Before using an HPLC column, the HPLC column is best to perform column performance tests and save the results as a reference for future evaluation of column performance changes. However, HPLC column should be noted that the column performance may be different due to the differences in the samples, liquid chromatography column, and column temperature; in addition, the column performance test is performed according to the conditions in the liquid chromatography column factory report ( the conditions used in the factory test are the best conditions), only in this way, the measured results are comparable.
Sample preparation of HPLC column: 1. Uses a liquid chromatography column to dissolve the sample. 2. Use a pre-treatment column to remove impurities in the sample that are strongly polar or irreversibly adsorbed with the column packing. Use a 0.45μm filter membrane to remove particulate impurities. 3. Preparation of liquid chromatography column: liquid chromatography is the mass exchange of sample components between the column packing and the liquid chromatography column to achieve the purpose of separation. Therefore, the liquid chromatography column is required to have the following characteristics: 4. The HPLC column has a certain ability to dissolve the sample, to ensure that the sample components will not precipitate in the column (or remain in the column for a long time). The liquid chromatographic column is somewhat inert and does not react chemically with the sample (except in special cases). 5. The viscosity of the HPLC column should be as small as possible, so as to obtain a good separation effect when using a longer analytical column; at the same time, reduce the pressure drop of the column and extend the service life of the liquid pump column viscosity). 6. The physical and chemical properties of the HPLC column should be compatible with the detector used. If a UV detector is used, it is best to use a solvent that has a low UV absorption. 7. The boiling point of the HPLC column should not be too low, otherwise, bubbles will be easily generated, which will make the experiment impossible.
After the HPLC column is prepared, it must be degassed. Removal of trace gases dissolved in the liquid chromatography column not only facilitates detection but also prevents trace oxygen in the liquid chromatography column from interacting with the sample. 8. Selection of HPLC column flow rate: since the column efficiency is a function of the linear flow rate of the HPLC column in the column, different flow rates can be used to obtain different column efficiency. For a specific HPLC column, the best efficiency should be pursued, and the best flow rate should be used. For a liquid chromatography column with an internal diameter of 4.6 mm, the flow rate is generally 1 ml/min.
For a column with an internal diameter of 4.0 mm, a flow rate of 0.8 ml/min is preferred. When the best flow rate is selected, the analysis time may be extended. The method of changing the washing intensity of the liquid chromatography column can be used to shorten the analysis time (for example, when using a reversed-phase column, the content of methanol or acetonitrile can be appropriately increased).
Notes on the use of liquid chromatography columns: a. Because methanol is cheap, the methanol system is recommended for reversed-phase columns (except where acetonitrile must be used). b. For normal phase columns, it is recommended to use petroleum ether with a boiling range of 30-60 °C or purified hexane as the liquid chromatography column. Unpurified hexane should not be used. It is better to use ultrapure water (resistivity greater than 18 megohms). Deionized water and double distilled water contain phenolic impurities, which may affect the analysis results. c. The aqueous HPLC column should be prepared well before the experiment, especially if the buffer solution is used as the liquid chromatography column in summer. Add good sodium azide to prevent bacterial growth. d. The liquid chromatography column requires a 0.45μm filter membrane to remove particulate impurities. e. Use HPLC grade solvent to prepare the HPLC column, use a suitable liquid chromatography column can prolong the service life of the liquid chromatography column, and improve column performance.