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Before the column is used, it is best to perform the performance test of the column and save the result as a reference for evaluating the performance change of the column in the future. However, it should be noted that the column performance may vary depending on the conditions of the sample, mobile phase, column temperature used, etc. In addition, the column performance test is performed according to the conditions in the column factory report (the conditions used in the factory test) It is the best condition), as long as the results of such tests are comparable.
1. Pretreatment of the sample:
a. It is best to use a mobile phase to dissolve the sample.
b. Use a pretreatment column to remove strong polarities in the sample or impurities that irreversibly adsorb with the column packing.
c. Use a 0.45 um filter membrane to remove particulate impurities.
2. Configuration of mobile phase:
Liquid chromatography is the separation of sample components between the column packing and the mobile phase for mass exchange. Therefore, the mobile phase is required to have the following characteristics:
a. Flow relative to the sample has a certain solubility, to ensure that the sample components will not precipitate in the column (or remain in the column for a long time).
b. The mobile phase is somewhat inert and does not react chemically with the sample (except in special cases).
c. The viscosity of the mobile phase should be as small as possible so that a good separation effect can be obtained when using a longer analytical column; at the same time, the column pressure drop is reduced and the service life of the hydraulic pump is extended (the method of increasing the temperature can be operated to lower the mobile phase) Viscosity).
d. The chemical nature of the mobile phase should be compatible with the detector used. If a UV detector is used, it is best to use a solvent with a lower UV absorption.
e. The boiling point of the mobile phase should not be too low, and it is responsible for the generation of bubbles, which may make the experiment impossible.
f. After the mobile phase is configured, degassing must be performed. Removal of trace gases dissolved in the mobile phase facilitates both detection and prevention of trace oxygen in the mobile phase from interacting with the sample.
3. Selection of mobile phase flow rate:
Since the column efficiency is a function of the linear flow rate of the mobile phase in the column, different column rates can be obtained using different flow rates. For the best column performance for a particular column, it is best to use the optimum flow rate. For a column with an inner diameter of 4.6 mm, the flow rate is generally 1 ml/min, and for an inner diameter of 4.0 mm column, the flow rate is preferably 0.8 ml/min.
When the optimum flow rate is selected, the analysis time may be extended. A method of changing the washing strength of the mobile phase can be employed to shorten the analysis time (for example, when the reverse phase column is used, the methanol or acetonitrile content can be appropriately increased).
This is the end of the introduction of Use of liquid chromatography column. I hope it can help you.