Then give to Caesar what is Caesar's, but give to God what is God's. Luke 20:22
Continuous Mùller's
sis for the segmented version of the technique, and flow injection analysis for the unsegmented one. The term analysis refers to operations on the material or, as proposed recently, the system under study (7). As such, it involves steps ranging from the planning of strategy to attacking a given analytical problem, to reporting and helping with the interpretation of final results. Considering that most continuous flow systems lack several of those steps, the analysis in the title is open to criticism. It should be recognized that what we are actually dealing with are sample processing systems that may incorporate more than one (never all) steps of analysis.
"Komplementàr Kolorimeter"
Continuous-flow procedures constitute one analytical answer to the increasing load being imposed on the practicing chemical analyst. The answer applies mainly to the problems posed when a large number of samples of similar nature have to be processed for the determination of a single species; such problems are common to clinical chemistry, environmental studies and control, and industrial processes and quality control. Continuous-flow procedures meet the need, in those cases, for shorter times and decreased operating costs per determination. By their use:
• a large number of samples can be processed with acceptable (many times highly competitive) precision and accuracy; • a meaningful statistical treatment of the data is possible; • human participation can be eliminated in many routine manipulations; and • better utilization of reagents is possible. The term analysis is frequently misunderstood in the vocabulary of analytical chemists. Its use in the title of this REPORT is a reluctant recognition of usage: continuous flow analy-
Figure 1 . Mùller's "Komplementàr Kolorimeter" (a) Glass cylinder in which the solution to be examined was placed; (b) concentric tube capable of vertical movement; (c) millimeter scale; (d) glass filter of color complementary to that of the solution. Observation was made from above against white light reflected by the adjustable mirror (e). If the inner tube was raised, the color of the solution predominated. If it was lowered to virtual contact at point (f), the color of the filter would be seen. A balance would be obtained at a certain intermediate point in which an approximately white color would be observed. Readout was obtained from scale (c) and determination based on comparison with standards. Reproduced from Reference 2 with permission 1312 A · ANALYTICAL CHEMISTRY, VOL. 53, NO. 12, OCTOBER 1981
Continuous-flow procedures often provide advantageous alternatives to wet chemical methods. The practice of wet chemical analysis has undergone several changes since the times when classical gravimetric and titrimetric methods monopolized the arsenal of practicing analytical chemists. Colorimetric instrumentation can be traced back to Mùller's "Komplementàr Kolorimeter" (Figure 1), and to Vierodt's description of an apparatus for obtaining absorption spectra for quantitative determinations (2). These contributions date back to 1853 and 1873, respectively. The introduction of the Beckman Model DU quartz photoelectric spectrometer, in the early 1940s, represented a quantum jump in instrumental development. About 15 years later Skeggs was responsible for another drastic change in the approach by which colorimetric determinations, and by extension practically all instrumental variations of wet chemical analysis, were performed. Skeggs's radical contribution was to introduce dynamic measurements instead of the classical "static" measurements using a cell or cuvette (3). The statement, "Scientific research consists in seeing what everyone else has seen, but thinking what no one else has thought" is attributed to Albert Szent-Gyorgyi, the Hungari0003-2700/81 /A351-1312$01.00/0 © 1981 American Chemical Society
Report Horacio A. Mottola Department of Chemistry Oklahoma State University Stillwater, Okla. 74078
Flow Analyses Revisited an biochemist who received the Nobel Prize for physiology and medicine in 1937; he was the first to isolate and characterize vitamin C as ascorbic acid. This statement seems to apply to every corner that we turn in tracing the development of analytical contin uous flow procedures. The elements of Skeggs's modular, continuous-flow concept that resulted in the workhorse of practically every clinical laboratory and most industrial analytical facili ties, the AutoAnalyzer developed and marketed by Technicon, existed in process control (4) before Skeggs adapted them to the analytical labora tory. An example of this is the Beckman Model 77 continuous-flow color imeter, recommended for monitoring the color of water, beverages, liquid chemicals, suspended solids (turbidimetry), and chlorine in water (4). Skeggs thought of what nobody else thought before. He proposed a novel manner of sample-reagent mixing and transport to detection, and empha sized the utility inherent in all modu lar set-ups.
rv I*
p^i
^i
Figure 2. Experimental set-up of Pungor et al. for continuous-flow analyses with graphite electrodes Ci and C2 = stopcocks; Κ = stirrer; Έ.Λ = indicating electrode; E2 = reference electrode; I = injection point. Reproduced from Pungor et al. (7) with permission
Argon Tank •*
His first full-length paper, however, established what became a condition, almost unchallenged for years, for the development of continuous-flow ana lyzers: the need for air segmentation. Demonstration of the fact that such procedures do not necessarily need air segmentation constitutes the latest landmark in the sequence of develop ments that led to wet chemical analy sis as practiced today. Although ana lytical chemists have been injecting samples into unsegmented flows (chromatography being a typical ex ample) for a long time, the real impact of unsegmented continuous-flow pro cedures was first felt in the middle 1970s. Szent-Gyorgyi's statement on scientific research again comes to The material covered in this REPORT was pre sented as part of the opening remarks at the sym posium "Flow Injection and Other Unsegmented Continuous Flow Sample Processing Systems," 182nd National Meeting of the American Chemi cal Society, New York, N.Y., Aug. 24, 1981.
Figure 3. Flow system used by Frantz and Hare for silica determination (a) Reagent reservoir; (b) 32-gauge Teflon tubing; (c) flow meter; (d) three-way valves; (e) sample injec tor; (f) micropipette; (g) 40-ft coil of Teflon tubing; (h) 6-mm colorimetric cell; (i) 60-mm colorimetric cell. Reproduced from Reference 8 with permission ANALYTICAL CHEMISTRY, VOL. 53, NO. 12, OCTOBER 1981 · 1313 A
mind when the present development of unsegmented continuous flow procedures is traced back to the papers of Ruzicka and Hansen (5) and Stewart, Beecher, and Hare (6). Ruzicka and Hansen as well as Stewart et al. did see what others missed: the direct challenge to the segmentation idea, the practical proof that unsegmented continuous flow under controlled dispersion is not only a viable alternative to the continuous air-segmented stream, but that it also offers distinctive advantages: faster determinations and less consumption of reagents. The prolific work of Ruzicka and Hansen's group in Denmark is primarily responsible for providing the impetus for the recognition and acceptance of unsegmented continuous-flow procedures by practicing analytical chemists and for stimulating interest in the theory and application of sample injection techniques into unsegmented flows. But one cannot ignore other pioneering work in unsegmented continuous-flow systems, such as that of Pungor et al. in Hungary (Figure 2) (7). This work in the late sixties and early seventies focused on evaluating the performance of electrochemical sensors in unsegmented continuous flow. The work of Frantz and Hare (8) describing the determination of silica to support experimental studies of hydrothermal mineral-solution equilib-
Probe
Figure 4. Closed-loop system for glucose determination by sensing dissolved oxygen electrochemically after the enzyme-catalyzed (glucose oxidase) oxidation of glucose The reaction occurs in the column labeled "Tragerfixierte Enzyme" containing glucose oxidase immobilized on a water-insoluble carrier. Reproduced from Bergmeyer and Hagen ( 10) with permission
Figure 5. Flow-through cell and flow-through loop for repetitive determinations by injecting the sample directly into the detection zone (a) Hypodermic syringe and sample injection port; (b) magnetic stirring bars; (c) peristaltic pump; (d) reagent solution reservoir. Reproduced from Eswara Dutt and Mottola ( 11) with permission
ria (Figure 3) also needs to be mentioned. This paper is a sequel to an earlier note by Hare (9) describing essentially the flow colorimeter of Figure 3 later used by Frantz and Hare (8). About the same time, Bergmeyer and Hagen (10) reported an enzymatic determination with a set-up including sample injection in a recirculating unsegmented flow system (Figure 4). Somewhat later, and a few months before the paper by Ruzicka and Hansen, we also described the use of an unsegmented-flow closed system with sample injection directly into the detection zone (Figure 5) (11). We can even set the clock 10 years earlier and find in a paper by Blaedel and Hicks (12) all the basic elements of determinations employing unsegmented continuous flow (Figure 6). A scrupulous literature search may very well find the elements of unsegmented continuous flow systems in even earlier contributions. All these contributions directly or indirectly tried to provide alternatives to achieve the goals for automatic wet chemical analysis already cited: more determinations per unit of time and less expensive determinations. Reaching this point we are reminded of the quotation, "What has been, that will be; what has been done, that will be done. Nothing is new under the sun" (Eccles. 1:9). Ruzicka and Hansen, however, must also be credited with finding a name for the new-born variety of determinations using unsegmented continuous flow. The approach that different authors employ varies somewhat but the term "flow injection analysis" coined by Ruzicka and Hansen (5) (Figure 7) is increasingly being used to encompass all these variations. Their latest contribution is the first monograph on
1314 A · ANALYTICAL CHEMISTRY, VOL. 53, NO. 12, OCTOBER 1981
flow injection procedures (13). A common characteristic of the procedures is the introduction of the sample by a discrete injection (better described as "intercalation" when a sliding valve is used) into a continuously flowing, unsegmented stream. The carrier stream transports the sample "plug" to the detection area in which the analytical signal is acquired. During sample transport, chemical reaction between the species to be determined and chemical components of the carrier stream provides additional interesting possibilities. For instance, one can cite the exploitation of hydrogen ion gradients for the potential simultaneous determinations of two or more species in a single sample plug (14). One example of an application without chemical reaction is the monitoring of fluoride ions of Slanina et al. (15). In any case, detection occurs in the unsegmented continuous-flow stream while the system is attaining equilibrium by a physical or a chemical process or both; thus, the procedures are kineticbased. Applications in continuous-flow systems have been primarily in wet chemistry. Recently, however, the possibility of adapting the basic concepts to gaseous solutions has opened new avenues (16) for this already widely accepted approach to repetitive determinations. Our own research guided by a better understanding of critical design has allowed us to demonstrate the possibility of determining gaseous species such as SO2 by reaction at a gas-liquid interface and monitoring of a color developed in the solution at the tailing part of the air sample plug. Judicious selection of tube diameter, sample volume, coil length and diameter (residence time), reactor material,
Figure 7. Typical flow diagram illustrating Ruzicka and Hansen's "flow injection analysis" The illustrative diagram is for the determination of phosphate and is reproduced from J. RUzicka and J. W. B. Stewart, Anal. Chim. Acta, 1975, 79, 79-91, with permission
Figure 6. Schematic outline of instrumental set-up for continuous measurement of reaction rates Absorbance of the reacting mixture is first recorded at the upstream cell and later, after a fixed time delay, again at the downstream cell. The ratio of absorbances in the cells and the travel time between those two cells provide the data for rate calculation. Reproduced from Blaedel and Hicks ( 12) with permission
and design of a special debubbler made this possible (17). There should be no doubt at this point that unsegmented continuousflow systems invite, more than many other approaches, the ingenuity of researchers: The single bead string reactor of van der Linden et al. (18) is a good example. More contributions displaying inventiveness may be expected in the near future as more and more chemists become aware of the potential of the approach. The versatility of unsegmented continuous flow is now well documented; still to come is a comprehensive description of the chemical and physical phenomena controlling "plug" dispersion, with emphasis on two parameters of analytical relevance: peak height (which dictates sensitivity) and time for return to baseline (which dictates determination rates). New and interesting approaches in this direction are starting to be documented (19-25). The recognition that chemical reactions may play an important role in the shape of signal profiles may lead to other important fundamental developments in the future. References (1) Pardue, H. L. "Abstracts of Papers," 182nd National Meeting of the American Chemical Society, New York, N.Y., August 1981; American Chemical Society: Washington, D.C., 1981; ANAL 2. (2) Szabadvâry, F. "History of Analytical
Chemistry"; Pergamon Press: Oxford, U.K., 1966; pp 337-43. (3) Skeggs, L. T., Jr. Am. J. Pathol. 1957, 28, 311-22. (4) Siggia, S. "Continuous Analysis of Chemical Process Systems"; John Wiley and Sons, Inc.: New York, 1959. (5) Ruzicka, J.; Hansen, Ε. Η. Anal. Chim. Acta 1975, 78,145-57. (6) Stewart, K. K.; Beecher, G. R.; Hare, P. E. Anal. Biochem. 1976, 70,167-73. (7) Pungor, E.; Fehér, Zs.; Nagy, G. Anal. Chim. Acta 1970,52, 47-54, and references therein. (8) Frantz, J. D.; Hare, P. E. "Carnegie Institution of Washington Year Book 72"; pp 700-6. (9) Hare, P. E. "Carnegie Institution of Washington Year Book 70"; pp 268-9. (10) Bergmeyer, H. U.; Hagen, A. Fresenius Z. Anal. Chem. 1972, 261, 333-6. (11) Eswara Dutt, V. V. S.; Mottola, H. A. Anal. Chem. 1975, 47, 357-9. (12) Blaedel, W. J.; Hicks, G. P. Anal. Chem. 1962,34,388-94. (13) Ruzicka, J.; Hansen, Ε. Η. "Flow In jection Analysis"; John Wiley & Sons: New York, 1981. (14) Betteridge, D.; Fields, B. Anal. Chem. 1978,50, 654-6. (15) Slanina, J.; Lingerak, W. Α.; Bakker, F. Anal. Chim. Acta 1980,117, 91-8. (16) Ramasamy, S. M.; Jabbar, M. S. Α.; Mottola, H. A. Anal. Chem. 1980,52, 2062-6. (17) Ramasamy, S. M.; Mottola, H. A. "Abstracts of Papers," 182nd National Meeting of the American Chemical Soci ety, New York, Ν. Υ., August 1981; American Chemical Society: Washing ton, D.C., 1981; ANAL 63. (18) Reijn, J. M.; van der Linden, W. E.; Poppe, H. Anal. Chim. Acta 1981,123, 229-37. (19) Vanderslice, J. T.; Stewart, Κ. Κ.; Rosenfeld, A. G.; Higgs, D. J. Talanta 1981,28,11-18. (20) Stewart, K. K.; Vanderslice, J. T.; Brown, J. F. "Abstracts of Papers," 182nd National Meeting of the American Chemical Society, New York, N.Y., Au gust 1981; American Chemical Society: Washington, D.C., 1981; ANAL 3. (21) Betteridge, D.; Goad, T. B.; Sly, T. J. "Abstracts of Papers," 182nd National Meeting of the American Chemical Soci ety, New York, N.Y., August 1981; Amer ican Chemical Society: Washington, D.C., 1981; ANAL 4.
1316 A · ANALYTICAL CHEMISTRY, VOL. 53, NO. 12, OCTOBER 1981
(22) Pardue, H. L.; Fields, B. Anal. Chim. Acta 1981,124, 39-63. (23) Pardue, H. L.; Fields, B. Anal. Chim. Acta 1981,124, 65-79. (24) Reijn, J. M.; Poppe, H.; van der Lin den, W. E. "Abstracts of Papers," 182nd National Meeting of the American Chemical Society, New York, N.Y., Au gust 1981; American Chemical Society: Washington, D.C., 1981, ANAL 5. (25) Painton, C. C ; Mottola, H. A. Anal. Chem. 1981,53,1713-15. The author acknowledges the support of the Na tional Science Foundation (Grant CHE-7923956).
Horacio A. Mottola was born in Bue nos Aires, Argentina, and received undergraduate and graduate educa tion at the University of Buenos Aires. He did postdoctoral work with H. Freiser (University of Arizona, Tucson). He has been associated with the chemistry department at Oklaho ma State University since 1967. Mottola's research interests include stud ies on the role of kinetics in analyti cal chemistry, analytical separations, continuous-flow systems, and analyt ical applications of photochromism.
