United States Environmental Protection Agency Environmental Monitoring Systems Laboratory P.O. Box 93478 Las Vegas NV 89193-3478 Pre-lssuance Copy April 1988 Research and Development r/EPA The Determination of pH by Flow Injection Analysis and by Fiber Optrode Analysis ------- THE DETERMINATION OF pH BY FLOW INJECTION ANALYSIS AND BY FIBER OPTRODE ANALYSIS by Stephen H. Pia, Donna P. Waltman, Daniel C. Hillman Lockheed Engineering and Management Services Company, Inc 1050 East Flamingo Road, Suite 120 Las Vegas, Nevada 89109 Contract Number 68-03-3249 '6 j Project Officer L. Edward M. Heithmar ^>v Quality Assurance and Methods Development Division P Environmental Monitoring Systems Laboratory ^ Las Vegas, Nevada 89193-3478 TO o ENVIRONMENTAL MONITORING SYSTEMS LABORATORY OFFICE OF RESEARCH AND DEVELOPMENT UNITED STATES ENVIRONMENTAL PROTECTION AGENCY LAS VEGAS, NEVADA 89193-3478 ------- NOTICE The information in this document has been funded wholly or in part by the United States Environmental Protection Agency under Contract 68-03-3249 to Lockheed-EMSCo. It has been subject to the Agency's peer and administrative review, and it has been approved for publication as an EPA document. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. ------- ABSTRACT Two new protocols for measuring pH have been developed. The first measures pH colorimetrically using a proprietary indicator dye mixture in a flow injection analysis (FIA) procedure. The second measures pH using a fiber optic chemical sensor (FOCS) specifically developed for pH determinations. The FOCS method measures pH by monitoring the fluorescence of a fluorescein derivative bonded to the distal end of a fiber optic cable, called an optrode. The FIA method currently has a precision and accuracy of about ±0.2 pH units and can measure 100 samples/hour. The matrix may affect the precision and accuracy. The FOCS method has a precision of ±0.05-0.20 pH units and an accuracy of ±0.1 to 0.6 pH units. The bias is largely due to inadequacy of the calibration model, which needs further development. About 10-60 samples/hour can be analyzed. The response time is dependent upon matrix. It varied from 10 seconds to 7 minutes in the solutions studied, with slowest response in dilute, poorly buffered samples. Finally, the characteristics of the FOCS Method will vary significantly with individual optrodes. The experimental results indicate that either flow injection analysis or fiber optic chemical sensor analysis could form the basis for an alternative to electrometric measurement of pH in certain circumstances. iii ------- IV ------- TABLE OF CONTENTS Abstract iii Figures vi Tables vii Acknowledgements ix 1.0 Introduction 1 2.0 Determination of pH by Flow Injection Analysis 2 2.1 Protocol Evaluation 2 2.2 Results 2 2.2.1 Calibration Curves 2 2.2.2 Accuracy 3 2.2.3 Precision 4 2.2.4 Sample Analysis Rate 5 2.2.5 Matrix Effects 5 2.3 Summary 9 3.0 Determination of pH by Fiber Optrode Analysis 10 3.1 Protocol Evaluation 10 3.2 Results 10 3.2.1 Calibration Curves 10 3.2.2 Accuracy 12 3.2.3 Precision 13 3.2.4 Sample Analysis Rate 15 3.2.5 Matrix Effects 15 3.2.6 Precision and Accuracy 17 3.3 Summary 25 4.0 Conclusions and Recommendations 27 5.0 References 29 Appendices A. Colorimetric Determination of pH by Flow Injection Analysis. . 31 B. Fluorometric Determination of pH with a Fiber Optic Chemical Sensor 38 ------- LIST OF FIGURES Number Page 2-1 Example of calibration curve for FIA pH Method 3 2-2 Example of output for FIA (0.5 AU full scale) 6 3-1 Examples of FOCS Response Curves - linear scale 11 3-2 Examples of FOCS Calibration Curves - logarithmic scale 11 3-3a Sample output from FOCS-3 in high ionic strength calibration buffers 18 3-3b Sample output from FOCS-6 in high ionic strength calibration buffers 19 3-3c Sample output from FOCS-8 in high ionic strength calibration buffers 20 3-4a Sample output from FOCS-3 in phosphate buffers 21 3-4b Sample output from FOCS-6 in phosphate buffers 22 3-4c Sample output from FOCS-8 in phosphate buffers 23 A-l Flow diagram for FIA System 37 B-l Block Diagram of Fluorometer 45 ------- LIST OF TABLES Number 2-1 2-2 2-3 ' 2-4 2-5 2-6 2-7 2-8 2-9 3-1 3-2 3-3 3-4 3-5 3-6 3-7 3-8 3-9 Calibration Line from Four Different Analysis Days Summary of Method Bias Summary of Method Precision Composition of Standards with Varying Ionic Strength Composition of Standards with Varying Buffering Capacity and Constant Ionic Strength Summary Data from Standards with Varying Ionic Strength Summary Data from Standards with Varying Buffering Capacity and Ionic Strength = 0.01 Summary Results for two Natural Samples FIA pH Method Analytical Characteristics FOCS Calibration Curve Data Summary of Method BIAS Summary of FOCS Methods Precision Summary of Duplicate Difference Data Composition of Standards with Varying Ionic Strength Composition of Standards with Varying Buffering Capacity and Constant Ionic Strength FOCS and pH Electrode Response Times for Phosphate Buffers and Lake Samples FN-9 Summary Data from Standards with Varying Ionic Strength Summary Data from Standards with Varying Buffering Capacity . . . Page 2 4 5 7 7 8 8 8 9 12 13 14 15 16 17 24 24 25 vn ------- LIST OF TABLES (Continued) Number Page 3-10 Summary Data for Two Natural Samples 25 3-11 FOCS pH Method Analytical Characteristics 25 4-1 Comparison of Analytical Characteristics for pH Electrode, FIA pH Method, and FOCS pH Method 27 vm ------- ACKNOWLEDGEMENTS We wish to acknowledge K. Street for supplying the mixed indicator used in the FIA method, D. R. Walt for supplying the fiber optic chemical sensors used in the optrode method, and N. R. Herron for technical assistance. The following persons are gratefully acknowledged for technical review of the manuscript: G. McKinney, K. Stetzenbach, and K. Street. ------- 1.0 INTRODUCTION An important parameter determined for acid deposition characterization and monitoring is pH. It has been measured during all of the EPA's Aquatic Effects Research Program Surface Water Surveys in both the field (on-site) and analyt- ical laboratories. Methodology for pH determinations involved both closed and open system measurements using glass pH electrodes (1). Laboratory measurements with pH electrodes were generally precise and accurate (±0.05 pH units), but proved to be time consuming and labor intensive, especially for the closed system measurements. When used in the field, electrodes and meters performed marginally (±0.5 to 1 pH unit). Future surveys and long term monitoring pro- jects will require field pH measurements using either in-situ or closed-system techniques. This necessitates the development of new methods for determining pH in the field. Recently, there have been a number of pH methods reported in the literature based on colon'metric techniques. Generally, two approaches have been taken. In the first, a chemical reagent (such as a dye) is bonded to a substrate and pH is monitored as a function of absorbance, transmittance, or fluorescence, at a specific wavelength (dependent on the reagent) (2-7). In the second approach, the chemical reagent is bonded directly to the distal end of an optical fiber (7-9). In this case, fluorescence is normally employed. An alternate, non- colorimetric approach to measuring pH involves the new technology of chemically sensitive field effect transistors (10). All of the aforementioned approaches are potentially applicable to field use. However, a full characterization has not been performed on any of them. Furthermore, they are often hampered by a limited pH range, complex response (calibration) curves, and unknown lifetime. In an initial step to develop a field method, this study was undertaken to fully characterize two colon'metric methods and check their suitability for field use. One colon'metric method being tested involves the use of an optical fiber coated with fluorescein, the fluorescence being pH dependent at 530 nm (11). The second involves the use of a dye mixture with a pH dependent absorb- ance (13) in a FIA method. ------- 2.0 DETERMINATION OF pH BY FLOW INJECTION ANALYSIS 2.1 PROTOCOL EVALUATION All experiments were performed using the protocol listed in Appendix A. In evaluating the protocol, the following issues were addressed: calibration curves, precision, accuracy, sample analysis rate, matrix effects, and natural lake sample analysis. The basic method analytical characteristics were deter- mined using stable, well characterized pH buffers. The effects of several matrix variables (ionic strength and buffering capacity) on the analytical characteristics were studied using acetate, phosphate, and sulfuric acid pH buffers. An early attempt to use carbonate buffers was abandoned due to the difficulty in preparing reproducible samples. The pH of dilute carbonate buffers drifted due to interaction with the atmosphere. Finally, the perform- ance of the method was tested with two, well-characterized natural lake samples. 2.2 RESULTS 2.2.1 Calibration Curves The response curves were studied by measuring the signal (mV, where 1000 mV = 0.5 absorbance units) of the calibration buffers and plotting the signal vs. the buffer pH. A sample calibration curve is shown in Figure 2-1. The data is noticeably non-linear above pH 6.8. For calibration purposes, the data between pH 3.6 and 6.8 is linear and can be fit acceptably using linear regression. The calibration line is reproducible from day-to-day. Table 2-1 contains the regression statistics from four calibration lines obtained on separate days over a two-week period. TABLE 2-1. CALIBRATION LINES FROM FOUR DIFFERENT ANALYSIS DAYS Line r^ Regression Line Equation N 1 2 3 4 0.996 0.994 0.994 0.989 Y = (-13015) pH H Y = (-13816) pH i Y = (-11815) pH H Y = (-12518) pH i K (963112) • (999+16) • (863114) - (903120) 5 5 5 5 ------- 600 EXAMPLE OF FIA CALIBRATION CURVE \ I o iD I u Q. 500 - 400 - 300 - 200 - 100 - Y = -130 X + 963 r = 0.996 3.0 5.0 BUFFER pH 7.0 Figure 2-1. Example of calibration curve for FIA pH Method. The non-linearity of the method above pH 6.8 is related to the indicator reagent currently being used. As the composition of the indicator reagent is proprietary, the composition or concentration of the dye mixture could not be varied in an attempt to increase the linearity above pH 6.8. Consequently, the method is limited to sample analysis in the pH range 3.6 to 6.8. 2.2.2 Accuracy The method accuracy was estimated by preparing a calibration curve and analyzing the series of pH calibration buffers twice over a two-hour time period. The bias was then defined as the difference between the average measured pH and the expected pH. Table 2-2 summarizes the bias observed on four days of analyses. ------- TABLE 2-2. SUMMARY OF METHOD BIAS Bias (pH Units) Buffer pH* Day 1 Day 2 Day 3 Day 4 Mean + Std. Dev. 3.60 4.00 5.00 6.00 6.80 7.00 7.20 -0.01 0.04 -0.10 0.12 -0.05 -0.23 -0.28 -0.10 0.03 0.08 0.10 -0.11 -0.36 -0.33 -0.03 0.01 -0.03 0.16 -0.11 -0.29 -0.37 -0.16 0.12 0.02 0.14 -0.13 -0.36 -0.40 -0. 0810.07 +0.0510.05 -0.01lO.08 +0.1310.03 -0. 1010.03 -0.3110. 06 -0.3510. 05 Composition of the buffers is given in the protocol (Appendix A). These are nominal pH values. Actual pH values may vary slightly and were determined daily by glass pH electrode as described in Appendix A. It is apparent that the bias is not constant over the entire calibrated range. Below pH 6, the bias is generally less than 0.1 pH unit. It appears though, that the sign of the bias is pH dependent (i.e., negative at pH 3.6, positive at pH 4, either at pH 5). This is indicative of a reproducible but not perfectly linear calibration curve. From pH 6 to 6.8, the bias shifts from +0.1 at pH 6 to -0.1 at pH 6.8. The larger, shifting bias at pH values in the area 6 to 6.8 is most likely due to non-linearity of the calibration curve as the pH approaches 7. Above pH 6.8, the bias increases to the extent that the method is not useful. 2.2.3 Precision An estimate for precision was obtained from replicate analyses of the cal- ibration buffers performed on four different days. The results are summarized in Table 2-3. The within-day and between-day precision estimates were obtained by ANOVA and are not significantly different. However, it is apparent that the analytical precision is pH dependent. As pH increases from 3.6 to 6.8, the between day precision improves from 0.21 to 0.05 and the within day precision to 0.01. At the same time the signal is decreasing from about 500 to 50, which rules out a decreasing signal/noise ratio as the cause of the larger variance at low pH values. Further investigation is necessary to determine the.cause of the reduced precision. Possibly, there is some physical or chemical interaction between the acid and indicator reagent that is responsible for the reduced precision. ------- TABLE 2-3. SUMMARY OF METHOD PRECISION Overall Precision Measured pH* (Standard Deviation) Within Between Buffer Day 1 Day 2 Day 3 Day 4 Day Day 1* 2 3 4 5 3.76±0.20 3.99±0.09 4.90±0.09 6.13±0.03 6.74±0.01 3.71±0.14 3.98±0.02 5.08±0.10 6.11±0.09 6.68±0.01 3.52±0.23 3.95±0.29 4.97±0.11 6.18±0.04 6.68±0.02 3.62±0.25 4.07±0.12 5.02±0.04 6.16±0.01 6.66±0.01 0.21 0.16 0.09 0.05 0.01 0.21 0.070 0.11 0.04 0.05 *Results for Buffer 1 are from quadruplicate analyses. Rest are from duplicate analyses. 2.2.4 Sample Analysis Rate The sample throughput for the current FIA set-up is 100 samples per hour. With the 200 uL sample loop size this analysis rate results in well resolved FIA peaks, as shown in the example output in Figure 2-2. No attempt was made to increase throughput for this initial evaluation. 2.2.5 Matrix Effects In pH electrode measurement systems, the sample matrix can often have a significant impact on the measurement. Measurements in low ionic strength, poorly buffered samples are often slow to stabilize (10 to 20 minutes per measurement is common) and variable (precisions vary up to ±0.5 pH unit). Accurate measurements can be obtained, but it takes extra care and can be very tedious. One of the driving forces behind developing new pH methods is the desire to eliminate the matrix effects. The effect of ionic strength and buffering capacity was investigated by analyzing a series of standards with varying ionic strength and buffering capacity (expressed as acid neutralizing capacity, ANC). A list of the standards used is given in Tables 2-4 and 2-5. The measurement data is summarized in Tables 2-6 and 2-7. From the data in Table 2-6, it is apparent that the analysis bias and precision was not affected by ionic strength. However, the bias for the sul- furic acid standards (though not affected by ionic strength) was much greater than expected. The expected pH was verified by an electrode measurement, so a preparation error is ruled out. The cause must be further investigated. The bias for the acetate and phosphate standards are similar to that observed for the calibration buffers (Table 2-2). The precision values are also similar but somewhat larger than those observed previously (see Table 4). ------- 1000—1 500— c o> CO Sample 1 2 3 4 5 PH 3.6 4.0 5.0 e.o 6.8 I 2 Time (min) Figure 2-2. Example of output from FIA (0.5 AU full scale). In addition to the synthetic samples analyzed, two natural lake samples were also analyzed. The samples, FN9 and FN10, are well characterized audit samples used internally. Each sample was analyzed several times over the course of the study. Summary results are given in Table 2-8. The results are in reasonable agreement with the reference values. The reference values for pH were obtained by pH electrode measurement during a recent EPA lake and stream survey. ------- TABLE 2-4. COMPOSITION OF STANDARDS WITH VARYING IONIC STRENGTH Sulfuric Acid Standards mN H2S04 Ionic Strength* Expected pH** No. 1 2 3 0.10 0.10 0.10 0.00015 0.0042 0.040 4.01 4.03 4.08 No. No. mM HOAc Acetate Standards mM NaOAc Ionic Strength* mM KH2P04 mM Na2HP04 Ionic Strength* Expected pH** 1 2 3 0.48 0.48 0.48 0.52 0.52 0.52 0.001 0.005 0.041 4.74 4.69 4.55 Expected pH** 1 2 3 0.25 0.25 0.25 0.25 0.25 0.25 0.001 0.005 0.041 7.15 7.10 6.95 *Ionic strength is adjusted with KC1 . **Expected pH values were calculated as described in references 14 and 15 and have been corrected for activity. TABLE 2-5. COMPOSITION OF STANDARDS WITH VARYING BUFFERING CAPACITY AND CONSTANT IONIC STRENGTH* Acetate Standards No. mM HOAc mM NaOAc Expected pH** ANC 1 2 3 0.035 0.48 5.0 0.065 0.52 5.0 4.86 4.66 4.62 65 520 5000 *Ionic strength is adjusted to 0.01 using KC1. **Expected pH values were calculated as described in references 14 and 15 and have been corrected for activity. ------- TABLE 2-6. SUMMARY DATA FROM STANDARDS WITH VARYING IONIC STRENGTH Standard Sulfur ic Acetate Phosphate Ionic strength 0.00015 0.0042 0.040 0.001 0.005 0.041 0.001 0.005 0.041 Expected pH 4.01 4.03 4.08 4.74 4.69 4.55 7.15 7.10 6.95 Measured pH electrode* 4.01 ± 0.05(10) 4.81 ± 0.15(11) 7.05 ± 0.10(15) PH FIA** 4.32±0.11 4.40±0.11 4.39±0.11 4.71±0.21 4.72±0.21 4.66±0.21 6.94±0.13 6.94±0.13 6.88±0.13 FIA bias +0.31 +0.37 +0.31 -0.03 +0.03 +0.11 -0.21 -0.16 -0.07 *Number of replicates over 5-day period indicated in parentheses. **Precision is estimated from ANOVA analysis of data from three days for sulfuric and acetate standards and two days for phosphate standard. TABLE 2-7. SUMMARY DATA FROM STANDARDS WITH VARYING BUFFERING CAPACITY AND IONIC STRENGTH = 0.01 Standard ANC Expected pH Measured pH pH electrode* FIA Acetate 65 520 5000 4.86 4.66 4.62 5.06 ± 0.11(11) 4.80 ± 0.07(13) 4.72 ± 0.02(13) 4.78+0.09(4) 4.80±0.08(4) 4.77±0.06(4) *Number of replicates over 5-day period indicated in parentheses. Sample TABLE 2-8. SUMMARY RESULTS FOR TWO NATURAL SAMPLES Reference pH Conductance (pS) ANC (Range) Measured pH FN9 FN10 50 30 140 4 6.8-7.3 5.0-5.3 6.73±0.07(3) 5.07±0.14(8) ------- 2.3 SUMMARY The determination of pH by FIA using an indicator dye reagent appears promising. A summary of analytical characteristics is presented in Table 2-9. TABLE 2-9. FIA pH METHOD ANALYTICAL CHARACTERISTICS Parameter Rate: 100 samples/hour pH Range: 3.6 to 6.8 Bias: 0.1-0.2 pH unit Precision: ± (0.05-0.2) pH unit The applicable pH range is suitable for most surface water analysis, but it needs to be expanded. This will necessitate changing the indicator dye com- position. Alternatively, it may be possible to increase the range by monitor- ing several wavelengths using a multiple wavelength detector. The precision and accuracy are acceptable for general work but may have a pH dependence. Again, this may be a result of the particular indicators chosen. The precision is also limited by the precision of the FIA system. Generally, the reproducibility of the FIA peaks is about 3 to 5 percent. When this precision is transformed onto the pH scale (x-axis), it is magnified by the logarithmic nature of the scale (e.g., a 5 percent precision at pH 4, 4±0.2, is about a 40 percent precision in hydrogen ion concentration). For this reason, it will be necessary to use high precision pumps and valves if the precision of the method is to be improved significantly. The precision and accuracy do not appear to be affected by the sample ionic strength or buffering capacity. There may be effects related to specific sample components and interactions with the indicator dyes. The analysis rate speaks for itself, 100 s/hr is very rapid for pH analyses. With further work, the rate could easily be doubled. In conclusion, the FIA pH method is currently applicable to rapid analysis (100 s/hr) of natural waters with a precision and accuracy of ±0.2 pH unit over the pH range 3.6 to 6.8. Further work is necessary in order to increase the applicable range, improve the figures of merit, and more fully investigate interferences. ------- 3.0 DETERMINATION OF pH BY FIBER OPTRODE ANALYSIS 3.1 PROTOCOL EVALUATION All experiments were performed using the protocol listed in Appendix B. In evaluating the protocol, the following issues were addressed: calibration curves, precision, accuracy, sample analysis rate, matrix effects, response times, and real sample analysis. The basic method analytical characteristics were determined using stable, well characterized pH buffers. The effects of several matrix variables (ionic strength, buffering capacity, and temperature) on the analytical characteristics were studied using sulfuric acid, acetate, and phosphate pH buffers. An early attempt to use carbonate buffers was abandoned due to the difficulty in obtaining reproducible samples. Finally, the perform- ance of the method was tested with two, well-character!'zed natural lake samples. The FOCS used in the protocol evaluation are prototypes produced by Dr. Walt of Tufts University. As such they are not commercially available. A complete description is given in Reference 10. The evaluation is meant to characterize and determine the applicability of the sensors to real world sample analyses. Three FOCS were tested, identified as FOCS-3, FOCS-6, and FOCS-8. 3.2 RESULTS 3.2.1 Calibration Curves Response curves for each FOCS were generated by plotting the emission intensity (expressed as mV) versus pH for the calibration buffers. Each FOCS had its own unique response curve, as seen in the examples pictured in Figure 3-1. Apparently, the manufacturing process is not 100 percent reproducible from optrode to optrode. The curves have similar shapes, though differing only in emission intensity. FOCS-6 and FOCS-8 most likely have a greater quantity of fluoroscein bonded to the fiber. A noteable feature of the curves is that they are not linear, which complicates the calibration procedure. A point to point fit was not used to reduce data because of its tedious nature. However, the response curve is essentially logarithmic over the pH range 3.5 to 7.5. If the log (emission signal) vs. the buffer pH is plotted, a reasonable linear fit is obtained. Examples of calibration lines using this procedure are given in Figure 3-2. There is still some non-linearity evident, but it is an improve- ment from the curves in Figure 3-1. An alternative to the linear model for calibration purposes is a polynomial expression model. However, the software to perform polynomial fits was not available in this laboratory. 10 ------- FOCS RESPONSE CURVES E 2 O 2 O O 2 g in tn o: O O 900 800 - 700 - 600 - 500 - 400 - 300 - 200 - 100 D FOCS-3 •f FOCS-6 o FOCS-8 3.0 5.0 7.0 9.0 BUFFER pH Figure 3-1. Examples of FOCS Response Curves - linear scale, 3.1 FOCS CALIBRATION CURVES 3 - 2.9 - 2.8 - 2.7 - 2.6 - 2.5 - 2.4 - 2.3 - D FOCS-3 : Y = 0.050 X + 2.1 , r - 0.97 + FOCS-6 : Y = 0.20 X + 1.5 , r = 1.00 O FOCS-8 : Y = 0.18 X -f 1.6 , r = 0.99 3.0 5.0 7.0 9.0 BUFFER pH Figure 3-2. Examples of FOCS Calibration Curves - logarithmic scale. 11 ------- The regression data for each FOCS calibration curve on several days are given in Table 3-1. It is noticeable that the calibration curve slope for the more sensitive probes (FOCS-6 and -8) drifts down with time, indicating that the sensitivity is dropping. This is evidence that the optrodes have a finite lifetime. Contrastingly, the low sensitivity optrode (FOCS-3) has a reproduc- ible response. The y-intercept is stable from day-to-day for. all optrodes because it is somewhat normalized by the instrument set-up procedure (i.e., the pH 4 standard is always set at 200 mV as part of calibration procedure). A likely explanation for the negative drift in FOCS-6 and -8 is that a certain quantity of the total bonded fluorescein is labile and is lost with use. After this initial loss, the remaining fluorescein is stable. The end result is that each FOCS must be calibrated every day. The calibration curves are stable for at least 2 to 3 hours. For routine use, the calibration curve would have to be monitored closely within a day (with QC samples) to detect excessive drift. TABLE 3-1. FOCS CALIBRATION CURVE DATA FOCS ID Day 3 1 2 3 7 10 6 1 2 3 7 8 1 2 3 7 Slope 0.050±0.006 0.051±0.006 0.061±0.007 0.050±0.007 0.050±0.005 0.195±0.007 0.177±0.008 0.173±0.008 0.16 ±0.01 0.18±0.01 0.1810.01 0.14±0.01 0.13±0.01 y-intercept 2.10±0.02 2.09±0.02 2.05±0.02 2.08±0.02 2.09±0.02 1.52±0.03 1.6010.03 1.60±0.03 1.6710.03 1.60+0.04 1.5910.04 1.7110.04 1.7710.04 r 0.97 0.97 0.97 0.97 0.98 1.00 1.00 0.99 0.99 0.99 0.99 0.99 0.98 3.2.2 Accuracy The method accuracy was estimated by first preparing a calibration curve then by analyzing the series of pH calibration buffers twice over a two- hour time period. The bias was then defined as the difference between the average measured pH and the expected pH. The data for three days of analysis are summarized in Table 3-2. The bias ranges from about 0.1 to 0.6 pH units for FOCS-3 and from 0.1 to 0.4 pH units for FOCS-6 and -8. This bias is due to the non-linearity of the calibration line. A better calibration procedure is necessary in order to improve the bias. Polynomial fits will be investigated when software is obtained. 12 ------- TABLE 3-2. SUMMARY OF METHOD BIAS Bias (pH units) FOCS ID Buffer pH* Day 1 Day 2 Day 3 Mean Std. Dev, 3 3.6 4.0 5.0 6.0 6.8 7.0 7.2 7.4 6 3.6 4.0 5.0 6.0 6.8 7.0 7.2 7.4 8 3.6 4.0 5.0 6.0 6.8 7.0 7.2 7.4 0.40 0.19 -0.45 -0.65 0.05 0.21 0.31 0.32 0.16 0.04 -0.11 -0.22 0.05 0.11 0.10 0.04 0.23 0.07 -0.21 -0.34 0.07 0.15 0.15 0.08 0.39 0.17 -0.41 -0.65 0.03 0.20 0.31 0.33 0.18 0.05 -0.13 -0.26 0.06 0.11 0.10 0.05 0.24 0.06 -0.20 -0.35 0.08 0.16 0.14 0.08 0.42 0.12 -0.40 -0.63 0.08 0.19 0.28 0.29 0.19 0.05 -0.14 -0.28 0.05 0.12 0.11 0.07 0.27 0.10 -0.26 -0.41 0.06 0.16 0.18 0.14 0.40 0.16 -0.42 -0.64 0.05 0.20 0.30 0.31 0.18 0.05 -0.13 -0.25 0.05 0.11 0.10 0.05 0.25 0.08 -0.22 -0.37 0.07 0.16 0.16 0.10 0.02 0.04 0.03 0.01 0.03 0.01 0.02 0.02 0.02 0.01 0.02 0.03 0.01 0.01 0.01 0.02 0.02 0.02 0.03 0.04 0.01 0.01 0.02 0.03 * Composition of the buffers is given in the protocol (Appendix B). These are nominal pH values. Actual pH values may vary sightly. 3.2.3 Precision An estimate for precision was obtained from duplicate analysis of the calibration buffers performed on several days. The results are summarized in Table 3-3. The within-day and between-day precision were estimated by ANOVA and are not significantly different. The precision is constant over the pH range 3.6-7.4 but is dependent on the particular optrode. The higher sensitiv- ity optrodes (FOCS-6 and FOCS-8) had precision of about 0.02 to 0.05 pH units while precision for the lower sensitivity optrode (FOCS-3) was 0.1 to 0.2 pH units. A change in precision with optrode aging was not evident. However, over a longer time frame, a decrease in precision would be expected as the op- trode sensitivity decreased. An estimate of optrode lifetime was not obtained in this study. 13 ------- TABLE 3-3. SUMMARY OF FOCS METHODS PRECISION Measured FOCS ID FOCS-8 FOCS-6 FOCS-8 Buffer* 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 Day 1 4.03+0.06 4.13±0.09 4.55±0.03 5.56±0.03 6.84±0.09 7.23+0.08 7.52±0.08 7.78±0.12 3.79±0.04 3.99±0.03 4.89±0.02 5.79±0.02 6.84±0.02 7.13±0.02 7.32±0.02 7.32±0.02 3.86±0.02 4.02+0.01 4.79±0.00 5.67±0.01 6.86±0.01 7.17±0.01 7.55±0.03 7.54±0.03 Day 2 4.02+0.12 4.12±0.09 4.59±0.11 5.37±0.08 6.82±0.15 7.22+0.12 7.52±0.06 7.78±0.12 3.81±0.01 4.00±0.01 4.87±0.01 5.75±0.01 6.85±0.02 7.14±0.01 7.32±0.01 7.32±0.01 3.87±0.03 4.01±0.04 4.79±0.01 5.67±0.00 6.87±0.00 7.18±0.00 7.33±0.03 7.54±0.02 4 4 4 5 6 7 7 7 3 4 4 5 6 7 7 7 Day 3 .05±0. .07±0. .60+0. .39±0. .87±0. .21±0. .49±0. .74±0. .82±0. .00±0. .86±0. .74±0. .84±0. .14±0. .32±0. .32±0. 3.89±0 4.05±0 4.74±0 5.61±0 6.85±0 7.18±0 7.35±0 7.60±0 pH Day 4 15 13 05 00 03 06 06 02 02 03 01 04 00 01 01 01 .03 .03 .03 .02 .02 .01 .05 .01 4. 4. 5. 6. 7. 7. 7. 3. 4. 5. 6. 7. 7. 7. 3 4 5 6 7 7 7 10±0. — 69±0. 44±0. 75±0. 19±0. 42±0. 80±0. 86±0. — 88±0. 73±0. 82±0. 12±0. 34±0. 34±0. .95±0 — .79+0 .62±0 .84+0 .16±0 .88+0 .60+0 26 15 11 12 18 17 17 03 06 05 08 06 03 03 .14 .03 .05 .06 .04 .03 .06 Preci (Std. With- in Day 5 Day 4.19+0.39 0.22 0.10 4.80±0.30 0.16 0.07 6.96±0.05 0.10 7.36±0.15 0.13 7.49+0.20 0.15 7.80+0.13 0.11 0.03 0.02 0.03 0.03 0.02 0.03 0.03 0.03 0.07 0.03 0.02 0.03 0.03 0.02 0.04 0.03 si on Dev. Be- tween Day 0.14 0.06 0.19 0.06 0.16 0.13 0.09 0.04 0.05 0.03 0.03 0.05 0.02 0.01 0.03 0.03 0.07 0.03 0.05 0.06 0.06 0.02 0.03 0.06 *A11 results are from duplicate analyses, except Buffer 7, for which N=4. One parameter not apparent from the precision data in Table 3-3 is method drift. In the calibration curve results, it was noted that there is a negative drift from day-to-day in the calibration curve slope. The drift within an analysis can be studied by examining the difference between duplicate analyses (approximately one hour between analyses). This data is summarized in Table 3-4. All of the mean differences are less than or equal to zero. The second pH value in a duplicate pair was determined to be less than the first pH value at P=0.05 level using the Wilcoxon signed rank test. For FOCS-6 and -8 the negative drift is within normal analytical constraints (< 0.05 pH unit) and is less than the analytical bias. The drift for FOCS-3 appears to be more severe than FOCS-6 and -8. However, this is an artifact from the lower sensitivity for FOCS-3 (the same drift in analytical signal will result in a larger pH difference for FOCS-3). 14 ------- TABLE 3-4. SUMMARY OF DUPLICATE DIFFERENCE DATA Delta* Standard pH FOCS-3 FOCS-6 FOCS-8 3.6 4.0 5.0 6.0 6.8 7.0 7.4 -0.28±0.18(5) -0.15±0.03(3) -0.16±0.17(5) -0.06±0.09(4) -0.12±0.07(5) -0.1610.09(5) -0.1310.090(5) -0.04±0.02(4) -0.03±0.02(3) -0.04±0.03(4) -0.04+0.02(4) -0.03±0.02(4) -0.0410.04(4) -0.04±0.04(4) -0.02±0.04(3) -0.03±0.03(3) 0.0010.04(4) -0.0210.04(4) -0.0210.04(4) -0.0210.03(4) -0.0210.05(4) *Delta = pH (Final) - pH (initial). Number of data points is given in ( ). Overall, the precision (expressed as a standard deviation) of FOCS optrodes ranges from 0.02 to 0.2 pH units. The precision will vary with the individual optrode sensitivity and will deteriorate as the optrode ages. 3.2.4 Sample Analysis Rate The measurement procedure using optrodes is manual. The measurement time per sample is about 1 minute (including rinse). However, this is matrix depen- dent. The analysis times in certain matrices may take 5 to 10 minutes. Consequently, analysis rates range from 10-60 samples/hour. More information on response times is given in 3.2.5. 3.2.5 Matrix Effects The effects of ionic strength and buffering capacity were investigated by analyzing a series of standards with varying ionic strength and buffering capacity. A list of the standards used is given in Tables 3-5 and 3-6. 3.2.5.1 Response Time-- Initial reports on the FOCS optrodes indicated a very rapid response time. For most samples tested this was true. Typical strip chart recorder output from the three FOCS optrodes tested are shown in Figures 3-3a, 3-3b, and 3-3c. In the measurement procedure the optrode is placed in a sample with the fluoro- meter shutter closed (strip chart baseline), the shutter is open, and the response monitored on the recorder. When the response is stable, the shutter is closed, the probe rinsed, and the process repeated for the next sample. The samples analyzed in Figure 3-3 are high ionic strength calibration standards. Results for the sulfuric acid, acetate, and most phosphate standards with varying ionic strength were similar. The response time is generally about 10 seconds, which includes the 8 second delay built into the fluorimeter. For the sulfuric acid standards (pH4), ionic strength did not have an effect on the response time. For the acetate buffers (pH 4.7), neither ionic strength nor buffering capacity had an effect on response. Also, for the phosphate buffers, 15 ------- TABLE 3-5. COMPOSITION OF STANDARDS WITH VARYING IONIC STRENGTH* Sulfuric Acid Standards No. mN H2S04 No. Ionic Strength Expected pH Measured pH (pH Electrode) 1 2 3 0.10 0.10 0.10 0.00015 0.0042 0.040 4.01 4.03 4.08 4.01 ± 0.05(10) Acetate Standards No. mM HOAc mM NaOAc Ionic Strength Expected pH Measured pH (pH Electrode) 1 2 3 0.48 0.48 0.48 0.52 0.52 0.52 0.001 0.005 0.041 4.74 4.69 4.55 4.81 ± 0.15 mM KH2P04 mM N32HP04 Ionic Strength Expected pH Measured pH (pH Electrode) 1 2 3 0.25 0.25 0.25 0.25 0.25 0.25 0.001 0.005 0.041 7.15 7.10 6.95 7.05 ± 0. 10(15) *Ionic strength is adjusted with KC1. Expected pH values were calculated as described in references 14 and 15 and have been corrected for activity. no ionic strength effect on response was observed. For comparison purposes, the response time for a pH electrode in the same samples is 0.5 to 2 minutes. Recordings for the phosphate buffers with varying buffering capacity are given in Figures 3-4a, b, and c. It is quite obvious that response time was affected significantly by buffering capacity. For the lowest buffering capacity phosphate sample, the response time was increased to about 3 minutes for two of the optrodes (FOCS-6 and FOCS-8). An even longer increase in response time for one of the lake samples is also seen in Figure 3-4. The response time data for the phosphate buffers and the natural lake samples (FN-9 and FN-10) is summarized in Table 3-7. Corresponding response times for pH electrode measurements in the same samples are also listed. It is interesting to note that the pH electrode response was faster than the optrode response in FN-9 but significantly slower in FN-10. The pH electrode response is expected to be slower in FN-10 (lower ionic strength and buffering capacity), but it is not obvious why the optrode response is opposite that of the electrode. From the data, it is apparent that the sample matrix can have a significant effect on the optrode response time. 16 ------- TABLE 3-6. COMPOSITION OF STANDARDS WITH VARYING BUFFERING CAPACITY AND CONSTANT IONIC STRENGTH* Acetate Standards No. mM HOAc mM NaOAc Expected pH Measured pH (pH Electrodes) ANC 1 2 3 0.035 0.48 5.0 0.065 0.52 5.0 4.86 4.66 4.62 5.06 ± 0.11 (11) 4.80 ± 0.08 (13) 4.72 ± 0.00 (13) 65 520 5000 Phosphate Standards No. mM KH2S04 mM Na2HP04 Expected pH Measured pH (pH Electrodes) ANC 1 2 3 0.025 0.25 2.5 0.025 0.25 2.5 7.18 7.15 7.06 6.49 ± 0.74 6.93 ± 0.07 7.06 ± 0.02 75 750 7500 * Ionic strength is adjusted to 0.01 using KC1. Expected pH values were calculated as described in references 14 and 15 and have been adjusted for activities. Buffering capacity, pH, specific components (i.e., particular anion or cation) and ionic strength may play an interrelated role, but further investigation is required. 3.2.5 Precision and Accuracy The analytical results from the analysis of the standards with varying ionic strength and buffering capacity are summarized in Tables 3-8 and 3-9, respectively. By comparing the precision and bias for the low ionic strength standards to that for the high ionic strength, highly buffered calibration standards, it is evident that the optrode performance is affected, especially for the more sensitive optrodes FOCS-6 and FOCS-8. Performance is worse in circumneutral (about pH 7) solutions with low ionic strength and low buffering capacity. This is very evident in the phosphate buffers. As noted previously, the response time in the phosphate buffers was long. Results for the natural samples were similar, as seen in Table 3-10. Overall it can be concluded that the FOCS performance can be influenced significantly by the sample matrix. Results in low ionic strength, poorly buffered samples seem to be affected the most. This will definitely have an impact on the applicability of FOCS to the determination of pH in natural surface waters. Further studies are necessary in order to determine the interferences and make final recommendations. 17 ------- 400- - ^ 300 — z g W 200 — o CO CO ^ 100- 7.4 3.6 4'° v^s 5.0 1 1 ' 6.0 6.8 1 1 ' 7.0 (** 1 ^«^ \ ' 1 0 2 4 6 8 10 12 TIME (minutes) Figure 3-3a. Sample output from FOCS-3 in high ionic strength calibration buffers (pH indicated on peaks). 18 ------- 700 — 600 — ^ 500 — £ _J O 400 — CO z O CO CO 5 300- Lil 200- 3.C n . ,ooJ 0 $ i '< \ 2 6.0 >^MA ) 5.I * r^ 6.8 7.0 7.4 k^> * | ' 1 ' | ' 1 4 6 8 10 Figure 3-3b. Sample output from FOCS-6 in high ionic strength calibration buffers (pH indicated on peaks). 19 ------- 900— - 800 — - 700 — - 600 — g 500 — 2 g ^ 400 — o CO CO UJ 300 — 200 — 100 — C i 3fl 1 i.8 4 i 2 Tl , kO &j 1 4 c /r 5.0 nit1! 7 7.0 6.0 • t+ AC r.4 r • i • i 6 8 10 -\ •. Figure 3-3c. Sample output from FOCS-8 in high ionic strength calibration buffers (pH indicated on peaks). 20 ------- 300 — £ < 200- O CO Z o CO 100 — CO 2 *^l^« G PB 2 PB 1 r**^ -x^~ PB 3 | . | i 246 FN 9 _ ^k. •— • ^ i^*S*-r~*-^~ FN 10 r* ^ ' 1 ' | ' 1 8 10 12 TIME (minutes) Figure 3-4a. Sample output from FOCS-3 in phosphate buffers (constant ionic strength, variables buffering capacity. See Table 3-6) and in two real samples. 21 ------- 800 — > E z g CO z g CO CO I 600 — 400 — PB 3 FN 9, PB2 PB 1 200 — 50 — 1 I I I 1 I 1 I I I I I ' FN10 6 8 10 12 14 TIME (minutes) Figure 3-4b. Sample output from FOCS-6 in phosphate buffers (constant ionic strength, variables buffering capacity. See Table 3-6) and in two real samples. 22 ------- 1000 — -800 — j 600 — < z g V) O 400- OT CO w 200 —\ 100 — 50 PB 3 PB 2^- PB 1 FN 10 I 2 I 6 I 8 I 10 I 12 I 14 I 16 18 TIME (minutes) Figure 3-4c. Sample output from FOCS-6 in phosphate buffers (constant ionic strength, variables buffering capacity. See Table 3-6) and in two real samples. 23 ------- TABLE 3-7. FOCS AND pH ELECTRODE RESPONSE TIMES FOR PHOSPHATE BUFFERS AND LAKE SAMPLE FN-9* Sample Nominal ANC Ionic Response (Minutes) pH (Meq/L) Strength FOCS-3FOCS-6FOCS-8Electrode 0.05 mM Phosphate 0.50 mM Phosphate 5.0 mM Phosphate FN-9 Lake Sample FN-10 Lake Sample 7.2 7.2 7.1 6.6 5.5 75.0 750 7500 140 4 0.01 0.01 0.01 * ** 0.2 0.2 0.2 2.4 0.2 3.2 1.2 0.4 6.9 0.2 2.2 0.5 0.3 5.6 0.2 4 2 1 2 15-20 *Conductance = 50 **Conductance = 30 TABLE 3-8. SUMMARY DATA FROM STANDARDS WITH VARYING IONIC STRENGTH Standard Sulfuric Acetate Phosphate Ionic Expected FOCS-3* Strength pH 0.00015 0.0042 0.040 0.001 0.005 0.051 0.001 0.005 0.051 4.01 4.03 4.08 4.74 4.69 4.55 7.15 7.10 6.95 PH 3.7±0.3 4.0±0.2 4.2±0.1 3.8±0.6 4.2±0.03 4.6±0.2 4.8±0.2 5.4±0.1 5.6±0.1 Bias -0.3 -0.03 +0.12 -0.9 -0.5 +0.05 -2.3 -1.7 -1.3 FOCS-6* pH 3.65±0.03 3.9±0.2 4.2±0.2 3.8±0.03 4.4±0.4 4.7±0.3 5.6±0.1 6.2±0.5 6.9±0.5 Bias -0.4 -0.1 +0.18 -0.9 -0.3 +0.2 -1.5 -0.9 -0.05 FOCS-8* PH 3.8±0.2 4.0±0.2 4.1±0.2 3.9±0.1 4.2±0.3 4.4±0.3 5.0±0.5 5.611.0 6.010.9 Bias -0.2 -0.03 +0.02 -0.8 -0.5 -0.2 -2.1 -1.5 -1.0 *Error estimate was determined from ANOVA of data from three days of analysis. 24 ------- TABLE 3-9. SUMMARY DATA FROM STANDARDS WITH VARYING BUFFERING CAPACITY* Standard Acetate Phosphate ANC (Meq/L) 65 520 5000 75 750 7500 Expected FOCS-3 PH 4 4 4 7 7 7 .86 .66 .62 .18 .15 .06 pH 4.5±0.6 4.3±0.4 4.2±0.2 5.1±0.1 6.0±0.4 6.2±0.2 Bias -0.4 -0.4 -0.4 -2.1 -1.2 -0.9 :================= FOCS-6 pH 4.59±0. 4.41±0. 4.39±0. 5.5±0. 6.1±0. 6.29±0. 2 04 04 2 1 04 Bias -0.3 -0.2 -0.2 -1.7 -1.0 -0.8 FOCS-8 pTT 4.57±0 4.42±0 4.41±0 5.5±0 6.2±0 1.4±0 .2 .1 .1 .2 .2 .1 Bias -0.3 -0.2 -0.2 -1.7 -0.9 -0.7 *Ionic strength is constant at 0.01. Precision estimates were obtained by ANOVA of data from three days of analysis. TABLE 3-10. SUMMARY DATA FOR TWO NATURAL SAMPLES Sample FN-9 FN-10 Conductance (MS) 50 30 ANC Meq/L 140 4 Reference PH (range) 6.8 - 7.3 5.0 - 5.3 FOCS-3 PH 6.4±0.3 4.3±0.3 FOCS-6 PH 6.5 ±0.2 4.56±0.04 FOCS-8 PH 6.5±0.1 4.4±0.1 3.3 SUMMARY The determination of pH by FOCS optrodes may be a useful tool but needs further investigation. A summary of analytical characteristics is presented in Table 3-11. TABLE 3-11. FOCS pH METHOD ANALYTICAL CHARACTERISTICS Parameter Rate: 10-60 samples/hour pH Range: 3.6-7.5 Bias: 0.1-0.6 pH unit Precision: 0.02-0.2 pH unit 25 ------- The large bias is primarily due to the calibration procedure. The response curve of the optrodes with pH is non-linear. This initial method attempted to linearize the response curve by plotting the log of the signal vs. pH. This transformation was only partially successful, as evidenced by the large and variable bias. A polynominal fit may .perform better and will be investigated when software is available. The precision is affected by the optrode sensitivity. Each optrode has a different sensitivity, with the more sensitive optrodes having better precision. However, the sensitivity of each optrode decreases with age or use. The aging process of the optrodes has not been fully investigated. In high ionic strength, well buffered samples, the response time of the optrodes is generally about 10 to 20 seconds. However, it is significantly affected by the sample matrix. For a dilute phosphate buffer, the response time was 3 minutes, while for a low ionic strength lake sample the response time was 7 minutes. It also appears that the response times for more sensitive optrodes are more affected by the matrix. Further investigation into the causes is necessary in order to determine the factors affecting optrode response times. An important aspect of the optrodes not studied here is the manufacturing process. It is apparent that the sensitivity, aging process, and response times will all be affected by the preparation procedure. These characteristics could be optimized in the preparation. However, it is even more important that the preparation be reproducible. The precision and accuracy of the optrode are also affected by the sample matrix. Both decrease as ionic strength and buffering capacity decrease. The matrix effect is also more severe at circumneutral pH values. Overall, the optrodes performed well for high ionic strength samples. Further investigation and developments are necessary, particularly in defining potential interferences and in optrode production. The interferences will have impact on the applicability of optrode measurements. At this time, optrodes for pH measurement are not suitable for routine analysis. More development must be done to improve response times and variability in response signal in dilute surface waters. 26 ------- 4.0 CONCLUSIONS AND RECOMMENDATIONS Protocols for two new techniques for measuring pH have been developed and characterized. One method measures pH colorimetrically using a mixture of indicator dyes in a flow injection analysis procedure (FIA pH method). The other method measures pH using a pH fiber optic chemical sensor (FOCS), or pH optrode. The FOCS method measures pH fluorometrically by monitoring the fluorescence of an indicator dye (fluorescein) bonded to the distal end of a fiber optic cable. The basic analytical characteristics are listed and compared to those for a pH electrode in Table 4-1. TABLE 4-1. COMPARISON OF ANALYTICAL CHARACTERISTICS FOR pH ELECTRODE, FIA pH METHOD, AND FOCS pH METHOD Parameter pH Rate (samples/hr) pH Range Bias (pH Unit) Precision (pH Unit) Electrode 4-20 3-10 ±0.05 ±0.05 Method FIA 100 3.6-6.8 ±0.01 ±(0.05 to 0.20) FOCS 10-60 3.6-7.5 ±(0.1 to 0.6) ±(0.02 to 0.2) For routine use in determining pH in natural surface waters, the FIA method compares very well with the pH electrode. It has acceptable precision and bias and is very rapid. Improvements in both precision and accuracy are possible by improving the FIA hardware (more accurate and precise solution handling using syringe pumps). Also it may be possible to increase the linearity and applicable pH range by modifying the indicator reagent mixture and/or monitoring more than one wavelength. Currently, the FIA procedure is not suitable for routine use because of the limited availability of the indicator reagent. Its nature is proprietary but should be published soon (16). Also, potential interferences should be investigated. For example, the bias for dilute acid standards (pH4) was larger than anticipated. The bias appeared to be related to the pH or acid used rather than ionic strength. Interferences from organic components in surface waters should also be studied. Finally, a larger set of real samples should be analyzed and compared to pH electrode data. 27 ------- The FOCS pH method was encouraging but not as well developed as the FIA method. The optrodes investigated were prototypes and consequently the analytical characteristics varied from optrode to optrode. The bias was dependent upon the optrode sensitivity and varied from 0.1 to 0.6 pH unit. The bias is magnified by the the inadequacy of the calibration model. The linear model chosen for the optrode does not explain all of the variation. A poly- nomial model will be tested when the software becomes available. Another optrode characteristic is a limited lifetime. Optrode sensitivity decreases from day-to-day. The actual realistic optrode lifetime and factors affecting it have not been determined and will require further studies. The precision of optrode measurements was excellent, varying from 0.02 to 0.20 pH units. Again, the precision is related to the optrode sensitivity, and hence optrode lifetime. Sensitive optrodes will have better precision. The sample matrix will definitely affect both the FOCS optrode signal and response time. Although response times are generally 10 to 20 seconds, the response times in dilute, low-buffering capacity samples may increase to 7 minutes. Additional studies are needed to further examine the response time and sample matrix. The variable response times would make the FOCS pH optrode impractical for use in an FIA system except for matrices in which its response time is well known. An improvement in calibration bias will have to be completed before the effect of sample matrix on precision and accuracy can be completely quantified. Although the use of FOCS pH optrodes is very exciting and encouraging, more work is necessary. Specifically, the manufacturing process will have to be improved in order to improve optrode production reproducibility, sensitivity, lifetime, and availability. The applicability for FOCs pH measurements will be dependent upon these improvements. In conclusion, of the two new techniques, the FIA method appears more promising. Upon further characterizing interferences and improving linearity, a method suitable for measuring pH in surface waters would be ready for use. The FOCS pH optrode will require manufacturing changes and further characteriza- tions in order for it to become analytically viable. 28 ------- 5.0 REFERENCES 1. Hillman, D.C., J.F. Potter, and S.J. Simon. 1986. National Surface Water Survey Eastern Lake Survey (Phase I - Synoptic Chemistry) Analytical Methods Manual. EPA-600/4-86/09, U.S. Environmental Protection Agency, Las Vegas, Nevada. 2. Peterson, J.I., S.R. Goldstein, and R.V. Fitzgerald. Fiber Optic pH Probe for Physiological Use. Anal. Chem. 52(6): 864-869, 1980. 3. Jones, T.P. and M.D. Porter. Optical pH Sensor Based on the Chemical Modification of a Porous Polymer Film. Anal. Chem. 60(5): 404-406, 1988. 4. Saari, L.A. and W.R. Seitz. pH Sensor Based on Immobilized Fluoresceinamine. Anal. Chem. 54(4): 821-823, 1982. 5. W.R. Seitz. Chemical Sensors Based on Fiber Optics. Anal. Chem. 56(1): 16A-34A, 1984. 6. Kirkbright, G.F., R. Narayanaswamy, and N.A. Weilti. Fiber Optic pH Probe Based on the Use of an Immobilized Col orimetric Indicator. Analyst 109: 1025-1028, 1984. 7. Woods, B.A., J. Ruzicka, G.D. Christian, and R.J. Charlson. Measurement of pH in Solutions of Low Buffering Capacity and Low Ionic Strength by Opto Sensing Flow Injection Analysis. Anal. Chem. 58(12): 2496-2502, 1986. 8. Ruzicka, J. and E.H. Hansen. Optosensing at Active Surfaces - A New Detection Principle in Flow Injection Analysis. Anal. Chim. Acta 173: 3-21, 1985. 9. Hirschfield, T. Remote and In-Situ Analysis. Fres. Z. Anal. Chem. 324: 618-624, 1986. 10. Munkholm, C., D.R. Walt, F.P. Milanovich, and S.M. Klainer. Polymer Modification of Fiber Optic Chemical Sensors as a Method of Enhancing Fluorescence Signal for pH Measurement. Anal. Chem. 58(7): 1427-1430, 1986. 11. Jordan, D.M., D.R. Walt, and F.P. Milanovich. Physiological pH Fiber Optic Chemical Sensor Based on Energy Transfer. Anal. Chem. 59(3): 437-439, 1987. 29 ------- 12. Benson, J. New Technology in pH Analysis. American Lab. 20(3): 60-62, 1988. 13. Street, K.W. A Wide Range Fiber Optic pH Sensor With a Linear Response. American Chemical Society Division of Analytical Chemistry, Paper No. 171, Fall, 1987. 14. Metcalf, R.C. the Accuracy of pH Determination in Glacial Melt-Waters. Zeitschrift fur Gletsch er Kunde and Glazialgcologie 20, 41-51, 1984. 15. Stumm, W. and J.J. Morgan. 1981: Aquatic Chemistry. An Introduction Emphasizing Chemical Equilibria in Natural Waters. New York, John Wiley and Sons. 16. Street, K.W. Personal Communication, April 1988. 30 ------- APPENDIX A COLORIMETRIC DETERMINATION OF pH BY FLOW INJECTION ANALYSIS 1.0 SCOPE AND APPLICATION 1.1 This method is applicable to the determination of pH in natural surface or ground waters. 1.2 This method is applicable to samples with a pH in the range 3.6 to 6.8 pH units. 100 samples per hour may be analyzed. 1.3 This method is not applicable to highly colored water which absorbs in the region of 555 nm. 2.0 SUMMARY OF METHOD Using flow injection analysis, a pH indicator reagent is mixed with sample and the absorbance of the mixture monitored at 555 nm. The pH indicator reagent is a proprietary mixture of pH indicator dyes whose absorbance at 555 nm is proportional to pH. The absorbance signal is approximately linear in the pH region 3.6 to 6.8 (Street, 1987). 3.0 INTERFERENCES Turbidity in the samples may interfere with the absorbance measurement. Samples that contain substances which absorb at 555 nm may not be analyzed by this method. 4.0 SAFETY The standards, samples, and reagents pose no particular hazard to the analyst. Protective clothing (safety glasses, laboratory coat, and gloves) must be worn when preparing or handling reagents. 5.0 APPARATUS AND EQUIPMENT 5.1 Flow injection analyzer set up as in Figure A-l. 5.2 pH Meter Digital meter capable of measuring pH to ±0.01 pH unit and equipped with automatic temperature compensation. 31 ------- 5.3 pH Electrodes High quality, low sodium glass pH and reference electrodes with free-flowing junctions. 6.0 REAGENTS AND CONSUMABLE MATERIALS 6.1 Ethanol (69 percent V/V) Dilute 69 mL ethanol (95 percent purity) to 100 mL with reagent water. 6.2 FIA pH Calibration Buffers (pH 3.6, 4, 5, 6, 6.8, 7, 7.2, and 7.4)* pH 3.60 - Pipet 3.15 mL l.ON HC1 and 62.5 mL 0.4M KHP into a 500 mL volumetric flask and dilute to the mark with reagent water. pH 4.00 - Pipet 0.500 mL 0.10N HC1 and 62.5 of 0.4M KHP into a 500 mL volumetric flask and dilute to the mark with reagent water. pH 5.00 - Pipet 11.30 mL l.ON NaOH and 62.5 of 0.4M KHP into a 500 mL volumetric flask and dilute to the mark with reagent water. pH 6.00 - Pipet 2.8 mL l.ON NaOH and 25.0 mL l.OM KH2P04 into a 500 mL volumetric flask and dilute to the mark with reagent water. pH 6.80 - Pipet 11.2 mL l.ON NaOH and 25.0 mL l.OM KH2P04 into a 500 mL volumetric flask and dilute to the mark with reagent water. pH 7.00 - Pipet 14.55 mL l.ON NaOH and 25.0 mL l.OM KH2P04 into a 500 mL volumetric flask and dilute to the mark with reagent water. pH 7.20 - Pipet 17.35 mL l.ON, NaOH and 25.0 mL l.OM KH2P04 into a 500 mL volumetric flask and dilute to the mark with reagent water. pH 7.4 - Pipet 20.0 mL l.ON HaOH and 25.0 mL l.OM KH2P04 into a 500 mL volumetric flask and dilute to the mark with reagent water. * Formulations from Bates, 1964. Calibrate the FIA pH buffers by the following procedure: a. Calibrate the pH meter and electrode with the pH 4 and 7 NBS-traceable buffers as directed by the manufacturer. b. Measure each of the FIA pH buffers in duplicate. The actual pH for the FIA buffers is taken as the mean of the duplicate measurements. 6.3 Hydrochloric Acid (1.00 N) Carefully add 8.35 mL of concentrated hydrochloric acid (HC1, 12N, ACS reagent grade or equivalent) to 75 mL reagent water. Cool and dilute to 100.0 mL with reagent water. Standardize as follows. 32 ------- a. Place 1 g sodium carbonate (Na2C03, primary standard grade), weighed to the nearest mg, into a 125-mL erhlenmeyer flask and dissolve in 30 to 40 ml water. Titrate with the HC1 to pH of about 5. Lift electrode and rinse into beaker. Boil solution gently for 3-5 minutes under a watch glass cover. Cool to room temperature. Rinse cover glass into beaker. Continue titration to a pH in the range of 4.3-4.7. Stop titration and record exact pH and acid volume. Very carefully add titrant to lower pH exactly 0.3 pH units and record volume. b. Calculate the HC1 normality by the following equation: wt. Na? COo (g) x 1000 HC1 N = £ ±-1 53 (g/eq) (2B-C) B = ml HC1 to reach first recorded pH C = ml HC1 to reach 0.3 pH units lower than first recorded pH c. Repeat twice more and calculate the average and relative standard deviation (rsd). The rsd must be less than 5 percent or the standardization must be repeated. 6.4 Hydrochloric Acid (0.10 N) Dilute 10.0 nt 1.0 N HC1 to 100.0 ml with reagent water. 6.5 Indicator Reagent (70 percent V/V) Dilute 7 mL concentrated reagent (proprietary formulation, obtained from K. Street) to 10 ml with 69 percent ethanol. 6.6 pH Meter Calibration Buffers (pH 4 and 7) Commercially available, NBS-traceable pH buffers at pH values of 4 and 7. 6.7 pH 4 QC Sample Dilute 1.00 mL 0.100 N ^$04 (commercially available and certified) to 1.000 L with reagent water. 6.8 Potassium Dihydrogen Phosphate (1.0 M) Dissolve 34.02 g potassium dihydrogen phosphate (KHgPO/i) in 200 mL reagent water and dilute to 250.0 mL with reagent water. 6.9 Potassium hydrogen phthalate (0.4000 M) Dissolve 20.42 g potassium hydrogen phthalate (C8H5K04 or KHP, primary standard grade) in reagent water and dilute to 250.0 mL with reagent water. 33 ------- 6.10 Reagent Water Water to prepare reagents and standards must conform to ASTM specifications for Type II water. (ASTM, 1984). 6.11 Sodium Hydroxide (1.0 N) Dissolve 4g sodium hydroxide (NaOH) in 75 ml reagent water. Cool and dilute to 100.0 mL with reagent water. Protect from the atmosphere. Standardize daily as described below. a. Pi pet 10.00 ml in NaOH into a 50 mL erhlenmeyer flask. Add one drop methyl orange indicator. Titrate with standardized 1.0 N HC1 to the end point (orange). b. Calculate the NaOH normality by the following equation: (ml HC1) (N HC1) NaOH N = mL (NaOH) c. Repeat twice more and calculate the average and rsd. If the rsd is greater than 5 percent, the standardization must be repeated. 7.0 SAMPLE COLLECTION, PRESERVATION, AND STORAGE Samples should be collected in amber polyethylene bottles. Once collected, store at 4°C and minimize atmospheric exposure. 8.0 CALIBRATION AND STANDARDIZATION 8.1 Analyze the FIA pH calibration buffers as described in Section 11.0. 8.2 Prepare a calibration curve by plotting the FIA peak height vs. buffer pH. Perform a linear regression on the data to obtain the coefficients of the calibration line. Data with a pH greater than 6.8 may not be on the linear portion. Do not use any data which is not on the linear portion of the curve. HT = m (pH) + b, where; HT = peak height pH = pH m = slope of the line b = y-intercept of the line 34 ------- 9.0 QUALITY CONTROL 9.1 Initial Calibration Verification (ICV) Analyze the pH4 QC sample immediately after calibration. If the measured value is not 4.0±0.1 pH units, halt analysis and recalibrate. 9.2 Continuing Calibration Verification (CCV) After every 10 samples and after the last sample, analyze the pH4 QC sample and the pH7 FIA calibration buffer. If either measured value is more than 0.2 pH unit from the expected value, halt the analysis. Recalibrate. All samples up to the last acceptable CCV must be reanalyzed. 9.3 Duplicate Analysis Every sample is analyzed in duplicate and the mean reported. 10.0 PROCEDURE 10.1 Set up the FIA manifold as shown in Figure 1. Place reagent lines (Rl and R2) and sample line (SI) in water and flush system for 10 minutes, checking for leaks and blocked lines. 10.2 Turn photometer power on and allow it to warm up for 15 minutes. Check operation as described in owner's manual. 10.3 Place the Rl line into reagent water and the R2 line into the indicator reagent. Connect the SI line to the auto-sampler. With pump on, let system equilibrate for 10 minutes. 10.4 Load the auto-sampler with the FIA calibration buffers, ICV, CCVs, and unknown samples. Record in notebook the analysis sequence. Analyze the sample following the manufacturer's directions for operation of the instrument. Record peak heights in notebook. Determine measured concentrations as described in Section 12. 10.5 At end of day, place Rl, R2, and SI lines in reagent water and flush system for 15 minutes. Remove from water and pump system dry. Turn power off and remove pump tubes. 11.0 CALCULATIONS From the calibration line constructed in Section 8.0, determine the sample concentration using the mean peak height. If the regression was performed, the sample concentration may be calculated by: Mean Peak Height - b pH measured = m 35 ------- 12.0 PRECISION AND ACCURACY In a preliminary, single laboratory study, the duplicate precision for a series of synthetic samples with pH values 3.6-6.8 ranged from ±0.05 to ±0.2 pH units. Analytical bias was ±0.1-0.2 pH units. For two natural lake samples, results were similar, as indicated in the table below. Sample Conductance (uS) ANC Reference pH* Measured pH FN9 50 140 6.65 ± 0.14 6.73+0.07(3) FN10 30 4 5.5 ± 0.2 5.07+0.14(8) *Reference values were obtained by pH electrode. FN10 reference measurements were unstable. Values ranged from 5.1 to 5.5 13.0 REFERENCES American Society for Testing and Materials, 1984. Annual Book of ASTM Standards, Vol. 11.01, Standard Specification for Reagent Water, D 1193-77 (Reapproved 1983). ASTM, Philadelphia, Pennsylvania. Bates, R.G. Determination of pH, Theory and Practice. John Wiley and Sons, Inc., New York, New York 1964. Street, K.W. A Wide Range Fiber Optic pH Sensor With a Linear Response. American Chemical Society Division of Analytical Chemistry, Paper No. 171, Fall, 1987. 36 ------- 30 CM MIXING COIL WASTE SAMPLE LOOP S1 INJECT VALVE (LOAD) WASTE DETECTOR (555 nm) PUMP R1: CARRIER, REAGENT WATER R2: INDICATOR REAGENT 81: SAMPLE ANALYSIS RATE: 100 SAMPLES/HR DETECTOR: 0.5 AU FULL SCALE SAMPLE LOOP: 200 MICROLITERS Figure A-l. Flow diagram for FIA System. 37 ------- APPENDIX B FLUOROMETRIC DETERMINATION OF pH WITH A FIBER OPTIC CHEMICAL SENSOR 1.0 SCOPE AND APPLICATION 1.1 This method is applicable to the determination of pH in natural surface or ground waters. It has not been tested for waste water or contaminated water samples. 1.2 This method is applicable to samples with a pH in the range 3.6 to 7.5 pH units. 1.3 This method is not applicable to waters containing compounds which fluoresce at 530 nm when excited at 485 nm. 2.0 SUMMARY OF METHOD A fiber optic chemical sensor (FOCS) for pH is constructed by immobilizing fluorescein to the distal end of a fiber optic (Mankholm, 1986). Fluorescein is a fluorescent compound which emits light at 530 nm when excited at 485 nm. The intensity of fluoresence at 530 nm is pH dependent. Sample pH is measured by dipping the probe into the sample and monitoring the fluorescence at 530 nm while exciting at 485 nm. 3.0 INTERFERENCES Interferences have not been completely investigated. However, any compound with fluorescence characteristics similar to flourescein (i.e., emits at 530 nm when excited at 485 nm) will interfere with the method. The FOCS response time is partially dependent upon the sample matrix. The FOCS must be tested in new matrices to ensure that a proper measurement time is used. 4.0 SAFETY The standards, samples, and reagents pose no particular hazard to the analyst. Protective clothing (safety glasses, laboratory coat, and gloves) must be worn when preparing or handling reagents. 38 ------- 5.0 APPARATUS AND EQUIPMENT 5.1 pH Meter Digital meter capable of measuring pH to ±0.01 pH unit and equipped with automatic temperature compensation. 5.2 pH Electrodes High quality, low sodium glass pH and reference electrodes with free-flowing junctions. 5.3 pH fiber optic chemical sensor - obtained from D. R. Walt at Tufts University. 5.4 Fluorimeter suitable for use with fiber optics. Must be capable of exciting at 485 nm and detecting emission at 530 nm. The protocol is written assuming a Douglas Portable Filter Fluorimeter is used. Other fluorimeters will necessitate changes in the exact measurement procedure (Section 10.0). 5.5 Strip chart recorder. 6.0 REAGENTS AND CONSUMABLE MATERIALS 6.1 FOCS pH Calibration Buffers (pH 3.6, 4, 5, 6, 6.8, 7, 7.2, and 7.4)* pH 3.60 - Pi pet 3.15 mL l.ON HC1 and 62.5 mL 0.4M KHP into a 500 mL volumetric flask and dilute to the mark with reagent water. pH 4.00 - Pi pet 0.500 mL 0.10N HC1 and 62.5 of 0.4M KHP into a 500 mL volumetric flask and dilute to the mark with reagent water. pH 5.00 - Pipet 11.30 mL l.ON NaOH and 62.5 of 0.4M KHP into a 500 mL volumetric flask and dilute to the mark with reagent water. pH 6.00 - Pipet 2.8 mL l.ON NaOH and 25.0 mL l.OM KH2P04 into a 500 mL volumetric flask and dilute to the mark with reagent water. pH 6.80 - Pipet 11.2 mL l.ON NaOH and 25.0 mL l.OM KH2P04 into a 500 mL volumetric flask and dilute to the mark with reagent water. pH 7.00 - Pipet 14.55 mL l.ON NaOH and 25.0 mL l.OM KH2P04 into a 500 mL volumetric flask and dilute to the mark with reagent water. pH 7.20 - Pipet 17.35 mL l.ON, NaoH and 25.0 mL l.OM KH2P04 into a 500 mL volumetric flask and dilute to the mark with reagent water. *Formulations from Bates, 1964. 39 ------- pH 7.4 - Pi pet 20.0 mL l.ON HaOH and 25.0 ml l.OM KH2P04 into a 500 ml volumetric flask and dilute to the mark with reagent water. Calibrate the FIA pH buffers by the following procedure: a. Calibrate the pH meter and electrode with the pH 4 and 7 NBS-traceable buffers as directed by the manufacturer. b. Measure each of the FIA pH buffers in duplicate. The actual pH for the FIA buffers is taken as the mean of the duplicate measurements. 6.2 Hydrochloric Acid (1.00 N) Carefully add 8.35 ml of concentrated hydrochloric acid (HC1, 12N, ACS reagent grade or equivalent) to 75 mL reagent water. Cool and dilute to 100.0 ml with reagent water. Standardize as described below. a. Place 1 g sodium carbonate (Na2C03, primary standard grade), weighed to the nearest mg, into a 125-mL erhlenmeyer flask and dissolve in 30 to 40 mL water. Titrate with the HC1 to pH of about 5. Lift electrode and rinse into beaker. Boil solution gently for 3-5 minutes under a watch glass cover. Cool to room temperature. Rinse cover glass into beaker. Continue titration to a pH in the range of 4.3-4.7. Stop titration and record exact pH and acid volume. Very carefully add titrant to lower pH exactly 0.3 pH units and record volume. b. Calculate the HC1 normality by the following equation: HC1 N = wt. Na2 C03 [g]) x 1000 53 (g/eq) (2B-C) B = mL HC1 to reach first recorded pH C = total mL to reach pH 0.3 pH units lower c. Repeat twice more and calculate the average and relative standard deviation (rsd). The rsd must be less than 5 percent or the standardization must be repeated. 6.3 Hydrochloric Acid (0.10 N) Dilute 10.0 mL 1.0 N HC1 to 100.0 mL with reagent water. 6.4 pH Meter Calibration Buffers (pH 4 and 7) Commercially available, NBS-traceable pH buffers at pH values of 4 and 7. 6.5 pH 4 QC Sample Dilute 1.00 mL 0.100 N H2S04 (commercially available and certified) to 1.000 L with reagent water. 40 ------- 6.6 Potassium Dihydrogen Phosphate (1.0 M) Dissolve 34.02 g potassium dihydrogen phosphate (KH2P04) in 200 ml reagent water and dilute to 250.0 mL with reagent water. 6.7 Potassium hydrogen phthalate (0.4000 M) Dissolve 20.42 g potassium hydrogen phthalate (CgHs^ or KHP, primary standard grade) in reagent water and dilute to 250.0 ml with reagent water. 6.8 Reagent Water Water to prepare reagents and standards must conform to ASTM specifica- tions for Type II water. (ASTM, 1984). 6.9 Sodium Hydroxide (1.0 N) Dissolve 4g sodium hydroxide (NaOH) in 75 mL reagent water. Cool and dilute to 100.0 mL with reagent water. Protect from the atmosphere. Standard- ize daily as described below. a. Pipet 10.00 mL in NaOH into a 50 mL erhlenmeyer flask. Add one drop methyl orange indicator. Titrate with standardized 1.0 N HC1 to the end point (orange). b. Calculate the NaOH normality by the following equation: NaOH N = (mL HCL) (N HC1) mL (NaOH) c. Repeat twice more and calculate the average and rsd. If the rsd is greater than 5 percent, the standardization must be repeated. 7.0 SAMPLE COLLECTION, PRESERVATION, AND STORAGE Samples should be collected in amber polyethylene bottles. Once collected, store at 4°C and minimize atmospheric exposure. 8.0 CALIBRATION AND STANDARDIZATION The response curve (signal vs. pH) for the pH FOCS is not linear. In an attempt to linearize the response curve, the logarithm of the response is plotted against pH. This transformation is only partially successful. Alternate calibration techniques are currently under investigation. 8.1 Analyze the FOCS pH calibration buffers as described in Section 10. 41 ------- 8.2 Prepare a calibration curve by plotting the log (response signal) vs. buffer pH. Perform a linear regression on the data to obtain the coefficients of the calibration line. log (R) = m (pH) + b, where; log (R) = log (Fluorometer response signal) pH = pH m = slope of the line b = y-intercept of the line 9.0 QUALITY CONTROL 9.1 Initial Calibration Verification (ICV) Analyze the pH4 QC sample immediately after calibration. If the measured value is not 4.0±0.1 pH units, halt analysis and recalibrate. 9.2 Continuing Calibration Verification (CCV) After every 10 samples and after the last sample, analyze the pH4 QC sample and the pH7 FOCS calibration buffer. If either measured value is more than 0.2 pH unit from the expected value, halt the analysis. Recalibrate. All samples up to the last acceptable CCV must be reanalyzed. 9.3 Duplicate Analysis Every sample is analyzed in duplicate and the mean reported. 10.0 PROCEDURE 10.1 Turn on the fluorometer power and allow to warm up for 15 minutes. 10.2 Refering to Figure B-l, set the voltage range to 20V and the Output 2 Mode to "Lamp". Set lamp intensity to constant power. Check the lamp voltage and adjust to 4.00 volts. Reset the Power 2 Mode to "Lock-In". 10.3 Connect the recorder to the Output 2 recorder jack and set the recorder span to 1 volt full scale. Turn power on and set the chart speed to 1 cm/min. 10.4 Connect a pH FOCS to the output of the Optical Block. 10.5 Set the fluorometer controls as follows: Set Shutter In/Out - in (blocking the light path) Set Output 2 - lock-in Set Lamp Intensity - constant power, lamp voltage: 4.00V Set Gain - set so that span between pH 4 and pH 7 buffers is between 0-1000 mV. 42 ------- Set Settling Time - 8.0 sec. Set Shutter Internal/External - Internal Set Detector - on Set Excitation Filter: 485 nm Set Emission Filter: 530 nm 10.6 Place FOCS into protective holder. 10.7 Fill three 150 mL beakers with water for rinsing the FOCS. Rinse the FOCS by dipping the FOCS three times in each beaker. 10.8 Blot dry the FOCS holder with laboratory tissue and dip into FOCS pH 4 buffer. Toggle the shutter in/out to out and adjust the FOCS output to 200 + 5 mV with the offset control. Toggle the shutter in/out to in and wait a few minutes. Toggle the shutter out and readjust the output to 200 mV if necessary. Toggle the shutter in and record the offset dial reading, and the pH 4 buffer reading. 10.9 Toggle the shutter out and set the recorder to the same mV reading as that on the meter. Should be 200 + 5 mV. Toggle the shutter in. 10.10 Blot dry the FOCS and dip three times into each rinse beaker. Blot dry the FOCS and begin analysis of samples, starting with the FOCS pH calibration buffers and ICV. Analyze unknown samples following required QC sample analysis frequency. Perform each measurement as follows: Step 1. Dip FOCS into sample, making sure the protective cable sheath is not in contact with the solution. Toggle the shutter out. Wait until the output is stable, i.e., not increasing consistently with time. For most samples, this takes about 10-20 seconds. Monitoring the strip chart recorder output assists in this task. Step 2. Tottle shutter in, remove FOCS from sample, blot dry, and rinse by dipping three times into each of the rinse beakers. 11.0 CALCULATIONS From the strip chart recorder output, measure the response signal for each standard, QC sample, and real sample. Construct a calibration curve as instructed in Section 8.0. Calculate the logarithm of each real sample response signal. From the calibration curve, determine the sample concentration. If the regression was performed, the sample concentration may be calculated by: pH measured = log (response signal) - b m 43 ------- 12.0 PRECISION AND ACCURACY In a preliminary, single laboratory study, the duplicate precision (standard deviation) for a series of synthetic samples with pH values from 3.6-7.5 ranged from ±0.02 - 0.2 pH units, depending upon the individual pH optrode. Analytical bias was 0.1 to 0.6 pH units. Bias is due primarily to the linear calibration fit (i.e., the curve is not perfectly linear). 13.0 REFERENCES American Society for Testing and Materials, 1984. Annual Book of ASTM Standards, Vol. 11.01, Standard Specification for Reagent Water, D 1193-77 (Reapproved 1983). ASTM, Philadelphia, Pennsylvania. Bates, R.G. Determination of pH, Theory and Practice. John Wiley and Sons, Inc., New York, New York 1964. Munkhom, C., D.R. Walt, P.P. Milanovich, and S.M. Klainer. Polymer Modifi- cation of Fiber Optic Chemical Sensors as a Method of Enhancing Fluorescence Signal for pH Measurement. Anal. Chem. 58(7): 1427-1430, 1986. 44 ------- Ouse POWER SWITCH 1 CONST. P D CONST. V © LAMP LIGHT BLOCK [fjl © D1io .5°8 ©I NTENSITY GAIN | OFFSE SETTING TIME VOLTAGE RANGE RECORDER LNAMP H20 OUTPUT (x) Cj LOCK IN cm METER SHUTTER IN INT OUT EX1 OUTPUT 2 FIBER OPTIC LIGHT GUIDE - OPTICAL j BLOCK T U DETECTOR ON 0 OFF DETECTOR r II SENSOR HOLDER FLUORIMETER SCHEMATIC Figure B-l. Block Diagram of Fluorometer, 45 ------- |