High Performance Liquid Chromatography (HPLC) is a technique used by chemists, researchers, and scientists to separate and analyze compounds present in a mixture. HPLC has become an essential tool in the field of analytical chemistry, pharmaceuticals, food and beverage, and environmental analysis. In this review, we will discuss the working of HPLC, its components, and the different types of HPLC techniques.
Before diving into the details of HPLC, let's understand the basics. HPLC is a type of liquid chromatography that uses high-pressure pumps to move the sample through a column packed with stationary phase material. The stationary phase material in HPLC is generally a fine solid particle with a size ranging from 3 to 10 micrometers. The sample mixture is injected into the column, and the different compounds present in the sample move through the column at different rates. The separation is based on the relative affinity of the compounds for the stationary phase and the mobile phase. Now, let's move on to the working of HPLC.
The Working of HPLC
Components of HPLC
The essential components of HPLC are:
- The mobile phase
- The stationary phase
- The pump
- The injector
- The detector
The Mobile Phase
The mobile phase is the solvent that carries the sample through the column. The mobile phase is selected based on the solubility of the sample mixture and the stationary phase material. The most commonly used mobile phases in HPLC are water, methanol, acetonitrile, and tetrahydrofuran. A mixture of two or more solvents can also be used as the mobile phase to improve the separation efficiency.
The Stationary Phase
The stationary phase is the material present in the column that interacts with the sample mixture. The stationary phase material can be polar, nonpolar, or both, depending on the separation required. The most commonly used stationary phase materials are silica, alumina, and polymers such as polystyrene divinylbenzene and polyvinyl alcohol. The stationary phase material is packed into the column in the form of small particles with a size ranging from 3 to 10 micrometers.
The Pump
The pump in HPLC is used to move the mobile phase through the column at high pressure. The pump can generate pressures ranging from 2000 to 8000 psi and can deliver a constant flow rate of the mobile phase. The high pressure generated by the pump helps to maintain the stationary phase material in the column and improves the separation efficiency.
The Different Types of HPLC Techniques
Reverse Phase Chromatography
Reverse Phase Chromatography is the most commonly used HPLC technique. In reverse phase chromatography, the stationary phase material is nonpolar, and the mobile phase is polar. The sample mixture is injected into the column, and the nonpolar compounds in the sample interact with the nonpolar stationary phase material. The polar compounds move faster through the column and are separated from the nonpolar compounds.
Normal Phase Chromatography
In normal phase chromatography, the stationary phase material is polar, and the mobile phase is nonpolar. The sample mixture is injected into the column, and the polar compounds in the sample interact with the polar stationary phase material. The nonpolar compounds move faster through the column and are separated from the polar compounds.
Gel Permeation Chromatography
Gel Permeation Chromatography is a technique used for the separation of large molecules such as proteins, polymers, and macromolecules. In this technique, the stationary phase material is a gel matrix with varying pore sizes. The sample mixture is injected into the column, and the large molecules get trapped in the gel matrix, while the small molecules move through the column at a faster rate.
Ion Exchange Chromatography
Ion Exchange Chromatography is a technique used for the separation of ionic compounds. In this technique, the stationary phase material is charged, and the mobile phase is a buffer solution with a pH above or below the pKa of the charged groups in the stationary phase material. The sample mixture is injected into the column, and the ionic compounds in the sample interact with the charged stationary phase material. The ionic compounds are separated based on their charge and affinity for the stationary phase material.
The Importance of HPLC Techniques
HPLC is a versatile and powerful analytical technique that has become an essential tool for chemists, researchers, and scientists. HPLC is used in various fields such as pharmaceuticals, food and beverage, environmental analysis, and forensic science. The accurate and precise separation and analysis of compounds present in a mixture are critical for the success of any research or analysis. HPLC techniques provide a reliable and efficient method for analyzing complex mixtures and identifying the compounds present in them.
| Component | Function |
| The mobile phase | Carries the sample through the column |
| The stationary phase | Interacts with the sample mixture |
| The pump | Moves the mobile phase through the column at high pressure |
| The injector | Injects the sample mixture into the column |
| The detector | Detects the separated compounds |