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
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IV
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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
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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
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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
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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
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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.
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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.
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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.
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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.
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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.
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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.
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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)
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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.
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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