E. H. Hansen and J. Ruzicka The Technical University of Denmark Chemistry Department A DK-2800 Lyngby Denmark
The Principles of Flow Injection Analysis as Demonstrated by Three Lab Exercises
T h e implementation of automated analytical procedures in students' lab courses is greatly impeded by the high costs of the commercially available equipment. Thus, only few students have the opportunity to familiarize themselves with these types of analysis which, nevertheless, are gaining access into more and more industrial, clinical, chemical, and academic laboratories. With the development of the new type of continuous flow analysis. Flow Iniection Anal~sis'.~,it is, however, possihle-for a limited expense-to make experimental setups which can effectively he used to demonstrate the elements and characteristics of continuous flow analysis. Flow Iniection Analvsis is based on iniection of a samole into a continuously moving, nonsegmented carrier stream, oronelled b v a oeristaltic oumo. which stream in itself mieht Lonstitute areagent. The &je& sample thereby forms a well defined zone which is then transported toward a detector. During this transport the sample solution is mixed with the carrier stream-and possibly also with other reagents added sequentially further downstream-and reacts with its comoonent(s) to form a snecies which can he measured in aflowihrouyh detector (ah:sorhance. electrode potential, etc.), the output heiny a peak which yields the nnnlyticnl result. This new concept of continuous flow analysis might be better understood if explained by pointing out its three principles: (21) Sample injrction; (11) Repralucihle timing: and (cj Controlled dispersion. The purpose of the sample injection is to place a well defined sample zone into the continuously moving stream in such a way that the movement of this stream is not disturbed. This is executed by using a specially constructed valve. The exactness of the iniection techniaue allows the concent of the "steady stntr signal" to he abandoned and the sampling freuuencv . . to he substantiallv inrreased. while the samolr and reagent consumptions are reduced. As the sienal is not to he read on the flat Dart of the resmnse curve, hut,n its steep ascending part, aiso a highly reproducible resident time of the sample in the system is essential, and this is readily accomplished in the absence of air as the flowing stream is totally non-compressible. Besides, as the sample does not pass through the pump on its way to the detector, its path through the system is well defined, and the dispersion of the sample zone and the resident time can he chosen a t will to suit exactly the requirements of the chemistry involved. The controlled dispersion of the sample zone which occurs during its passage through the system toward the detector results in a response curve the peak shape of which is characteristic of the Flow Injection system. Expectedly, the sample zone broadens as i t moves downstream and changes from the orieinallv s h a ~ eto a more svmmetrical and " . asvmmetrical . eventual Gaussian form. By changing the flow parameters, the dispersion can he mani~ulatedeasilv to suit the reauirements of particular analytic& procedure so that optimum response is obtained a t minimum time and reagent expense. At our Department a 3-week (5 hrlday) course in instrumental analysis is compulsory in the fourth semester for all undergraduate students majoring in chemistry. We have for the Inst three years included three l-day exercises in Flow Injection ~ n a l y s i s For . practical reasons all three exercises have been run with spectrophotometric detection. One is the ~~~
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determination of chloride, another the determination of phosphate, and the third one a titration of acid samples with base. Around each exercise has been built a small "case story." In this paper the three exercises are described in detail, and having been met with enthusiasm hy the students a t our lab course, it is our hope that others will find them equally useful.
Apparatus The peristaltic pump was an Ismatec, Model Mini-S-840 (Ismatec, Switzerland) aperated with color-coded Ismatec pump tubes of suitable internal diameters to obtain the desired flow rates (see Figs. 2,4, and 5). Any pump which delivers a regular, pulsefree flowmight be used. The spectrophotometer was either a Corningmodel254or a Corning model 252 (with filters) equipped with a Hellma flow-through cell, type 178.12 (volume 18 &I,light path 10 mm; Hellma, Germany). In fact, any type of spectrophotometer can be used provided that it can accommodate a flow-throughcell preferably with a volume of less than 2040-&I. The recorder was a Servograph REC 51, furnished with an REA 112 sensitivity unit (Radiometer, Denmark). The manifolds (Figs. 2, 4, and 5) were made from polyethylene tubings (0.50 and 0.75 mm i.d.) of nearly similar outer diameter. The coils were made by winding the appropriate lengths of tubings around small methacrylate cylinders. These and the other components were glued onto small pieces of Lego toy blocks (Lego, Denmark) so that the individual components could be conveniently attached toa Lego board. The connections between different tubes were made by small perspex blocks with conical holes so that the tubes could he fitted interlockingly. The mixing (gradient) chamber3 was machined from Perspexe, consisting of two parts, a Lower circular unit of 13mm inner diameter housing a Teflon%overed magnetic stirring bar 7 mm long, and an upper part with a dome-shaped inner cavity (see Fig. 5). The total volume of the chamher war 0.98 ml, the hole for the incoming solution beine drilled at the base of the lower nart and the outlet beine drilled in the center of the uoner oart. conicnllv The , . hoth holes heine n~~ -~ ,shaied. ~, chnrnbrr was attarhed ra 8 Leyo bond which was then placed on a rnagoetw stirring talh; i n prartae it na5 round rhac mixing was equally effective if the stirrer was operated anywhere in the range 60-240 rpm. The injection port was a precisely made rotary valve4consisting of two parts (Fig. 1):a Perspex house (a) and a revolving, interchangable Teflonmcore (2 andlor 3). Core 2 was furnished with avolumetric bore of constant volume (30 ul, i.e., a 25 mm long and 1.3 mm wide hole) while core 3 was machined in such a way that a piece of polyethylene tubing of any desired length and internal diameter could he attached to it thereby serving as a sample loop (L) of well defined volume (here 75-200&l).The house (1) was furnished with a bypass (B) of higher hydrodynamic flow resistance than the sample path (i.e., 20 cm 0.5 mm i.d. polyethylene tubing when used with core 2 and 125 cm of 0.5 cm polyethylene tubing-wound as a coil-when used with core 3). Thus, while the sample is being injected by a syringe (S) into the sample path (with overflow a), the carrier solution (h) is continuously bypassing the injection port through B to c. Only when the valve has been turned, thestream in the bypass comes toastandstill and the precisely metered amount of the sample is injected by the carrier stream from the sample path into the reaction line (c). The
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' ' Ruzieka. J. and Hansen. E. H.. Anal. Chim. Acto.. 99 (1978) . . 37.
Betteridge, D., Anal. Chem., 50 (1978) 832A. Ruzieka, J., Hansen, E. H., and Mosbaek, H., Anol. Chim. Aeta, 92 - - 11877) - - . , ,7% ---. &Ruzicka,J., Hansen, E. H., Mosbaek, H., andKrug, P. J., Anol. Chem. 49 (1977) 1858. \
Volume 56, Number 10, October 1979 / 677
sample lines (L)were made from polyethylene tubing of 1mm id., the "dead volume" of the core 3-consisting of the holes drilled in the Teflon* hlock-amounting t o 20 PI. Since the coefficients of expansion of the Perapex@and Teflon" terials are different. excessive temoerature chanees mieht lead to
Reagents All reagents were of AR grade. The distilled water used was degassed by means of a water pump in order to avoid formation of any air bubbles during the experiment. For the determination of phosphate, a solution of 0.005 M ammoniumheptamolybdate (6.1793 g/l) in 0.4 M nitric acid was used together with a 0.7 %aqueous solution of ascorbic acid to which was added 1% glycerine. The standards were prepared by successive dilutions of a 100 ppm phosphate standard solution. The reagent solution used far the determination of chloride was prepared hy dissolving 0.626 g of mereury(II)thiocyanate, 30.3 g of iron(III)nitrate, 4.72 g of concentrated nitric acid and 150 ml of methanol in water, making the final volume up to 1liter. The standards were made by suitable dilution of a stock solution containing 1000 ppm CI. For the titrationprocedure a 1.10-'Mpotassium hydroxidesolution, carbonate-free, was prepared from a 0.1 M Titrisol standard solution (Merck, West Germany) by further dilution with redistilled water, to which was added hromthymol blue in &10-90 concentration (1rnl per 500 ml base of a solution containing 0.4 g of bromthymol blue and 25 ml of 96% ethanol diluted to 100 ml with redistilled water). The hydrochloric acid standards in the range 8.0.10-3 M to 1.10-' M were also prepared from a Titrisol solution.
Using solutions containing 5,10,20, and 30 ppm CL (prepared by dissolving NaC1) we arranged the samples in 3 diagrams, one depicting a river which runs into the sea, another a delta, and one illustrating a fresh water reservoir into which seawater seeps through the bottom in the middle. The analytical procedure is based on the following reactions:
The manifold used is shown in Figure 2. (Instead of using this manifold, the phosphate manifold (Fig. 4) might be employed, simply by pumpm:&rier stream through hoth pump tubes.~l'hr earner st&, conrams HgrSVN,, nnu F r ~ l l i , Thechioridr . d t h c i n j r r t ~ sample d rrscts with Hr13'Kl.. .. lihrrntme S C X - nnirh in turn with FPIIIII forms t h r~r d - h m d complrx LC," Fw'~CN,Y',the intcnwt\ of which i 3 ~CIISU ~T pP r ~J ~ r c , ~ ~ h o t ~ ~at! ~480 ~ ~ nm. t ~ i The r n l neight lv of the recorded absorbance peak is then proportional to the concentration of chloride in the sample, cf. Fig. 3. Besides Fe(SCN)z+, other (higher) complex ions might he formed; thus, thecalibration curve cannot he expected to be linear over larger spans of concentrations. After having started the instruments, the students are asked t o exercise the injection technique (injected volume 30 pl), and prepare aqueous chloride standard solutions (5, 10,20,30, and 40 ppm CL), while the reagent solution is pre-mixed and ready for use. Then they are to calibrate the instrument (note the toxic waste t o he collected in a separate bottle), and finally run the samples, all injected in triplicate. The results are calculated on the basis of the calibration graph and plotted on the survey chart. The reproducibility of the method is estimated by calculating the standard deviation on the peaks obtained by injecting a 20 ppm C1 standard solution 10 times.
Exercises Determination of Chloride In this exercise the students are to analyze 49 chloride containing samples which are simulated to he collected in an area where seawater and fresh water mix. Each samole is numbered and depicted in a square survey area ehart, 7 times 7 samples. Thus, by drawing isomnrentration . ~ ~ .rmves on the chart the mixine characteristics can he dctrrmnwd. Senndtrr is told rc, c w m i n 15.000 p p m CI. the samples all hm.tng h e n prrhluted ,50(1 tinbps in g-rdrr tce .~commodarrthe analytical readouts ~
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Figure 3.Determination of chloride in the range 5 to 75 ppm Ci with the system shown in Figure 2. in order to demonstrate the reproducibilityof measurement. each sample was injected in quadruplicate. Injected volume 30 p1, sampling rate ~ 1 2 sampleslhr. 0 The fast scan of the 30 ppm sample (R3d and the 75 ppm sampie (Rlr) on the right shows the extent of the carryover (less than 1% ) if samples are injected in a span of 30 sec (difference between S1 and 5%).
~ i g u r e1. injectionvalve consistingof a statn (1) and a rotor (2 or 3).While rotor 2 has a fixed volumetric bore of 30 fi1 rotor 3 can be furnished with interchangeable sampie loops (L) allowing the injection of different volumes. The sample is introduced manually by means of the syringe S, the excess of sampie being drained through overtlow a. While in the sampling position the canier stream bypasses the rotor through the shunt (6).After turning the rotor to the injection position, the precisely measured sample zone is swept by the carrier stream into the system because the bypassconduit b s a higher hydrodynamic flow resistance lhen the sample path.
Figure 2. Flow diagram for the spechophotomehicdetermination of chloride. S, point of sample injection: W, waste: tube length in cm: tube internaldiameter in mm.
678 / Journal of Chemical Education
MOLYBDATE
0.6
ASCORBIC ACID
0.6
Figure 4 Flow amgram for lhe specnopnotomew8cdetermlnat M of phO~phate S, polnt of samp e nlect.on. W, waste t ~ o lengtn e ogwen n cm. wn le the t.W internal diameter is stated in mm
Flgdre 5 Manllold far spectropnolometrcac &base t waloon S palm ol sample mjen on. G mwng c u m o n om wncn a control ed a lut on gradlent at me mleeted Sample can be created. The output from the mixing chamber is monitored in a flow-through cell situated as closely as possible behind G.
Determination of Phosphate In this exercise the students are to analyze for phosphate a series of 30 20-ml aqueous fractions simulating the output from a ehromatographic column (range 0-25 ppm P-PO6 arranged in a more or less Gaussian type distribution pattern). On the hasis of the analytical results the total amount ofP-PO4 which was adsorbed on the column and the number of theoretical plates in the column are to he determined. The analytical procedure is based on the following reactions:
Lthe manifold used being shown in Figure 4. The carrier stream contains molybdate and ascorbic acid. Since a mixture of these two components is not stable, they are mixed in the system before the sample injection port. Phosphate forms with molybdate a heteropolyacid in which molybdenum can he reduced from oxidation state 6 to 5 with ascorbic acid farming a strongly blue-colored complex which can be measured speetrophotometrieally a t 660 nm. While the first reaction is fast. the second one is relstivelv slow: however. the nreeise timing of the low Injection system secuies that an eaactfrac%on of the heteropolyacid is reduced. Thus, the recorded ahsorhanee peak is proportional t o the concentration of phosphate present in the sample. Since the reagent solution itself exhibits a slight absorbance a t the wavelength used, it is necessary to determine the blank value (by injecting water). After start of the instruments, the students are asked to prepare aaueous nhosnhate standard solutions (0. 5. lo. 15. 20. 25. and 30 tr61A a i d-~ekhr&x+ ?-,~) . ~ -~~, , .-the instrument - ~ - ~lini'eked , ,~ v o l u k 30 ,uli. .The- 30~ samples are then each injected in triplicate and their contents calculated on the basis of the calibration graph. The totalp-PO4content may then be found either by summing up over all 30 20-ml sample fractions, or by measuring the area under the (Gaussian type) elution curve as obtained in a graph where the y-axis is concentration of solute (P-PO4) and the x-axis is effluent (ml, i.e., fraction number). The latter might be preferable since this graph has to be constructed anvwav ~~, , in order to determine the retention volume.. V.... and the width of t he elution curve at its haw. U', which two parameters are usrd 11, c&ulnte the numlwr of theoretical plate% ,t .n = 161V, LV+lnfwmation, which thestudcntz haw to find thcmrelws in the literature, e.g?j. The repraducihility of the method is determined as previously described, i.e., by injecting a standard solution 10 times and calculating the standard deviation. ~
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Titrationof Strong Acid with Strong Base In this exercise the students are t o analyze a series of samples of strong acid (HCI) by continuous titration with strong base (NaOH). The samples are simulated tooriginate from a process stream which has to be maintained a t a constant level. During a hreak-down period the acid concentration decreases, and hy analyzing 16 samples taken st regular intervals the normal and abnormal acid con~utrationshave to he determined (we prepared 13 "normal" samples of a concentration around 6.5.10-'M and 3 "break-down"samples of a concentration of -2.10-2M). Additionally the students have t o answer some questions related to the theory of the titration procedure. The quantitative determination of the acid concentration is based upon titration of the aeid (HCI) with base (NaOH), the acidsamples being injected into a Flow Injection system comprising a mixing chamber (Fig. 5) in which a controlled dilution gradient of the samples can be created. Ascarrier stream is used a l.0-lfl-3 M NaOH solution containing the indicator bromthymol blue. When an acid sample of sufficiently high concentration is injected, the indicator in the mixing chamber will undergo a color change from blue to yellow. Yet, by the continuous addition of carrier stream the aeid sample will he continuously diluted, and when its concentration has decreased to the level of the carrier stream the color will change to hlue aeain: " . that is. the color chanee will indicate the euuivalence point, and the time elapsed between the two color changes, t., will yield a measure of the concentration of the acid. The actual detection of the color changes is effected by continuous monitoring of the carrier stream in a flow-through cell a t 620 nm. Thus, in this particular ease it is the width of the recorded peak which yields the analytical readout, and as previously derived,3, it can he shown that t. (min) is given by the following expression:
v
t., =-In 10 log Co
- -v In 10 log C N ~ O H
(1)
where Co is the concentration of acid a t time 1 = 0 (i.e., a t that point
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SCAN Figwe 6. Titration of a stmm acid bv a shono base in the svstem shown in Fia. 5. wllh Oromthymo. blue as and catar Cond'llons are g "en n thetext. Sample concenlration. from left to rqhl: 8.10-~&?1.10-'M 2.10"t.t 4.10WM 6. 1WZM8.10 ' A t and 1.10-'MHCI. The equ,valence t me. &,ploneo u e r s ~ s log Co(MHCI)yields a straight line over the entire Concentration range
when all of the injected sample is situated in the mixing chamber); V is the volume of the mixing chamber (ml); and C N ~ OisHthe coneentration of the carrier stream. A plot oft., against log Co-as obtained by injecting a series of aeid standards-wdl thus yield a straight line with a slope of V/o In 10. Hence, on the basis of this calibration curve one can by measuring t,, of a given sample determine its initial concentration Co', which is related to Co (concentration of the sample in the chamber a t t = 0) according to: Co=- SvCd (2) v u is the volumetric flow velocity (mllmin);
where S v is the injected sample volume. Substituting eqn. (2) into eqn. (1) yields:
v
v
+v
t., = - In 10 log ~d - - In 10 log C N ~ O H- in 10l o g 3
v
(3)
Extrapolated tot,, = 0, eqn. (1) or (3) will directly yield the detection limit of the procedure. From eqn. (3) it furthermore appears that if samples of constant concentration (Co') but different volume (Sv) are injected into a carrier stream of fixed concentration (CN~OH). a plot of tW against log S v will yield a straight line, similarly of slope Vlu (In 10). After having switched on the instruments the students are asked t o prepare the acid standard solutions (8.0.10-3, 1.0.10-2, 2.0.10W, 4.0.10-2,6.0.10-2,8.0.10-2and l.O.lOWIM) and then run a calibration curve bv iniectine 125 ul of these standards. each in dunlicate (with the injekioh portrtfurnishedwith the rotor 3, kgure 1, the sample loop ( L ) being 16 cm of 1.0 mm i.d. polyethylene tubing). Measuring transmittance, the color changes of the indicator during the titration procedure-which will be from the hlue base color to the yellow acid color and back to blue again-will he registered as an increase and then a decrease in the recorded signal. All peaks will be of the same heieht, but the width will varv as a function of Cn.(and for constant ~ v , - o f~ d )Figure , 6 (asmall negative signal appears a t the endof the titration procedure which is due to a slight adsorption of the indicator in the acid farm on the walls of the system). In the calibration curve, tSqis plotted on the ordinate against log Coor log C d o n the abscissa. t., b i n ) is directly read off the recorder paper as the peak width b (inmml-measuring the peak width of all peaks at the same level, approximately half way between the base line and the top of thepeak-that is, b = t,.u,, where ci, is the recorder speed (mmlmin). After having determined o (-1.70 mllmin) by collecting the waste over a fixed time interval, the students are asked firstly to calculate thevolume of the mixing chamber (from the slope of the calihration curve) using eqn. (1) or (3). Furthermore, the detection limit is t o be determined (far,t = 0) and compared with the calculated value (eqn. (1) - (3)). The 16 samples are then injected, each once, and the normal and hreak-down levels determined. By means of a set of polyethylene sample loops of identical internal diameter (1 mm) hut of different lengths, that is, volumes (75, lM), 150, and 200 @I),the students are finally asked to inject samples of the 1.0.10-'M HCI standard (each sample twice) and prove that plotting t,, against log S v yields astraight line, cf. eqn. (3). (Note to the nominal values of the sample loop volumes are to he added the dead volume of the injection port, i.e., 20 PI.) Also V is to he calculated from this graph and compared with the previously found result. Fritz, J. S. and Schenk, G . H., "Quantitative Analytical Chemistry," 3rd Ed., Allyn and Bacon Inc., Boston, USA, Ch. 18. Volume 56. Number 10, October 1979 I 679
As in all the exercises, the system is washed thoruughly hrfure vlosr-d,mn by pumping disrilled water through it. Conclusion Besides the positive response from the students, our experience in the lab has been that during the course very little of sample and reagent solutions are used, because Flow Injection actually is a microtechnique. As the exercises are run in sessions of three successive days on a rotating basis, it is only on the first day of each session that intensive supervision is required. Because the readout is available within 15 see after sample injeetian-except for the titration procedure-an amazing number of analyses can be performed within a short span of time compared to manual techniques. It should he added
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680 / Journal of Chemical Education
that a number of more sdvaneed exercises has been developed, comprising new principles such as stop-flow, merging zones, solvent extraction and enzymatic assays. A fully automated system controlled by a microcomputer (PET) has recently been developed as part of an advanced research program. Acknowledgment T h e authors wish t o express their appreciation t o H a n s Mosbaek, Eva Thale, a n d Inge Marie Johansen of this Department and t o Jaap-Willem Hutter, visitor from t h e Technical University of Twente i n T h e Netherlands, for assistance i n setting u p and testing t h e Flow Injection exercises.
