Table of Contents
What is High-performance liquid chromatography (HPLC)?
HPLC, sometimes referred to as high-performance liquid chromatography, is an analytical method used to separate, distinguish, or quantify each component in a mixture.
Utilising the fundamentals of column chromatography, the mixture is separated, and spectroscopy is used to identify and quantify it.
The development of column chromatography from low-pressure suitable glass columns to high-pressure suitable metal columns occurred in the 1960s.
Thus, HPLC may be thought of as a greatly enhanced variation of column liquid chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres.
Principle of High-Performance Liquid Chromatography (HPLC)
- In a separation column between a stationary and a mobile phase, the purification happens.
- A separation column contains a granular substance with very tiny porous particles as the stationary phase.
- On the other hand, the mobile phase is a solvent or solvent combination that is pushed through the separation column under high pressure.
- The sample is fed into the mobile phase flow from the pump to the separation column through a valve with a linked sample loop, such as a tiny tube or a stainless steel capillary.
- As a result of interactions with the stationary phase, the various components of the sample are maintained to variable degrees, which causes them to migrate across the column at various speeds.
- After exiting the column, each chemical is identified by an appropriate detector, which sends a signal to the computer's HPLC programme.
- A chromatogram is obtained in the computer's HPLC software at the conclusion of this procedure or run.
- The various compounds may be identified and measured using the chromatogram.
Instrumentation of High-Performance Liquid Chromatography (HPLC)
The Pump
- The pump system was developed as a result of HPLC development.
- The pump creates a flow of eluent from the solvent reservoir into the system and is positioned in the liquid chromatography system's uppermost stream.
- A "standard" need of pumps is the ability to generate high pressure, but it should also be able to deliver a constant pressure under any circumstance as well as a predictable and repeatable flow rate.
- The majority of pumps used in modern LC systems (also known as reciprocating pumps) move a motor-driven piston back and forth to create flow. This piston action causes "pulses" to be produced.
Injector
- The pump is located adjacent to an injector.
- The sample is injected into the eluent flow using the simplest approach, which involves a syringe.
- The most common injection technique relies on sampling loops.
- The usage of the autosampler (auto-injector) technology, which enables repeated injections at certain scheduled times, is also quite common.
Column
- Inside of the column is where the separation is done.
- Instead of glass columns, modern columns are frequently manufactured in stainless steel casing.
- As opposed to calcium carbonate, silica or polymer gels are the more common packing materials utilised.
- Eluents ranging from acidic to basic solvents are utilised for LC.
- Since stainless steel is resistant to a wide range of solvents, stainless steel is typically used for column housing.
Detector
- Analyte separation is carried out inside the column, and the achieved separation is monitored by a detector.
- When an analyte is not present, the eluent's composition remains constant. While the eluent's composition is altered by the analyte's presence. Measurement of these differences is what a detector performs.
- This variation is tracked via an electrical signal. There are several detectors to choose from.
Recorder
- Since the change in eluent is being detected by a detector as an electrical signal, it is still not apparent to the human eye.
- Pen (paper) chart recorders were widely utilised in earlier times. A computer-based data processor (integrator) is more popular these days.
- There are many different kinds of data processors, ranging from simple systems with an integrated printer and word processor to those with software that is specifically made for an LC system and includes features like peak-fitting, baseline correction, automatic concentration calculation, molecular weight determination, etc. in addition to data acquisition.
Degasser
- The eluent used for LC analysis may contain gases that are invisible to the human eye, such as oxygen.
- An unstable baseline results from the detection of gas as noise in the eluent.
- Degasser removes gases using specific polymer membrane tubing.
- The surface of the polymer tube has many, very tiny holes that permit air to pass through but do not permit liquid to do the same.
Column Heater
- The column temperature frequently has a significant impact on the LC separation.
- Maintaining constant temperature conditions is crucial for getting reproducible outcomes.
- At higher temperatures (50 to 80°C), improved resolutions may also be attained for specific analyses, such as sugar and organic acid.
- As a result, columns are often kept within the column heater or oven.
Types of High-Performance Liquid Chromatography (HPLC)
1. Normal phase:
Silica is an example of a polar column packing, whereas water is a non-polar mobile phase. It is employed for chiral compounds, geometric isomers, cis-trans isomers, and water-sensitive substances.
2. Reverse phase:
Non-polar column packing, such C18, is used, and water and a miscible solvent, like methanol, make up the mobile phase. It may be used to samples that are polar, non-polar, ionizable, and ionic.
3. Ion exchange:
Ionic groups are present in column packing, and buffer serves as the mobile phase. It is employed to divide cations from anions.
4. Size exclusion:
In a porous media, molecules diffuse into the pores and are segregated based on how big they are compared to the pores. Large molecules elute first, followed by smaller ones.
Applications of High-Performance Liquid Chromatography (HPLC)
The HPLC technique has advanced to the point that it can now be used in practically every field of chemistry, biochemistry, and pharmacy.
- Analysis of drugs
- Analysis of synthetic polymers
- Pollutant analysis in environmental analytics
- Drug detection in biological matrices
- Isolation of valuable products
- Controlling the purity and quality of important chemicals and industrial items
- biopolymer separation and purification, such as those of enzymes or nucleic acids
- purifying of water
- Trace component pre-concentration
- Exchange of ligands in chromatography
- Protein ion-exchange chromatography
- Chromatography of oligosaccharides and carbohydrates at high pH
Advantages of High-Performance Liquid Chromatography (HPLC)
- Speed
- Efficiency
- Accuracy
- Versatile and incredibly accurate at recognising and quantifying chemical constituents.
Limitations
- Cost: Despite its benefits, HPLC can be pricey since it needs a lot of expensive organics.
- Complexity
- Some substances are difficult to identify using HPLC because they are permanently adsorbed, and others are just weakly detectable.
- Gas chromatography is more effective in separating volatile compounds.
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