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Anal. Chem. 1991, 63,1680-1685
Principles of Stopped-Flow Sequential Injection Analysis and Its Application to the Kinetic Determination of Traces of a Proteolytic Enzyme Jaromir Ruzicka* and Thomas Gubeli'
Department of Chemistry, BG-IO,University of Washington, Seattle, Washington 98195
The concept of sequentlal InJectIonanatyds is deacrlbed, and an appllcatlon of thls novel technique to an assay of traces of a proteolytlc enzyme Is demonstrated. The automated method Is based on a stopped-flow procedure wlth fluorescence detection. lnstead of using the conventbnal constant flow rate, a slnusoldal flow pattern, as generated by a robust cam-drlven piston pump, has been applied. The valldatkm of the novel flow lnjectlon system uslng a nonreactive fluorescent compound ylelded hlghly reproduclble results over a concentratton range of 3 orders of magnitude. The callbratkm graph for the enzyme actlvltles In the range 5-75 pu mL-' could be Rned a second-order polymnlal. The detecHon lknn Is 2.5 pu mL-l, which corresponds to 7.2 ng mL-' pure active enzyme.
INTRODUCTION The demand for mechanically simple and robust flow injection methodology, as advanced by the needs of process analytical chemistry, has been the driving force behind developing a sequential injection technique and its important component, the sinusoidal flow pump. While in a conventional flow injection technique (1)a sample zone is injected into a flowing carrier and auxiliary reagents are merged with it on the way to the detector, the sequential injection analysis (SIA) (2) is based on a different approach. By using a selector (rather than injection) valve, the wash solution, sample zone, and reagent zone(s) are sequentially aspirated into a channel (Figure 1). In this way, a stack of well-defined zones is obtained, which is then mixed in a preprogrammed way by flow reversal while being injected into a suitable reactor and detector. For the kinetic-based assay described in this work, a selected section of the mutually interdispersed reagent and sample zones is arrested within the observation field of the detector by stopping the flow. This allows measurement of the reaction rate during the stopped-flow period, where the changing signal reflects the rate of the chemical reaction (Figure 2). The benefit of the stopped-flow injection technique, elimination of interfering phenomena such as variable blank values, lag phases, and nonlinear sections of the reaction rate curves, has been documented previously (3-5), yet this work for the first time deals with SIA methodology with the aim of exploring its potential and limitations. The reasons for introducing the SIA technique are its advantages: (1) regardless of the number of solutions (sample, standard, reagent, and wash), SIA uses only a single valve and a single pump; (2) once configured, the components and associated flow channel do not need physical restructuring, since sample and/or reagent volumes are determined solely by the length of the piston stroke; (3) the degree of zone dispersion (sample and reagent intermixing) can be finely tuned by the length Present address: Department of Pharmacy, University of Bern, Baltzerstrasse 5, CH-3012 Bern, Switzerland.
and number of flow reversals, (4) the reaction time (inside or outside the detector) can be adjusted through the duration of a stopped-flowperiod. Finally, the SIA configuration allow us to exploit the advantages of a sinusoidal flow piston pump (6) (inertness, durability, reproducibility, and absence of check valves). The kinetic assay described below is of interest since there is a documented need for a sensitive, reliable, and automated assay of traces of proteases present as airborne particles, since these enzymes have a high allergenic potential to cause asthmatic reactions, when inhaled even at very low concentrations. While these hazards no longer exist for the consumer, since the detergent enzyme granules are surrounded by a protective coating, in the enzyme production, a method for monitoring of the working environment is still much needed. Therefore, the American Conference of Governmental Industrial Hygienists (ACGIH) has established a ceiling limit of 0.06 pg of enzyme m-3 for pure airborne dust (7). Although an automated, sensitive determination of traces of protease based on colorimetric detection and a preconcentration technique has been described in our previous work (B), the preconcentration step is too time consuming and the experimental setup is deemed to be too complex for implementation in a process environment. Therefore, SIA-based fluorescence monitoring has been chosen for this study, since it provides higher sensitivity and a wider dynamic range (9, IO). The fluorescent reagent used in the present paper was a tripeptide containing 7-amino-4-methylcoumarin(AMC) as a fluorophore. Similar AMC amides have been proposed for the sensitive determination of subtilisins (11),and therefore, the present work may serve as a model for their assay by SIA. EXPERIMENTAL SECTION Reagents. All chemicals used were of analytical grade. For evaluating the system parameters in the absence of chemical reactions, fluorescein as a marker has been used along with the following solutions. The wash solution was 0.05 M borate buffer of pH 8.5. The standard fluorescein (Sigma, St. Louis, MO) solutions were prepared by serial dilution of a 1mg mL-' stock solution of fluorescein sodium in the buffer above. For enzyme assays, a subtilisin from Novo-Nordisk, Copenhagen, Denmark (Savinase),obtained as a powder with an activity of 3.46 Novo internal unitsjg, was used. (One Novo unit equals 50 international units.) The enzyme powder contained 1%pure active enzyme. Standards covering the range 5-75 pu mL-' were prepared in 0.1 M Tris-HC1 buffer of pH 8.3 containing 0.01 M CaClz (Sigma) and stored at 4 "C when not in use. The wash solution was the Tris buffer described above, which was degassed [ before using. 7 - [ N - (a-Succinyl)-~-alanyl-~-alanyl-~-(phenylalanyl)]amino]-4-methylcoumarin(Bachem Bioscience Inc., Philadelphia, PA) was used as the substrate. A working solution (0.5 mM) was made by dissolving the substrate in NJ-dimethylformamide (Sigma) and by appropriate dilution with [ Tris-HC1 buffer. An alternative substrate, 7 - [ N -(a-methoxysuccinyl-~-alanyl-~-alanyl-~-prolyl-~-methionyl)]amino]-4-
methylcoumarin (Bachem)was not available in sufficientlyhigh stability and was therefore not used. All substrate solutions were stored at -4 "C.
0003-2700/9 110363-1680$02.50/0 0 1991 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1991
-
reagent (3) sample (2) wash (1)
WASH
S,
S
Valve
R
1
Zone Sequencing S , S
P
Zone Injection
R
*
Flgure 1. (top) Diagram of the sequentialinjection system, comprising a piston pump, holding coil (HC), reaction coils (RC1 and RC2), an eight-port selector valve (with four ports in use), and a detector (D). W is the waste and S, is the sample aspiration line. (bottom) Schematic diagram of the sequenced zone structure and of the mutual zone penetration during and following flow reversal and injection downstream toward a detector. S,, sample carryover: S, sample zone; R, reagent zone; PI product to be monitored.
Fl0.w Direction
Valve Position
t
-m Fn
z
START!
TIME
___)
Figure 2. Overlay of three stopped-flow response curves, obtained by injecting 25, 50, and 75 mu of enzyme/mL, and by chart recording the analog output of the fluorometer. Synchronization of the piston and valve positions during reversed (REV) and forward (FWD) piston travel along with data collection during a stopped-flow interval (STOP) is controlled by means of a computer and appropriate software. Sample is loaded with the valve in position 2 and substrate in position 3. Note that data collection occurs during the 25-s period at the end of the stopped-flow interval.
Instrumentation and Procedures. The sequential injection manifold as shown in Figure 1was constructed from the following components: a dual-piston,computer-controllable, sinusoidal flow pump (Alitea, US, Medina, WA); an eight-port electrically actuated selector valve (ModelCSD8P, Valco Instruments, Houston, TX); and a Hewlett-Packard 1046A spectrofluorometer with a xenon-arc lamp, equipped with a 5-pL quartz flow-through cell, which was modified by replacing the inlet and outlet stainless steel tubing with 0.5"-i.d. Teflon tubes to minimize flow resistance. The cell compartment was thermostated at 26 "C. The tripeptide substrate used is highly fluorescent, as is its hydrolytic (AMC). The excitationand product, 7-amino-4-methylcoumarin emission spectra overlap to a certain extent so that at the chosen substrate level of 0.5 mM the fluorescence background is too high when the measurement is performed at the maxima of the AMC spectra (A,, 342 nm; A,, 438 nm). However, if excited at 370 nm and measured at 470 nm, the background becomes acceptable, since at these wavelengths the relative fluorescence intensity of AMC is 850-fold higher than that of the substrate. Also, a cut-off
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fiiter of 408 nm and a lamp flash frequency of 110 Hz were used. The analog output from the fluorometer with a full scale of 1V was digitalized at a rate of 10 Hz with 12-bit resolution by the analog-to-digitalconverter of a data acquisition and control interface (Lab Data Systems, Seattle, WA). An IBM compatible lap-top computer (Toshiba 3100SX) with software for data acquisition and pump and valve control was linked to the flow system by using the above interface. The selector valve was fitted with two reaction coils (RC1 of 0.81 mm i.d., 120 cm long; and RC2 of 0.50 mm i.d., 50 cm long) and a holding coil (HC of 1.32 mm i.d., 100 cm long), made of Teflon tubing. These dimensions were selected by considering that the combined sample and reagent volume should not exceed 25% of the stroke volume of 2 mL. The holding coil, having a volume of 1.4 mL, serves as a buffer, which prevents sample from entering the syringe. This apparently large volume is needed, since even prior to flow reversal the combined stacked up sample/reagent zones may, due to the parabolic profile of the injected zones, occupy twice as much as the originally injected volumes. The reaction coil 1, having a volume of 600 pL, is designed to accommodatethe entire reagent zone and the non-cross-contaminated sample zone. Finally, reaction coil 2 was kept as short as physically possible (100 pL), given by the distance between the flow cell and the pump. Since the combined volume of the two reaction coils was approximately one-third of the available stroke volume, complete washout of the flow channel was guaranteed. The measuring cycle (Figures 1and 2) comprised the following operations: (1) aspiration of carrier/wash solution with selector valve in position 1,while the piston made a large reverse step; (2) aspiration of sample solution with selector valve in position 2, while the piston made a small reverse step; (3) aspiration of reagent solution with valve in position 3 using a small piston reverse step; (4) forward piston move with valve in position 4 with the stroke length adjusted such that a preselected section of the interdispersed sample/reagent zone was transported into the detector; (5) stopped-flow period for reaction rate monitoring; and (6) forward large piston stroke that expelled all sample, reagent, and carrier/wash solutions from the valve and from the flow channel. Sequential sampling differs from the conventional sample injection, as performed by means of a two position injection valve, in several respects. First, the injected sample volume is controlled by the length of the reverse piston stroke. This results in flexibility in selecting the injected volume, in contrast to valve injection, where the sample volume can be changed only by reconfiguring the sample loop. Second, a sample carryover will occur any time the next sample is aspirated, since the portion of previously aspirated sample left in the sample aspiration line (Sc,Figure 1) inevitably is introduced into the channel. Besides minimizing this volume by keeping the aspiration line (S,) short and narrow, several techniques can be used to avoid this problem. Of these, two have been used in this work (1) in experiments with fluorescein marker, the cross-contaminated sample zone was expelled to waste through the auxiliary waste line; (2) in enzymatic assays, a sufficientlylarge sample volume was aspirated, so that the element of sample fluid selected for the reaction rate measurement was situated in the uncontaminated (frontal) section of the dispersed sample zone. The sinusoidal flow pump (6) is a cam-driven piston pump (Figure 3A) that generates a variable flow rate that can be calculated from the equation Q = 2R?r2r2vsin a. The flow rate, Q, is therefore dependent upon the radius of the cam, R, the radius of the piston, F, the frequency of the pump in hertz, Y, and the angle of rotation, a. The pump prototype used in this work had the following parameters: R = 15.4 mm, F = 5.74 mm. With a cam rotation speed of v = 4.04 X Hz, correspondingto a pump setting of 30, the maximum flow rate is 2.40 mL/min, while the full load/discharge cycle, which is 360' of continuous cam turn, lasts 4.13 min. Such a load cycle is, however, not practical to use because it encompasses too wide a range of flow rates and lasts too long. Also, since a stopped-flow interval has to be executed for the reaction rate measurement, the cam movement must be computer controlled and is therefore easy to operate discontinuously and even in the forward/reverse direction within a selected arc. In the present pump configuration, cam movement within the arc of 30-142' yielded a stroke volume of 2.0 mL. Thus, by
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1991
A
A
270"
syringe 180"
-t .-cnKcn
H 100 sec E
90"
Q=2Rn2r2vsina C
B
Cam Position (degrees)
+ 2.40
Scan + Figure 4. (left) Peak profiles obtained by injecting in triplicate a 1 ppm (A), 0.5 ppm (B), and 0.1 ppm (C) fluorescein sodium solution. (right) Peak profile recorded on an expanded time scale, whereas the baseline during the aspiration step (1, Figure 2) is not shown. I n these experiments, the cam was moving between 30 and 142' in reverse (aspirate) and forward (inject and measure) directions, without a
stop-flow interval.
Time Figure 3. (A) Operational principle and main parameters of the sinusoidal flow pump. Q , flow rate; R , cam diameter; r , syringe diameter; v, frequency: a,angle of rotation. (B) Modified sinusoidal flow pattern as used in this work (for details, see text).
starting the cam at 30' in the reverse mode (Figure 3B), 1.6 mL of wash solution was aspirated within 46 s, while the distribution valve was in position 1 (Figure3A, lined area). The cam movement was stopped and the valve was switched to position 2. The reverse motion of the piston was resumed for a few degrees until 230 p L of sample solution was aspirated (Figure 3A, clear segment). The cam movement was stopped again, the valve was advanced to position 3, and cam movement in the reverse direction was resumed to aspirate 160 p L of reagent solution (Figure 3B, hatched area). Thereafter, the pump was stopped, the valve was switched to position 4, and forward movement of the cam, which was at 142' at this point, was initiated, so that the interdispersing zones moved toward and into the detector. The flow was then stopped for 40 s while the reaction rate was measured. Finally, the forward cam movement was resumed until the starting point of 30' was reached. At that point, all of the reaction mixture and the originally aspirated wash solution had been expelled to waste. The total analysis time was 160 s. There are two important details of this procedure. First, a relatively large sample volume of 230 pL has to be aspirated in order to avoid the contribution of the cross-contaminatedsection of the sample zone (S,,Figure l),which was approximately 30 pL, to the reaction rate measurement. Second, it is important to program the pump in such a way that it is stopped whenever the valve is being turned; otherwise, a pressure buildup and ensuing flow surge will adversely affect the reproducibility of the measurement. The software used to control the pump and valve movement allowed this synchronization to be made, by programming all events on a time basis given by the computer clock. However, each analysis cycle was terminated individuallyby an internal pump mechanism, which sensed when the cam was at the starting position of 30'. This technique prevents any timing errors that otherwise might accumulate during a long series of measurement. RESULTS AND DISCUSSION Synchronized injection of sample and reagent zones followed by flow reversal and subsequent highly controlled movement of the interdispersing zones into the detector is a necessary condition for the reproducible execution of SIA. In order to design a proper system configuration and validate the physical performance of the sequential injection apparatus, a nonreactive compound, fluorescein, was used. Fluorescein sodium standard solutions in the concentration range 0.01-10 ppm,
prepared in 0.05 M borate buffer of pH 8.5, were injected in triplicate by using the same buffer as a carrier/wash solution. The excitation wavelength was 488 nm and the emission wavelength 510 nm. The reproducibility of these injections based on peak height was (ppm/RSD) 0.01/0.97%,0.1/0.67%, 0.5/0.75%, 1/0.51%,5/0.26%,and lO/O.43%. The resulting calibration curve was linear over the whole range of injected standards (r = 0.998). An example of triplicate injection of three standards (Figure 4, left) is shown along with a peak profile recorded at a broader time scale (Figure 4, right), to document that the SI peaks do not differ from those obtained by classical flow injection. These results show that the sequential analyzer is able to perform sample injection, zone dispersion, and its transport from the injector to the detector in a highly reproducible manner. Implementation of the stopped-flow method requires a high repeatibility of capturing a selected portion of the interdispersed zones within the detector. This is accomplished by controlling the length of the stop-delay time, during which the zones intermingle and move from reactor coil 1, through the valve, and through reactor 2 into the detector. To determine such a delay time and the reproducibility of the zone positioning, the profiles of the overlapping zones were investigated by injecting a 0.01 mM AMC solution, alternately as "sample" (S) and as "reagent" (R), by using Tris buffer (0.1 M, pH 8.3) as the carrier/wash stream. The individually recorded curves were overlaid as shown in Figure 5. The AMC solution, which serves in this experiment as a marker dye (fluorescein could have been used as well), shows how the injected reagent and sample will be spread during the passage through the flow channel and to what degree these two zones will overlap. Analogous to double-injection FIA (I) an isodispersion point (ID)is observed, for which DA = DR (where DA = CoA/CA and DR = COR/CR, D being the dispersion coefficient, Co the concentration of the analyte (A) and of reagent (R) in the originally injected solutions, and C the concentration in the element of fluid corresponding to the isodispersion point). Thus, if the element of fluid corresponding to IDis arrested in the detector, the reagent/sample concentration ratio is the same as the one of the solutions originally injected. Therefore, if (?R/@s was for example 101, the same reagent/sample ratio will be maintained a t the isodispersion point. Then, considering the curves of Figure 5A, a t time tl, the reagent/sample ratio would be 201 since the response of the R curve is twice the response of the S curve at that point. Conversely, at time t5,only a 2 5 reagent/sample ratio will be obtained, even when the injected reagent is 10
ANALYTICAL CHEMISTRY, VOL. 63,NO. 17, SEPTEMBER 1, 1991
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Table I. Reproducibility of the Measurement of Reaction Rates delay time, s
method A reaction raten RSD,b ?& 6.1 3.1 3.5 2.0 6.4
976.3 1132.5 900.5 654.3 336.1
111 113 115 117 119
corr coeffe
delay time, s
0.9993 0.9993 0.9989 0.9994 0.9990
73 74 75 76 77
method B reaction raten RSD,b % 736.7 798.8 744.5 542.4 372.4
corr coeffe
3.4 1.2 3.9 0.7 0.9
0.9998 0.9995 0.9999 0.9998 0.9997
a Fluorescence units per minute, mean of three repetitive measurements. n = 3. Mean correlation coefficient (r) of linear regression of reaction rate cruves, n = 3.
A
t
-
s
c m
w
Time
t
A
-
I -
c
cn
w
.-s
-
-a
-
E
-
L
4
u)
Time
-
Flgurr 5. Overlap of reagent (R) and sample (S) zones as simulated by sequential injections of AMS solutions of equal concentration, showing the suitable thne zones (t,-t6) surrounding the Isdispersion point I, (for details, see text). Note that the sample peak S, which is subjected to a larger flow reversal, is more symmetrical than the reagent peak R.
times more concentrated than the injected sample solution since the response of the S curve is 4 times as much as of the R curve at that point. The benefit of this gradient interplay is that by selecting a certain delay time the enzyme/substrate ratio can be finely tuned via computer control-in analogy with the conventional stopped-flow FIA technique. The question is, however, whether the steepness of the sample and reagent profiles allows a reproducible reaction rate measurement to be performed. To answer this question, two systems were configured system B, as described above and mainly used in this work, and system A, which had a larger internal volume (RC1 i.d. = 0.81 mm, L = 100 cm; RC2 i.d. = 0.81 mm, L = 35 cm; cam start at 60") and which was operated at a lower flow rate. A comparison of corresponding curves obtained with the fluorescent tracer (0.01 mM AMC solution) for system A (Figure 5A) and system B (Figure 5B) shows much broader peaks and a wider range of t values for system A, which then should presumably yield more reproducible results. Having thus experimentally established the range of time delay values (tl-ts) that will yield useful reaction rate measurements in both system configurations, a series of reaction rate curves was recorded with the aim of optimizing the enzyme substrate ratio by identifying an optimum time delay value. Therefore, by using Tris buffer (0.1 M, pH 8.3) as the carrier/wash solution, 0.5 mM 7-[N-[(a-succinyl)-~-alanyl-~-
e
13
,/"
0
14
,'
0
E
H
-
20 sec
a Y ,
.
.
.
.
Time
.
.
.
.
.
4
Flgure 8. Effect of different delay times t , - t , on the slope of the reaction rate curves, as caused by varylng the substrate enzyme ratbs (for details, see text).
alanyl-~-(phenylalanyl)]amino]-4methylcoumarinin the same buffer was used as the reagent, and a 50 pu mL-' enzyme solution as a sample, a series of curves with increasing delay times tl-t5 was recorded. In each of these experiments, the intermixed zones of the sample and reagent were allowed to settle for 15 s after reaching the detector, and then data were collected for 25 s (Figure 2). After the stopped-time period of 40 s had been completed, the system was flushed with the carrier/wash solution. The reaction rate curves obtained with both systems were overlaid as shown in Figure 6, and the reproducibility of reaction rate measurements is summarized in Table I. The reaction rates were calculated according to zero-order kinetics by linear least-squares regression of data obtained during the fixed time interval of 25 s during which 250 data points have been collected. System A gave the highest values by stopping the flow at 113 s of time delay, whereas for system B a delay time of 74 s yielded the highest reaction rate. These results showed that the performance of the system was not improved by using a method with broader zones and 13% lower flow rates. Calibration curves for low enzyme activities covering the range from 5 to 75 pu mL1were obtained using system A and a time delay of 113 s. Standards were prepared in Tris buffer (0.1 M, pH 8.3) and injected in triplicate. All calibration
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1991
graphs were exponential and could therefore be fitted with a second-order polynomial (r = 0.9995). Plotting the square root of the response values versus enzyme activity gave a linear calibration graph with a correlation coefficient of r = 0.997. The precision was 5.9% for a 75 pu mL-’ standard and 6.4% for a 5 pu mL-’ standard (n = 3). The detection limit, defined in the usual way as 3 RSD values above the background noise level, was 2.5 pu mL-’ corresponding to 7.2 ng mL-’ pure active enzyme. Finally, the Michaelis-Menten constant was determined by varying the substrate concentration and using a 25 pu mL-’ enzyme solution. Substrate standard solutions in the range from 0.25 to 2 mM in 0.1 M Tris buffer of pH 8.3, containing 10% DMF to increase substrate solubility, were injected in duplicate. By using a Lineweaver-Burk plot, the Michaelis constant, K,, corrected for dispersion, was calculated to be 0.557 mM. The linear regression equation had a correlation coefficient of r = 0.999, This K, value is comparable to those obtained with other AMC substrates ( l l ) where , it was found for Z-Gly-Gly-Leu-MCA BPN subtilisin K, = 0.55 mM and for ZGly-Gly-Leu-MCA Carlsberg K, = 0.65 mM, the overall range of K, values for fluorogenic substrates being 0.55-1.8 mM depending on the sequence of amino acids to be hydrolyzed. The reasons for selecting the position and 25-5 duration interval on the SIA response w e should be briefly dimwed. Its selection was based on investigation of the individual sections of the response curve for linearity, by collecting 10 data pointa/s throughout the entire measurement period. This approach is consistent with the one adopted in the previously published stopped-flow injection reaction rate method (3), where a selected portion of the FIA response curve was used to measure the reaction rate constant of the well-studied chemical reaction of oxidation of crotonic acid by permanganate. It was established a t that time that the radial mixing of sample and reagent is complete within 150 ms, yet it is necessary to let the carrier stream relax for a few seconds after the pump had been stopped to avoid influence of liquid rearrangement within the flow cell. Concentration gradients within the flow cell would, of course, distort a proper rate law behavior. However, by identifying the linear section of the reaction rate curve, which is observed shortly after the flow has been stopped, and by using only data collected within that section, a very good agreement between the published values of reaction rate constants, as obtained by a classical batch procedure, with those obtained by flow injection was confmed. The value of the previous FIA work on permanganate oxidation is that it serves as a reference method for the SIA-based reaction rate measurement conducted in this work, since the conditions for sample/ reagent mixing are similar because channel dimension flow rates and flow cell volumes are of the same order of magnitude and the obtained response curves have similar characteristics. Further evidence of completeness of radial mixing can be gleaned from comparison of peak shapes obtained in the present study and in the FIA stopped-flow reference method (3). Due to the flow reversal inherent in the SIA technique, the peak shapes obtained here (Figures 5 and 6) are approaching the Gaussian form, which according to the tank-in series model indicates that the elementa of fluid passed through at least 10 mixing stages. The result of such an intense mixing process along with the use of a large excess of substrate (AMC) assured that only about 1% of the substrate present in the flow cell has been enzymatically converted during the 40-5 stopped-flow interval at the time delay intervals selected here. Therefore, it can be safely assumed that zero kinetic conditions existed and Michaelis-Menten formalism was applied. An additional proof of this is the observation that the reaction velocity when
plotted in double-reciprocal form (Lineweavel-Burk) yielded a straight line. Considering that injection of a fluorescein tracer also yielded a strictly linear response, it may be speculated that slight nonlinearity of the calibration curve for enzyme content is due to the presence of enzyme impurities of another class that are also capable of cleaving the substrate, yielding a fluorescence product other than the molecules of AMC.
CONCLUSION This work confirms the applicability of SIA for reactionrate-based assays and highlighta the mechanical simplicity and versatility of this novel technique, since the degree of mixing and the length of the reaction time as well as the sample and reagent volumes may be changed without physical reconfiguration of the flow channel by means of programming the piston and valve movements. On the negative side, SIA yields a lower sampling frequency than conventional flow injection methods and requires specialized, moderately sophisticated software to control analyzer functions and data collection. The repeatability of the sequential injection technique, as demonstrated by experiments with fluorescein and by reaction rate measurements (Table I), is comparable to or even better than conventional flow injection methods. Although this is only the second work dealing with the principle of SIA, its relation to conventional flow injection methodology is emerging. Though the injection and propulsion components used are quite different, the underlying principles remain the same: well-defined sample and reagent zones are injected and merged, the reaction products are formed within well-defined areas of composite concentration gradients, and the transient signal yields reproducible analytical results. This is why previous experience with stopped-flow FIA-based kinetic assays applies also to SIA. One may ask why SIA, a seemingly straightforward extension of FIA, has not been tried before. First, injection of a sample solution by means by a “dead-end” selector valve posed a problem of material carryover between different samples. Second, the mutually interdispersed sample/reagent concentration gradients must be captured within the detector with a high degree of reproducibility. Third, the precisely synchronized sequencing can be reproducibly executed by computer control only by means of a specifically designed software. Finally, although any single channel computercontrollable pump could in principle be used for SIA, the use of a piston device is preferred since piston pumps deliver solutions more reproducibly than peristaltic pumps. The novel configuration of a piston pump used in this work-a camdriven sinusoidal flow generating pump-has been shown (6) to provide solution delivery with the repeatability necessary for the present purpose. For a rational design of the sequential injection analyzer, the degree of sample dispersion must be considered as the main design guideline and much fundamental work remains to be done in this area, both experimentally and theoretically since the sequential injection method is more complex than the conventional flow injection. The mathematical model of zone merging during the flow reversal does not exist nor has it been investigated experimentally. The reaction product is formed at a reagent/sample interface and therefore the monitored peaks appear narrower than the composite samplelreagent zone is. For this reason, we can expect that the change of injected volumes will af€ect the response curve shape more profoundly than has been observed in conventional flow injection. Future work should therefore be aimed a t understanding better the complex kinetic processes taking place within the flow channel, in which stacked zones are subject to mutual interdispersion while they react. Ultimately, a set of rules will emerge that will allow more efficient optimization
Anal. Chem. 1991, 63, 1685-1691
of system parameters, rather than by the trial-and-error approach adopted in this study. Being completely computer controllable and compatible, SIA should lend itself ideally to the simplex optimization studied.
(6) Ruzlcka, J.; Marshall, G. D.; CMsUan, 0. D. Anal. Chem. 1000, 62,
1861. (7) American Conference of Governmental Industrlal Hygienists. We&old Llmlt Values and Bbkglcai Exposure Indlces; Clnclnnatl, OH, 1988. (8) Well. T.; Ruzicka, J.; Christian, G. D. Telenta, In press. (9) Z a b u , K.; Yamaglshi, K.; Ohkura, Y. Chem. phann. Bull. 1088, 36 (1I), 4488. (10) Kelly, T. A.; Christian, G. D. Anal. Chem. 1082, 54. 1444. (11) Kenaoka. Y.; Takahashl, T.; Nakayama. H.; Tanlzawa, K. Chem. phann. BUN. 1085, 33(4), 1721.
ACKNOWLEDGMENT Cecille Grataloup's technical assistance is gratefully acknowledged along with G. D. Christian's kind interest in this project. Registry No. Subtilisin, 9014-01-1. LITERATURE CITED Hensen, E. H. Fbw In/ecmOn A n a m , 2nd ed.: Wlley 8
Ruzlcka, J.; Sons: New York, 1988. Rurlcka, J.: Marshall, G. Anal. Chlm. Acta 1090, 237. 329. Hungerford, J. M.; Christian, G. D.; Rurlcka, J.; Glddlngs. J. c. A M / . Chem. 1085, 57, 1794. Masoom, M.; Wwsfokl, P. J. Anal. Chlm. Acm 1086, 179, 217. Monde. H. A. K ~ H C o ~ ~ ~ ~wileyla Sons: ~ New York, 1988.
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RECEIVED for review February 20,1991. Accepted May 30, 1991. We express our gratitude to Novo-Nordisk,Denmark, for supporting this project by purchasing necessary materials and reagents and to the National Science Foundation of Switzerland for funding of T.G. during his stay a t the University of Washington, as well as to the National Institute of General Services (Grant No. SSS-6 (b) 1R43 GM b y Medical ; 45087-01) for partial financial assistance.
Factor Analysis of the Thermogravimetry of Rubber Blends by Singular Value Decomposition and Variance Minimization C. H.Lochmiiller* and S. J. Breiner Department of Chemistry, Duke University, Durham, North Carolina 27706 M. N. Koel
Institute of Chemistry, Academy of Sciences of the Republic of Estonia, Tallinn, Estonia, USSR 200026
M . A. Elomaa Department of Polymer Chemistry, University of Helsinki, Meritullinkatu l A , SF-001 70 Helsinki, Finland
Thermogravlmetry of a wries of rubber blends and mixtures of chkroprene rubber (CR), MclClkneacryknlbRe rubber (NBR), and common rubber addnlves was performed under fbwlng He from 200 to 550 OC/mIn. The resuits were analyzed by udng factor analytical methods. Singular value decompodilon of the derivative data (DTG) matrix Indicates thai, upon bbnding wlth even small a"tsof NBR, CR loses most of its dlsiinctlve thermal character, leaving a blend conslstlng partially of native NBR and a composite species unlike either of the two siartlng materials. Removal of NBR and CR Influences from the curves was accomplished by udng a constrained variance mlnlmlzatlon procedure. The r e d i s are consistent W h an argumeni that new species are generated during blending.
INTRODUCTION Applications of thermogravimetry (TG) are quite varied, due, in part, to the conceptually simple nature of such determinations. The resulting data are correspondingly simple, though the processes leading to the changes in mass of the sample may be extremely complicated. It is in the interest of economy to limit this introduction to the approaches common to elastomers and especially to rubbers. Practitioners of TG in the rubber industries have found many uses for it including the determination of thermal 0003-2700/91/0363-1685$02.50/0
properties, investigations of reaction kinetics (e.g., ref 11, and quantitation in systems where the composition is approximately known and an assay is desired. Typical quantitative uses include the determination of moisture, volatile organics, oil, filler, and plasticizer concentrations (2). In such determinations, the material is typically heated at a preprogrammed rate (ca 20-150 OC/min) and the desired quantity is calculated from the mass loss corresponding to the pyrolysis (or evaporation) of the component of interest. This approach is recognized as simple and reasonably accurate, and the use of TG in compositional determination recently has been the object of standardization by the ASTM (3). Certain determinations, such as that for carbon black, depend on the ability to modify the atmosphere during the TG run (e.g., refs 4 and 5). A persistent problem in TG and particularly with polymer samples is reproducibility. The many known influence on TG response and the difficulties with their control are reviewed elsewhere (6-9). The use of samples finely divided with a cutting blade seems to alleviate many of these problems (IO). In the work reported here, sample sizes of 1-2 mg were found to be optimal for reproducing TG features. The question addressed in this paper is whether TG curves can provide more than simple, bulk-compositional determinations-i.e., whether information about structural details of the elastomers is reflected in such curves. If TG curves do contain such information, it might be revealed when the results are viewed by using multivariate techniques in the proper context. If, for example, one is interested in subtle 0 1991 American Chemical Society
