Elementary Knowledge of Using a Column
1. The installation of the HPLC column
Use a filtered and degassed mobile phase (without buffered salt) to thoroughly rinse the pump and tubing of the chromatograph, making sure that the system does not contain air bubbles.
According to the flow direction of mobile phase indicated on the column, connect the column inlet with the pump pipe, do not connect the outlet with anything.
The flow rate of the pump was set to 0.1 mL/min or less, and it was gradually raised to the normal flow rate (t>5 min).
When the solvent flows from the outlet of the column, set the flow rate to 0 and connect the column outlet with the detector.
The amino column and the cyan column can be used either in the normal phase environment or in the reverse phase environment. When we need to change from the reverse phase to the normal phase or on the contrary, we need to flush the system with 20-30 times volume of THF as a transition solvent, or it is easy to make the pump block, appear the phenomenon of abnormal pressure.
2. Precautions for using the column
(1) The Mobile Phase:
A.The solvent must be HPLC grade, the reagent is as high as possible.
B.Does not have a chemical reaction with the stationary phase, viscosity is small, and the sample has a suitable solubility, requiring k in the range of 1 to 10 (available range) or 2 to 5 (the best range), when k value is too small, it is not conducive to separation; when the k value is too large, the sample may precipitate in the mobile phase.
C.The mobile phase must be filtered and degassed before use.
D.Make sure that the solvents are miscible.
E.The mobile phase must match with the detector, if you were using a UV detector, can not use the solvent that the cut-off wavelength is greater than the detection wavelength.
Plenty of impurities will greatly reduce the performance of the column. when we need to replace the mobile phase, make sure that the solvent is miscible. If the solvent is not miscible with the solvent in the column, then use a transition solvent, otherwise, it will cause permanent damage to the column. When the salt or buffer separate out from the mobile phase, it will also cause permanent damage to the column. The solubility of the sample in the mobile phase should be checked before each injection, and if possible, try to use the mobile phase to dissolve the sample.
(2) The Stationary Phase:
A.The pH of the mobile phase should be maintained between 2.0 and 8.0.
B.If necessary, use pre-saturated columns and guard columns.
C.When using an amino column, avoid containing aldehydes and ketones as much as possible in the mobile phase and the sample.
(3) Back pressure and flow rate:
A.Keep the back pressure below 3500 psi.
B.Avoid any sudden pressure changes.
C.If the back pressure is high (>3500 psi), the column can be rinsed reversely, but the flow rate should be 1/5-1/2 of the normal flow rate.
D.If the test cell needs to be degassed, a back pressure regulator can be used.
In order to maximize the life of the column, the flow rate can be adjusted according to the particle size of the column packing and the inner diameter of the column, making the back pressure less than 3500 psi. If the back pressure does not exceed the limit, the flow rate of the column can be set arbitrarily.
The relationship of Particle size, Inner diameter, Flow rate and back pressure.
(4) Loading amount: The maximum sample volume of the column depends on the size of the column, the conditions and the type of sample, so it is difficult to give a definite value. While the maximum injection volume is relatively easy to determine. The injection volume is too large or the sample concentration is too high will cause peak broadening or peak fusion.
Column size and Maximum sample volume