pH Optrode Calibration and Theory
Initial Start Up
1. Start OOISensors software.
2. Go to Configure  Hardware and select the appropriate hardware configuration.
Click “OK”.
3. Test that the software is accessing spectrometer by clicking on the “Scan” switch.
Spectra should appear in the upper graph window.
Setup for pH Sensor
1. Under Configure Spectrometer, activate channels with pH sensors and enter
their analysis wavelengths: HA (acid peak), A (base peak) and Baseline. (The
isobestic point is not currently used in the OOISensors pH module).
Tip: If you aren’t sure what these wavelengths are, measure the absorbance in
OOIBase32 to determine the peaks and where an appropriate baseline is. The
baseline should be beyond the last peak (no absorbance should be present).
These wavelength values are used in all of the computations and must be
entered correctly.
2. Close the configuration panel with the “OK” button.
3. Unscrew the tip of the probe (both the transmission and reflection probe have a
screw tip). Place a single piece of the film inside the probe and screw the tip back
on. Be careful not to over tighten the tip, this can break the mirror in the
transmission probe and break the film in the reflection probe.
4. Place the probe in the first pH sample and make sure there are no bubbles in the
light path of the probe.
Calibrating the pH Sensor
1. Open the pH calibration window from the “Calibrate” pull-down menu.
2. Choose spectrometer channel for calibration.
3. Enter values for “pH for Low Buffer”, “pH for High Buffer”, “Nominal pK”. The
slope value should be set to 1. The pH for the low buffer and high buffer should
be within the linear range of the indicator being used. For example, for phenol red
the low and high buffer should be pH 6.5 and 8.5 or pH 7 and 8. These numbers
are used in the calculations and should be entered correctly. Each indicator has a
different linear range and should only be used within that range for
measurements.
4. “Integration Period”, “Average” and “Boxcar Smoothing” are displayed for the
active channel. These values can be modified and will be used in the rest of the
application if accepted.
Page 1 of 8
pH Optrode Calibration and Theory
Tip: Click on the yellow scan button and optimize the integration time based on the
spectra on the righthand side of the screen. It is recommended to have the
maximum peak around 2500 counts. The average should definitely be greater
than 20. The boxcar should be around 10 to 20.
5. Block the light path and click “Dark”. Intensity (counts) values for all
wavelengths of interest will be stored.
6. Place the optrode in a “Low pH” sample and let equilibrate for 10 minutes. Then
click on the Low pH button to take its measurement. The “Low pH” is your
reference point for your absorbance measurements and should be less than the
lowest value in the linear range of the indicator. For example, for phenol red a
good reference pH would be 5. Intensity (Counts) values for all wavelengths of
interest will be stored. These values are used as the reference values in the
absorbance calculations.
7. Place the optrode in a “High pH” sample and wait 10 minutes. The high value
should also be outside the linear range of the indicator. For phenol red, pH 10
would be appropriate. After stabilizing, click on the “High pH” button and take
its measurement. An absorbance (AU) curve will be stored in the graph (Red
line). Values for all wavelengths of interest will be stored as well.
8. Place the optrode in a “Low Buffer” sample and wait 10 minutes. The low buffer
should be the same number entered earlier. Click on the “Low Buffer” button to
take its measurement. An absorbance (AU) curve will be stored in the graph
(Blue line). Values for all wavelengths of interest will be stored as well.
9. Place the optrode in a “High Buffer” sample (pH 8 for phenol red) and wait 10
minutes. Click on the “High Buffer” button to take its measurement. An
absorbance (AU) curve will be stored in the graph (Green line). Values for all
wavelengths of interest will be stored as well.
Tip: The absorbance graphs for each measurement will appear in the left-hand
graph. The intensity measurements will be in the right-hand graph. Pay
attention to the intensity measurements. A sudden overall drop in intensity
may be attributed to a bubble (this is especially true when using the
transmission probe). If an increase is noticed where there is a saturation of the
detector, the integration time must be dropped and the calibration restarted
from step 5.
10. Click on the “Update Channel Calibration” button.
11. From this point and on, when you click the “Scan” button, intensity and
absorbance curves for the samples will be displayed (black line) along with the
values for wavelengths of interest. All pH values (for the different models) will
be calculated and displayed in the lower right-hand corner.
12. At this point, choose which model you would like reported on the main
OOISensors screen. Do this by selecting the model in the upper left-hand corner.
Tip: In general, models 2 and 3 work best for all of the indicators.
Page 2 of 8
pH Optrode Calibration and Theory
13. Close the calibration screen.
14. Go to Configure  Spectrometer and select the model that you want to be
displayed in the upper right-hand corner on the main screen (it should be the same
model chosen on the calibration page). Click on “OK”.
15. On the main screen, switch to “Scan On” in the lower right-hand corner. In the
upper left-hand will be reported the pH value for your sample. The absorbance
curve will appear in the upper graph. Be sure to rescale to see the spectra fully.
Tip: Be sure to shut down the software correctly so the software retains these
calibration values. If the software or computer crash, then the values will be
lost and you will have to recalibrate the optrode.
Page 3 of 8
pH Optrode Calibration and Theory
Theory of operation and calculations
HA  H   A 
pH   log[ H  ]
K
[ A  ][ H  ]
[ HA]
pK   log K  log[ HA]  log[ A ]  log[ H  ]

pH  pK  log[ A ]  log[ HA]
or
pH  pK  log
Page 4 of 8
[ A ]
[ HA]
pH Optrode Calibration and Theory

Model # 1. Looks at base peak only. No baseline correction. Uses nominal pK
and slope.
[ A ] 
[ A ] Abs
[ A ]HighpHAbs
[ HA]  1  [ A  ]
If

[A ]
 0 then pH  0
[ HA]
else
1
[ A ]


pH  pK  
log

[ HA]
 2.302585 

Model # 2. Looks at base peak only. Corrects for baseline.
[ A ] 
[ A ] Abs  BaselineAb s
[ A ]HighpHAbs  HighpHBase lineAbs
[ HA]  1  [ A  ]
If

[A ]
 0 then pH  0
[ HA]
else
1
[ A ]


pH  pK  
 log
[ HA]
 2.302585 
Page 5 of 8
pH Optrode Calibration and Theory

Model # 3. Looks at base peak only. Uses the values of two buffers to predict
absorbance of [A] High pH and [HA] High pH.
 pH
LowH 10 LowBuffer
 pH
HighH  10 High Bu ffer
correct for baseline
[ A ]LowBufferAbs  [ A ]LowBufferAbs  LowBufferBaselineAbs
[ A ]HighBufferAbs  [ A ]HighBufferAbs  HighBufferBaselineAb s
calculate theoretical maximum absorbance at peak

[ A ]HighpHAbs 
LowH * [ A ]LowBufferAbs * [ A ]HighBufferAbs  HighH * [ A ]LowBufferAbs * [ A ]HighBufferAbs
LowH * [ A ]LowBufferAbs  HighH * [ A ]HighBufferAbs
calculate pK from the LowBuffer
[ A ]LowBufferAbs

,
[A ] 
[ A ]HighpHAbs
if
[ A ]
1
[ A ]
else pKlow  0
 0 then pKlow  pH lowBuffer 
log
[ HA]
2.302585
[ HA]
calculate pK from the HighBuffer
[ A ]HighBufferAbs

,
[A ] 
[ A ]HighpHAbs
if
[ HA]  1  [ A ]
[ HA]  1  [ A ]
[ A ]
1
[ A ]
else pKhigh  0
 0 then pKhigh  pH HighBuffer 
log
[ HA]
2.302585
[ HA]
use the average
if pKhigh  0 and pKlow  0 then pK 
[ A ] 
If
Page 6 of 8
[ A ]abs  BaselineAb s
,
[ A ]HighpHAbs
pKhigh  pKlow
2
[ HA]  1  [ A ]
[ A ]
1
[ A ]


 0 then pH  0 else pH  pK  
log

[ HA]
[ HA]
 2.302585 
pH Optrode Calibration and Theory

Model # 4. In addition to previous, it uses both acid and base peaks
 pH
LowH 10 LowBuffer
 pH
HighH  10 High Bu ffer
correct for baseline
[ A ]LowBufferAbs  [ A ]LowBufferAbs  LowBufferBaselineAbs
[ A ]HighBufferAbs  [ A ]HighBufferAbs  HighBufferBaselineAb s
[ HA]LowBufferAbs  [ HA]LowBufferAbs  LowBufferBaselineAbs
[ HA]HighBufferAbs  [ HA]HighBufferAbs  HighBuffer BaselineAb s
calculate theoretical maximum absorbance at peak
LowH * [ A ]LowBufferAbs * [ A ]HighBufferAbs  HighH * [ A ]LowBufferAbs * [ A ]HighBufferAbs

[ A ]HighpHAbs 
LowH * [ A ]LowBufferAbs  HighH * [ A ]HighBufferAbs
LowH * [ HA]LowBufferAbs * [ HA]HighBufferAbs  HighH * [ HA]LowBufferAbs * [ HA]HighBufferAbs
[ HA]HighpHAbs 
LowH * [ HA]LowBufferAbs  HighH * [ HA]HighBufferAbs
DyeCons tan t 
 [ HA]HighpHAbs
[ A ]HighpHAbs
if [ HA]HighpHAbs  [ HA] Abs  BaselineAb s then Ratio  5000 else
Ratio 
If

DyeCons tan t
[ HA]Abs  BaselineAb s  [ HA]HighpHAbs
Ratio  0.0005
 or 
Ratio  5000
 then
pH  0
else
log Ratio 
2.302585
pH  pK  Ratio
Ratio 
Page 7 of 8
pH Optrode Calibration and Theory
Model #4 Revised:
Looks at base peak only. Corrects for baseline.
[ A ] 
[ A ] Abs  BaselineAb s
[ A ]HighpHAbs  HighpHBase lineAbs
[ HA]  1  [ A  ]
Then calculate the pK and slope values from
1
[ A ]


pH  pK  Slope * 
 log
[ HA]
 2.302585 
where the two points you are using are the low buffer and high buffer
Then proceed to calculation
If

[A ]
 0 then pH  0
[ HA]
else
1
[ A ]


pH  pK  Slope * 
log

[ HA]
 2.302585 
Page 8 of 8
pH Optrode Calibration and Theory