1. Ensure the instrument is in good condition. If necessary, replace washing solutions and waste liquids, clean injection needles, replace injection liners and spacers, and clean nozzles, etc.
2. Ensure the chromatography column to be used is in good condition, with a clean column head. If necessary, cut off 1-2 cm, and ensure the column efficiency is normal.
3. Understand the items to be detected, including control, purity, residual solvents, etc.
4. Understand the characteristics of the sample, such as solubility, stability, etc. For example, whether it is stable in the presence of water or alcohol, or whether it is prone to hydrolysis or ester exchange. Avoid using water, methanol, or ethanol as diluents for these compounds, and prefer acetonitrile as a diluent. Determine whether the compound is stable in acid or alkaline conditions. For example, Boc structures are not acid-resistant and require new liners. Determine whether the compound is stable at high temperatures, etc.
5. Determine the detection method based on the chemical structure and physical properties of the compound. If the compound has UV absorption, liquid chromatography (LC) is preferred for detection. If the compound does not have UV absorption, contains some carbon and hydrogen, and has good thermal stability, gas chromatography (GC) is preferred. When the limit of detection is low (several tens of ppm), LC-MS can be used for detection. Common compounds suitable for GC include those with a molecular weight of less than 150 and a boiling point of less than 350℃ (cannot be used for GC if it is greater than 400℃), containing a four-membered ring, five-membered ring, six-membered ring structure, and some bridged and fused-ring structures. If the compound is a salt, it needs to be injected after being freed. If the compound contains one or more carboxyl groups, its boiling point will increase significantly, and it may not be suitable for GC. If the compound contains both amino and carboxyl groups (such as amino acids), it generally has a high boiling point and cannot be detected by GC.

II. Method development ideas

1. If the compound is not highly polar, use the general methods for HP-5 and DB-624 (12 min or 20 min) for preliminary screening, with a longer running time preferred to ensure that all substances are completely eluted. Observe the peak height, peak shape, and separation degree.
2. If the peak height is too low, increase the peak height by changing the injection volume or increasing the sample concentration (1000-6000). The sample concentration for GC is generally greater than 20 mg/mL. Alternatively, adjust the split ratio to increase the response value (the larger the split ratio, the easier it is to cause split discrimination).
3. If the peak shape is not good, increase the column flow rate appropriately, increase the split ratio, and increase the tail blow gas flow rate to make the peak shape narrow and sharp. If there is front or tailing in the peak shape, replace the chromatography column with a higher polarity. If there is significant tailing of early eluting components, ensure that there is no gas leakage in the chromatography column installation. Then, lower the injection port temperature by 50°C or adjust the initial temperature of the program to be 10-25°C lower than the boiling point of the solvent (slower heating rate).
4. When the peak shape is good but the separation is not sufficient, and the two peaks are not separated, the column temperature can be adjusted, the flow rate can be lowered, and the temperature program can be adjusted to increase the ramp rate slowly.
5. When the peak height, shape, and separation are all acceptable, the durability of the method should be examined. The temperature of the injection port can be changed to check for substance residues at low temperatures and substance decomposition at high temperatures. The flow rate can be adjusted to see if it greatly affects the peak retention time, and if the impact is too great, it may lead to reduced separation. Finally, the LOQ, solution stability, and other confirmation methods should be performed.