High performance liquid chromatography (HPLC) has become a very versatile and powerful separation and analytical method over the years. It is an advanced form of liquid chromatography (LC).
Instead of introducing the solvent into the column and allowing it to drip down under the influence of gravity, in HPLC the sample is forced through the column under high pressures of nearly 400 atm, resulting in faster and more efficient separation.
This technique is also called high pressure liquid chromatography.
The Principle of HPLC
HPLC follows the same basic principle as chromatography. Different components in the sample have varying affinities to the adsorbent material. This causes a difference in the flow rate for each component which leads to their separation as they come out of the column. The only difference is that the speed and sensitivity of HPLC is much higher than that of LC due to the application of a high pressure.
The magnitude of pressure applied depends on several factors such as the length and diameter of the column, flow rate, size of particles in the stationary phase, and mobile phase composition.
The Components of HPLC
Columns: HPLC columns are normally made of stainless steel and are 50 - 300mm long with an internal diameter of 2 - 5mm. They are filled with the adsorbents (stationary phase) of particle size 3 – 10µm.
Sample Injector: The sample is injected into the column by an injector which is capable of handling sample volumes in the range of 0.1 - 100mL under high pressures of up to 4000psi.
Reservoir: The solvent or the mobile phase is placed in a glass reservoir. It is usually a blend of polar and non-polar liquids whose concentrations depend on the sample composition.
Pump: The solvent in the mobile phase is aspirated by a pump from the reservoir and forced through the HPLC column and then the detector.
Detector: The detector in a HPLC system is located at the end of the column and it detects the components of the sample that elute from the column. Different types of detectors such as fluorescence, mass-spectrometric, UV-spectroscopic, and electrochemical detectors are used.
Data collection systems: The signal from the detector is received by recorders which are used to process, store, and reproduce chromatographic data. The data is interpreted and integrated by a computer which produces a user-friendly chromatograph.
The Technique of HPLC
The key steps in the HPLC separation technique are as follows:
- Injection of the liquid sample into the column containing the stationary phase.
- Individual sample components are forced down the tube by high pressure from the pump.
- Components are separated under the influence of various chemical/physical interactions with the particles in the stationary phase.
- The separated analytes are identified by the detector present at the end of the column.
- The detector measures the concentration of the components.
- Data from the detector is processed and a chromatogram is produced.
The Applications of HPLC
HPLC is widely used in the following applications:
- Qualitative analysis - Separation of thermally unstable chemical and biological compounds, e.g., drugs (aspirin and ibuprofen), salts (sodium chloride), proteins (egg white or blood), organic chemicals (polystyrene and polyethylene), herbal medicines and plant extracts.
- Quantitative analysis - To determine the concentration of a compound in a sample by measuring the height and area of the chromatographic peak.
- Preparation of pure substances for clinical and toxicology studies and in organic synthesis. This is also called preparative chromatography.
- Trace analysis – this is the analysis of compounds present in very low concentrations in a sample. This is very useful in pharmaceutical, toxicology, environmental, and biological studies.
Reviewed by Afsaneh Khetrapal, BSc (Hons)