“High-Performance Liquid Chromatography Principle, Procedure and Application Basic Note – 2020”.
Today in this post I’m going to teach you about “HPLC Chromatography (High-Performance Liquid chromatography)” which is also known as “High-Pressure Liquid Chromatography”.
There are so many blogs available on the internet but not basic notes. Here I’m trying to solve this problem and give a basic note on HPLC chromatography.
In this part, we will cover the introduction of HPLC Chromatography in detail.
What is HPLC Chromatography?
It means high-performance liquid chromatography which is mainly used in the separation, identification, and quantification of the mixture of compounds.
Now you think separation and identification but how?
Suppose we have a mixture of any plant extract containing lots of different components and here we are required to separate out a particular single component, here HPLC chromatography plays a very important role.
We can easily separate out the components which we require.
Here, the sample may be a blood sample, bacterial toxins, fungi toxins, protein mixture, plant extract or it’s maybe anything and we are just required to separate out any single component with the high-performance liquid chromatography.
Suppose we have a mixture which contains different components.
Here, first, we separate the component then identify if it is protein and then quantify it in how much amount it’s present in the sample mixture.
These all processes are done by HPLC Chromatography.
HPLC Chromatography is a type of liquid chromatography.
Why is HPLC called High-Performance Liquid Chromatography??
Now you may think what is the meaning of high-performance liquid chromatography, and why it’s called HPLC, what’s the reason? The reason is simple.
In HPLC the purification process is done on the basis of Polarity.
The polar components are water-loving and known as Hydrophilic. Nonpolar Components are water non-loving, known as Hydrophobic.
The purification process is based on the “Polarity”.
Definition of HPLC Chromatography.
High-Performance Liquid Chromatography is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture.
It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material.
Who invented high-performance liquid chromatography?
In 1903, The Russian botanist “Mikhail Tsvet” is considered to have invented this technique and he called this technique first-time Chromatography.
He invented this technique when he reported separation of different plant pigments into a series of colored bands on a packed column.
What is the principle of high-performance liquid chromatography?
HPLC chromatography is used to separate the compound that is dissolved in a sample mixture with the help of pressure.
Mobile phase solution contains 90% of water and 10% of an organic solvent, which is hydrophilic in nature, now this solution with the help of a pressure pump directly applies to the stationary phase.
Between both of these, the sample injector part is available. Here, the sample is injected with the help of injection and then the sample and mobile phase now both are allowed to run on the stationary phase.
Here as the stationary phase, silica gel is used. The stationary phase is a packed tube where individual components of the sample are transported along with the help of pressure. little bit of gravitational force also plays a role in it.
In the initial stage Buffer is passed and when the buffer passes from the column and is detected on the detector, it shows a straight line on the graph, which is known as Baseline. It’s a very important part of practical for equilibration of the system.
Here the stationary phase is hydrophobic in nature and our mobile phase is hydrophilic in nature.
Now here the nature of the sample is unknown, here the composition of the mixture which is hydrophilic in nature is easily eluted out from the column.
Because the stationary phase is hydrophobic in nature and that’s why they do not make any kind of bond with it and directly pass out from the column. Then, it gets detected on the detector and shows a peak on the data system of the hydrophilic molecules.
Here the collector is also connected with a detector where a sample of the hydrophilic molecule is collected.
Now the components of the mixture, which have hydrophilic nature, make the bond with resin and that’s why they move very slowly from the column.
Now the concentration gradient is applying.
Concentration gradient means the concentration of the water is now low and the content of the organic solvent is high. It gradually increases during the process. Once if the concentration of water is 10% and the concentration of organic solvent is 90%, now the nature of the mobile phase totally changes and now it’s known as hydrophobic in nature.
Now, the molecule makes the bond with resin. They easily pass out from the column, detected on the detector, and give a peak on the system of the hydrophobic molecule. Now, this also collects out through the collector, and results are shown in the graph, and this graph is called a Chromatograph.
Components of HPLC Chromatography?
1. Stationary phase
2. Mobile phase
5. Sample Injection
7. Data system
1. Stationary Phase
In the HPLC instrument, the stationary phase is a column. It’s a tube-like structure with insight having a solid adsorbent material.
Here as a stationary phase, silica gel is commonly used solid absorbent material which is hydrophilic in nature.
We run a mobile phase and sample with the help of a pressure pump on the column and separate out our desired sample.
It’s hydrophobic in nature because the amount of water is low.
2. Mobile Phase
In HPLC a liquid solution is used as a mobile phase which contains 90% of water and 10% of organic solvent.
It’s hydrophilic in nature because the amount of water or ratio of water is high compared to organic solvent.
In HPLC Chromatography, the sample may be a blood sample, Bacterial toxins, Protein mixture, Plant extract It may be anything.
There are a total of 3 types of pump available which is used in HPLC Chromatography.
1. Reciprocating piston pump.
2. Syringe Pump
3. Constant Pressure Pump.
The reciprocating piston pump is currently used in 90% of commercially available HPLC chromatography systems.
This pump is consisting of a sample chamber in which the solvent is pumped by the forth motion of a motor-driven piston.
2. Syringe Pump.
Syringe pumps provide a very constant flow rate.
3. Constant Pressure Pump.
The pressure from a gas cylinder delivered through a large piston drives a mobile phase.
5. Sample Injection.
In HPLC, there are several devices available either for auto or manual injection of the sample.
Device: 1. Stop flow Injector
2. Septum Injector
3. Rheodyne Injector
(List of Detectors use in HPLC)
1. UV Detector
2. Fluorescence Detector
3. Electro-Chemical Detector
4. Mass Spectrometric
5. Refractive Index Detector
6. Photodiode array Detector
7. IR Detector
1. UV Detector
UV Detector is the most common type of detector for liquid chromatography.
It’s working on the basis of the electronic transition within molecules.
2. Refractive Index Detector
Refractive Index Detector is nearly a universal detector but it has one disadvantage, that is it has a low detection limit.
Passes visible light through compartments, reference and sample.
What is the application of HPLC Chromatography?
In Pharmacy HPLC Chromatography is widely used for easy identification of clinically relevant molecules for the purpose of large scale processing and purification.
It’s used in the quality control department of pharmaceutical companies.
It is also used in the determination of product shelf-life.
Tablet dissolution of pharmaceutical dosage forms.
2. Consumer Product
Maintain the quality and consistency of soft drink product types.
Ensuring the sugar level in fruity juices.
For identification of individual components in the sample.
For measurement of the concentration or amount of compound in a sample.
For the preparation of pure compounds.
The HPLC Chromatography is used for the analysis of antibiotics.
Use in the detection of endogenous neuropeptides in the brain extracellular fluids.
Phenol determination from river water.
For the identification of diphenhydramine.
Advantages and Disadvantages of HPLC Chromatography.
Advantages of HPLC chromatography.
1. Speed (Minutes)
2. High Resolution
Disadvantages of HPLC chromatography.