High-Pressure Liquid Chromatography (HPLC)
Definition: High-Pressure Liquid Chromatography (HPLC) is a separation technique in which a liquid or properly dissolved solid sample is passed through a column at high pressure. It is also referred to as High-Performance Liquid Chromatography and is used to separate, identify or quantify each component in a mixture.
Principle of HPLC (High-Performance liquid Chromatography)
The basic principle of HPLC is that it separates a sample into its constituent parts based on the relative affinities of distinct molecules for the mobile phase and the stationary phase used in the separation.
The distribution of the analyte between a mobile phase (eluent) and a stationary phase (packing material of the column) is the basis for HPLC separation. The molecules are retarded while passing through the stationary phase, depending on the chemical structure of the analyte. The duration a sample spends “on-column” is determined by the unique intermolecular interactions between its molecules and the packing material. As a consequence, the constituents of a sample get eluted at different times, and hence the separation is achieved.
High Performance Liquid Chromatography Instrumentation
- Solvent reservoir: Solvent reservoir is also known as mobile phase reservoir. The high viscous solvent is discouraged to use as it takes much more time to travel through column, and high pressure is required for the viscous solvent. These leads to peak broadening, and hence better not to use such sovent. The choice of solvent depends on the nature of sample and the sensitivity of the detector.
- Pump: The pump’s function is to propel a liquid (known as the mobile phase) through the liquid chromatograph at a set flow rate, which is measured in milliliters per minute (mL/min). A pump can deliver a constant mobile phase composition (isocratic) or a rising mobile phase composition (gradient) during the chromatographic experiment.
- Injector: The injector is used to insert the liquid sample into the mobile phase’s flow stream. Sample quantities range from 5 to 20 microliters (L).
- HPLC Column: It is known as the heart of the chromatograph. The column length generally varies from 5 cm to 30 cm,and its diameter ranges from 2-50 mm. Mostly, stainless steel is used as materials for the construction of the tubing, while Silica and alumina particle is used as packing materials. The mobile phase is aspirated from the solvent resorvoir and forced through the system’s column and detector by a pump.
- Detector: The detector detects individual molecules leaving the column and delivers an output to a recorder or computer, resulting in a liquid chromatogram.
- Computer: It takes the signal from the detector and makes use of it to decide the time of elution (retention time) of the sample components (qualitative analysis) and the quantity of pattern (quantitative analysis).
Application of High Performance Liquid Chromatography
High-Performance Liquid Chromatography is used for both qualitative and quantitative analysis. Some of the major applications of HPLC are listed below:
- Pharmacetical applications for the analysis of drugs
- Purification of water
- Ligand-exchange chromatography
- Used for analyzing air and water pollutants
- Used for analyzing complex mixtures in chemistry and biochemistry research
- Used in quality control to ensure the quality of raw materrials
- Ion-exchange Chromatography of proteins
- Separation and analysis of non-volatile or thermally unstable compounds
Advantages of High Performance Liquid Chromatography
- High resolution
- Quick analysis
- Separation of volatile and non-volatile components
Disadvantages of HPLC:
- High cost
- Relatively difficult to operate
FAQs
What is the difference between high pressure and low-pressure liquid chromatography?
There’s no relative comparison between high pressure and low-pressure liquid chromatography. The term “high” in high-pressure liquid chromatography is used as the high pressure is required to pass the mobile phase through a column packed with the stationary phase.
What is High-performance Liquid Chromatography?
High-Performance Liquid Chromatography (HPLC) is a separation technique in which a liquid or properly dissolved solid sample is passed through a column at high pressure
What are the types of HPLC?
There are 4 types of HPLC: Normal phase, Reverse phase, Size-exclusion, and Ion-exchange HPLC.
What is the basic principle of HPLC?
The basic principle of HPLC is that it separates a sample into its constituent parts based on the relative affinities of distinct molecules for the mobile phase and the stationary phase used in the separation.
