CORRESPONDENCE
Exchange of Comments: Pumping Pressure and Reagent Consumption in Flow Injection Analysis Sir: Should we choose not to comment on Margoshes article
(I), where it is argued that “an unsegmented continuous flow analysis system will either consume considerably more reagent than an [air] segmented system or else will require very high pumping pressures”, we would reinforce the author’s misleading claim that the concept of air-segmented streams still remains the best and only practical basis for continuous flow systems. Therefore, reluctantly, we enter into a discussion, which although unwelcome for a single manufacturer, will in the final analysis promote the future development of continuous flow systems. I t is widely believed that there are only two ways to prevent carryover in continuous flow systems, Le., either by air segmentation (2), or by the use of a turbulent flow which, by yielding a flat velocity profile, should result in a lesser sample zone dispersion than the laminar flow where the velocity profile is parabolic. True enough, in order to sustain a certain turbulent flow pattern (as defined by Reynolds number, Re = 127Q/d, where Q is the pumping rate in cm’/s and d is the diameter of the pump tube in cm), a decrease in the pumping rate Q by a factor of 10 requires such a reduction of the tube diameter that the pumping pressure must be increased by 10000 (Ref. 1,eq.3). So far, Margoshes’ argument is “correct”, that is, if one uses the formula for pressure in the laminar region to calculate the pressure in the contended turbulent flow. Yet a crucial question remains; which is the minimum Reynolds number a t which the sample zone dispersion-and therefore also the carryover-is still acceptable? The author presents no experimental proof of his own, but lists in Table I ( 1 ) the systems with acceptable carryover which have Re between 340 and 150. Then he proceeds further to a paper of Lane and Sirs ( 3 ) where he finds in Figure 4 “a narrow indicator band for a Reynolds number of 165 and a peak with extended tailing a t a lower solution flow with a Reynolds number of 2.7”. This leads him to the conclusion that the minimum conceivable Re for unsegmented streams “can be as low as 150”, therefore, ostensibly requiring either high pumping rates or large pumping pressures. There is, however, no reason to believe that the value of Re = 150 is the limiting one. First, the actual experimental values of Figure 4 of Lane and Sirs ( 3 )read just opposite to what Margoshes claims. Next, more importantly, there exist, in addition to air segmentation or turbulence, a number of other tools which can be utilized to limit the dispersion of the sample zone. In the series of papers dealing with our approach to unsegmented flow analysis, flow injection analysis (4-131, these factors are especially treated in the theoretical Part X (13), but in the present context one of them-the molecular diffusion-has a particular relevance and will therefore be discussed in detail. I t will be shown below, both theoretically and b y experiment, that the flow injection analysis, utilizing a nonsegmented laminar flow of reagent, can be operated: ( a ) the better the lower Re is, with a limit of Re = 0.1; ( b ) a t Re = 20 a t a rate of 120 determinations per hour a t the normally used pumping pressure even in lines of 0.5-mm i.d.; (c) with a typical sample volume of 30 FL and volumes as small as 4 pL; and ( d ) with 1858
A N A L Y T I C A L CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
a reagent consumption which is smaller than that consumed of the AutoAnalyzer system.
THEORETICAL BACKGROUND There exist a number of interesting works dealing with dispersion of a sample zone as it flows through a narrow open tube. This type of specialized hydrodynamics has many important applications in flow velocity measurements (e.g. in pipes and veins (3)) and has also been studied for the purpose of estimating diffusion constants (14). The measure of the dispersion of the sample zone is the dispersion number 6 = D t / L 2 , a dimensionless group (where D is the axial dispersion coefficient in cme/s, t is the time in s, and L is the tube length in cm) often used in chemical reactor engineering. The concentration gradient within the sample zone in the axial direction is in the first approximation described by a stoichiastic variable of the Gaussian type which as modified by Taylor ( 1 4 ) is expressed by the following equation:
where C is the concentration a t distance X , M is the mass of material concentrated a t point L = 0 a t time t = 0, n is the radius of the tube and 6 is the dispersion number. The approximations made here differ from the real experimental conditions of continuous flow in two important ways: (a) the injected material is not concentrated in an infinitely small volume at the point of injection; (b) the resulting peak shape IS not Gaussian (Le., symmetrical) unless a sufficiently long time is allowed to pass from t = 0. These deviations are treated in detail elsewhere (13),yet, for the present purpose this model is satisfactory to illuminate the relation between the Reynolds number and the dispersion number 6. The works of Aris ( I j ) , Levenspiel (16) and notably Taylor ( 1 4 . 17, 18) describe quantitatively the dispersion process in both laminar and turbulent flow. Their conclusions are graphically summarized in Figure 1in which three flow regions and twci minima of the dispersion number value are seen. True enough, a t Re 2 2000, a lower dispersion of the sample zone is found due to turbulent flow. There is a discontinuous region with “incipient” turbulence at 1000 5 Re 5 2000, but, most interestingly, there is yet another minimum a t Re = 0.1. Therefore, in the region of laminar flow, a decrease in pumping rate will result in a decrease of peak spreading and lesser carryover. The minimum at the dispersion number-Reynolds number curve was discovered and quantitatively described by Taylor (14) and used by him for measurement of the molecular diffusion. The minimum is caused by “Taylor‘s effect” due to which in the region a t Re z 0.1 the parabolic velocity profile created by the forward movement of the carrier stream is just averaged by the radial molecular diffusion. There is a certain minimum time needed for this effect to become pronounced, and its full exploitation would require too slow pumping rates to be practical. Yet in 2-m long tubes
Table I. Comparison of Reagent Consumption in the Determination of Phosphate Characteristics Reagent consumption in mg per sample System Technicona Flow injectionb
I Flow injection
Molybdate 2.8 29.7 16.9 2.2
Ascorbic acid 4.9 24.0 12.9 1.8
Nitric or sulfuric* acid 31.0* 121.1 64.9 9.1
Sampling rate, samples h-l
...
50
Sample volume, PL 430
360 440 34
225 420 100
500 500 30
Reynolds No.
I1 a
Ref. 19.
Ref. 5.
I
d
Figure 2. Flow diagram for t h e determination of phosphate by flow injection. S, point of injection; W, waste: the tube length is given in cm, while t h e tube internal diameter is stated in mm
Figure 1. The dispersion number 6 as a function of the Reynolds number (Re)for aqueous solution in the laminar, transient, and turbulent regions
of flow. T h e model applies for narrow open tubes
a pronounced effect of the molecular diffusion will be observed already after 6 s in a 0.5-mm i.d. tube, 14 s in a 0.75-mm i.d. tube, and 25 s in a 1.00-mm i.d. tube, corresponding t o R e 5 30, R e 5 68, and R e 5 92, respectively. These values are naturally approximate ones, as they will be affected by temperature, diffusion coefficients, and viscosity; yet t h e general trend is obvious and t h e above considerations demonstrate t h e beneficial effect of low pumping rates. A detailed study of flow patterns in open tubes using t h e color indicator technique has been compared with computer simulated curves, compared with t h e works of Taylor, Levenspiel, and Aris, and used t o develop a theory of flow injection analysis (13). In this way it has become possible t o optimize t h e conditions for t h e analytical procedures based on nonsegmented continuous flow.
EXPERIMENTAL VERIFICATION The original procedures for the determination of phosphate (5),criticized by Margoshes ( I ) , has been optimized and compared with the most recent AutoAnalyzer method (19). For confirmation, also the chloride method (7) has been miniaturized and used as an example of how very small sample volumes can successfully be analyzed. Apparatus. T h e Peristatic P u m p , MP-13 GJ-4 (Isomatec S.A., Ziirich, Switzerland),was operated at speed 10 with suitable pump tube diameters to obtain the desired flow rates (see Figures 2 and 4). T h e Spectrophotometer, Corning model 254, was furnished with a Hellma flow through cell type OS 178.12, volume 18 pL. The end of the reaction tube was inserted into the cell as close as possible to the tubular optical path in order to minimize the dead volume. T h e Recorder was Servograph REC 51 furnished with a REA 112 sensitivity unit (Radiometer AS., Copenhagen, Denmark). T h e Manifold was made from polyethylene tubing and Leg0 toy building blocks as described earlier (7-10). T h e Injection Port was a precisely made rotary valve with a bore with a volume of 30 pL (18, 10, and 4.5 p L resp.) and furnished with a bypass of a higher flow resistance (see manifolds Figures 2 and 4). Thus while the sample is being injected by a syringe into the volumetric bore, the carrier solution is continuously bypassing the injection port. Only when the valve has been turned, the stream in the bypass comes to a standstill and the precise amount of the sample solution is injected by the carrier
_ _ ____ “ ^
___.
sei\-
Figure 3. Determination of phosphate in the range 5 to 40 pg P/mL. Each sample injected in quadruplicate, injected volume 30 pL sampling ) 40 rate 100 samples/h. T h e fast scan of 20 pg P sample ( R Z 0 and pg P sample (R4,Jon the right shows the extent of the carryover (iess than 1 YO)if samples are injected at a time span of 35 s (difference between S,and S,)
stream from the volumetric bore into the reaction line. Reagents. All reagents were of AR grade and redistilled water was used throughout. For the determination of phosphate, a solution of 0.005 M ammoniumheptamolybdate in 0.4 M nitric acid was used together with a 0.570 aqueous solution of ascorbic acid to which was added 0.270 glycerine. The standards were prepared by successive dilutions of a 100 ppm phosphate stock solution (0.4390 g of anhydrous potassium dihydrogenphosphate per liter). The reagent solution used for the determination of chloride was prepared by dissolving 0.626 g of mercury(I1) thiocyanate, 30.3 g of iron(II1) nitrate, 4.72 g of concentrated nitric acid and 150 mL of methanol in water, making the final volume up to 1 L (7). The standards were made by suitable dilution of a stock solution containing 1000 ppm C1 (1.648 g of sodium chloride per liter).
RESULTS T h e determination of phosphate was carried out in the modified manifold shown in Figure 2 which differs from t h e original one ( 5 )in reduced tube diameter (0.75 mm) and tube length (0.5 m) as well as injected sample volume (30 pL). This miniaturization and t h e decrease of t h e pumping rate by a factor of 18 results in a dramatic decrease of t h e reagent consumption per analysis (Table I) at the same time yielding, however, a satisfactory calibration curve (Figure 3) at a sampling rate of ca. 100 determinations per hour. The extent of carryover at the rate of 120 samples/h is readily seen in Figure 3, showing a sample peak recorded a t a high paper velocity, by observing the time span between two subsequent ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
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Table 11. Comparison of Reagent Consumption in the Determination of Chloride Characteristics Reagent Consumption per sample Sampling Hg(SCN),, Fe(NO,),, HNO,, Methanol, Reynolds rate, mg mg mg PL No. samples h-I
System Tec hnicona Flow injectionb I Flow injection I1 Ref. 20.
1.00 0.75 1.62
48.5 36.4 78.2
7.6 5.7 12.2
0.19
9.1
1.4
Sample volume, PL
390
182
60 80 200
400
45
25
120
30
240 180
90 68
Ref. 7. m d r
A,
U
Figure 4. Flow dagram for the determination of chloride by flow injection. S, point of injection; W, waste, tube length in cm; tube internal diameter
in mm
-
A
I
_-
7
CB
::Ah-
CL-
?.
I Oi5.
-
3 1
t
I
SCAN-
Flgure 5. Determination of chloride in the range 5 to 75 ppm CI. Each sample injected in quadruplicate. Injected volume 18 pL, sampling rate 120 samples/h. T h e fast scan of 30 ppm sample ( R S 0and ) 75 ppm sample ( R 7 5 on ) the right shows the extent of the carryover (less than 1 % ) if samples are injected in a span of 3 s (difference between S ,
and S 2 ) injections (SI time of injection of the recorded sample, Sztime of the next sample to be injected) and the time of which 1% sample color is still present in the flow cell. T h e determination of chloride was carried out in the manifold shown in Figure 4 which again differs from the original one (7) by an overall miniaturization. T h e sample volume was 18 FL, the consumption of materials was again lower than in the corresponding AutoAnalyzer procedure (20) (Table 11) and the sampling rate was 120 determinations per hour (Figure 5). T h e sample volumes can be reduced still further. This is demonstrated in Figure 6, where the 50 ppm chloride sample is analyzed in the manifold depicted in Figure 4, by injecting 30, 18, 10, and 4.5 pL of sample solution and by recording the peaks a t a high paper speed. Obviously, ultramicrosystems, if needed, can be designed.
DISCUSSION T h e e c o n o m y of reagent and s a m p l e c o n s u m p t i o n as compared for the AutoAnalyzer and the Flow Injection method is clearly in favor of the nonsegmented flow. Obviously, other analytical procedures can be modified using the same approach. Thus recently, a total nitrogen method ( 6 , 8 ) ,based on ammonia measurement through formation of indophenol blue, which requires sequential reagent addition in a more complex manifold, has been miniaturized. It consumes 30 or 13 fiL of sample solution and an average 60% of the reagents used in the corresponding AutoAnalyzer procedure. Also, the 1860
ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
Flgure 6. A rapd scan of sgnals obtained by injecting 50 ppm CI sample of the following volumes: (a) 30 pL, (b) 18 pL, (c) 10 pL, and (d) 4.5 FL. The manifold used is that depicted in Figure 4
most recently published enzymatic determination of glucose (11) in blood serum by flow injection analysis requires lower amounts of enzyme per determination than the corresponding AutoAnalyzer method (3.12 gU of glucose dehydrogenase vs. 12.6 gU in the AutoAnalyzer procedure). T h e economy of t i m e has two aspects: the sampling rate and the resident time of the sample in the automated system. In the first works on flow injection, high pumping rates were used to achieve a high sampling frequency, often in excess of 250 samples/h. As such rates seldomly are needed and might be difficult to mantain in the routine laboratory, rates in the range of 90 to 120 determinations per hour were instead adopted-still well ahead of the standard AutoAnalyzer procedures. The resident time of the sample in the system, which is also closely connected with the duration of the start-up, wash, and close-down procedures, is the second aspect of the time economy of any automated analytical equipment. Thus, if we compare, e.g., the phosphate procedures, it can be seen that the readout on the flow injection system is available 14 s after the sample has been injected (Figure 3), while in the corresponding AutoAnalyzer procedure 150 s elapses between the aspiration of the sample and the steady-state readout. In this context it is interesting to point out that it takes 9 s in the AutoAnalyzer system for the sample zone to be aspirated, to be transported to and through the pump and to the point where it becomes segmented by air. This is a time span comparable to that needed in the flow injection system to obtain the readout. This aspect and especially the extent of dispersion in the nonsegmented conduit of the AutoAnalyzer is yet another interesting point to compare. Finally, while advantages of the flow injection system having been pointed out, its drawbacks should be mentioned. T h e main limitation is obviously the rate of the chemical reaction employed for a particular analysis. This is a true limitation, as long as one wishes to operate without an automated sample changer in a working cycle with only one sample in the reaction
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. T h e 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 CITED 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 Jose Krug’ 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. T h e 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. T h e 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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