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Column Usage Experience
Before using a column, perform a column performance test and save the results as a reference for future evaluation of column performance changes. However, note that column performance may vary depending on the sample, mobile phase, column temperature, and other conditions used. Additionally, column performance tests should be conducted under the conditions specified in the column's factory report (the conditions used in factory testing are optimal). Only then will the results be comparable.
1. Sample pretreatment: a. Dissolve the sample using the mobile phase. b. Use a pre-treatment column to remove strongly polar impurities or impurities that irreversibly adsorb to the column packing. c. Use a 0.45μm filter membrane to remove particulate impurities.

2. Mobile phase preparation: Liquid chromatography achieves separation through mass exchange between the sample components and the column packing and mobile phase. Therefore, the mobile phase must meet the following requirements: a. The mobile phase should have a certain solubility for the sample to ensure that sample components do not precipitate (or remain in the column for an extended period). b. The mobile phase should be relatively inert and not react chemically with the sample (except in special cases). c. The viscosity of the mobile phase should be as low as possible to achieve good separation with longer analytical columns while reducing column pressure drop and extending the life of the liquid pump (temperature increase can be used to reduce viscosity). d. The physicochemical properties of the mobile phase should be compatible with the detector used. For example, when using a UV detector, use solvents with low UV absorption. e. The boiling point of the mobile phase should not be too low, as this can easily cause bubbles, disrupting the experiment. f. After preparing the mobile phase, degassing is essential. Removing trace gases dissolved in the mobile phase benefits detection and prevents trace oxygen from reacting with the sample.

3. Mobile phase flow rate selection: Since column efficiency is a function of the linear velocity of the mobile phase in the column, different flow rates yield different column efficiencies. For a specific column, to achieve optimal column efficiency, use the optimal flow rate. For a column with an internal diameter of 4.6mm, a flow rate of 1ml/min is generally recommended; for a 4.0mm column, 0.8ml/min is ideal. Using the optimal flow rate may extend analysis time. To shorten analysis time, adjust the elution strength of the mobile phase (e.g., for reverse-phase columns, appropriately increase the content of methanol or acetonitrile).
Note: a. Due to its affordability, methanol is recommended for reverse-phase columns (unless acetonitrile is necessary). b. For normal-phase columns, petroleum ether with a boiling range of 30-60°C or purified hexane is recommended as the mobile phase; unpurified hexane should not be used. Use ultrapure water (resistivity greater than 18 MΩ), as deionized and double-distilled water may contain phenolic impurities that could affect analysis results. c. Aqueous mobile phases are best prepared just before the experiment, especially in summer when using buffer solutions as the mobile phase overnight is not advisable. Add sodium azide to prevent bacterial growth. d. Filter the mobile phase through a 0.45 μm membrane to remove particulate impurities. e. Use HPLC-grade solvents to prepare the mobile phase. Using a suitable mobile phase can extend column life and improve column performance.
The purpose of flushing the column is simply to use an organic solvent, but viscosity should not be too high. Organic solvents prevent bacterial growth, and flushing the column aims to prevent bacterial growth from clogging the instrument system and column. Generally, flushing with methanol and acetonitrile interchangeably is acceptable, but acetonitrile is more expensive than methanol.
Reasons for Retention Time Changes
Column head collapse: During use, the packing settles, creating a small void at the column inlet, leading to poor separation.
Remedy: Unscrew the column head, take a small amount of the same packing, moisten it with methanol, and fill the void repeatedly. Then reassemble the column head and flush with solvent for 1-2 hours to equilibrate.
Summary: Proper use of buffer salts is essential to prevent salt precipitation and extend column life. In short, filter before use and flush after use. (Source: Experiment and Analysis)