High Performance liquid Chromatography also known as HPLC is a widely used instrument for analysis in pharmaceutical, chemical and food industries. It has powerful ability to identify and quantify the complex mixtures and this makes it an essential instrument for analytical laboratories. This article will provide detailed knowledge on major components of HPLC and its working.
Each component of the compound mixture interacts differently with the stationary phase. This behavior of compounds decides their flow within the column and separates them.
What is HPLC?
HPLC system is an advanced version of column chromatography that is used to separate, identify and quantify the components from a mixture. A very high pressure is used to push the sample into the column having a solid stationary phase within it that separates the mixture into its components.Each component of the compound mixture interacts differently with the stationary phase. This behavior of compounds decides their flow within the column and separates them.
Key Components of the HPLC System
Solvent Reservoir: This is a container that holds the mobile phase and is a mixture of organic solvents like methanol, acetonitrile and water.Pump: It helps to deliver the mobile phase from the reservoir to the HPLC system at a very high pressure. The flow rate of mobile phase plays an important role in separation of compounds.
Injector: Injector is an injection system to introduce the sample into the system. It can be done manually or automatically in automated systems.
Column: Column is the heart of the HPLC system. The column is like a metal pipe, filled with the solid stationary phase and separates the compounds in the mixture.
Detector: It identifies and quantifies the separated compounds as they elute from the column.
Data System: The signals produced by the detector are processed by software and create a chromatogram as an output.
Principle of HPLC
In HPLC chromatography, column plays a significant role in the separation of different compounds because it contains stationary phase. The stationary phase is a bad of polar or non-polar particles according to the type of column. Polar and non-polar columns are used according to the nature of the sample to be analyzed.Mechanical pumps are used to pump the mobile phase into the system and injector introduces the sample into the mobile phase which enters the column at a constant flow rate. The mobile phase acts as a carrier in the whole process.
When a mixture of compounds enters a column, it separates the compounds on the basis of their polarity. If the stationary phase is non-polar then it attracts the non-polar compounds and a polar compound elutes first then a non-polar compound and if the stationary phase is polar then non-polar compound elutes first.
Detectors are used to determine the separated compounds by ultraviolet absorption. The light absorption depends upon the concentration of the compound in the mobile phase. These response signals are recorded by computer software in the form of peaks and purity of the compound is calculated by the peak area of different compounds. Computer systems and software should be validated.
Retention Time: The Key to Separation
Each component from the mixture comes out or elutes from the column at a specific time and it is known as the retention time of the compound. This time is determined by the interaction between compound and stationary phase in the presence of the mobile phase.The compound that interacts more with the stationary phase will elute later and the compound that interacts less with the stationary phase eludes earning and is detected by the detector as per their respective elution. Their time of elution from the column is plotted on the chart in the form of a chromatogram.
Types of HPLC Systems
In different types of HPLC systems, only columns are different and the whole HPLC system remains the same. In all types of HPLC systems, columns are changed to have a different separation mechanism. On the basis of the separation mechanism HPLC systems are of the following four types.1. Normal Phase: In normal phase HPLC, the stationary phase remains polar usually silica and the mobile phase remains non-polar usually hexane. Normal phase HPLC is best for separating polar compounds.
2. Reverse Phase: In the reverse phase HPLC system stationary phase remains non-polar usually C8 and C18 and the mobile phase remains polar usually a mixture of water, methanol and acetonitrile. This is the most commonly used method for the analysis of pharmaceutical compounds. It is the ideal method for separating non-polar or moderately polar compounds.
3. Ion Exchange: As the name shows, this separates compounds on the basis of their charge. Generally, it is used for the analysis of biochemical compounds like proteins, amino acids and nucleotides.
4. Size Exclusion: The size of the molecules of compounds is the base of the size exclusion separation mechanism. It separates the compounds on the basis of their molecular size. Large molecules elute first and small molecules elude later. It is used for analysis of proteins and polymers.
Types of HPLC Detectors
HPLC Detector measures the concentration of the components as they exist from the column. The type of detector is selected on the basis of the properties of the compound to be analyzed. Following are some widely used detectors.
1. UV-Visible Detector: A UV detector is used for the analysis of compounds that absorb UV light. These are commonly used in the analysis of pharmaceutical compounds.
2. Refractive Index Detector: The RI detector measures the change in the refractive index of the solution passing through it.
3. Fluorescence Detector: These are highly sensitive detectors used to detect compounds that have fluorescence.
4. Conductivity Detector: The conductivity detector measures the conductivity of the solution and is used for ionic compounds.
5. Mass Spectrometry Detector: These have high specificity and are generally used in advanced research in the pharmaceutical industry.
1. Pharmaceutical Industry: It is the most used instrument in the pharmaceutical industry for the identification and quantification of drug compounds. Assay, content uniformity, dissolution and stability studies are done by HPLC in pharmaceuticals.
2. Environmental Analysis: Pollutants in water and soil are detected by HPLC. Residues of pesticides are also monitored using this technique.
3. Food and Beverage Industry: The quantification of additives and preservatives in food materials and the percentage of alcohol in beverages are analyzed using HPLC. The nutritional value analysis of food material in food industries is also done by this technique.
4. Clinical and Forensic Science: Drug testing and biological floods and toxicology analysis are done in clinical and forensic science by HPLC.
2. It has excellent sensitivity and resolution and can be automated to run 24x7 without any human intervention.
3. It is able to detect and quantify a wide range of components.
4. It is fast and has reproducible results.
2. Operators require training before use
3. It is not suitable for volatile compounds
4. Mobile phase preparation and column maintenance are time-consuming processes.
1. UV-Visible Detector: A UV detector is used for the analysis of compounds that absorb UV light. These are commonly used in the analysis of pharmaceutical compounds.
2. Refractive Index Detector: The RI detector measures the change in the refractive index of the solution passing through it.
3. Fluorescence Detector: These are highly sensitive detectors used to detect compounds that have fluorescence.
4. Conductivity Detector: The conductivity detector measures the conductivity of the solution and is used for ionic compounds.
5. Mass Spectrometry Detector: These have high specificity and are generally used in advanced research in the pharmaceutical industry.
Application of HPLC
HPLC is used in various industries for analysis due to its accuracy, reproducibility and flexibility.1. Pharmaceutical Industry: It is the most used instrument in the pharmaceutical industry for the identification and quantification of drug compounds. Assay, content uniformity, dissolution and stability studies are done by HPLC in pharmaceuticals.
2. Environmental Analysis: Pollutants in water and soil are detected by HPLC. Residues of pesticides are also monitored using this technique.
3. Food and Beverage Industry: The quantification of additives and preservatives in food materials and the percentage of alcohol in beverages are analyzed using HPLC. The nutritional value analysis of food material in food industries is also done by this technique.
4. Clinical and Forensic Science: Drug testing and biological floods and toxicology analysis are done in clinical and forensic science by HPLC.
Advantage of HPLC
1. HPLC is a highly precise and accurate technique.2. It has excellent sensitivity and resolution and can be automated to run 24x7 without any human intervention.
3. It is able to detect and quantify a wide range of components.
4. It is fast and has reproducible results.
Limitations of HPLC
1. It has a very high initial cost.2. Operators require training before use
3. It is not suitable for volatile compounds
4. Mobile phase preparation and column maintenance are time-consuming processes.
Thank's a lot Choudhary; Tell me hoe to expand capability of HPLC for amino acids analysis
ReplyDeleteThank you, it is written precisely
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