How to use liquid chromatography column
Before using the LC column, it is best to test the performance of the column and save the results as a reference for evaluating the performance changes of the column in the future. However, be aware that: LC column performance may vary depending on the sample, active phase, column temperature, 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 are the best conditions), and only then can the measured results be comparable.
1. Pretreatment of the sample:
a. It is best to use the active phase to dissolve the sample.
b. Use the pretreatment column to remove the strong polarity in the sample or the irreversible adsorption of impurities with the column packing.
c. Filter the particulate impurities by using a 0.45 μm filter membrane.
2. Preparation of the activity phase:
Liquid chromatography is the separation of sample components in the mass exchange between the column packing and the active phase, so the active phase is required to have the following characteristics:
a, the activity relative to the sample has a certain solubility, to ensure that the sample components will not precipitate in the column (or stored in the column for a long time).
b. The active phase is inert and does not react chemically with the sample (except in special cases).
c. The viscosity of the active 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 liquid pump is prolonged (the viscosity of the active phase can be reduced by using the advanced temperature method). ).
d. The physical and chemical properties of the active 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 active phase should not be too low, otherwise bubbles will be easily generated, which may make the experiment impossible.
f. After the activity phase is prepared, it must be degassed. In addition to the trace gases dissolved in the active phase, it is beneficial for detection and also prevents trace oxygen in the active phase from interacting with the sample.
3. Selection of the active phase flow rate:
Since the column efficiency is a function of the linear flow rate of the active 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 selected to be 1 ml/min, and for a column having an inner diameter of 4.0 mm, a flow rate of 0.8 ml/min is preferred.
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