An Automated Fast Reaction-Rate System for Quantitative Phosphate Determinations in the Millisecond Range A. C. Javier, S. R. Crouch,' and H. V. Malmstadt Unioersity of Illinois, Department of Chemistry and Chemical Engineering, Urbana, 111. 61801 A new approach has been developed for precise and accurate quantitative determinations of phosphate with analysis times in the millisecond range. The method utilizes a completely automated stopped-flow system with direct digital readout of initial reaction rates to measure the initial rate of formation of 12molybdophosphoric acid from phosphate and Mo(VI) in acid medium. This rapid reaction yields digital rate data proportional to the phosphate concentration about 100 milliseconds after the reagents are automatically mixed. The phosphate concentration from 0.1 ppm and up can be read out directly on the digital meter or a printer. With the automatic sampling system to deliver phosphate samples to the mixing chamber several thousand samples or standards can be analyzed per hour. An average of ten results can be read out in about 10 seconds, including all sample handling, and the relative standard deviations of the averages are less than 1%. Results are presented to demonstrate the relative freedom from interferences, the high sensitivity and the general applicability of the technique.
STOPPING
TO D R A I N
I TO V
PM
DIGITAL CONTROL RATE METER SYSTEM A
M I X I N G CHAMBER
1 I
SAMPLE
$, I I
DOUBLE 3-WAY STOPCOCK
I - STOPCOCK MOTOR
I I
REAGENTS
I I
I
ANALYTICAL METHODS which utilize the measurement of the initial rate of a chemical reaction have gained much popularity in recent years. Automated instrumentation ( I , 2 ) has made possible the precise and accurate measurement of initial rates on a routine basis. For analytical reactions which are very slow or which have unfavorable equilibrium constants, the advantages of the kinetic approach are obvious. Quantitative initial rate measurements can be obtained in a matter of seconds even on reactions which take days to go to completion. In addition to shorter analysis times there are other important characteristics of reaction rate measurements which suggest that initial rate methods should be advantageous even for rapid analytical reactions which go to completion in a matter of seconds or less. For example, initial rate measurements can often be made very quickly after the reaction is initiated, before slower interfering reactions have begun. Hence, even for very rapid reactions the advantage of kinetic differences can be taken to achieve analytical selectivity and to eliminate interfering substances. One of the most important characteristics of the reaction rate method is that it involves a relative measurement. The absolute value of the parameter (absorbance, cell potential, fluorescence, etc.) chosen to monitor the reaction, does not have to be measured accurately. It is only necessary to measure the rate of change of the parameter with time accurately. Hence, even for very rapid reactions, the reaction rate method can offer freedom from those interferences which contribute to the absolute value of the parameter (turbidity, dirty cells, junction potentials, other fluorescing materials, etc.), but which do not enter the chemical reaction and do Present address, Department of Chemistry, Michigan State University, East Lansing. Mich. (1) G. E. James and H. L. Pardue, ANAL.CHEM., 40,797 (1968). (2) E. Cordos, S . R. Crouch, and H. V. Malmstadt, ibid.,p 1812.
I 2 - M L SYRINGES
I
L Y
SYRINGE DRIVE MOTOR
MICROSWITCH
Figure 1. Schematic diagram of automatic stopped-flow system not contribute to the rate of change of the parameter with time. A new approach to the determination of phosphate based on a reaction which is complete in seconds is presented here. The rate of formation of 1Zmolybdophosphoric acid from phosphate and Mo(V1) is measured with a completely automatic spectrophotometric stopped-flow system with digital rate readout. The measurement time is typically 0.3 second and the total analysis time about 0.7 second. For high precision and accuracy an average of ten results can be read out in about 10 seconds, including the cell flushing between samples. Standard deviations of the averaged ten results are less than 1
z.
MEASUREMENT SYSTEM
A block diagram of the automated spectrophotometric stopped-flow system is shown in Figure 1. The basic sample introduction system is based on a high-precision rapid-injection and automatic refill pipet (3). The sequence of operations is begun either by manually pressing the start button or by automatic programming. The unit can be used for single reactions or can operate continuously. Sequence of Events. Activating the start cycle causes the stopcock motor to turn the double three-way stopcock 90' clockwise so that the sample and reagent solutions can be drawn into the system. As soon as the refill tubes are opened, (3) H. V. Malmstadt and H. H. Pardue, ANAL.CHEM.,34, 299
(1962).
VOL. 41,NO. 2, FEBRUARY 1969
0
239
Figure 2. Typical reaction rate curves for phosphate and Mo(V1) in acid solution C H N O=~ 0.5714, Cno(v1, = 2.0 X 10-2M, and CPO,~-= 4.0 X 10-4~
the drive motor turns the cam counterclockwise one-half cycle and the spring-loaded syringe plungers follow, thereby drawing sample and reagents into their respective syringes. The cam is set so that the stroke of the 2-ml syringes fills each syringe with 0.25 ml of new solution. The stopcock then turns in a counterclockwise direction opening the path of the syringes for delivery to the mixing chamber. Then the drive motor turns the cam clockwise one-half cycle which rapidly drives the 0.25 ml of each solution out of the syringes and the solutions are forced into the mixing chamber and observation cell. The system can be used with two modes of stopping the flow of solution. For continuous and completely automated operation, a check valve connected to the drainage port operates to open at a certain pressure. The flow of the solution through the observation cell causes a build-up of pressure in the system which opens the check valve. In this mode, the syringe plunger rests against the stop bar and is nonoperative. At the end of the drive cycle for introducing the reagents, the check valve rapidly closes which stops the flow in the observation chamber. As soon as the measurement is complete, the cycle can automatically begin again, or the system can be operated manually by the start button if only one reaction is desired. In the second method of halting the flow, the solution from the mixing chamber flows through the observation cell into the stopping syringe and the flow is rapidly stopped when the stop-syringe plunger hits the stop block. Release of waste solution is then done by operating a valve manually. This is suitable for single reactions. Sample System. The sample pick-up system is a motor driven turntable which rotates the sample beakers into position under a dip tube of polyethylene. The dip tube raises for the sample cell to move into position and then lowers to draw in sample. The sample system can be operated manually or programmed to operate in synchronization with the stopped-flow system. An entire cycle of the apparatus takes less than one second. Flow System. The flow system consists of the reagent sample syringes, the syringe drive and stopcock motors, the mixing chambers, the observation cell, and the stop-valve assembly. The several important characteristics of the flow system that influence the premeasurement time are discussed below. 240
ANALYTICAL CHEMISTRY
FLOWVELOCITY.To achieve adequate time resolution, the flow velocity through the mixing chamber and observation cell must be rapid enough to ensure efficient mixing, and the flow must be stopped as suddenly as possible. In this system the flow velocity is determined by the speed of the syringe drive motor and the impedances to liquid flow throughout the system. The drive motor and stopcock motor have been described previously (3). By using a 72-RPM motor to drive the syringes, a linear flow velocity of approximately 1 m/sec was obtained. Although this flow rate is satisfactory for the reaction system under investigation, faster reactions would require a higher flow velocity. The lower velocity is sufficient, however, to ensure that turbulent flow occurs through the mixing chamber. The sample and reagent syringes are 2-ml gas-tight syringes. MIXINGCHAMBER,The mixing chamber must provide fast and efficient mixing of the two solutions. A two-stage mixing chamber based on the design of Gibson and Milnes ( 4 ) was used in this work. The dead time of the system, which is the time interval from the initial contact of the solutions in the mixing chamber to the point at which observation can be made, depends on the efficiency of the stopping device and the flow velocity. To demonstrate the combined effect of these factors, optical studies of an acid-base reaction with phenolphthalein indicator were made which showed that complete replacement of solution in the observation cell and attainment of equilibrium transmission are complete in 20 milliseconds. In this system, the observation cell was located one cm from the end of the mixer. OBSERVATION CELL. The observation cell should be of small volume to minimize over-a11 dead volume in the system. However, the observation cell must be of sufficient path length to provide the necessary sensitivity for the spectrophotometric detection system. In this system, the 2-mm diameter cylindrical observation cell has a 2-cm pathlength and a total volume of 0.06 ml. STOPPINGDEVICE, One method of stopping the flow of solution is to use a 2-ml syringe and a stop bar. To minimize dead volume, the stopping syringe should be arranged as close to the observation cell as possible. For this work, the stop syringe was 1 cm from the end of the observation cell. In the automatic mode, the manual valve was opened, the stop syringe plunger pushed to the stop block, and the stainless steel check valve then acted as a convenient automatic valve. The check valve was mounted 5-cm away from the end of the observation cell. Observation and Readout System. The spectrophotometric observation and readout system consisted of a stable, high intensity, continuous light source, a high resolution monochromator, the observation cell, a photomultiplier tube, a high impedance operational amplifier current-voltage converter, and a digital rate meter which was recently described (2). Because many of the considerations involving the spectrophotometric system are the same as in ordinary spectrophotometry, only those items which must be specially considered for use in the stopped-flow system are described here. INCIDENT LIGHTTO OBSERVATION CELL. Because of the small cell volume used in the observation system, the incident light level into the cell must be specially considered. The amount of light reaching the cell depends on the intensity of the continuous radiation source, the light losses in the monochromator, the slit width, the light losses between the monochromator and the observation cell, and the area of the en(4) Q.H. Gibson and L.Milnes, Biochem. J., 91,161 (1964).
trance window to the cell. The amount of light incident to the cell must be high enough that statistical fluctuations in the photomultiplier output are small compared to the measured output due to the reaction. A simple calculation based on counting statistics reveals that for a 1% fluctuation level compared to the measured changes in current, the incident light level must be sufficient to give a photomultiplier current of about 10-7 ampere, when the 100% transmittance is established for this system. The light source was a tungsten lamp with ribbon filament (GE 18A/T 10/1-6V) run from a stable 18-A, 6.3-V dc power supply. For wavelength isolation, the Heath EU-700 grating monochromator was employed. A slit width of about 250 microns allowed enough light to the photocathode such that 100% T was equivalent to 5 X 10-7 A photomultiplier current. DETECTOR AND READOUT.The detector is a 1P28 photomultiplier tube, the output of which is directed to a high-inputimpedance-operational amplifier (Nexus LFT-1). A ratemeter ( 2 ) performed integration of the slopes of the rate curves over a preselected period, and typically the integration period was 100 milliseconds. The Heath Universal Digital Instrument, Model EU-805X, provided digital data proportional to the slopes of rate curves and proportional to concentration. PHOSPHATE DETERMINATION The rate of formation of 12-molybdophosphoric acid from phosphate and Mo(V1) in acid solution was found to obey the following rate law ( 5 ) .
where [Mo(VI)] = total concentration of Mo(V1). At acid concentrations less than 0.5M a simpler relationship exists,
Within this range of acid concentration, the reaction rate is faster which is desirable for rapid analysis. By keeping the Mo(V1) in large excess at all times, the reaction rate is made directly proportional to phosphate concentration. Figure 2 shows typical rate curves for the formation of the heteropoly complex. The low frequency noise superimposed on the signal is evident and is one source of error in the analysis. The rapid rate at which measurements are taken eliminates the possibility of using an electronic filter which could produce distortion of the signal. Integration over the linear portion of the rate curve averages out the error from the signal fluctuations. Increased precision is also obtained by taking several measurements and averaging. For example, by taking ten measurements per sample and averaging, a three-fold increase in precision is possible as compared to single measurements. Experimental Conditions. The basic considerations in the choice of experimental parameters result from the mechanistic investigation of the rate of formation of 12-MPA ( 5 ) . Two-tenths molar nitric acid is used with low phosphate concentration (up to micromolar range) to ensure fast reaction. This enables analysis with integration times of 100 milliseconds. Acid concentrations higher than 0.5M can be used with millimolar phosphate concentrations. The concentration of Mo(V1) is kept at least one-hundred fold in excess of the phosphate concentration to ensure that the formation of the complex is favored. ( 5 ) A. C. Javier, S. R. Crouch, and H. V. Malmstadt, ANAL.CHEM., 40, 1922 (1968).
At the onset of the reaction, the noise on the reactionrate curve is greater due to equilibration processes. A delay time of 100-200 milliseconds is allowed before integration of the slopes of the rate curves is made. Hence, measurements are made only when steady initial rates are obtained. To prevent errors due to temperature fluctuations, all measurements are made at the same temperature. A water bath is maintained at about 25 f 0.1 "C and water is circulated through the water jacket surrounding the observation cell, and the reagents and sample are maintained at this temperature. Blood Serum Determinations. The present method is conveniently applied to blood serum determination. Deproteinization is accomplished with 9 % (w/v) trichloroacetic acid (6). Neutralization is done using a pH meter and combination microelectrode so that the serum samples are in the same pH range as the phosphate standards, Reagents. All reagent solutions are prepared from analytical grade chemicals in deionized water. A 100-ppm P stock solution is prepared from 0.43925 gram of ovendried KH2POI made up to one liter of aqueous solution. This solution is stable and can be kept for months. The working standards, prepared by dilution of the stock, are also stable and used from week to week, A 0.1M aqueous stock solution of Mo(V1) is prepared by weighing out 24.1964 grams of Na2Mo04.2H20to make up one liter of solution. This stock solution is also stable over several months. Working solutions of 4 x 10-2M and 5 X 10-2M Mo(V1) in 0.4M H N 0 3 are prepared at least once a week. Both the stock and the working solution are kept in polyethylene bottles to prevent leaching of silicon from the volumetric glassware. The deproteinization reagent is a 9 solution of trichloroacetic acid by weight. One normal sodium hydroxide is prepared from Acculute standard volumetric reagent. For the interference studies a stock solution of 5000 ppm As is prepared by dissolving 0.6603 gram AszORand diluting to 100 ml with deionized water. 100 ml of 5000 ppm Si is prepared from 5.058 grams NazSiOs~9Hz0.The stock and working silicate solutions were all stored in polyethylene bottles. Preparation of Blood Samples. Two-tenths milliliters of blood serum are treated with 1.8 ml of TCA and centrifuged or filtered (6) to separate the coagulated protein. The supernatant liquid is neutralized with 1N NaOH and made up to 5 ml in a volumetric flask. A 2-ml aliquot of the neutralized solution is further diluted to 5 ml and used for analysis. This is enough sample for about 15 rate measurements and the flushing of the mixer and cell. RESULTS AND DISCUSSION Analytical Curves. As shown in Tables I and 11, the digital rate information is linear over a wide range of concentration. With the set of experimental parameters used, 0.10 ppm is the lowest concentration that can be accurately measured. This limit, however, could be extended by changing the experimental conditions such as the concentration of reagents, the integration time, the slit width of the monochromator, etc. For the higher range of concentration, a direct concentration readout was obtained by adjusting the slit width of the monochromator. For the set of data in Table 11, the slit width was adjusted such that for a 2.5-ppm P standard, the ratemeter output was 250 mV with an integration time of 0.1 second. For the lower range of phosphate concentrations, the experimental parameters were adjusted to give a proportionality constant of 400. The choice of a proportionality factor higher than 100 is for increased precision. The precision of the method (6) S. R. Crouch and H. V. Malmstadt, ibid., 39, 1090 (1967). VOL. 41, NO. 2, FEBRUARY 1969
241
Table I. Automatic Reaction-Rate Results for Low Phosphate Concentrations
zRelative P, pprn
Average readout, mV
standard deviations
0.10 0.20 0.25 0.40 0.505 0.75
41.1 80.7 101 .o 161.1 200.0 301.6
2.15 0.53 0.33 0.34 0.27 0.42
*The % relative standard deviation between averages of 10 results. b The 0.5-ppm standard was used to set the readout. Table 11. Automatic Reaction-Rate Results for High Phosphate Concentrations
zRelative p, PPm
Average readout, mV
standard deviations
0.50 1.00 1.25 1.50 1.75 2.00 2.25 2.505 3.00 3.50 4.00 4.50 5.00
54 103 125 149 172 200 226 251 302 352 40 1 45 1 501
1.01 0.79 0.85 1 .05 0.97 0.54 0.40 0.42 0.79 0.95 0.67 0.44 0.21
,,The % relative standard deviation between averages of 10 results. * The 2.50-ppm standard was used to set the readout for direct digital concentration data. TaMe 111. Automatic Reaction-Rate Results for the Determination of Phosphorus in Blood Serum (Concentration in mg/ml) Addeda
...
19.4 38.8
Total
Foundb
Recovery
...
2.84 22.3 42.7
100.5 102.2
22.2 41.6
...
a Standard phosphate solutions added to blood serum originally containing 2.84 mg/100 ml. Manufacturer’s reported values (averages of lo00 determinations).
2 . 6 f 0 . 2 mg P/100 ml (Fiske-Subbarow). 2 . 7 f 0 . 2 mg P/100 ml (AutoAnalyzer).
Table IV. Effect of Interferences on Automatic Phosphate Results
Interferent 50 ppm As
500 ppm As 40 ppm Si a The results.
242
P, ppm
Average readout, mV
0.5 0.5 0.5 0.5
50.7 50.2 50.0 49.9
zRelative
zError
standard deviations
...
1.56 2.00 0.82 1.67
0.99 1.38
1.57
obtained from averaging successive groups of 10 determinations is shown in both tables. An average of about 2-3% relative standard deviation (for single determinations) and less than 1% relative standard deviation between averages of 10 results is easily obtained on a routine basis. For a certain set of experimental conditions (reagent concentrations, delay time, integration time, slit width, etc.), the linear range is limited by the pre-equilibration time. For slower reactions, a longer pre-equilibration time (delay time) is necessary. A longer integration time can also be used with the slower reactions which results in increased averaging. The effect of the delay time and integration period on the precision can be seen by comparing the results for 0.5 ppm P in Tables I and 11. The poorer precision in Table I1 results from the shorter integration and delay times, which were optimized for higher phosphate concentrations. Analysis of Blood Serum. Results of the application of the automatic rate method to the determination of phosphate in blood serum is shown in Table 111. The percentage recovery is good. The sensitivity of the present method is favorable to the small serum sample normally available for analysis in clinical laboratories. A 0 . 2 4 aliquot of blood serum is enough for about 100 rate measurements plus the dead volume used in flushing the reaction cell. Increased precision is obtained by averaging a large number of rate measurements in a relatively short analysis time (about 10 seconds for 10 determinations). Interference Study. Two of the most troublesome interferences in existing colorimetric phosphate methods are arsenate and silicate. Elimination of the arsenate and silicate interferences, as well as interference from germanate and tungstate, is often done by selective extraction of the 12-molybdophosphoric acid (7, 8) with an organic solvent, which is an inconvenient and time consuming procedure. Interference studies utilizing the new rapid reaction-rate procedure show that arsenate is not an interferent (see Table IV). The rate of formation of the arsenate heteropoly acid is very slow compared to the rate of formation of the phosphate heteropoly acid. A concentration of arsenate 1000 times higher than the phosphate concentration produces a negligible change in the phosphate results. Silicate interference is not completely eliminated in the new reaction-rate method. However, a silicate concentration 80 times higher than the phosphate concentration can be talerated as seen from the data in Table IV, a situation which is not possible in the classical colorimetric methods. These results illustrate another advantage of this new reaction-rate method. The difference in reaction rate between arsenate and phosphate and between silicate and phosphate (with Mo(V1)) eliminates the interference of arsenate and silicate without any chemical treatment. The data on the interference study were taken with short integration times, hence, the precision is comparable to that for the 0.5 ppm P sample in Table 11. The low phosphate concentration used in the interference study was chosen to illustrate that even under very unfavorable conditions (low phosphate concentration, short integration time), the classical interferences can be tolerated in the present method. The results would be better under optimum conditions. Conclusions. A new fast reaction-rate procedure has been introduced for the determination of phosphate. The new
z relative standard deviation between averages of 10 (7) M. A. DeSesa and L. B. Rogers, ANAL.CHEM., 26,1381 (1954). (8) W. S. Zaugg and R. J. Knox, ibid.,38, 1759 (1966). ANALYTICAL CHEMISTRY
phosphate procedure is simple, very rapid and is capable of high accuracy and precision. In addition, the method is relatively free from common phosphate interferences. The pH, concentration of reagents, and integration time can be manipulated for analysis of a wide range of phosphate concentrations. The results of blood serum analyses indicate the suitability of the new procedure to the analysis of biological samples. In addition, the method should be applicable to other phosphate-containing samples.
Although the determination of phosphate was chosen to illustrate the application of fast reaction-rate measurements for routine analyses, many other applications are possible. There are many fast reactions that could be utilized for analyses by the millisecond-range rate measurements. The investigation of other rapid reactions suitable for analytical procedures is in progress. RECEIVED for review September 27, 1968. Accepted November 12, 1968.
Determination of Ethanolamides in Mixtures by Differential Saponification Rates Fred H. Lohman and Theresa F. Mulligan The Procter & Gamble Company, Cincinnati, Ohio The fatty alkanolamides can be saponified in alcoholic KOH to give two moles of weak base for every mole of KOH reacting with one mole of the amides. This serves to distinguish them from amines, amine soaps, amine esters, and the ester function of amide esters, and other by-products present in the commercial alkanolamides which yield only one mole of weak base per mole of KOH consumed during saponification. This formation of extra base has been used to follow the saponification of mixtures of commercial monoand diethanolamide and makes possible the application of the differential reaction rate technique to these systems. The pseudo-first-order rate constant for lauric diethanolamide was found to be 70 times that of lauric monoethanolamide, which condition makes the determination of these two materials by this technique inherently precise.
fication of mono- and diethanolamides. Consequently, a method based on differential saponification rates seemed feasible. Siggia, Hanna, and Serencha (7) reported on the analysis of mixtures of primary amides by a differential hydrolysis rate technique ; however, their technique of distilling the ammonia from the reaction mixture as it is formed and titrating the distillate is not applicable to the ethanolamides for the amines formed are not readily volatile. The chemical reactions to be considered in the saponification of commercial ethanolamides in alcoholic KOH and subsequent titration of the weak bases produced are as follows:
THECOMMERCIAL
RCON(CHzCHzOOCR)2 30H- = 3 RCOO"(CH~CHZOH)~ (2)
ETHANOLAMIDES, especially the diethanolamides, are mixtures of some complexity; they contain, in addition to the amide, appreciable amounts of the free amine, amine soap, amide esters, amidoamines, and possibly also amine esters ( I ) . The specifications on these materials generally recognize these facts (2). The presence of these other functional groups thus precludes the use of the standard hydrolytic methods for amides unless corrections can be made for contributions by these other constituents (3). The ion exchange method for nonionic content gives a reliable figure for the total amide content of such materials; however, it does not distinguish between different kinds of amides such as the mono- and diethanolamides in mixtures. Similarly, the published infrared methods (4, 5 ) do not provide for the determination of the individual alkanolamides in mixtures. The differential reaction rate technique has been used rather extensively for determining the individual components of binary mixtures of compounds having the same functional group. The work of Livengood and Johnson (3) and Ranny et al. (6) indicated a considerable difference in the rates of saponi-
(1) H. L. Sanders, J . Amer. Oil Chem. SOC.,35,548 (1958). (2) Toilet Goods Association Standards, Spec. No. 80, Washington, D. C., June 27, 1960. (3) S . M. Livengood and C . H. Johnson, Proceedings of the
Chemical Specialties Manufacturers Association Symposium on Analytical Methods for Surfactants, Hollywood, Fla., Dec. 1957 p 113. (4) M. M. Miller, ANAL.CHEM.,30, 1884 (1958). (5) M. F. Mallery, ibid., p 1884. (6) M. Ranny, J. Prachor, and J. Novak, Prumysl Potrmin, 14, 211 (1963).
RCON(CHzCHz0H)z
+ OH-
=
RCOO-
+
+ HN(CHrCH20H)z
(1)
+
+ H+ = HzN+(CHzCHzOH)z RCOO- + H+ = RCOOH HZN+(CHzCH*OH)z + OH- = HN(CH2CH20H)z + H?O RCOOH + OH- = RCOO- + HzO HN(CH2CH20H)z
(3) (4)
(5)
(6)
The first equation represents the saponification of the amide itself, and the second, the complete saponification of the amide ester. Equations 3 and 4 represent the conversion of the weakly basic components of the sample to their conjugate acids. Equations 5 and 6 represent the neutralization of these conjugate acids. The significant thing about all these reactions is that 5 and 6 both yield one mole of weak base for every mole of strong base consumed, whereas the saponification of the amide constituents (Equations 1 and 2) yields an extra mole of weak base for every mole of amide present. Thus, if the basic components of a sample are first converted to their conjugate acids before saponifying the mixture in alcoholic KOH, a determination of the milliequivalents of base over and above that added as KOH would allow a determination of the milliequivalents of amide saponified. ( 7 ) S. Siggia, J. G. Hanna, and N. M. Serencha, ANAL.CHEM., 36, 277 (1964). VOL. 41, NO. 2, FEBRUARY 1969
243
