High Performance Liquid Chromatography | Gases | Coregas NZ

High performance liquid chromatography

HPLC is commonly used in pharmaceutical research and development applications such as drug discovery. Clinical laboratories also rely on HPLC for blood and urine sample analysis.

HPLC is used to separate chemicals in a liquid sample to allow qualitative and quantitative analysis in the detector which follows the chromatography column. Mass spectrometer (LC-MS), photodiode array (PDA) and UV/visible light spectroscopy detectors are the most common ones to use in combination with HPLC, although fluorescenceFID and other detectors may also be used. The technique finds common application in many branches of organic chemistry, such as pharmaceutical production quality control, drug discovery R&D and forensic testing, and clinical diagnosis.

In the LC-MS system, at the point where the liquid mobile phases leave the HPLC column, the liquid sample is sprayed, or nebulised, to produce micro-droplets. These rapidly evaporate to release ionised analyte molecules, which are then separated and detected by the mass spectrometer. High-flow sprays require a high flow of nitrogen to assist with nebulization of the sample. Coregas Nitrogen 5.0 is recommended as a LG-MS nebuliser gas.

HPLC column mounted at the front of the instrument to allow access to change the column

HPLC has many similarities to gas chromatography. Both use a column packed with an adsorbent material to separate the components of the sample, which is passed through the column in the mobile phase. The main difference is that HPLC operates in the liquid phase and the carrier is a liquid solvent (typically water mixed with methanol or acetonitrile), whereas the carrier for the mobile phase in gas chromatography will be a gas, such as helium. Helium is also used in HPLC for degassing the mobile phase solvents.

Success factors

Helium degassing is the key to HPLC success

When HPLC solvents are mixed, air bubbles may form which can cause problems in HPLC analysis. This problem arises because pure solvents equilibrate with atmospheric air in the laboratory environment. When pure solvents are mixed, the solubility of air may be less than it was in the pure solvents and the excess air will cause gas bubbles. Furthermore, the solvent will be fully saturated with air, so the slightest changes in pressure may cause outgassing, where rough surfaces produce nucleation sites and the creation of bubbles.

Low pressure solvent mix is highly prone to air bubble formation. In low-pressure mixing, the solvents are mixed at atmospheric pressure and outgassing can take place at any subsequent stage in the flow path. With high-pressure mixing, solvents first pass through the pumps, then mixing takes place under high pressure in the mixing chamber. The mixture may have been saturated with air at atmospheric pressure, but at elevated pressure outgassing is prevented. With high-pressure solvent mixing, bubble formation, if any, can take place when the mobile phase exits the column and returns to atmospheric pressure.

The consequences of a “gassy solvent” may become evident with:

  • Unstable and noisy baselines
  • Reduced check valve performance
  • Excessive pressure, which can lead to pump failure
  • Spurious peaks as air bubbles pass through the detector
  • Lower flow rate precision of the pump as air bubbles cause cavitation
  • Flow transfer of the mobile phase through the HPLC column due to creation of air bubble dead volumes

All the above issues can be prevented by degassing the mobile phase. It is not necessary to remove all the dissolved air; it is sufficient to reduce the amount of dissolved air below the supersaturation level in the mobile phase.

There are three common degassing techniques and the use of helium gas is widespread. Helium purging can remove up to 80% of the dissolved air. Vacuum degassing removes approximately 60% of the dissolved air. One option is to apply vacuum during filtration of the mobile phase through a 0.45 or 0.22μm porosity membrane filter. On–line vacuum degassing is available on most commercial available systems. Sonication using ultrasonic baths is also used extensively. As a stand-alone technique, it is limited to remove only up to 30% of the dissolved air and so is generally used in combination with helium or vacuum degassing.