Precautions for the use of liquid chromatography columns Before using the column, it is best to test the performance of the column and save the results as a reference for future evaluation of column performance changes. However, it should be noted that the column performance may vary depending on the conditions of the sample, mobile phase, column temperature, etc.; in addition, the column performance test is performed according to the conditions in the column factory report (factory test) The conditions are the best conditions), and only then, the measured results 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 to produce irreversible adsorption impurities with the column packing.
c. The particulate impurities were removed by filtration using a 0.45 Î¼m filter membrane.

2. Preparation 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, the flow relative 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 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 life of the liquid pump is prolonged (the temperature can be lowered 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 a lower UV absorption.
e, the boiling point of the mobile phase should not be too low, otherwise it will easily generate bubbles, 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 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 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.

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 using a reverse phase column, the content of methanol or acetonitrile can be appropriately increased).

note:

a. Since methanol is inexpensive, it is recommended to use a methanol system for reversed-phase columns (except where acetonitrile must be used).
b. For the normal phase column, it is recommended to use petroleum ether with a boiling range of 30-60 Â° C or purified hexane as the mobile phase. Unpurified hexane should not be used. It is best to use ultrapure water (resistivity greater than 18 megohms) for water, and phenolic impurities in deionized water and double distilled water, which may affect the analysis results.
c. The aqueous mobile phase is most *prepared before the experiment, especially in summer, using a buffer solution as the mobile phase. It is best to add sodium azide to prevent bacterial growth.
d. The mobile phase was filtered using a 0.45 Î¼m filter to remove particulate impurities.
e. Prepare the mobile phase with HPLC-grade solvents and extend the life of the column with a suitable mobile phase

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