Solubility saturation shake-flask method

Avdeef, A., Berger, C. M., Brownell, C. pH-metric solubility. 2. Correlation between the acid-base titration and the saturation shake-flask solubility-pH methods. Pharm. Res. 2000, 17, 85-89. 

The shake-flask method is based on the phase solubility technique that was developed 40 years ago and is still the most reliable and widely used method for solubility measurement today (Higuchi and Connors, 1965). The method can be divided into hve steps sample preparation, equilibration, separation of phases, analysis of the saturated solution and residual solid, and data analysis and interpretation (Yalkowsky and Banerjee, 1992, Winnike, 2005). 

In our laboratory, saturation shake-flask solubility is routinely performed at pH 6.8 and pH 1.0 (for acids only) 0.5 mL of buffer is added to about 2 mg of sample in a small borosilicate vial (2 mL) and sonicated for 3 minutes using a sonicating bath. The suspension is then incubated at 25.0°C for 20 hours in a thermostated water bath at a shaking speed of200 cycles/min.The phases are separated by decantation/centrifugation.The supernatant is carefully collected and the solute quantified using the analytical method defined previously. Before each injection, a blank is performed to avoid phantom peaks due to carryover or impurities present in the system. After the centrifugation it is advisable to re-check the pH of the medium. 

Another common source of uncertainty in solubility determinations is when the compound is highly ionized (>99%) at the pH considered. This usually happens when the pH is more than 2 units above the pKa for an acid or than 2 log units lower than the pKa for a base. In this case the solubility pH profile is very steep around that pH, which results in a higher uncertainty on the result. This is the case of Diclofenac at pH 6.8, where we observed some differences within our measurements and between our data and literature values. This limitation is, however, valid for both dissolution template titration method and the saturation shake-flask. 

The solubility of the compound will typically be studied by creating a saturated solution, adding an excess of material under study to a known amount of solvent until dissolution ceases. The sample will then be shaken under controlled room temperature (20-25 °C) typically for 24-48 h. After this time, the solution will be assumed to have achieved a thermodynamic equilibrium, and the liquid will be saturated. It is important to be certain that some solute remains, to ensure that the liquid is completely saturated. The liquid is then assayed to determine the exact amount of material that went into solution. This value is the thermodynamic solubility of the molecule. This is typically called the shake flask method. ... 

The experimental approaches are similar to those for solubility, i.e., employing shake flask or generator-column techniques. Concentrations in both the water and octanol phases may be determined after equilibration. Both phases can then be analyzed by the instrumental methods discussed above and the partition coefficient is calculated from the concentration ratio Q/Cw. This is actually the ratio of solute concentration in octanol saturated with water to that in water saturated with octanol. 

Sutton and Calder (9) have also measured the solubilities of several alkylbenzenes in distilled water and in seawater by a method based on GC. Saturated solutions were prepared by equilibrating water with aromatic vapor in an all-glass apparatus consisting of a 1-L Erlenmeyer flask with an insert tube. The insert tube was used to store the compound. It was capped with a ground-glass stopper. The liquid hydrocarbon did not come into contact with the water except through a perforation in the insert, which allowed hydrocarbon vapors to enter the headspace above the water in the flask. The flask was placed in a constant-temperature shaking bath controlled at 25.0 dt 0.1°C. The water was equilibrated for 48 hr prior to analysis. The solubilities were determined by solvent extraction of the saturated solutions with subsequent analyses of the extracts by GC. 

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