Drug Analysis - Liquid Chromatography Optimization

For the optimization of liquid chromatography, there are many optimization directions that can be chosen. Among them, the ones that have a greater impact are the mobility system, chromatographic column, pH value, etc., which are explained from the following aspects:

1. Detection wavelength

Use the maximum absorption wavelength of the main component as the detection wavelength, and be careful to avoid end absorption; if the maximum absorption wavelength of impurities is significantly different from the main component, you can use dual wavelengths or add a correction factor to correct it.

2. Injection volume

This is not clearly defined, usually 10μl, 20μl, and also 50μl, 100μl, but pay attention to the maximum quantitative range of the injection quantitative loop;

3. Mobile phase system

(1) If you create a new method, there is no special suggestion for this. You can choose it by yourself. You can consider your own operating habits and ease of operation;

(2) If there is a mobile phase system reference, you can optimize it based on the mobile phase system, or you can replace the system;

4. pH

Regarding the choice of pH, the textbook recommendation is to calculate pKa, pH, etc. based on the structure of the substance, and then determine the pH value of the mobile phase system. However, in actual situations, where multiple impurities need to be separated using one method, and the properties of the impurities are confusing, the calculated pH value is generally not applicable.

So I have to make a habit of preparing acidic, neutral and alkaline systems under a fixed mobile phase system, and examine the sensitivity of each impurity to the pH value; then gradually narrow the pH range, and finally determine the pH of the mobile phase.

5. Chromatographic column

For chromatographic columns, my practical experience is the same as pH selection. One method needs to separate many impurities, and it seems not applicable to select chromatographic columns based on polarity. Based on experience, the commonly used chromatographic columns with different fillers are summarized as follows:

(1) C18 column: Central air-conditioning type, suitable for everything, the first choice, but the C18 columns currently on the market vary greatly, and you can screen columns from different manufacturers before determining the method;

(2) C8 column: suitable for macromolecular substances, and will be retained earlier than C18;

(3) C4 column: suitable for separating substances with larger molecular weight, and will be retained earlier than C8;

(4) Chiral column: suitable for isomers;

(5) hilic chromatography column: suitable for components with no retention or small retention time;

(6) Amino column: suitable for analysis of sugars, absorbed amino acids, and amide impurities;

(7) Cyano column: This is a very complicated chromatographic column. Components that are extremely retained on the C18 column will be less retained on the cyano column; components that are very weakly retained on the C18 column will be less retained on the cyano column. stronger;

(8) Phenyl column: suitable for aromatic compounds and some highly polar compounds;

6. Elution gradient: adjust by yourself

7. Flow rate: adjust by yourself

8. Column temperature: adjust by yourself

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