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The first stage minimizes only the positions of solvent molecules and ions, and the second stage is an unrestrained minimization of all the atoms in the simulation cell

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Signs of contamination or degradation: Cloudiness or turbidity (potential bacterial growth or precipitation) Visible particles or crystals (potential contamination or degradation products) Discolouration or browning (potential oxidative degradation) Visible mould growth (rare, but indicates severe contamination) Unpleasant or unusual odour (potential bacterial contamination) Action: If any of these signs are observed, discard the vial immediately