Installation and Preparation of HPLC Columns

To understand how to install and use an HPLC column, you first need to understand what a column is made of.

HPLC column is a column packed with a stationary phase to separate the mixed components. It consists of a column tube, a pressure cap, a ferrule (seal ring), a sieve plate (filter), a joint, a screw, and the like. The HPLC column joints at both ends of the HPLC column are equipped with sieve plates, which are sintered stainless steel or titanium alloy with a pore size of 0.2~20μm (5~10μm). Depending on the particle size of the filler, the purpose is to prevent the filler from leaking out.

Installation of HPLC columns

  1. Thoroughly flush the chromatograph’s pump and tubing with the filtered and degassed mobile phase (without buffer salts), making sure that the system is free of air bubbles.
  2. According to the flow direction indicated on the column, the column inlet is connected to the pump end line, and the column outlet is not connected.
  3. The flow rate of the pump is set to 0.1 mL/min or lower, and the flow rate is slowly raised to the normal flow rate (t>5 min).
  4. When the solvent is free to flow out from the outlet end of the column, set the flow rate to 0 and connect the column outlet to the line at the detector end.
  5. Balance the column with 10-30 column volumes of mobile phase and normal flow rate.
  6. The amino column and cyano column can be used in both normal phase and reverse phase environments. When it is necessary to change from the reverse phase to a positive phase or positive phase to the reverse phase, it is necessary to use 20-30 column volumes of THF as a transition solvent to flush the system. Otherwise, the check valve of the pump is easily blocked, and pressure abnormality occurs.

hplc-column

Preparation for the Use of HPLC Columns

Before using the HPLC columns, it is best to test the performance of the column and save the results as a reference for future evaluation of the HPLC column’s 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 impurities that irreversibly adsorb with the column packing.
    c. Use a 0.45 um filter membrane to remove particulate impurities.

  2. Selection of mobile phase flow rate

    Since the column efficiency is a function of the linear flow rate of the mobile phase in the HPLC columns, 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 a reverse phase HPLC column is used, the content of methanol or acetonitrile can be appropriately increased).