The components of a sample mix can significantly affect the separation and resolution of compounds during capillary electrophoresis CE analysis. Factors such as the size, charge, and shape of the molecules, as well as the composition of the buffer and the capillary, can influence the separation efficiency and resolution. To optimize the separation and resolution of a mixture containing two or more compounds using capillary electrophoresis, follow this protocol:1. Selection of the appropriate capillary:Choose a capillary with an appropriate inner diameter ID and length. A smaller ID can provide better separation efficiency, while a longer capillary can improve resolution. The most common capillary dimensions for CE are 50-100 m ID and 30-100 cm in length.2. Buffer selection and optimization:Select a suitable buffer system based on the pKa values of the compounds and the desired pH range. The buffer should provide adequate buffering capacity and maintain a stable pH throughout the analysis. Commonly used buffers include phosphate, borate, and Tris-HCl. Optimize the buffer concentration usually between 10-50 mM to achieve the desired separation and resolution.3. pH optimization:Adjust the pH of the buffer solution to optimize the separation and resolution of the compounds. The pH should be chosen based on the pI isoelectric point of the compounds, as this can affect their charge and, consequently, their migration behavior. A pH above the pI will result in a negatively charged compound, while a pH below the pI will result in a positively charged compound.4. Addition of additives:In some cases, the addition of additives to the buffer solution can improve separation and resolution. Common additives include surfactants, chiral selectors, and organic solvents. Surfactants can be used to separate compounds based on their hydrophobicity, while chiral selectors can be used to separate enantiomers. Organic solvents can be used to modify the solubility and viscosity of the buffer solution, which can affect the separation efficiency.5. Optimization of the separation voltage:Adjust the separation voltage to optimize the separation and resolution of the compounds. Higher voltages can result in faster migration times but may also lead to increased Joule heating, which can negatively affect the separation efficiency. The optimal voltage will depend on the specific compounds and buffer system being used.6. Temperature control:Maintain a stable temperature during the analysis, as temperature fluctuations can affect the migration behavior of the compounds. Most CE instruments have built-in temperature control systems, but it is essential to ensure that the temperature remains constant throughout the analysis.7. Sample preparation and injection:Proper sample preparation is crucial for achieving optimal separation and resolution. Ensure that the sample is free of particulates and contaminants, as these can cause peak broadening and poor resolution. Use an appropriate sample injection method e.g., pressure injection or electrokinetic injection and optimize the injection time and pressure/voltage to achieve the desired sample loading.8. Data analysis and optimization:Analyze the electropherograms obtained from the CE analysis to evaluate the separation and resolution of the compounds. Optimize the experimental conditions iteratively, if necessary, to achieve the desired separation and resolution.By following this protocol and adjusting the various parameters as needed, it is possible to optimize the separation and resolution of a mixture containing two or more compounds using capillary electrophoresis.