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

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                     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

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                                     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.

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                                    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

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         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).

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     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

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                                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

-------
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-
^ 300 —
z
g
W 200 —
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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

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700 —
600 —
^ 500 —
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_J
O 400 —
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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
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CO
UJ
300 —

200 —
100 —
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1
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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

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300 —
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< 200-
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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
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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

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  1000 —
  -800 —
j  600 —
<
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V)

O  400-
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  200 —\




   100 —


    50
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PB 1
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              I
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                6
I
8
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10
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12
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14
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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

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    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

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     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

-------
     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.


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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.
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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.

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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
                                       43

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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,




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