A Comparison of the Integrity 10 Reaction Station IR Measurement Probes - Application Note
Introduction
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| Figure 1. Cole-Parmer Integrity 10 Reaction Station |
Phase diagrams are fundamental to the design and development of optimized crystallization processes, ensuring the production of high-quality crystals. The measurement of a sample solution’s solubility and nucleation point can take a long time when using classical techniques. In the case of proteins this is a particular problem as the determination cannot be carried out simultaneously by one single method.
In this application note a Cole-Parmer Integrity 10 Reaction Station, equipped with three different infrared transmission detectors, will use the turbidity measurement technique to determine the nucleation and solubility points of solution of lysozyme in a sodium acetate buffer, with a comparison being made on the % transmission (%T) data recorded by each probe.
This application note will also compare the current small in-situ IR probe and a large in-situ probe when used to determine the nucleation and solubility points of a viscous solution.
Experimental Methods
Nucleation and Solubility Point Determination - IR Probe Comparison
A sample of lysozyme was dissolved in 0.1 M sodium acetate buffer pH 5.0 and 4% (wt) sodium chloride (NaCl) to give a solution with a concentration of 20 mg/ml.
The solution was heated and cooled in the Cole-Parmer Integrity 10 Reaction Station in a controlled manner to determine the nucleation and solubility points. Turbidity measurements were collected using the optional Cole-Parmer ATS10360 non-intrusive IR probe , the optional Cole-Parmer ATS10230 small in-situ IR probe and the optional Cole-Parmer ATS10394 large in-situ IR probe .
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| Figure 2. Image of an in-situ IR probe and a diagram of the probe’s measurement principle |
The Determination of the Nucleation and Solubility Points of a Viscous Solution
A 70% (w/w) sucrose solution was prepared to give a sample with a viscosity of approximately 480 cP(1).
The solution was heated and cooled in the Cole-Parmer Integrity 10 Reaction Station in a controlled manner to determine the nucleation and solubility points. Turbidity measurements were collected using the optional Cole-Parmer ATS10230 small in-situ IR probe and the optional Cole-Parmer ATS10394 large in-situ IR probe.
Results
Nucleation and Solubility Point Determination - IR Probe Comparison
The solubility point was defined as the point at which the %T reached a stable plateau and the nucleation point was defined as the first point at which a sustained drop in %T was measured. The %T data recorded with each IR probe is shown in Figure 3.
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| Figure 3. Turbidity change of lysozyme solution with temperature (20 mg/ml). |
| IR Probe | Nucleation Point (℃) | Solubility Point (℃) |
| Non Intrusive ATS10360 | 5.9 | 35.2 |
| Small in-situ ATS10230 | 5.6 | 35.0 |
| Large in-situ ATS10395 | 5.4 | 35.1 |
| Std Deviation | 0.25 | 0.10 |
Table 1. Determined nucleation and solubility points with different IR probes.
The experimental results show no significant differences when the nucleation and solubility points of the three different probes are compared. The standard deviation of the nucleation point is 0.25 °C and the standard deviation of the solubility point is 0.1 °C. As solubility is defined by thermodynamic principles, only a small variation in the observed results would be expected, but as the nucleation point is affected by experimental conditions such as the stirring rate, cooling rate, measuring probes etc. a greater variation in results would be expected.
The Determination of the Nucleation and Solubility Point of a Viscous Solution
An assessment of the nucleation and solubility points of a 70% sucrose solution was made and the %T values from each probe are shown in Figure 4.
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| Figure 4. Turbidity change of lysozyme solution with (20 mg/ml); ATS10230 & ATS10394. |
| IR Probe | Nucleation Point (℃) | Solubility Point (℃) |
| Small in-situ ATS10230 | Not Detected | Not Detected |
| Large in-situ ATS10394 | 28.2 | Not Detected |
Table 2. Determined nucleation and solubility points with different in-situ IR probes.
The %T data from the two in-situ IR probes shows that the Cole-Parmer ATS10230 small intrusive IR probe is unable to detect the nucleation or solubility point of the 70% sucrose solution while the Cole-Parmer ATS10394 large intrusive IR probe is only able to detect the nucleation point.
Conclusion
In the low viscosity lysozyme sample no significant difference was observed in the nucleation and solubility points that were determined with the IR probes tested during this study.
In the high viscosity sucrose sample, the solubility point could not be determined with either of the IR probes tested. This is most likely due to inefficient mixing of the sample which meant that the sucrose crystals were not efficiently cleared from the light path of the probe. The nucleation point of the sucrose sample was only detectable with the large in-situ probe. The most likely explanation for this is again related to the inefficient mixing of the sample and the comparatively small opening to the light path found on the small in-situ probe. Further investigation work is required where more vigorous stirring mechanisms are employed to ensure the sample solutions are mixed effectively.
Acknowledgements
The content of this application note is adapted, with permission, from a report entitled Electrothermal* Integrity 10 - “IR – Probes” written by Prof. Dr. J. Ulrich***.
* Since this application note was written, the name of the product has changed to Cole-Parmer Integrity 10 Reaction Station.
**Martin-Luther-Universitität Halle-Wittenberg, Zentrum für Ingenieurwissenschaften, Verfahrenstechnik / TVT, D-06099 Halle (Saale), Germany
References
1. Neil L. Pennington, Charles W. Baker; Sugar, a user's guide to sucrose, (Chapman and Hall, London, 1990), p. 52.
