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Smart pH Cuvettes Instructions
Overview
The Fiber Optic pH Sensor system consists of the following:
Smart pH Cuvettes (1 cm x 1 cm PMMA or quartz) and/or patches
Ocean Optics VIS-NIR spectrometer (or Jaz Sensor module)
SpectraSuite software for reading values
Light source (LS-1 Tungsten Halogen Light Source with a blue filter or a white LED)
CUV-UV Cuvette Holder
Connecting Fibers
Calibration requires recording spectra in high and low pH samples, as well as in at least one pH
standard solution (such as a NIST-traceable buffer).
The Smart pH Cuvettes use a sol gel sensing material coated onto the inner walls of a cuvette. The
immobilized indicator dye(s) are encapsulated into the sol gel matrix, allowing for the diffusion of
ions while preventing leaching of the dye. The cuvettes provide very accurate measurements in the
biological range, from pH 5 to 9. Advantages of these cuvettes over traditional potentiometric devices
include faster response time, easy storage and no maintenance, and low cost. These are especially
useful for monitoring low conductivity samples such as boiler water, where electrode devices fail.
Ocean Optics’ fully integrated pH systems provide full spectral analysis to help eliminate errors from
dye leaching or from changes in turbidity, temperature, and ionic strength. Inherent calibration based
on the physical properties of the immobilized indicator dye eliminates the need for frequent
calibration. The ratiometric algorithm provides accurate and reproducible measurements at a high
resolution.
Smart pH Cuvettes are 1 cm by 1 cm and are made of Poly(methyl methacrylate) (PMMA) or quartz.
The PMMA material has a temperature range of -5 °C to 70 °C, while quartz is available for specific
applications such as high-temperature measurements. The cuvettes are compatible with aqueous
solutions, ethanol/methanol solutions, ammonia, peroxides, sodium hypochlorite solutions, while the
quartz cuvettes are also compatible with concentrated acids (nitric, sulfuric, acetic, etc) and acetone.
Although PMMA cuvettes are designed to be disposable, they can be used multiple times. The lifetime
depends on the extent of exposure to high pH levels and harsh chemicals. See
Storage/Lifetime for more information.
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Smart Cuvettes Instructions
Cuvette Storage/Lifetime
Smart Cuvettes can be stored dry at room temperature for any amount of time. As they are used, the
cuvettes may slowly leach indicator dye from the sensing material. As a rule,
absorbance at pH 11 falls below 0.1, the cuvette should be discarded and replaced
reference of pH 1). The cuvette’s lifetime depends on frequency of use, harshness of the samples it is
exposed to, the temperature of samples, and other environmental factors.
once the maximum
(assumes a
Nature of Samples
The analyte solutions being measured should have a pH within the biological range (pH 5-9) for
accurate readings. Data obtained from analyte solutions that register values above or below this range
should not be considered valid within the specifications of the cuvette. Concentrated acids and acetone
will ruin the plastic cuvettes, so these types of chemicals should be avoided. Aqueous solutions,
ethanol/methanol solutions, peroxides, ammonia, and sodium hypochlorite solutions are all compatible
with the sensor material and the plastic cuvette. Samples should be optically transparent, having no
turbidity or sediment present. It is also ideal to have analyte solutions that are colorless, though
colored liquids can be compensated for.
Response time is dependent on the ionic strength of the solution, with higher salinity samples
responding notably faster. For example, using the calibration buffers of pH 5 – 8 will show a 90%
response in 10 seconds or less, but when pure D.I. water is being measured, more time is needed to
equilibrate at a final value. Make sure that the cuvette is filled to a sufficient level with the analyte
solution. If the liquid level is at or below the optical path, the data will not be valid. To ensure there is
sufficient liquid, it is recommended to fill the cuvette about 70% its height. Likewise, more accurate
results will be obtained if the cuvette is rinsed once or twice with the analyte solution after calibration.
This removes any residual buffer solution that may contaminate your sample. Note that once the
cuvette has been affixed into the cuvette holder, it should not be moved or removed until all
measurements have been completed.
When immersed in solution, the film dyes may leach very slowly over time and will have to be
replaced. The film response rate is limited by diffusion of ions into the material, therefore increasing
stirring speed and ionic strength tend to increase the response rate.
pH Smart Cuvette Set Up
The following procedures describe how to connect and calibrate pH Smart Cuvettes using a VIS-NIR
spectrometer, a light source and SpectraSuite software. See your spectrometer and SpectraSuite
manual for more detailed installation information.
Installing the pH Sensor System
► Procedure
Perform the steps below to install the pH Sensor components:
1. Install SpectraSuite on your computer.
2. Connect the spectrometer to your computer using the supplied USB cable.
3. Install the light source as specified in its instructions.
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Smart Cuvettes Instructions
4. Attach the fibers between the spectrometer, cuvette holder, and light source.
5. Turn on the light source and allow it to warm up for the period specified in the light source
instructions.
Caution
Make sure that the cuvette says fastened in the cuvette holder with the tightening
screw and that it does not move until all measurements have been completed.
Any movement will change the optical signal, disrupting the quality of the
measurement.
Calibrating the pH Sensor System
The Smart Cuvettes include a pre-calibrated pK value determined at the factory. This value was
originally obtained at 22°C, and it is recalculated using the temperature compensation algorithm based
on the temperature that was entered in SpectraSuite’s Calibration Wizard. Using the Factory
Calibration method is ideal for being able to start making pH measurements quickly, though it is less
accurate than performing an Independent Calibration. The specifications listed for the cuvettes assume
a complete Independent Calibration, as this eliminates the errors seen from temperature and other
environmental differences.
Using Factory Calibration
► Procedure
1. Open SpectraSuite and select File | New | New Sol Gel pH Measurement.
2. Click the Calibration Wizard button to begin the calibration.
3. Select the spectrometer to use and click
4. Select
Use Factory Calibration and click Next. The Experimental Parameters screen appears.
Next.
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Smart Cuvettes Instructions
5. Enter your Experimental parameters: Acquisition Wavelength, Baseline Wavelength, and
approximate
Ambient Temperature. Click Set, then click Next.
Note
For Smart pH Cuvettes that perform in the biological range (pH 5 – 9), the
Acquisition Wavelength is 620nm and the Baseline Wavelength is 750nm.
6. Enter the value for pK that came with your Smart pH Cuvette. Then click Next.
7. Take a low pH reference spectrum at pH 1.0. To do this, fill the cuvette with pH 1 buffer.
Allow it to sit for 5 seconds, then remove the buffer. Refill the cuvette with a fresh sample.
Acquire. The program adjusts the integration time to prevent saturation. When
Click
complete, click
8. Take a dark spectrum. To do this, block the light source and click
Then click
9. Unblock the light source.
10. Take a high reference spectrum for pH 11.0. To do this, remove the pH 1 buffer and fill the
cuvette with pH 11 buffer. Allow this to sit for 10 seconds, then remove the buffer and refill
the cuvette with a fresh sample. When complete, click
11. Depending on the value for pK you previously entered, the wizard will ask you to add pH 5 or
pH 8 buffer. For pK values less than 6.5, pH 8 is used; for pK value greater than 6.5, pH 5 is
used. Remove the pH 11 buffer and fill the cuvette with the requested pH buffer. Allow it to
sit for 10 seconds, remove the buffer and refill the cuvette with a fresh sample. Click
and then click
Next.
Acquire Dark Spectrum.
Next.
Next.
Acquire,
Finish.
12. You are now ready to take pH measurements. See Taking pH Measurements.
Performing an Independent Calibration
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► Procedure
1. Open SpectraSuite and select File | New | New Sol Gel pH Measurement.
2. Click the Calibration Wizard button to begin the calibration.
Smart Cuvettes Instructions
3. Select the spectrometer to use and click
Next. The Experimental Parameters screen appears.
click
4. Enter your Experimental parameters:
approximate
Ambient Temperature. Click Set, then click Next.
Next. Select Perform Independent Calibration and
Acquisition Wavelength, Baseline Wavelength, and
Note
For Smart pH Cuvettes that perform in the biological range (pH 5 – 9), the
Acquisition Wavelength is 620nm and the Baseline Wavelength is 750nm.
5. Take a low pH reference spectrum at pH 1.0. To do this, fill the cuvette with pH 1 buffer.
Allow it to sit for 5 seconds, then remove the buffer. Refill the cuvette with a fresh sample.
Acquire. The program adjusts the integration time to prevent saturation. When
Click
complete, click
6. Take a dark spectrum. To do this, block the light source and click
Then click
7. Unblock the light source.
8. Take a high reference spectrum for pH 11.0. To do this, remove the pH 1 buffer and fill the
cuvette with pH 11 buffer. Allow this to sit for 10 seconds, then remove the buffer and refill
the cuvette with a fresh sample. When complete, click
Next.
Acquire Dark Spectrum.
Next.
Next.
9. Follow the wizard and repeat Step 8 for pH buffers 5, 6, 7, and 8 (follow on-screen prompts).
Then, click
10. You are now ready to take pH measurements. See Taking pH Measurements.
Finish.
Taking pH Measurements
Now that you have finished calibration, you can take pH measurements in the biological range.
► Procedure
1. Fill the cuvette with the analyte solution for pH measurement in the biological range. The pH
value appears on the screen in the
Current pH field (upper right corner).
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2. Press the Run/Stop button to toggle data acquisition appearing in the lower table on the
screen. Data is recorded at the time interval you specify in the
Time Interval (sec) field.
3. Click the Reset button to clear the table and restart the run time.
4. Click the Export button to open a window to save your data in a format that can be opened
with Microsoft Excel or a text program such as Wordpad. The exported data file contains all
of the variables that you have entered and have been calculated, along with a time stamp for
data acquisition and save, the time-resolved pH data, and complete spectra for all reference
and calibration buffers used.
5. Click the Export Calibration button to open a window to save your calibration data. This will
create a file containing the reference spectra and other variables that can later be loaded via
the Calibration Wizard, allowing for very quick setup.
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Algorithms Used
pH Calculation
Smart Cuvettes Instructions
…where Abs
SlopepKpH
is the sample absorbance at 620nm with baseline correction, and Abs
Sample
log*
Abs
11
Sample
AbsAbs
SamplepH
is the
pH11
absorbance at pH 11 at 620nm with baseline correction.
Temperature Compensation
When you select Use Factory Calibration in SpectraSuite, the value for pK is adjusted via the van’t
Hoff equation based on the current temperature you entered:
11
*480
log
12
epKpK
*480
log
12
epHpH
TT
12
11
TT
12
Resetting pK and Slope
An x-y plot is made using data obtained from intermediate buffers 5 through 8. The x-axis is of the
term:
log
Abs
11
Sample
AbsAbs
SamplepH
…for each of the buffers. The y-axis shows the pH value of the buffers. This generates a plot such as
the one shown below:
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Performing a linear fit gives a line with pK equal to the y-intercept and slope equal to the slope. In the
example chart above, the new pK value would be 6.2977 and the new slope value would be 1.7248.
Specifications
Specification Value
Size and Materials 1 cm x 1 cm PMMA cuvette; 1 cm x 1 cm quartz cuvettes
available for specific applications such as high-temperature
measurements
pH Range Biological range (5-9)
Temperature range -5 to 70 °C for PMMA cuvettes
Accuracy <1% of reading
Resolution 0.01 pH
Response Time 90% step response in 10 seconds
Standardization User needs 2 buffers (pH 1 and 11)
Factory Calibration User needs 1 buffer (pH 5 or 8)
User Complete Calibration Option Users have the option to perform their own complete calibration,
requiring 4 buffers (pH 5, 6, 7, and 8)
Sensory Signal Absorbance at 620nm and 750nm
Expendable Parts PMMA cuvettes are semi-disposable, -- they can be used
multiple times, but they are not intended for long-term
permanent use
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Smart Cuvettes Instructions
Specification Value
Usage Lifetime Potential of being used as many as 50 or more measurements.
Lifespan depends on extent of exposure to high pH levels and
harsh chemicals. Discard after maximum absorbance at pH 11
falls below 0.1 (assumes reference of pH 1).
Recommended Spectrometer
Configuration
Any VIS/NIR spectrometer can be used. Either LS-1 (with blue
filter) or white LED can be used as light source.
Temperature Compensation Factory calibrated pK values are adjusted based on the user-
inputted temperature via the van’t Hoff equation (see Algorithms
Used). Factory coefficients are generated at 22°C.
Chemical Compatibility:
PMMA
Aqueous solutions, ethanol/methanol solutions, ammonia,
peroxides, sodium hypochlorite solutions
Glass
All listed for PMMA plus concentrated acids (nitric, sulfuric,
acetic, etc) and acetone
Chemicals to Avoid for PMMA Concentrated acids (nitric, sulfuric, acetic, etc), acetone
Sterilization:
PMMA
Glass
Gamma irradiation and Ethylene Oxide
Autoclavable, Gamma irradiation, and Ethylene Oxide TBD
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