Before using the column (Agilent column), perform a column performance test and save the results as a reference for evaluating the performance of the column in the future. However, it should be noted that the column performance may be different due to the differences in the samples, mobile phase, column temperature and other conditions used. In addition, the column performance test is performed according to the conditions in the chromatographic column factory report (used in the factory test Conditions are the best conditions), only in this way, the measured results are comparable.
1. Sample pretreatment:
a. It is best to use the mobile phase to dissolve the sample.
b. Use a pre-treatment column to remove impurities that are strongly polarized in the sample or produce irreversible adsorption with the column packing.
4. Use 0.45µm filter membrane to remove particulate impurities.
2. Preparation of mobile phase:
Liquid chromatography is the mass exchange of sample components between the column packing and the mobile phase to achieve the purpose of separation, so the mobile phase is required to have the following characteristics:
a. The mobile phase has a certain dissolving capacity 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 inert and does not react with the sample (except in special cases).
c. The viscosity of the mobile phase 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 (the temperature can be used to reduce the viscosity of the mobile phase ).
d. The physical and chemical properties 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 low UV absorption.
e. The boiling point of the mobile phase should not be too low, otherwise bubbles will easily be generated, which will make the experiment impossible.
f. After the mobile phase is prepared, it must be degassed. Removal of trace gases dissolved in the mobile phase is not only conducive to detection, but also prevents 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 efficiencies can be obtained using different flow rates. For a specific chromatographic column, to pursue better column efficiency, try to use a better flow rate. For a column with an inner diameter of 4.6mm, the flow rate is generally 1ml / min. For a column with an inner diameter of 4.0mm, a flow rate of 0.8ml / min is preferred.
When a better flow rate is selected, the analysis time may be extended. The method of changing the washing intensity of the mobile phase 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).