Mechanism analysis of the decrease in low-temperature solubility of biological buffer CAPS

In biochemical experiments, CAPS buffer, as an important alkaline buffer, is widely used in Western blotting, enzyme catalyzed reactions, and HPLC separation due to its stable pKa value (about 10.4), good water solubility (up to 11.07 mg/mL at 25 ℃), and low cell membrane permeability. However, experimenters often find that the solubility of CAPS significantly decreases at low temperatures (such as 4 ℃ or -20 ℃), leading to difficulties in buffer preparation or uneven concentration, which in turn affects the reliability of experimental results. This article will analyze this phenomenon from three levels: molecular mechanisms, environmental factors, and experimental operations, and propose targeted optimization solutions.

Molecular mechanism of low temperature solubility decrease

The dissolution process of CAPS is essentially the process of its molecules forming a hydration layer with water molecules through hydrogen bonding. At room temperature, the sulfonic acid group (- SO3H) and amino group (- NH -) in CAPS molecules combine with water molecules through polar interactions to form stable solute solvent complexes. However, as the temperature decreases, the thermal motion of water molecules weakens, and the hydrogen bonding network tends to become rigid, resulting in a decrease in the binding energy between CAPS molecules and water molecules. Experimental data shows that the solubility of CAPS decreases by about 30% at 4 ℃ compared to 25 ℃, which is closely related to changes in the dynamic properties of water molecules.

In addition, the crystallization behavior of CAPS undergoes significant changes at low temperatures. At room temperature, CAPS molecules are dispersed in a disordered state in solution; When the temperature drops below the critical point, molecules form an ordered lattice structure through hydrophobic interactions and π - π stacking. This phase transition process will further reduce the solubility of CAPS and even lead to the precipitation of undissolved solid particles. For example, when preparing CAPS buffer solution, if the solvent is not sufficiently preheated, white flocculent precipitates can often be observed, which is a direct manifestation of low-temperature induced crystallization.

The synergistic effect of environmental factors on solubility

In solvent ion strength experiments, deionized water is often used to prepare CAPS buffer solution. However, if there are residual metal ions (such as Ca ² ⁺, Mg ² ⁺) in the water, they will form complexes with the sulfonic acid groups in CAPS molecules, reducing their effective solubility. At low temperatures, the hydration of ions is enhanced, the stability of the complex is improved, and further exacerbating the decrease in solubility. For example, in a solution containing 0.1 mM Ca ² ⁺, the solubility of CAPS at 4 ℃ is reduced by 15% compared to pure water system.

The solubility of CAPS is closely related to its dissociation state due to pH fluctuations. When the pH is below pKa (10.4), CAPS molecules exist in protonated form (- SO3H) with high solubility; When the pH approaches or exceeds pKa, the solubility of the deprotonated form (- SO ∝⁻) significantly decreases. Under low temperature conditions, the pH value of the buffer solution may drift due to the dissolution of CO ₂ or hydrolysis of impurities, indirectly affecting the dissolution behavior of CAPS.

By understanding the molecular mechanism and environmental factors that contribute to the decrease in low-temperature solubility of CAPS, researchers can optimize the preparation process and storage conditions in a targeted manner to ensure the stability of the buffer performance. In the future, with the development of new zwitterionic buffering agents, the limitations of CAPS in low-temperature experiments are expected to be fundamentally resolved.

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