Principles of liquid chromatography sample analysis! Based on my many years of analysis experience!

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To analyze a sample, you don’t just try to separate it with various mobile phases as soon as you get it, but you first start by analyzing the sample. Whether it is a polymer compound, high viscosity, biologically active biomolecules, whether some or all of the sample components can be ionized (according to these conditions, you can select the chromatographic column, mobile phase, column temperature, flow rate, ion pair reagent, whether pretreatment is required, etc.)... Detailed analysis: First, you should understand the solubility properties of the sample, determine the molecular weight of the sample, possible molecular structure and analytical characteristics, and finally select the HPLC separation mode to complete the analysis of the sample. 　　The solubility of the sample is based on the solubility of the sample in the organic solvent. It can be initially judged whether the sample is a non-polar compound or a polar compound. If the sample is dissolved in a non-polar solvent, it indicates that the sample is a non-polar compound. Adsorption chromatography, normal phase partition chromatography, and normal phase sound chromatography can usually be selected for analysis. Bitter samples are soluble in polar solvents or miscible polar solvents, indicating that the sample is a polar compound, and reversed-phase partition chromatography or the more widely used reversed-phase bonded phase chromatography is usually used for analysis. If the sample is dissolved in the aqueous phase, you can first check the pH value of the aqueous solution. If it is neutral, it indicates a non-ionic component, and it can often be analyzed by reversed-phase (or normal-phase) bonded phase chromatography. If the pH is weakly acidic, the method of inhibiting sample ionization can be used, adding sulfuric acid and phosphoric acid to the mobile phase to adjust the pH to 2 to 3, and then using reversed-phase bonded phase chromatography for analysis. If the pH is weakly alkaline, cationic counterions can be added to the mobile phase and then analyzed by ion pair chromatography. If the pH is strongly acidic or alkaline, ion chromatography can be used for analysis. Due to the slow removal of ion pair reagents in the stationary phase, when changing the mobile phase, sometimes a long period of equilibrium is required; and due to the purity of the ion pair reagents, baseline fluctuations and interference problems in ion pairs are common. 　　The optimization condition is to produce the most separated peaks in the final chromatogram. Knowing the chemical structure of a compound, knowing whether an acid or base is present, may result in better separation when the pH value of the mobile phase = or close to the average pKa value of the mixture. Optimize K first; as the capacity factor increases, the spectral peak broadens and the peak shape becomes shorter. (When all spectral peaks match 1