Six Key Points to Keep in Mind When Using Liquid Chromatography!

From Strong to Weak

Start the experiment with 90% acetonitrile (or methanol)/water (or buffer solution), which can quickly yield separation results. Then adjust the ratio of organic solvent (acetonitrile or methanol) based on the peak appearance.

Threefold Rule

For every 10% reduction in organic solvent (methanol or acetonitrile), the retention factor increases by approximately threefold. This is known as the threefold rule, a smart and efficient method. While adjusting, pay attention to the separation of each peak.

Coarse to Fine Adjustment

When separation reaches a certain degree, reduce the organic solvent adjustment from 10% to 5%, and then gradually decrease the adjustment rate following this rule until the separation of components no longer changes.

Characteristics of Mobile Phases
An ideal liquid chromatography mobile phase solvent should have low viscosity, good compatibility with the detector, be easy to obtain in pure form, and have low toxicity. When selecting a mobile phase, consider the following:
- i. The mobile phase should not alter the properties of the packing material. Low cross-linked ion exchange resins and exclusion chromatography packing may swell or shrink when exposed to certain organic phases, changing the nature of the column bed. Alkaline mobile phases should not be used in silica-based column systems, and acidic mobile phases should not be used in adsorbents like alumina or magnesia.
- ii. Purity. The lifetime of a column is influenced by the amount of mobile phase passed through, especially when impurities accumulate on the column.
- iii. Compatibility with the detector. For UV detectors, the mobile phase should have no or very low absorption at the detection wavelength. When using a refractive index detector, choose a solvent with a refractive index that differs significantly from the sample to increase sensitivity.
- iv. Low viscosity . High viscosity solvents affect solute diffusion and mass transfer, lowering column efficiency and increasing column pressure drop, which lengthens separation time. Ideally, select mobile phases with boiling points below 100°C.
- v. Proper sample solubility. Poor solubility can cause sample precipitation at the column head, which affects purification and damages the column.
- vi. Easy sample recovery. Select volatile solvents.
pH of the Mobile Phase
When separating weak acids (3 ≤ pKa ≤ 7) or weak bases (7 ≤ pKa ≤ 8) in reversed-phase chromatography, adjusting the pH of the mobile phase can inhibit ionization of the sample components, increasing retention on the stationary phase and improving peak shape. This technique is called reversed-phase ion suppression. For weak acids, the smaller the pH, the larger the retention factor. For weak bases, the opposite is true. For weak acid samples, add a small amount of weak acid to the mobile phase, commonly 50 mmol/L phosphate buffer or 1% acetic acid solution; for weak base samples, use 50 mmol/L phosphate buffer or 30 mmol/L triethylamine solution. Adding organic amines to the mobile phase can reduce tailing by minimizing interactions with residual silanol groups, serving as a tailing suppressor.
Triethylamine: A volatile, colorless liquid with an ammonia-like odor, triethylamine [(CH3CH2)3N] is a base that forms salts soluble in water when reacted with inorganic acids. It is used in rubber vulcanization accelerators, surfactants, bactericides, solvents, and in the synthesis of quaternary ammonium compounds.
Choosing Buffer pH
To choose an appropriate buffer pH, first determine the analyte’s pKa. A pH two units above or below the pKa ensures good, sharp peaks. From the Henderson-Hasselbalch equation, pH = pKa + log([A-]/[HA]), we know that when the pH is two units above or below the pKa, 99% of the compound will exist in one form, allowing for sharp peaks.
Degassing the Mobile Phase
The mobile phase used in HPLC must be degassed to prevent bubbles, which can affect pump operation, reduce column efficiency, and destabilize the detector’s baseline, making detection impossible (e.g., increased noise, baseline instability). Dissolved gases may react with the sample, mobile phase, or stationary phase. Oxygen dissolved in some solvents can form UV-absorbing complexes, causing baseline drift or ghost peaks during gradient elution. Degassing methods include heating, vacuum, ultrasonication, or helium sparging.
Filtering the Mobile Phase
All solvents must be filtered through a 0.45 µm (or 0.22 µm) membrane before use to remove particulate impurities. Use separate membranes for organic and aqueous phases.
Storing the Mobile Phase
Store the mobile phase in glass, PTFE, or stainless steel containers, not plastic, as many organic solvents (e.g., methanol, acetic acid) can leach plasticizers from plastic, contaminating the solvent.
Handling Halogenated Solvents
Halogenated solvents can contain acidic impurities that react with the stainless steel in HPLC systems. They should be freshly prepared, especially if mixed with ethers, which can produce corrosive byproducts.

Selecting the Mobile Phase Solvent

The selected mobile phase solvent must be chemically stable and not react with the stationary phase or sample components.
The solvent must not interfere with the detector’s operation.
In preparative separations, the solvent should be easy to remove without hindering component recovery.
The solvent's viscosity should be low to ensure appropriate pressure drop across the column.
The solvent should be cost-effective, readily available, safe to use, and of high purity.

Precautions for Using Mobile Phase Solvents

Always use HPLC-grade solvents. Filter the mobile phase through a 0.45 µm membrane and degas before use to remove particles and air bubbles.
Use the mobile phase at room temperature to avoid baseline drift and bubble formation.
Use HPLC-grade reagents and freshly distilled water, not deionized or purified water, to avoid ghost peaks.
Use low or high pH values with caution as they can chemically damage the column’s packing material.
Rinse the system with water after using a buffer-containing mobile phase, and never leave the buffer in the system overnight.
Prepare and filter buffer solutions before use to avoid precipitation.
For gradient runs, ensure that the mobile phases are miscible to avoid precipitation of salts.
Use isopropanol as an intermediate solvent when switching between incompatible mobile phases.

Storage of Mobile Phases

Store the mobile phase in glass, PTFE, or stainless steel containers, tightly sealed to prevent evaporation and contamination. Phosphate or acetate buffers should be freshly prepared and used within three days.