19A - CV - Experiments

19A - CV - Experiments#

Notebook 19A contains instructions for mandatory experiments

Goals - Overview

Week 17: Understand the design limitations and build a simple model of a potentiostat
Week 18: Build the potentiostat and write code for voltammetric measurements
Week 19: Run voltammetric measurements with different techniques and samples

Experiments - Week 19

19A: CV - Experiments
19o: Optional - Experiments

Goals#

Run Cyclic Voltammetry experiments on samples with Paracetamol and Aspirin

Evaluate your experiments

Structure#

Background Preparation

Anticipate: Classroom, Recommended as Preparation!

Simulate: Classroom, Recommended as Preparation!

Implement and Investigate: Classroom

Requirements#

❗❗❗ = Mandatory

🏆 = Entirely optional - It is interesting and leads to a deeper understanding.

🏆🏆 = Optional. Recommended - It can be re-visited later.

🏆🏆🏆 = Recommended - It is relevant now.

🏆🏆🏆🐐 = Strongly recommended - It might be challenging, but it is surely worth the effort!

Note: The number of trophies (🏆) indicates the level of recomendation for the optional tasks. It does not correlate with the level of difficulty.

❗❗❗ Background#

Phosphate-buffered Saline#

Phosphate-buffered Saline, PBS, and in this course colloquially - the Buffer, is widely used in biological and biochemical experiments to maintain a stable pH and osmotic balance, replicating the conditions in living organisms.

🧪PBS solution - pH 7.4 - Ingredients

139 mM \( \text{NaCl} \) (Sodium Chloride) provides the ionic strength of the buffer, mimicking physiological conditions.

2.7 mM \( \text{KCl} \) (Potassium Chloride) adds potassium ions to the buffer, similar to what’s found in biological systems.

10 mM \( \text{Na}_2\text{HPO}_4 \) (Disodium Hydrogen Phosphate) is a buffering agent that helps maintain the pH at 7.4.

1.8 mM \( \text{KH}_2\text{PO}_4 \) (Monopotassium Dihydrogen Phosphate) balances the pH along with \( \text{Na}_2\text{HPO}_4 \), ensuring the solution is stable and physiological.

in \( \text{H}_2\text{O} \) (Water)

This solution is used for preparing samples provided for the measurements.

Samples#

We selected two common over-the-counter analgesics for your measurements.

Paracetamol - 0.5mg/ml in PBS

Samples with Paracetamol were prepared using a generic 500mg Paracetamol tablet purchased in Kruidvat. The concentration of 0.5 mg/ml in PBS was chosen for its optimal performance across a wide range of experimental parameters.
Aspirin - 2.5mg/ml in PBS

Samples with Aspirin were prepared using a generic 500mg Acetylsalicylzuur (acetylsalicylic acid) tablet purchased in Kruidvat. In this case, a concentration of 2.5mg/ml in PBS - close to the saturation level in aqueous solutions in normal conditions was chosen for the experiments.

💡 Constructing waveforms using np.arange() and np.concatenate()#

We would like to propose yet another approach to generating a waveform for cyclic voltammetry with np.arange() and np.concatenate(). We believe that this approach is more intuitive than the ones proposed before, like the method using the Triangle, but feel free to use any technique that gives a correct waveform.

Breaking down a CV measurement cycle into segments separated by vertices can simplify construction of custom waveforms. In a conventional, one-cycle, voltammetry measurement, the cell potential starts at Ucell=0 (vertex0), and is swept to the highest positive cell potential Umax (vertex1). In the next phase, Ucell is swept to Umin (vertex2) - typically a negative value. In the last phase, the cell potential is brought back to zero (Vertex3), so this waveform consists of three segments. The choice for the vertices and for their order is dictated by the goals of the measurement, so if you decide to conduct advanced measurements, you might need to design a suitable waveform for each case.

In Anticipate 2.2, we listed some parameters for a recommended measurement of Paracetamol, where you will find a list of vertices for the first cycle. The optimal waveform for the measurement of Aspirin might be slightly different, but re-using the one recommended for Paracetamol is a good starting point.

In the proposed technique, Ustep is the control parameter. The NUM_SAMPLES is relaxed and derived from the resulting length of the waveform, e.g. len(waveform). If your Ustep>5mV, you can safely work with upto 3 cycles without overloading the Pico, but it’s still a good idea to print the length of your final waveform to confirm that it’s not too long.

Example for constructing a conventional waveform for Cyclic Voltammetry using np.arange() and np.concatenate()

Each segment of the waveform can be generated using np.arange(), for example:

segment1 = np.arange(vertex0, vertex1, step) which takes the limiting values of that segment, e.g. (0, Umax)
and a suitable spacing step = segment1[i+1]-segment[i] between the values e.g. step = Ustep. Note that it’s positive for sweeps going more positive, and negative for sweeps going more negative.

All segments can be combined into the waveform representing one complete cycle using np.concatenate(), for example;

fullcycle = np.concatenate((segment1, segment2, ...))

Similarly, for multiple cycles, the final waveform is a concatenation of the full cycles.

If the starting point of the measurement deviates from the starting point of the default cycle, which is the case for Paracetamol, a custom segment e.g. prep = np.arange(Ubegin, 0, Ustep) can be prepended to the full waveform, for example;

customwaveform = np.concatenate((prep, waveform))

❗❗❗ Anticipate#

A1. Limits of PBS for Voltammetry#

Based on your measurements of the Buffer in week 18, what is the practical cell potential window (Umin and Umax) for measurements of samples in the PBS solution?

# Notes

A2. Establish the settings for the measurements#

A2.1. Parameters for CV of Paracetamol 0.5mg/ml#

Consider our recommended settings for measuring Paracetamol with Cyclic Voltammetry.
Recommended settings
1. Ucell - vertices across the cycle.
  1. U0 = Ubegin = -0.2V –> Start the sweep from slightly below the zero potential
  2. U1 = Umax = 1.4V –> Sweep to the maximum positive cell potential
  3. U2 = Umin = -0.5V –> Sweep from the maximum positive to the maximum negative cell potential
  4. U3 = Uend = 0V —> Sweep from the maximum negative cell potential to zero potential
    Note that only the first cycle starts from \(U_{begin} \neq 0\). The subsequent cycles continue where the preceding cycle ends, so in this case at Ucell = 0.
2. Ustep = 5mV
3. rate = 100mV/s
4. Icell - suggested minimum range of detection - the peak values are expected within the limits
  1. Icellmax = 150 uA (expected upper limit)
  2. Icellmin = -50 uA (expected lower limit)
5. Ncycles = 3
This configuration will generate 2320 samples and take around 2 minutes. For testing, consider using a faster configuration with a higher rate or a larger Ustep.
Set or derive the remaining parameters required to configure your potentiostat (some might be optional - depending on your design choices), for example:
1. DELAY_MS = ??? - derived from rate - a value for sleep_ms() between steps.
2. wait = ??? - equilibration time
3. Navg = ???
4. Rf = ???
5. C = ???
6. gain = ??? - AMP0 (and/or AMP1) attenuation factor
7. … all other remaining design specific parameters

# Notes

A2.2. Parameters for CV of Aspirin 2.5mg/ml#

Choose a set of parameters for measuring this sample for the first time, so without any prior knowledge of the optimal settings. You may return to this question after gaining some insights and intuition from the measurements with Paracetamol.

# Notes

A3. Prepare waveforms#

Prepare waveforms for the measurements anticipated in A2.1 and A2.2.

# Notes

A4. Reproducibility and efficient use of resources#

The screen-printed sensors used in our experiments are single-use sensors, so the measurement results are considered to be correct only during the first cycle(s) of your analysis. However, the measurements of the Buffer do not degrade the electrodes significantly, so they can be repeated multiple times for testing and recording a reference signal before performing the actual measurement of each sample.

Plan your measurements with single-use sensors.

💡Hint: Re-using sensors

In some scenarios, the electrodes with only a few runs can be restored to provide an acceptable level of accuracy for further testing by flushing them with warm water, running multiple cycles of Voltammetry with the Buffer across a wide cell potential range, and optionally, even gently cleaning them with a cellulose tissue. However, such treated electrodes aren’t generally considered suitable for reproducible measurements, so if critical cases arise and your sensors have degraded, please request a new sensor.

# Notes

❗❗❗ Implement and Investigate#

💡 Testing before the measurements

Test your waveform and the chosen configuration using a sensor reserved for testing before performing an actual measurement of samples with a new sensor.

Before moving to a new sensor (which are sparse), do a test run with a used sensor and try with the buffer and then with the paracetamol. See whether you already observe an additional peak in the voltammogram of the used sensor. If it’s not yet working, you can do more testing without ruining a new sensor. If your configuration is already correct, continue to I1, for the actual measurement. Proceed similarily in I2.

I1. Cyclic Voltammetry - Paracetamol 0.5mg/ml#

Record a Voltammogram of the Paracetamol 0.5mg/ml sample with a Cyclic Voltammetry. Acquire at least one full cycle that captures Paracetamol’s signature peaks. Consider using the recommended parameters. Use only one single-use sensor for this sample.

I2. Cyclic Voltammetry - Aspirin 2.5mg/ml#

Record a Voltammogram of the Aspirin 2.5mg/ml sample with a Cyclic Voltammetry. Acquire at least one full cycle that captures the signature of Aspirin. Consider using one sensor for the initial measurement with rough estimates of the parameters and finding a more optimal configuration, and another sensor for the measurement with optimised settings.

# Notes