Anal. Chem. 1982, 5 4 , 43-46
43
GC detection method may have a broad range of unique applications in the field of trace organic analysis.
(9) Ithakisslos, D. S. J. Chromatogr. Scl. 1980, 18, 88. (10) DI Corcla, A.; Samperl, R.; Capponi, G. J . Chromatogr. 1978, 760,
ACKNOWLEDGMENT
(11) Aue, W. A,; Kaplla, S. J. Chromatogr. 1980, 788,1. (12) Karasek, F. W.; Malcan, A,; Tatone, 0. S. J. Chromatogr. 1975, 110,
The authors wish to thank Martin Williams and Steve Watson for their help in the construction of the spectrometer, John Frame and Fred Schuetze for their help with interfacing the miornnrnrnaanr tn th,e drift tllhe Inc., Austin, "*., "--", nnd ---l?racor, T X , for the use of the Tracor 560 capilla ry chromatographl. "*a"
I"'.,~"~~""VU~"*
-
LITEIRATURE CITED Revercomb, H. E.; Mason. E. A. Anal. Chem. 1975, 4 7 , 970. Cohen, M. J.; Karasek, F. W. J. Chromatogr. Sci. 1970, 8 , 330. Karasek, F. W.; Keller, R. A. J . Chromatogr. Sci. 1972, 70, 626. Karasek, F. W.; Kim, S. H. J. Chromatogr. 1974, 99, 257. Karasek, F. W.; Hill, H. H., Jr.; Kim, S. H.; Rokushika, S. J. Chromatogr. 1977, 135, 329. (6) Cram, S. P.; Chesler, S. N. J. Chromatogr. Scl. 1973, 7 1 . 391. (7) Boggs. G. 0.; Tou. J. C. Anal. Chem. 1978, 4 8 , 1351. (8) Ramstad, T.; Nestrick, 7'. J.; Tou, J. C. J. Chromatogr. Sci. 1978, 16, 240.
(1) (2) (3) (4) (5)
147. 295. (13) Karasek, F. VV.; Kane, D. M. J. Chromatogr. Sci. 1972, 10, 673. (14) Searles, S. K.; Sleck, L. W. J . Chem. Phys. 1970, 53, 794. 115) Karasek. F. W.; Denny, D. W.; DeDecker, E. H. Anal. Chem. 1974, 4 6 , 970. (16) Griffin, G. W.; Dzldic, I.; Carroll, D. I.; Stillwell, R. N.; Hornlng, E. C. Anal. Chem. 1973, 45, 1204. (17) Munson, M. S. B.; Field, F. H. J. Am. Chem. SOC.1987, 89, 1047.
RECEIVED for review June 25, 1981. Accepted September 28, 1981. The authors wish to acknowledge the National Science Foundation (Grant No. CHE 77-25743), The National Institutes of Health (Grant No. GM 24970), and the donors of the R'esearch Fund, administered by the American Chemical Society, for support during this research.
Potentiometric Detection System for Flow Injection Titrimetry S. F. Slmpson and F. J. Holler* Department of Chemistry, Universl@ of Kentmky, Lexington, Kentucky 40506
A new potentlometrlc detection system for use In flow Injectlon tltrlmetry was desllgned and Investlgated. An antllogrlthmlc ampllfler provldw a slgnal relateid to proton conceiitratlon and enables manual selectlon of system sensltlvlty. A descrlptlon of the mlcroelectrodes used as potentlometrlc sensors Is provlded. Preclslon wlthln a set of tltratlons averaged 0.8% RSD and unknowns were determlned with an average error of 4 % .
Since its introduction in 1975 ( I ) , flow injection analysis (FIA) has generated considerable interest among researchers (2-6). The flexibility of the technique is exemplified by its use in enzymatic determinations (3,extraction (8), and dialysis (9). Since FIA requires only a srnall sample (20-200 p L ) , it is a potentially useful tool for routine analysis in clinical laboratories. Rapid sampling and analysis rates inherent in FIA coupled with a variety of methods of detection further increase the range and adaptability of the technique. A number of authors have studied titrations in detail using FIA methodology (3, 119-12). One important difference between flow injection titrimetry (FIT)and other FIA procedures is the use of peak width rather than peak height for the quantitation of analytical data. As shown by Ruzicka and co-workers ( l l ) ,peak width measured in units of time is linearly related to the logarithm of the concentration of analyte. In order to u w peak width a13 the experimental measurable, a large dispersion effect a t the sample-carrier stream interface is advantageous because it serves to increase precision. In FIA, dispersion is usually created by placing a mixer between the sample injector and the detector. However, a recent publication suggests that FIA titrations may be performed without the use of a gradient mixer (12). In addition, the high accuracy and precision of electronic timers
greatly facilitates the precise measurement of peak widths. As a result, the precision of the data is limited by other factors such as the sensitivity of the detector and the precision of the flow rate. When applicable, potentiometric detectors have been used in FIA with favorable results (3, 6, 7, 1 1 , 13-16), and the performance of ion-selective electrodes has been evaluated in previous reports (13,17). Although potentiometric detectors offer advantages which complement the FIA method (simplicity, high specificity, and ruggedness), they are not without limitations. One limitation is the instability of base line voltage levels. Pulsing of the stream resulting from the peristaltic pumps often used in FIA systems tends to generate static charges within the stream. These charges affect the stability of the reference and indicator electrode signals. Consequently, a. stable base line voltage may be achieved only with difficulty, which lowers sensitivity and raises detection limits. In this report, we present a simple, inexpensive electronic detection system for w e with potentiometric sensors in flowing streams. The instrument may be easily adjusted to compensate for small drifts, and it has provision for manual selection of sensitivity. This allows the operator to choose a detection limit which is consistent with a given set of experimental conditions. The operatioin of the instrument was evaluated by titrating NaOH with HC1. Samples of the base were introduced into a stream of HC1 using a rotary injection valve (18). Long-term and short-term studies of precision were conducted to determine day to day reproducibility. Titrations of pseudounknowns served as checks on the accuracy of the instrument. The results of these studies are detailed below.
EXPERIMENTAL SECTION Apparatus. A block diagram of the experimental arrangement is displayed in Figure 1. The sample injection block was a
0003-2700/82/0354-0043$01,25/00 1981 American Chemical Society
44
ANALYTICAL CHEMISTRY, VOL. 54, NO. 1, JANUARY 1982
ELECTRODE ANTIMONY
pi
SYRINGE
) GLASS
ELECTRODE INJECTION BLOCK
MAGNETIC STIRRER SAMPLE
WASTE
Figure 1. System schematic for FIT with potentiometric detection.
Fd--r-l
Ag WIRE ANTIMONY
CAPILLARY GLASS ELECTRODE
ANTIMONY ELECTRODE
H I crn
Figure 2. Sample and reference microelectrodes for the FIT detection system.
machined rotary valve similar to that described by Ruzicka (18). The bore of the valve delivered a sample of 50 pL. The mixing chamber is made of glass and is roughly conical in shape. The solution enters tangentially a t the base of the chamber to promote efficient mixing and exits through a port located a t the apex of the chamber. A micro Teflon stirring bar was sealed inside the chamber during its fabrication. The total volume of the chamber was found to be 0.73 mL as determined by differential weighing with water. The glass and antimony electrodes used in the detection system are shown in Figure 2. The glass indicator electrode was constructed by sealing a small section of 0.5 mm i.d. Corning 015 pH glass into the cell as shown in the figure. The glass tubing serves both as the sensing element and the flow channel. A rubber serum cap holds the Ag-AgC1 internal reference in place and seals the electrode chamber. The antimony reference electrode is housed in a cube of Plexiglas. A hole drilled through the length of the cube serves as the flow channel. Stainless steel capillary tubes were sealed with epoxy a t the entrance and exit porta to support connection to the Tygon tubing of the flow system. Another hole was drilled perpendicular to the flow channel, and a small cylinder of antimony was sealed into the intersection of the two holes so that the face of the antimony cylinder was in contact with the flowing solution. A layer of mercury was deposited on the antimony to provide connection between the electrode and the copper coaxial cable of the detection system. All connections between the components of the flow system were made with 0.5 mm i.d. Tygon tubing. A flow rate of 1.5 mL/min was provided by a Buchler peristaltic pump. A block diagram of the detection system is shown in Figure 3 and a detailed circuit diagram is presented in Figure 4. Signals from both the reference and pH electrodes are amplified and buffered by a high impedance instrumentation amplifier (OAl, OA2, and OA3). Operational amplifier OA4 provides dc offset and further amplifies the signal from the twin T notch filter which is tuned to 60 Hz to minimize line pickup. Offset is provided with a precision &lo V reference constructed from a National LH0070 +10 V reference, an inverting amplifier, and a 50-kQpotentiom-
Flgure 3. Block diagram of detection system: I, indicator electrode; R, referenceelectrode; IA, instrumentation amplifier; OS, offset; Z, Summing amplifier; AL, antilog amplifier; D, differentiation amplifier; REF, variable dc reference; C, comparator: G, gate; OSC, precision osciilator; CTR, counter.
eter. The transfer function of the antilog circuit provides a voltage more nearly related to [H+] than to pH. The offset adjustment allows the operator to adjust the sensitivity of the circuit by placing the base line signal a t the desired point on the exponential, thus effectively determining the detection limit of the system. The exponential signal that results from a rapid increase in [H+] is differentiated by the circuit of OA6 which produces the characteristic spikes a t the beginning and end of the neutralization process (Figure 5). Two comparators (OA9 and OA10) follow the differentiator and each has a manually selectable precision voltage level as one input. Small drifts in the circuit may be compensated for by setting the start comparator reference level a t a point which is not crossed by the differentiator when the system experiences small drifts. When the derivative signal crosses the reference levels, the precision 1 kHz pulses pass through the gate circuit to the counter (Intersil Model 7226A Universal Counter Kit) as shown. One can select any point at which to stop the titration by proper adjustment of the stop comparator reference level. The output of the counter directly displays the time required for the neutralization process. Reagents. The carrier stream reagent was 0.001 M HCI. A stock solution of sodium hydroxide (Baker Reagent Grade) was prepared and standardized against potassium hydrogen phthalate (Fischer Primary Standard Grade). Subsequent standards and two pseudounknowns were prepared by sequential dilution of the stock solution. Double distilled water was used in the preparation of all solutions. Procedure. Before each experimental session, HCl reagent was permitted to flow through the system for 30 min. After the response of the electrode stabilized, the sensitivity was selected by adjusting the dc offset (OA4, Figure 4). The output of the exponentiator (OA5, Figure 4) was observed during several successive titrations of the most dilute base anticipated during the experimental session. While the preliminary titrations were being carried out, the offset was adjusted to yield sufficient sensitivity ana minimum noise. Once determined, the optimum value was maintained during subsequent titrations. A digital voltmeter (Keithly Model 130) was used to monitor the output of OA5 to ensure that the base line did not drift appreciably. An exponentiator output value of 0.20 V was selected and maintained for the concentration range used in this study. Prior to a titration, the reset switch shown in Figure 4 was depressed to clear the counter and arm the gate. After the sample was injected, it passed into the mixing chamber as shown in Figure 1 where it was mixed with the reagent stream to form a concentration gradient. As the gradient passed the indicator electrode, the voltage output of the electrode shifted, thus activating the counter. As the trailing concentration gradient swept through the flow channel of the sensor, the voltage level returned to the base line and halted the counter. The neutralization time was then recorded. Following a series of titrations of standard solutions, linear working curves were obtained by plotting time vs. log concentration.
RESULTS The responses of the antimony electrode and the glass electrode with respect to pH were evaluated. Response curves were prepared by plotting the output of the electrode as a function of pH. The response of the antimony electrode was non-Nernstian and its response curve exhibited small devia-
ANALYTICAL CHEMISTRY, VOL. 54, NO. 1, JANUARY 1982 45
6
- IO'V
Flgure 4. Schematlc dlagrcrm of detection system. Operational amplifiers 1, 2, and 3 are LM 351 type while all others are TL084 type;
denotes
precision resistors. ELECTRODE
I.A.
A -
>-
Table I. Reproducibility of Working Curves day slope SE intercept 1 2 8 4 5
ANTILOG OUTPUT
av a
60.1
.C
0.7'
Average value
i
130.6 132.4 134.6 132.8 139.2
1.050 0.546 0.839 1.97 1.43
60.12 59..84 60.55 59..94 63..55
R
1.2
134
0.9997 0.9999 0.9997 0.9986 0.9995
* 3'
standard deviation.
Table 11. Precision of Single Samples
START
a
concn, M
time,a s
0.01 56 0.03 13 0.0626 0.125 0.250 0.500 1.00
22.5 f 0.4 44.0 i 0.3 63.8 f 0.1 83.4 0.3 101.6 i 0.9 119.6 0.2 137.9 0.6
STOP
Flgure 5. Waveform profiles during titration. Exponential base line signal at point X ylelds high sensitiiity while signail at point Y yields lower
sensitivity. tions from linearity. These deviations are of little consequence in this system since the reference electrode is always exposed to the same reagent sollution (Figure 1). The antimony reference sensor compensates for voltage fluctuations caused by small changes in the flow velocity. The robust nature arid simple construction of the antimony electrode make it nearly ideal for flowing streams and for situations in which high accuracy is not required (19). The response of the glass electrode was linear over the range of p H values as expected. Because of its linear response, chemical inertness, and advantages common to glass electrodes, it was chosen as the indicator ebectrode. The shape and small size of the electrode allowed i t to be placed near the mixer, thus permitting detection of the concentration
a
Time
i
* * *
%
RSD
1.84 0.62 0.16 0.32 0.85 0.16 0.44
standard deviation.
gradient with maximum sensitivity. System Performance. Calibration curves constructed with time plotted as a function of log concentration were analyzed by use of linear regression techniques (Table I). The standard error of the estimate (S,) was used as a measure of goodness of fit. The average SEvalue of 1.25 s for the five curves listed indicated excellent linearity over approximately a 2-decade concentration range. Data obtained from five experimental sessions (Table I) were used to test the system for reproducibility over a more extended period of time. A comparison of the slopes indicates that the system responds reproducibly on a day to day basis. The two pseudounknowns prepared from the stock solution were titrated as a check on the accuracy of the instrument. Concentration values for the unknowns were calculated by
46
Anal. Chem.
using the experimental titration time and the calibration curve determined by regression analysis. The average percent error for these unknowns over five experimental sessions was found t o be 4%. The precision of a single measurement was studied at each concentration by making four injections for each sample. A summary of the data is presented in Table 11. Values for relative standard deviation (RSD) for this work ranged from 8% RSD to less than 0.01% RSD with an average of 0.8% RSD.
CONCLUSIONS Results obtained with this system indicate its potential as
a detector for flow injection titrimetry. The measurements themselves are time measurements which can be made precisely and accurately. Once the system is calibrated, samples can be titrated rapidly since speed of analysis is governed by the return to base line of the electrode signal. Implementation of a high-quality antilog circuit may provide some improvement in the stability of the base line and thus yield an overall improvement in the performance of the system.
ACKNOWLEDGMENT The authors thank W. C. Mateyka of the University of Kentucky glass shop for his craftsmanship and helpful suggestions.
46-49
LITERATURE CITED Ruzicka, J.; Hansen, E. H. Anal. Chlm. Acta 1975, 57, 145. Ruzicka, J.; Hansen, E. H. Anal. Chlm. Acta 1978, 9 9 , 37. Ruzlcka, J.; Hansen, E. H. Anal. Chlm. Acta 1980, 7 7 4 , 19. Betterldge, D. Anal. Chem. 1978, 50, 632 A. Ranger, C. B. Anal. Chem. 1981, 53, 21 A. Ruzicka, J.; Hansen, E. H. "Flow Inlection Analysis"; Wiley: New York, 1981. Ruzicka, J.; Hansen, E. H.; Ghose, A. K.; Mottoia, H. A. Anal. Chem. 1979, 51, 199. Karlberg, B.; Theiander, S. Anal. Cbim. Acta 1978, 98, 1. Hansen, E. H.; Ruzicka, J. Anal. Chlm. Acta 1976, 8 7 , 353. Astrom, 0. Anal. Chlm. Acta 1979, 105, 67. Ruzicka, J.; Hansen, E. H.; Mosbaek, H. Anal. Chim. Acta 1977, 92, 235. Ramsing, A. U.; Ruzicka, J.; Hansen, E. H. Anal. Chlm. Acta 1981, 129, 1. FQher, 2s.; Nagy, G.; TBth, K.; Pungor, E. Anal. Cbim. Acta 1978, 9 8 , 193. Hansen, E. H.; Ghose, A. K.; Ruzicka, J. Analyst(London) 1977, 102, 705. Zagatto, E. A.; Reis, B. F.; Fllho, H. Bergamin; Krug, F. J. Anal. Chim. Acta 1979. 109. 45. Ruzicka, J.: Hansen, E. H.; Zagatto, E. A. Anal. Chlm. Acta 1977, 88, 1. Slanina, J.; Lingerak, W. A.; Bakker, F. Anal. Chlm. Acto 1980, 777, 91. Ruzicka, J.; Hansen, E. H. J. Chem. Educ. 1979, 56, 677. Bates, R. G. "Determination of pH: Theory and Practice": Wiiey: New York, 1973.
RF,CEWE~ for review May 14,1981. Accepted October 13,1981. S.F.S. is grateful to the Ashland Oil Co. for a summer fellowship.
Interference in Determination of Ammonia with the Hypoehlorite-Alkaline Phenol Method of Berthelot T. T. Ngo," A. P. H. Phan, C. F. Yam,' and H. M. Lenhoff Department of Developmental and Cell Elology, Unlversity of California, Irvlne, Irvine, Callfornia 927 17
The blue color resultlng from the formatlon of indophenol In the Berthelol method of determlnlng ammonla was suppressed by prlmary and secondary amlnes, sulfldes, thlols, and ascorbic acid, and to a lesser extent by tertiary amlnes. We postulate that nucleophlllc addltlons of amlnes, thiols, and other nucleophlles to the qulnold lntermedlates of the Berthelot reactlon decrease the formatlon of Indophenol. It Is also posslble that reduclng agents deplete hypochlorlte to suboptlmal levels.
another study (11),we noted that a number of commonly used buffers such as Tris [tris(hydroxymethyl)aminomethane], glycylglycine, imidazole, bicine, tricine, etc. interfered with ammonia determination by suppressing formation of the blue color in the Bethelot reaction (11). In view of the wide-spread use of this reaction for determining ammonia in analytical laboratories, hospitals, and industrial facilities, we have systematically investigated commonly used substances that might affect color development in the Berthelot reaction. We present the results of this survey and some possible mechanisms of color suppression.
T h e formation of the deep blue color of indophenol from hypochlorite-alkaline phenol solution and ammonia was first reported by Berthelot more than a century ago (1). Since then this reaction has been widely used for determining ammonia (2-9). A number of modifications of the basic Berthelot reaction have been made, such as changes in the order of adding the reagents (7) and substituting different catalysts to accelerate the formation of indophenol. The following catalysts have been used manganese(I1) ion, sodium nitroprusside, and sodium pentacyanonitrosylferate (7-10). A few substances, such as copper, zinc, iron, bromide, hydroxylamine, and paminophenol(4) were reported to interfere with ammonia determination by the Berthelot reaction. In
Chemicals. Chemicals were obtained from commercial sources and used without further purifications. Abbreviations: Bicine, Nfl-bis(2-hydroxyethy1)glycine;Caps, (3-cyclohexylamino)propanesulfonicacid: Hepes, N-(Z-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid; Mes, 2 - ( N morpho1ino)propanesulfonic acid; Pipes, piperazine-NJVt-bis(2ethanesulfonic acid; Taps, (3-((tris(hydroxylmethyl))methyl)amino)propanesulfonate;Mops, 3-(N-morpholino)propanesulfonic acid, Tes, N-(tris(hydroxymethyl)methyl)-2-aminoethesulf; Tricine, N-(tris(hydroxymethy1)methyl)glycine; Tris, tris(hydroxymethy1)aminomethane. Reagents. Two reagents were required for ammonia determination by Berthelot alkaline phenol hypochlorite method: (reagent I) 10.00 g/L phenol, 0.05 g/L sodium nitroprusside; (reagent XI) 5.00 g/L sodium hydroxide; 0.42 g/L sodium hypochlorite. A solution of NHICl (0.5 mmol/L) was prepared daily in sodium phosphate buffer (50 mmol/L), pH 7.0. All compounds to be
EXPERIMENTAL SECTION
Current address: Clinical Research Institute of Montreal, 110 Pine Av. W., Montreal, Quebec, Canada.
0003-2700/82/0354-0046$01.25/00 1981 American Chemical Society
