In High-performance liquid chromatography (HPLC) method, the mobile phase is of prime importance.
It is the component which takes the actual process of separation under the influence of pressure over the stationary phase.
The HPLC mobile phase is a bit of concern in comparison to mobile phase of other chromatography techniques due to the following reasons.
♠ Any alteration in mobile phase composition can lead to wide fluctuation, noise and disturbances in the analytic recordings.
♠ Presence of minute particles in the mobile phase can block the column leading to interruption of the process.
♠ Presence of air bubbles in the mobile phase can vary the pressure applied by the pump on the mobile phase. Even chances of reading fluctuation are high.
HPLC mobile phase requirements:
♦ The mobile phase has to be of a defined composition.
♦ The mobile phase should be particle free. Hence the mobile phase once prepared should be filtered through 0.2μ filter paper.
♦ Internal air bubbles in the mobile phase should be absent. This can be done by solvent or mobile phase degassing technique.
♦ HPLC mobile phase has to stored in a cool place. This avoids any alteration within.
♦ The pH of the buffer has to be fixed and checked every time you start a fresh session of the HPLC procedure.
Types in HPLC mobile phases:
The mobile phase used in HPLC are two types and they are the isocratic type and the other is the gradient type.
In isocratic type, the mobile phase has a single solvent or more than one solvent yet the ratio of the composition is fixed throughout the HPLC analysis.
In gradient type, the number of solvents in the mobile phase is two or more. Here their ratio’s in the mobile phase composition Vary with time.
HPLC mobile phase preparation:
HPLC mobile phase has to be prepared with care to avoid errors. First, see the type of mobile phase, i.e., is it of isocratic type or gradient type.
In isocratic type, the solvent remains the same throughout the process of HPLC. Hence a single solvent may be used along with other solid components dissolved in it.
If gradient type, since the mobile phase composition changes with time, the solvents are individually connected to different pumps and the composition is controlled by the system itself.
Take the solvents as mentioned in standard protocol or procedure fixed by previous standardization. These solvents are to be of HPLC grade.
Select other solid components to be dissolved (HPLC grade) after weighing them properly to make a stock solution of proper molarity. These stock solution can be used to make mobile phase frequently for a continuous run of HPLC.
Mix the solid in water for HPLC or a small amount of mobile phase solvent itself in volumetric flasks to make a stock solution.
Then take a small volume of mobile phase solvent into a measuring cylinder preferably of one liter. Add predetermined volumes of solid component stock solution using a pipette and make up the final volume with the solvent as mentioned.
Then fix the pH of the buffer using highly concentrated acid or base to minimize the volume of their addition in pH fixation.
Then filter the whole mobile phase.
HPLC mobile phase filtration:
Mobile phase filtration has to be effective to remove any type of particulate content. For this filtration through a 0.02μ filter is recommended.
Filtration has to be done using vacuum degassing. For this
1. Take vacuum degassing filtration flask. Wash it thoroughly using water for HPLC and then finally with the solvent used for mobile phase.
2. Place a new 0.02μ filter membrane in the junction of reservoir and vacuum flask.
3. Connect the set up to the vacuum pump and switch it on.
4. From the top of the flask, pour the prepared HPLC mobile phase into the reservoir and allow it to collect in the vacuum flask after passing through filter paper.
5. Try to run the vacuum pump at lower pressure for effective filtration.
Once the filtration is done, take the mobile phase into a bottle/beaker. Then subject the HPLC mobile to degassing using an ultrasonicator or even by agitated vacuum degassing during filtration.
Agitated vacuum degassing during filtration includes running the filtration procedure with vacuum flask placed over a magnetic stirrer and also a magnetic bead inside the container.
Check the video below for agitated vacuum degassing.
A final suggestion is to try to use always freshly prepared HPLC mobile phase during the experimentation for better results.