Comphrensive information on HPLC (High Performance Liquid Chromatography)
This article describes about Comphrensive information on HPLC (High Performance Liquid Chromatography) technique.
What is mean by HPLC and it’s Principle ?
HPLC stands for High Performance Liquid Chromatography, is a technique used to separate, identify and quantify the components in a sample mixture. It is a sensitive, accurate, and precise Technique that can detect and quantify even trace amounts of compounds in a sample.
What are the Different Types of Detectors which are used in HPLC?
- UV Detector
- PDA Detectors
- Fluorescent Detector
- Conductivity Detector
- Refractive Index Detector
- Light Scattering Detector
What is mean by Column which is used in HPLC ?
In High Performance Liquid Chromatography (HPLC), a column is used to separate, identify & quantify the components of a mixture.
The column is a long, narrow tube packed with a stationary phase material (e.g. silica or polymer beads) that interacts with the mobile phase (e.g. liquid solvent) to separate the mixture’s components based on their physical and chemical properties.
The sample is injected into the mobile phase, which carries it through the column. The components of the sample interact with the stationary phase differently, causing them to be separated and eluted from the column at different times.
The eluent is then analyzed by a detector, and the resulting data is used to identify and quantify the components of the mixture.
The design and composition of the HPLC column can have a significant impact on the separation performance and overall effectiveness of the chromatographic analysis.
Image 02 : HPLC Column
What is mean by Stationary phase and Mobile Phase in HPLC ?
Stationary phase remains fixed in place i.e. in column while the mobile phase carries the components of the mixture through the medium being used.
The Stationary Phase :
Stationary phase remains fixed in place i.e. in column and it works by selectively adsorbing or partitioning the sample components based on their chemical and physical properties, such as size and polarity.
The Mobile Phase,
Mobile phase is a solution which carries it through the column, The components of the sample interact with the stationary phase differently, causing them to be separated and eluted from the column at different times.
What is mean by Suitability test in HPLC and What is its importance ?
A suitability test in High Performance Liquid Chromatography (HPLC) is a quality control measure that evaluate the performance of the HPLC system before beginning a new chemical analysis. The test is conducted to ensure that the system is working properly and that the separation of the components in the sample will be done in accurate and précised way.
Suitability tests typically involve injecting a standard sample mixture into the HPLC system and analyzing its behavior under certain conditions, such as a specific column and mobile phase. The results of the test are then compared to established criteria or acceptance limits to determine if the system is suitable for the analysis.
Some common parameters that are evaluated during an HPLC suitability test include resolution, peak symmetry, retention time, and peak area. By performing a suitability test, analysts can ensure that the HPLC system is operating optimally, reducing the risk of errors and ensuring reliable and accurate results for the sample being analyzed.
What is mean by Retention Time in HPLC ?
Retention Time in High Performance Liquid Chromatography (HPLC) means, time taken for a particular analyte (Component that is being analyzed) to elute or pass through the chromatographic column from the time it is injected into the system until it is detected by the detector. In short it can say as It is the time elapsed from the beginning of the sample injection to the detection of the compound.
The retention time of a compound is dependent on various factors including column Type, Mobile phase composition, Temperature, and the physicochemical properties of the compound itself.
What is mean by Relative Retention Time in HPLC ?
Relative retention time (RRT) in HPLC is a measure of the retention time of a specific compound in relation to another compound in the same chromatographic system. It is the proportion of the retention times of two compounds using identical chromatographic conditions
RRT can be used as a relative measure of compound retention, but it cannot be used to compare results obtained using different chromatographic conditions.
In short Formula for calculating the RRT is : Standard Retention Time / Sample Retention Time.
What are the components in HPLC?
High performance liquid chromatography (HPLC) is a chromatographic technique that utilizes a high pressure pump to pass a liquid mobile phase through a stationary phase column. The components of an HPLC system typically include:
- Solvent Reservoirs: The solvent reservoirs hold the solvents used to prepare the mobile phase.
- Degasser: The degasser removes any air bubbles from the solvent to ensure a stable and consistent flow rate.
- Pump: The pump delivers the mobile phase through the column at a constant flow rate.
- Injector: The injector introduces the sample into the mobile phase stream.
- Column: The column contains the stationary phase, which interacts with the sample components to separate them based on their physicochemical properties.
- Detector: The detector detects the separated sample components as they elute from the column.
- Temperature control: The temperature control system maintains the temperature of the column and other components to ensure stable chromatographic conditions.
- Pressure regulator: The pressure regulator controls the pressure of the mobile phase to ensure consistent flow rate and separation performance.
- Waste container: The waste container collects the mobile phase and sample waste after separation.
What is mean by Normal phase chromatography ?
Normal phase chromatography (NPC) is a type of liquid chromatography technique used for separating and purifying the molecules based on their physicochemical properties. In Normal phase chromatography, Stationary phase is Polar and Mobile phase is Non-polar.
During normal phase chromatography, mobile phase with sample is passed through the column. Molecules that are more polar will interact more strongly with polar stationary phase, and retained longer and elute later. In other side, more non-polar molecules will interact less strongly with stationary phase, and elute earlier.
Generally Normal phase chromatography is commonly used for the separation of polar to moderately non polar molecules.
What is mean by Reverse phase chromatography ?
Reverse phase chromatography (RPC) is a type of liquid chromatography technique used for separating and purifying the molecules based on their physicochemical properties. In reverse phase chromatography Stationary phase is Non-polar and Mobile phase is Polar.
During reverse phase chromatography, mobile phase and sample is passed through the column. Molecules that are more hydrophobic will interact more strongly with non-polar stationary phase, and will be retained longer and elute later. In contrast, more polar molecules will interact less strongly with stationary phase, and elute earlier.
Generally Reverse phase chromatography is commonly used for the separation of non-polar to moderately polar molecules.
What are the Types of Elution Techniques in HPLC?
There are the two types of Elution techniques in HPLC and those are as below
- Isocratic Elution Technique
- Gradient Elution Technique.
Furthermore Gradient Elution Techniques categorized as
- Linear Gradient Technique
- Step Gradient Technique
What is mean by Isocratic Elution Techniques in HPLC ?
An Isocratic isocratic elution technique is in which the solvent composition is remains constant throughout the separation process. In other words, the mobile phase is composed of a single solvent or a mixture of solvents with a constant ratio and concentration.
Isocratic gradient systems are generally simpler and easier to operate than gradient systems and They are often used for routine analyses of samples that contain only a few components or for analytical methods that do not require high resolution. This type of gradient may not provide the same level of resolution for the complex mixtures
What is mean by Gradient Elution Technique in HPLC?
Gradient program in HPLC refers to the method of changing the composition of the mobile phase during the separation process to optimize the separation of analytes in a sample.
What is mean by Liner Gradient Elution Technique in HPLC ?
What is mean by Step Gradient Elution Technique in HPLC ?
In a step gradient, the concentration of one solvent component is kept constant for a certain period of time and then abruptly changed to a new concentration for another period of time. This process is repeated several times throughout the analysis, creating a series of steps in the mobile phase composition.
For example, in a two component gradient system with solvent A and solvent B, the gradient might start with 100% solvent A and then switch to 90% solvent A and 10% solvent B after a certain amount of time. After another period of time, the gradient might switch again to 80% solvent A and 20% solvent B, and so on.
Step gradients are useful for optimizing the separation of complex mixtures with different chemical properties However, they are less commonly used than linear gradients in HPLC.
What are the Different Terminogly used for HPLC?
Different terminology used for HPLC and that are as, Liquid Chromatography/Soild Liquid chromatography /High performance Liquid Chromatography/ High pressure liquid chromatography.
The explanation is mentioned is as below.
Why HPLC is called as Liquid chromatography ?
As the Liquid material (Water/Solvent) used as a mobile phase in this chromatography technique so this technique is called the Gas chromatography .
Why HPLC is called as Solid Liquid Chromatography ?
In this Chromatography Technique, Stationary phase is in Soild state and Mobile phase is in Liquid state, so this technique is also termed as Soild liquid Chromatography.
Why HPLC is called as High Performance liquid Chromatography ?
As Resolution power (Ability to distinguish between two component) of this Techniques is very good,hence it is called as high performance and Liquid material (Water/Solvent) used as a mobile phase hence it is called Liquid chromatography.
Why HPLC is called as High Pressure liquid Chromatography ?
Mobile phase travels through the Column with the constant high pressure by the utilizing of the available pump in the instrument during its chemical analysis hence it is called as high pressure and Liquid material (Water/Solvent) used as a mobile phase hence it is called as liquid chromatography.
