Three points of operation of high performance liquid chromatography

First, degas

      Degassing the mobile phase is a very effective measure to avoid problems with the HPLC system and to get an ideal data. No bubbles are expected to be present in the HPLC system. The HPLC pump generates a lot of force when transporting the liquid. Since the compression ratio of the gas is much larger than that of the liquid, when the bubble is present, you will observe the instantaneous flow rate reduction and the system pressure drop. If the bubble is large enough, the liquid pump will not be able to deliver any solvent and if the pressure is below the pre-set pressure limit, the pump will stop working. Some pumps are designed to eliminate air bubbles well, while others are designed to stop when bubbles are present.

       When a bubble passes through the infusion pump, the bubble is usually dissolved in the mobile phase solution as the system pressure is high, as the mobile phase passes through the column. However, when the detector flow cell is reached, the system pressure returns to atmospheric pressure, so bubbles may appear again in the detector flow cell, and irregular burrs may appear on the chromatogram. To solve this problem, some instrument companies have designed a back pressure controller that provides enough pressure at the detector outlet to keep the bubbles dissolved in the mobile phase until they flow out of the detector. Of course, this pressure should not exceed the pressure limit that the flow cell can withstand, otherwise the detector may be damaged.

       Noise glitch in the liquid chromatogram of an ultraviolet/visible (UV/VIS) detector is usually a sign that bubbles enter and pass through the flow cell. Some detectors are also very sensitive to the presence of air, but exhibit different signs than UV/VIS. For example, it has been reported that the presence of dissolved oxygen in the mobile phase may cause some compounds to be lost when using a fluorescent (FL) detector. Fluorescence. In addition, an electrochemical (EC) detector that detects the change in current caused by a redox reaction on the surface of the electrode by the substance to be tested is also very sensitive to the presence of dissolved oxygen in the mobile phase. In addition, the presence of bubbles sometimes causes the retention time to not be reproduced.

       Therefore, care must be taken to eliminate air in the mobile phase and to prevent air from penetrating into the mobile phase by piping (such as PTFE tubing).

       These concerns can be avoided or minimized if proper attention is paid to the removal of dissolved air from the mobile phase prior to use. Commonly used degassing methods are as follows:

1. Blow the degassing method. By using the characteristic that the solubility of helium in the liquid is lower than that of air, it can be effectively removed from the mobile phase by discharging the liquid into the mobile phase storage container at a flow rate of about 60 mL/min for 10 to 15 minutes under a pressure of 0.1 MPa. Can eliminate nearly 80% of oxygen. With a highly efficient distributed jet flow device, a volume of helium gas can remove an equal volume of almost all of the gas from the mobile phase. This means that 1L of helium can complete the exhaust operation through the 1L mobile phase. Although this degassing method is good, our domestic radon price is relatively high, and few laboratories use this method.

2. Heat the reflux method. This method has a better degassing effect. Pay attention to the cooling efficiency of the condensation tower during operation, otherwise the solvent will be lost and the proportion of the mixed mobile phase will change.

3. Vacuum degassing method. This method can remove the dissolved gas by using a vacuum pump and reducing the pressure to 0.05 to 0.07 MPa. However, since vacuum degassing changes the composition of the mixed solvent, which affects the reproducibility of the experiment, it is often used for simple analysis of a single solvent system.

4. Ultrasonic degassing method. The mobile phase to be degassed was placed in an ultrasonic cleaner and shaken with an ultrasonic wave for 10 to 20 minutes. This method has the worst degassing effect.

5. Online degassing method. Now the HPLC instrument of the product can be equipped with an online degasser. Online degassing is simple to use, low in failure and effective. It is recommended to purchase the instrument when it is purchased. Some companies are optional, so it should be confirmed with the company when talking about the configuration.