5 key points to remember about liquid chromatography! ??

#01 Mobile Phase Selection and Handling in Liquid Chromatography

1. Characteristics of mobile phase requirements: low viscosity, good compatibility with detectors, easy availability of pure products, and low toxicity.

2. pH value selection: For weakly acidic or weakly alkaline samples, adjust the pH value of the mobile phase to increase the retention of components on the stationary phase and improve the peak shape.

3. Selection of buffer: Determine the pH value of the buffer according to the pKa of the analyte, so that the compound exists in one form and obtains good sharp peaks.

4. Degassed mobile phase: Remove dissolved gases in the mobile phase in advance to avoid the impact of bubbles on the system and analysis results.

5. Filter the mobile phase: All solvents should be filtered through a 0.45μm (or 0.22μm) filter membrane to remove impurity particles.

6. Storage of mobile phase: It should be stored in glass, Teflon or stainless steel containers. Avoid storage in plastic containers to prevent contamination and oxidation.

7. Pay special attention to the problem of halogenated organic solvents: they may contain acidic impurities, easily react with stainless steel, may produce corrosive products when mixed with ethers, and may produce crystallization when mixed with reactive organic solvents.

# 02 Selection of mobile phase solvent

??Chemical stability: The selected mobile phase solvent must have a certain chemical stability and not react with the stationary phase and sample components.

??Does not interfere with the detector: The solvent should not interfere with the work of the detector. Choose a mobile phase that does not affect the normal operation of the detector.

??Easy to remove: The solvent should be easy to remove without interfering with the recovery of the separated components.

??Small viscosity: The viscosity of the solvent should be small to ensure appropriate column pressure drop.

??Practicality: Solvents should be cheap, easily available, safe to use, and of high purity.

#03 Precautions when using mobile phase solvents

??Filtration and degassing: Before use, the mobile phase must be filtered and degassed with a 0.45μm filter membrane to remove particles and dust.

??Use after returning to room temperature: Use the mobile phase after returning to room temperature to avoid unstable baseline levels.

??Selection of reagents: Use HPLC grade reagents, such as water, use double distilled water, and the aqueous mobile phase needs to be replaced frequently.

??Clean the flow path: HPLC-grade reagents should be used as HPLC mobile phase reagents. The mobile phase solvent must clean the flow path frequently.

??Gradient change: In gradient experiments, pay attention to the mutual solubility of the mobile phase to avoid the problem of buffer salt precipitation.

# 04 Storage of Liquid Chromatography Mobile Phases

??Storage container: Mobile phase is generally stored in glass, Teflon or stainless steel containers and cannot be stored in plastic containers.

??Phosphate and acetate buffers: should be prepared as freshly as possible and do not store.

# 05 Triple Rule

For every 10% reduction in the amount of organic solvent (methanol or acetonitrile), the retention factor increases approximately three times, which is the three-fold rule. This is a smart and labor-saving method. During the adjustment process, pay attention to the separation of each peak.

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