This onset time includes the time for the solution to reach saturation (Ω = 1) with respect to ikaite and the time between reaching the Ω = 1 Cyclopamine concentration level and the onset of precipitation (usually at a much higher Ω value). Therefore, τ should be controlled by both thermodynamic and kinetic effects. While ikaite is precipitated from the solution, CO2 is released, which leads to a decrease in solution pH. This rapid change in pH could have been used to ascertain the onset of precipitation. However, during

the experiment, pH in the solution was kept constant by the addition of NaOH. Therefore, the change in NaOH volume added into the reactor vessel was used to determine τ as indicated in Fig. 2. In order to obtain a higher accuracy, τ was determined from the deviation of NaOH volume change (∆V) relative to the time interval (∆t = 2 min). The point where the slope ∆V/∆t started to increase was considered as the onset of ikaite precipitation. Immediately after the crystals were precipitated, indicated by the change in the volume of NaOH addition (Section 2.3), around 2 mL of the well-stirred solution was sampled together with the crystals by means of a pipette

and quickly transferred to a glass petri dish. The morphology of the crystals was characterized using a microscope (Zeiss, Axiovert 200M) with an objective of 63 × magnification. The phase identification of the crystals was done by means of Raman microscopy. Selleck Fulvestrant This method can be used to reliably distinguish between the various polymorphs of calcium carbonate (Nehrke et al., 2012 and Tlili et al., 2001). The confocal Raman microscope (WITec®, Ulm, Germany) was equipped with a diode laser (532 nm) and an Olympus® 20 × Teflon coated water submersible objective. During the Raman measurements, crystals were maintained

in the original solution and placed Cyclin-dependent kinase 3 in a glass petri dish, which was kept cold using an ice-water bath. The evolution of the IAP of Ca2 + and CO32 − in the solution under different experimental conditions was calculated by using the chemical equilibrium model Visual-Minteq 3.0 (Gustafsson, 2011) modified by the implementation of Ksp, ikaite according to Bischoff et al. (1993). The solubility constant of ikaite (Ksp, ikaite) was derived from log Ksp, ikaite = 0.15981 − 2011.1 / T, where T = K ( Bischoff et al., 1993). Since most equilibrium constants (including Ksp, ikaite) at high salinities and low temperatures are not well known, extrapolations of functional relationships had to be used. The input parameters for each run were the same as used in the experiments, and the model was run in the function of “titration”, simulating the experimental pumping of CaCl2 and NaHCO3 into the working solution.