All manganese solutions were prepared from a Fisher Scientific Company 1000 ppm manganese atomic absorption standard. Procedure. Sixty mL of distilled water, river water, or seawater were placed in a 100-mL volumetric flask and spiked with 10 pg of Mn. Two drops of bromophenyl blue indicator in 50% ethanol were added and the pH was adjusted to about 3.5 with 0.5 N HC1. Three mL of 5% ATMDTC were then added and the solution was allowed to sit for 2 to 3 min. After the addition of 10 mL of MIBK, the volumetric flasks were shaken vigorously on a horizontal shaker for 3 min. The phases were then allowed to separate and distilled water was added to bring the MIBK into the neck of the flask. The analysis was then done directly on the MIBK extract or on the treated solution as described in the
