Matters Needing Attention and Configuration of Mobile Phase in HPLC Column
In the chromatographic analysis, choose the best chromatographic conditions to achieve the most ideal separation is an important task for chromatographers, and it is also one of the tasks to achieve the establishment and optimization of HPLC analysis methods.
The flow velocity of the HPLC column
The column effect is a function of the linear flow rate of the mobile phase in the column, different HPLC column effects can be obtained by using different flow rates. For a particular column, the best column efficiency should be pursued, preferably using the best flow rate.
The flow rate of a column with an inner diameter of 4.6 is generally 1 ml/min, for column with an inner diameter of 4.0 mm, the flow rate of 0.8 ml/min is preferred. When the optimal flow rate is selected, the analysis time may be prolonged.
The method of changing the washing intensity of the mobile phase can be used to shorten the analysis time, such as when using reversed phase column, the content of methanol or acetonitrile can be increased appropriately.
Matters needing attention in the configuration of the mobile phase
Because methanol is cheap, the methanol system is recommended for the reverse phase column. For positive phase columns, petroleum ethers with a boiling range of 30-60 degrees Celsius or purified ethane are recommended as mobile phases, and ethane without purification shall not be used.
It is best to use purified water, deionized water, and double steam water containing phenolic impurities, which may affect the analysis results. HPLC liquid phase is prepared by using the solvent of grade, and the use of a suitable mobile phase can prolong the service life of the chromatographic column and improve the performance of the column.
Configuration of mobile phase in HPLC column
Liquid chromatography is the separation of the sample components by the mass exchange between the column filler and mobile phase, so the mobile phase is required to have the following characteristics:
1. The mobile phase has certain solubility relative to the sample, which ensures the sample components will not precipitate in the column or remain in the column for a long time;
2. The mobile phase provides with inertia and does not produce a chemical reaction with the sample;
3. The viscosity of the mobile phase should be as small as possible so that the separation effect can be obtained when the longer analytical column is used, and reduce the column pressure drop and prolong the service life of the liquid pump;
4. The physicochemical properties of the mobile phase should be adapted to the detector used;
5. The mobile phase boiling point should not be too low, otherwise, it is easy to produce bubbles, resulting in the experiment cannot be carried out;
6. After the configuration of the mobile phase, the process of degassing must be carried out. Removing the trace gas dissolved in the mobile phase is not only beneficial to monitoring but also can prevent the trace oxygen in the mobile phase from acting on the sample.