Report
Since Skeggs' classic work (1) it has been generally assumed that air segmentation of the flowing stream and attainment of a "steady state" signal are essential parts of continuous flow analysis (CFA). Recently, Rùzicka and Hansen (2) in Copenhagen and Stewart et al. (3-5) in Washington, D.C., have independently performed similar experiments of injecting the sample directly into the carrier stream, and have proved that analysis without air segmentation is not only possible but also advantageous. They have termed it flow injection analysis (FIA); this is the name used in this REPORT rather than "nonsegmented continuous flow analysis", which has wider implications. Skeggs may justly be considered the Henry Ford of analysis because of his revolutionary conception of continuous flow analysis. The improvement in sample throughput coupled with its accuracy, reproducibility, and reliability has resulted in its widespread use. However, the basic thinking behind the approach was to perform normal chemical procedures on a conveyor belt principle. Thus, in a colorimetric procedure, appropriate amounts of sample and reagent are brought together and mixed by successive inversions until a "steady state" is reached when its absorbance is measured. Air segmentation is used to prevent carryover of samples and to assist in the mixing; the proportionating pump,
oratory equipment, easily manufactured or purchased as a complete unit. The available options are discussed in the next section. Experimental The basic apparatus is shown as a block diagram in Figure 1. The essential parts are: a means to propel the carrier, a sample injection system, a detector and something to display the output, and linking tubing. There are many ways in which these components can be manufactured or combined. One of the simplest is what might be termed the "basic LEGO" model devised by Rûzicka and Hansen (6). The basic components are made of Plexiglas, tubing is 0.4-1-mm i.d. polypropylene, and connections are made by pushing the tubing into slightly tapered holes in the components. The whole, mounted neatly on a LEGO board (Figure 2), is ideally suited for teaching and occasional determination. This apparatus was developed by improving the connections and inlet system (7). The fully developed system is commercially available from BIFOK (Figure 3). Stewart used modified HPLC apparatus operated at 500 psi and with 0.2-0.4-mm i.d. tubing (3-5). In our laboratory we have used stainless steel tubing (0.8 mm i.d.), Swagelok connections and a standard chromatography valve, and controlled the injection and data processing with a microprocessor. One of our objec-
How Infection D. Betteridge Chemistry Department University College of Swansea Swansea SA2 8PP, UK
through which the sample reactant and buffer solutions are drawn, is a vital part of the apparatus. By contrast in FIA, there is no air segmentation, the sample is introduced as a plug via a valve or syringe, mixing is mainly by diffusion-controlled processes, and the response curves do not reach the steady state plateau, but have the form of sharp peaks. The absence of air segmentation leads to a higher sample throughput. The presence of a sample-carrier interface, over which concentration gradients develop during the course of analysis has opened up new analytical possibilities for continuous flow analysis. The reproducibility is good, and there is no sample carry-over. There is no need to introduce and remove air bubbles, and an expensive high-quality pump is not necessary. The requisite apparatus can be easily assembled from existing standard lab-
832 A · ANALYTICAL CHEMISTRY, VOL. 50, NO. 9, AUGUST 1978
tives is to improve the precision of 1-2%, which is easily obtained with all of the apparatus mentioned by a factor of 10. Other combinations can give perfectly satisfactory results, which indicates that wide variations in the important parameters can be tolerated. However, as shown in the section on theory, there are optimum conditions for operation, and on both theoretical and practical grounds a tubing of 0.5 mm i.d. is best. Larger diameter tubing leads to increasing dispersion, whereas with the smaller diameter the tubing may easily be blocked and high-pressure pumps are required for operation. The remaining discussion is concerned with specific components. Pressure Head. In most applications 0.5-1-mm i.d. tubing and flow rates of 0.5-5 mL min - 1 are employed so that the flow can be maintained by 0003-2700/78/0350-832A$01.00/0 © 1978 American Chemical Society
a peristaltic pump or constant head device. With a fast revolving multiroller pump, pulsation is never a problem—a consequence of having no compressible air bubbles. With some pumps it is a minor problem that can easily be suppressed (8). Stewart has encountered and overcome pulsation in high-pressure systems (9). Injection Port. The first of the inlet systems devised by Rûzicka and Hansen to gain widespread acceptance is the simple flap valve illustrated in Figure 4a. Typically, the sample volume is 0.1-0.2 cc. Because of the pressure surge it is desirable to have a length of tubing between the port and the carrier reservoir to act as a buffer. For the last year Rûzicka and Hansen have preferred a simple rotary valve (Figure 4b) that dispenses 30-100 ML; this is incorporated in the BIFOK apparatus. Both valves have a repeatability of 1% and permit sampling every 10 s. With a chromatography valve, samples of 6 μΐ-, have been rou tine in our laboratory. Detectors. In the earliest experi ments, in which spectrophotometric determinations were employed, a standard tubular flow cell (vol 18 ^L) and spectrophotometer were used to detect the sample peak after colour development. This remains a conve nient method, although the standard flow cell can be replaced with a Plexi glas block 4 X 1 X 1 cm through which a hole ca. 1 mm has been drilled and
Injection
Output
Waste
Carrier Reagent Detector
Figure 1 . Block diagram of basic FIA apparatus
alysis Figure 2. " B a s i c LEGO" apparatus with potentiometric detector
polished. This enables push-pull con nections to be made with polypropyl ene tubing, which fits into a standard cuvette holder, giving a flow cell that retains the flow pattern and has an ef fective volume of 8 μι.. Several novel detectors have been developed for use with FIA; these are also cheap and easy to assemble. Ion-Selective Electrodes. Rûzicka and Hansen have mounted an ion-selective electrode (ISE) and a reference electrode in a tubular container held at an angle of ca. 30°. The effluent stream flows onto the indicator electrode and then onto the reference electrode. An outflow tube is positioned so that the reference electrode is in a pool of effluent. Signals are obtained that are proportional to the function measured, provided the gap between the electrodes is gauged correctly (10). Furthermore, it is possible to use a series of electrodes and a cas-
Carrier pumped from reader's left to right, through rotary valve (Figure 3B) and ISE detector as described in text. Peak integrator situated above voltmeter
Figure 3. BIFOK FIA system A, pump; B, rotary valve; C, Plexiglas block connectors and polypropylene tubing (see inset); D, detector
ANALYTICAL CHEMISTRY, VOL. 50, NO. 9, AUGUST 1978 · 833 A
In addition to these, detectors that are in routine use for monitoring HPLC effluent may be used. Air bubbles generally interfere and must be removed. This can often be achieved by having the cell on an incline rather than horizontal.
Disposable Syringe
Solvent Extraction
Sample
Karlberg et al. have elegantly demonstrated that solvent extraction by FIA is a most attractive procedure (15). By use of a teepiece with a carefully positioned platinum side tube, the aqueous carrier containing the sample is interspersed alternately with very small segments of organic solvent. Extraction takes place across the aqueous-nonaqueous interface, and the two phases are separated after extraction by a second teepiece. Wall drag causes convection in both phases, increasing the rapidity of attaining equilibrium concentration distribution.
1 mm i.d.
0.5 mm i.d. Septum
Dialysis Figure 4. Simple valves for sample injection (a) Cross section of flap valve. Carrier flows through A. On depression of syringe, septum is forced down, and sample occupies resulting depression. Septum regains original position and forces sample into carrier stream. Constructed from Plexiglas blocks held together by nylon screws, (b) Rotary valve. Sample placed in wider tube of known volume. When rotated through 9 0 ° , carrier diverted through larger diameter tubing, and sample enters carrier stream
ceding effluent to obtain a measure of several species in the same sample. Under these dynamic conditions it is not necessarily the case that the potential is that which would be obtained if a conventional measurement were made. It may also be true that the sample matrix affects the rate of equilibration. However, in all cases so far reported, in which the calibrants have been properly matched samples, good calibration curves have been obtained. Potentiometric Detectors. In principle it is possible to detect a sample peak by differential potentiometry, with one electrode in the carrier stream before the point of injection. The difficulty is that the potential difference is very sensitive to the radial position of the electrode downstream from the injection point. If it just pokes through the side of the tubing, it is not very sensitive to concentration changes. (It seems as though there is a "dead" layer on the walls of the tube.) If it is placed midstream it interferes with the flow characteristics and gives irreproducible results. This led Nagy et al. to abandon FIA and resort to a mixing chamber (11). However, the difficulties have been overcome by Karlberg and Thelander, who have placed the last part of the tubing in a vertical position inside a small cup. The indicator electrode is a platinum wire centrally placed in the mouth of the tube, and a reference electrode is placed in the cup (12).
Photometric/Refractometric Detector. A flow cell has been manufactured with a 3-cm path length, a light emitting diode (LED) at one end, and a photodiode at the other (13). The total component cost including the associated electronics is less than $25. Insofar as the LED can be selected to cover most of the visible spectrum, it makes a spectrophotometer redundant. The sensitivity extends to the ppb region. Because the sample plug has a parabolic head and tail, it acts as a lens if there is a difference in refractive index between carrier and sample. By virtue of the design of the detector, where the light beam is along the axis of flow, it functions as a differential refractometer as well as a photometric detector. It is capable of better discrimination than an Abbe refractometer and is almost as sensitive as a conventional differential refractometer. Photometric detectors with the light beam normal to the flow may also function as refractometers, as is evidenced by the small negative peaks exhibited on many FIA peaks. However, this does not pose any problems in practice. Dielectric Detector. A conductimetric detector based on a semiconductor oscillator chip has been exhibited (14). It is cheap, but the optimum design has yet to be achieved. However, it has digital output, which is advantageous if a microprocessor is to be used to process the results.
A simple but effective dialyser, consisting of a dialysing membrane clamped between two Plexiglas blocks, has been reported and proved quite satisfactory for analysis of blood samples, etc. (6). Applications and Theory
The applications of FIA known to the author are summarised in Table I, page 842 A. Determinations that have been demonstrated in workshops are included, since it is probable that full accounts will soon be published. The order is roughly chronological, thus emphasising the trend to lower flow rates and shorter lengths of tubing, which has accompanied improvement in understanding of the theoretical principles underlying the method. The range of conditions under which satisfactory results can be obtained emphasises the robustness of the technique. However, a knowledge of the theory aids in the selection of optimum conditions; therefore, it will be briefly reviewed. First, two conceptual errors that have given rise to needless controversy must be disposed of. One relates to the notion that turbulent flow is essential. This is stated in the early papers of Rûzicka et al. (2, 16), but they soon realised that laminar flow is preferable. In fact, a Reynolds number (RE) of 2000, which is usually taken to be the onset of turbulent conditions, corresponds to a flow rate in 1-mm diameter tubing of 93 mL min - 1 . Therefore, it is unlikely that FIA has ever been carried out with turbulent flow. However, incipient turbulence can be caused by projections and coiled tubes; in the high-pressure systems an RE of 2000 is approached. Thus, the
A N A L Y T I C A L CHEMISTRY, V O L . 5 0 , NO. 9, AUGUST
1978 ·
835 A
point has been a matter of dispute (39, 40). The other error arose from the initial stress laid on the mechanical aspect of flow injection, which was emphasised by the terminology adopted. Critics have not been slow to point out that some forms of nonsegmented CFA predate 1974 (7). However, the analytical significance of maintaining the integrity of the sample plug and allowing mixing to be controlled by diffusion processes had escaped earlier workers. This is illustrated by Nagy et al. who performed the experiment of injecting a sample into a flowing stream in a large diameter open tube with the object of measuring the potential of the sample downstream— clearly FIA (11). Unfortunately, they used electrodes that protruded into the stream. This gave rise to erratic results, from which they concluded that the open tube must be abandoned in favour of a mixing chamber. They noted a further disadvantage of the open tube: "The reproducibility of the measurements was impeded by a concentration gradient perpendicular to the flow." It is the essence of flow-injection analysis that this concentration gradient is a key source of analytical information and that it is essential to maintain the integrity of the plug and to limit mixing to diffusion-controlled processes. Rûzicka and Hansen's first experiments were not performed with this in mind. They thought, in accord with conventional concepts, that turbulent flow assisted in dispersion of the sample and that the great increase in sample throughput and simplicity of apparatus were the most appealing features of the method. However, they soon recognised that turbulent flow was not needed. A hint that the mixing process was diffusion controlled was provided by a set of key experiments (24) and was strengthened by
the discovery of Sir Geoffrey Taylor's paper, "Dispersion of Soluble Matter in Solvent Flowing Slowly Through a Tube" (41). Further study has led to a statement of theory that is almost definitive and is certainly the starting point for all concerned with the method (7). It makes clear that what is really important about FIA is the chemistry that happens between the points of injection and detection, not the improved mechanics of injection and throughput. One can see how confusion and controversy arose, and the historian of science can only note that yet again we have an instance of the inventors not realising immediately the totality of what they had discovered and of others being within a whisker of getting there first. Basic Principles. Between the points of injection and detection the sample plug will have been physically dispersed to some degree, and in addition some chemical reactions may have taken place. The peak detected will reflect both processes. The physical dispersion is brought about partly by longitudinal flow which gives rise to parabolic head and tail and partly by radial diffusion (Figure 5). (It is analogous to chromatography without partitioning and without a stationary phase to disturb the flow pattern.) The relative importance of these processes depends upon the flow rate, the radius of the tube, the time of the analysis, and the magnitude of the diffusion coefficient. The precise relationships are given in Table II, which summarises the treatment of Rûzicka and Hansen (7). Basic Models and Equations. The peak obtained generally fits the Ccurve equation (Table II), but to evaluate the role of the empirical parameters, it is necessary to develop other equations. One is due to Taylor, who elucidated the significance of diffusion
(41). In any flowing stream in a pipe the velocity at the walls is zero, and that at the centre is twice the mean. Thus, if a sample plug is placed in the stream, it assumes a parabolic shape by the process of convection. If this were the only means of dispersion, some of the sample would stick to the walls, and the plug would have an infinitely long tail. However, the molecules can diffuse away from (and back to) the walls and thus gain some longitudinal motion as they join the main stream. The diffusion can be longitudinal, i.e., in the direction of flow or radial—perpendicular to the direction of flow. Taylor showed that while these processes always occur, dispersion by longitudinal diffusion can be ignored relative to that caused by the main flow pattern, whereas radial diffusion is always important in narrow tubes, and at low flow rates it may even be the major mechanism for dispersion. Under diffusion-controlled conditions, the peak shape is Gaussian. FIA generally is carried out just outside Taylor conditions, but Taylor's argument leads to the following practical conclusions: mixing will be complete without recourse to mechanical stirring; the concentration gradients in a given sample plug are both reproducible and predictable; and the peak shape will be influenced by differences in the sample and carrier matrices, because the whole of the plug, matrix and analyte, is diffusing into the carrier. Another useful equation is that for the "tank-in-series" model. This is exactly analogous to the concept of the height equivalent to a theoretical plate model of chromatography. It postulates that the tube between the points of injection and detection consists of a number, N, of imaginary tanks. These are in series and the simple mathematical models used to account for the distribution of a sample
Direction of Flow
Figure 5. Diagramatic representation of effects of convection and radial diffusion on concentration profiles of samples monitored at a suitable distance downstream from injection (a) No dispersion, (b) Dispersion predominantly by convection, (c) Dispersion by convection and diffusion, (d) Dispersion predominantly by diffusion
836 A ·
A N A L Y T I C A L CHEMISTRY, V O L . 5 0 , N O . 9, AUGUST 1978
tank-in-series model is used for all ex cept the narrow tube for which Tay lor's equation is most appropriate. The practical conclusions to be drawn from the detailed studies with respect to flow rate and tube length are shown in the table. It was also found that the optimum for the tube diameter is 0.5 Ψ 0.2 mm i.d. In this range a reason able flow rate can be maintained with out difficulty, reagent consumption is not excessive, and a variety of chem istries is available to the analyst. With limited dispersion the sample integrity is maintained to a high de gree. This is the ideal when FIA is
introduced into the first tank can be used to account for the physical dis persion of sample as a function of time. The actual "size" of the tank is a function of tube dimensions and flow rate. The shape of the distribu tion predicted by the tank-in-series model is shown in Figure 6. For high Ν it becomes Gaussian, i.e., indistin guishable from Taylor's model, but for Ν = 1 a very different shape is found. The application of this model to FIA has led to some radical developments that are described below. Dispersion. An empirical definition of dispersion is given in Table II. The
used as a sample inlet system for the measurement of pH, pCa, etc., of the sample. The justification of measuring pH, etc., this way lies in the small sample size (ca. 30 μι,), the short time of measurement (ca. 10 s) as the "steady state signal" is not required, and the possibility of performing sev eral determinations in sequence on the same sample. The theory suggests that in order to gain maximum sensitivity and minimum analysis time, it is bet ter to use a short narrow tube than a mixing chamber. With the mixing chamber and the typical parameters shown in Table II, the analysis time
Table II. Basic Equations and Guidelines for FIA comment
equation
BASIC MODELS AND EQUATIONS ( a ) general curve for FIA the Ccurve (b) dispersion Is controlled by radial diffusion. Taylor's equation (c) tank in series there are Ν imaginary tanks between the points of injection and detection
C =
^
g-(1-t/Tp/4i
2vb C-
M
1
f, V f / forN= 1
(i) 6 = Dt/L2 (ii) holds when Τ > r V 3 . 8 2 Dc
_-(1-;rt*/i.*4i
(i) Τ = rVr,and for W > 10, δ = 1/2 Τ1! Ν (ii) reactor volume = VR = QUFif r is constant (iii) for low Ν dispersion is skew, and for high W, it is Gaussian; therefore, long narrow tubes result in symmetric peaks, and shorter tubes give asymmetric peak. If the tube is equivalent to 1 tank, there is an exponential rise and fall
( W - 1)!
C = - e- 1 0 % interferes, but FIA preferable to static method ·
25.
glucose
pot (enzymatic oxidation)
buffer, pH 6 pass through immobilised glucose oxidase in a reactor
2.5
26.
NO;
N O ; electrode
buffer, pH 9.5
2
30 100
-
+
29
27.
C I , Br", l'
titration (Ag )
(DH2O (ii) NaN0 3 (0.1 M), AgNO3
