Scientist comparing pH measurements with optical sensorsAccuracy is everything, especially when it comes to measuring pH levels. That’s where optical pH sensing systems come into play. pH monitoring is used in various industries such as bioprocessing, environmental monitoring, pharmaceutical research and development, and food and beverage to ensure crucial factors such as safety, efficacy, and quality. But here’s the catch: not all optical monitoring methods are created equal. Learn the differences between them to discover which one delivers the utmost accuracy and efficiency in monitoring.

 

Optical pH sensing systems technology relies on specialized films that incorporate fluorescent indicator chemicals. These indicators are securely held within hydrophilic host polymers, making sure that they only measure the specific ions in the solution they are meant to detect.  By measuring how pH impacts the fluorescence of these indicators, we can accurately determine the pH of the solution.

 

Two Methods Can be Used to Measure pH Using Optical Sensors: RFM or DLR

 

Two competing methods are used to measure the effect of pH on the commercially available sensing films. These methods include Ratiometric Fluorescence Magnitude (RFM) and Dual-Lifetime Referencing (DLR). The competing methods use different optical processing hardware and signal treatments which yield subtle, yet significant, differences in system designs and performance.

Ratiometric Fluorescence method is where two or more intensities of wavelengths of excitation or emission spectrum are measured to detect changes in environment. Usually, a probe is used that is sensitive to an environmental parameter. With the RFM method, the sensing film contains a single fluorescent indicator which is photo-excited with two different wavelengths of light during which the magnitude of the resulting fluorescent signal for each excitation wavelength is obtained. One of the two wavelengths (Ex. λ1) excites both the protonated and de-protonated forms of the indicator with equal efficiency resulting in a fluorescence signal with a magnitude proportional to total amount of indicator available. The second wavelength of excitation (Ex. λ2) only excites the de-protonated form of the indicator. Knowing the total amount of indicator and that portion of it that is de-protonated allows the use of a modified form of the Henderson-Hasselbalch expression applied to a ratio of the two signal magnitudes (SMR) to determine the solution pH.

The DLR method uses sensing films that incorporate two luminescent compounds. These compounds include a pH sensitive fluorescent indicator with a short excited-state lifetime and a pH insensitive luminescent material with a long excited-state lifetime. Both compounds can be excited at the same wavelength and have emission spectra that overlap. This feature allows the collective lifetime of the luminescence from the sensing film to be determined by modulating the excitation light source and measuring the relative phase shift of the modulated luminescent emission.

Since the solution pH sets the intensity of the pH sensitive fluorophore, pH values that reduce it yield an increase in the observed signal phase while increases decrease the observed phase relative to the excitation signal. Similar to the RFM method, the relationship between phase shift and pH can be explained using a version of the Henderson-Hasselbalch equation.

The Biggest Advantage of Using RFM Technology is Ongoing Accuracy

 

One big advantage of the RFM method over DLR is that it can automatically adjust for changes in the optical signal, such as indicator loss due to photobleaching or leaching, reducing measurement drift. This is done by comparing fluorescent signals obtained for the deprotonated form of the indicator, to the signal from the first wavelength of excitation, under which both the protonated and de-protonated forms contribute to the observed emission signal. 

 

Like the RFM, the DLR is mostly immune to signal variations that stem from changes in optical coupling efficiency (the transfer of light from one medium or component to another) but unlike RFM, DLR does not adequately compensate for signal changes that occur because of photobleaching or indicator leaching when one of the two luminescent materials is lost at a higher rate. When that happens, the calibration drifts over time leading to inaccuracies in the measured values of pH.  For example, if the sensing dye of a DLR sensing film bleaches at a greater rate than the reference dye, the resulting phase shift will be higher than the film before the bleaching at the same value of pH. The result of the shift in phase will be an error in the reported pH of the solution.

Another advantage to RFM over DLR is in its insensitivity to solution temperature. The long-lived fluorophores typically used in the DLR approach can show a temperature sensitivity that requires the use of temperature input to correct for the effect of temperature on the lifetime of the reference fluorophore. Failure to account for this effect of temperature will lead to measurement error. Since the fluorophores used in the RFM approach have no temperature sensitivity over the prescribed range of operation, there is no need for temperature measurements and no effects of temperature on measurement accuracy.

 

Choosing the Best Technology is the First Step in Accurate and Precise Measurements

Polestar Technologies uses RFM technology in our optical sensors to monitor pH levels. By using this advanced method, our sensors ensure more accurate and precise measurements, providing confidence and reliability in pH monitoring applications. We offer a wide range of sensor designs including disposables with built-in sensors ensuring the perfect optical sensor will suit your needs, including even for the most difficult requirements.

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