- The role played by the mobile phase in liquid chromatography
The retention of the target compound in liquid chromatography is determined by the interaction strength between the compound and the mobile phase and stationary phase. The mobile phase acts as the “blood” of the entire liquid chromatography separation process, exhibiting affinity towards the components and engaging in competition with the stationary phase for the components. Therefore, the selection of the mobile phase directly affects the degree of separation of the components.
2. Principles for selecting the mobile phase:
The solvent used as the mobile phase should be immiscible with the stationary phase and should maintain the stability of the chromatographic column.
The solvent used in the mobile phase should have high purity to prevent impurities in the mobile phase from accumulating on the chromatographic column, leading to a decrease in column performance.
- Detection baseline value
For example, when using a UV detector, the mobile phase used should have no or minimal absorption at the detection wavelength. When using a differential detector, a solvent with a significant difference in refractive index compared to the sample should be selected as the mobile phase.
- Viscosity and boiling point.
The viscosity and boiling point of the solvent should be appropriate.
Using a low-viscosity solvent can reduce the mass transfer resistance of the solute, which is beneficial for improving column efficiency. However, solvents with excessively low viscosity, such as ether, should not be used as they can easily form bubbles in the chromatographic column or detector, affecting separation. On the other hand, high-viscosity solvents can affect solute diffusion and mass transfer, leading to reduced column efficiency, increased column pressure, and prolonged separation time.
For mobile phases used in sample preparation and purification, it is advisable to choose solvents with lower boiling points. This facilitates the removal of the solvent from the collected liquid after the column, using methods such as distillation, which is beneficial for sample purification.
- Solubility of the sample.
If the solubility of the sample in the mobile phase is poor, the sample may deposit at the head of the chromatographic column, resulting in a decrease in column efficiency.
Efforts should be made to avoid the use of solvents with significant toxicity to minimize environmental pollution and ensure the safety of laboratory personnel.
3. Commonly used mobile phases.
Reverse phase chromatography is the preferred separation mode for most sample analyses. This mode generally offers better universality, convenience, and ease of obtaining satisfactory separation results compared to other liquid chromatography modes. In reverse phase chromatography, non-polar/weakly polar bonded stationary phase columns are commonly used, such as C18 columns, phenyl columns, and so on.
The selection of mobile phases typically starts with an aqueous phase as the base solvent, which may include the addition of certain amounts of acids, bases, inorganic salt buffers, and so on. Then, a certain amount of polar solvents that are miscible with water, such as methanol (proton donor), acetonitrile (proton acceptor), tetrahydrofuran (dipolar solvent), and others, are added. The polarity of these solvents, as well as their K values (partition coefficients), significantly impact separation selectivity. The table below lists the polarity parameters p’ of some commonly used solvents.
/ Commonly used mobile phase properties in high performance liquid chromatography /
|Solvent||UV wavelength limit/nm||Refractive index||Boiling point/℃||Viscosity (25℃)/mPa·S||Solvent Polarity Parameter p’||Solvent Strength Parameter|
|Methyl Ethyl Ketone||330||1.379||80||0.4||4.7||0.51|
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