High Performance Liquid Chromatography
What is high performance liquid chromatography?
High Performance Liquid Chromatography (HPLC) is an advanced analytical technique used to separate, identify, and quantify the individual components within a complex chemical mixture. This method is widely employed in various fields, including pharmaceuticals, environmental testing, food and beverage analysis, and biochemical research, due to its precision, efficiency, and versatility.
Liquid chromatography operates on the principle of partitioning components between two fundamental phases: the mobile phase and the stationary phase. The mobile phase is a liquid solvent or a mixture of solvents that flows continuously through the system, carrying the sample mixture with it. The stationary phase, on the other hand, is a solid or a liquid-coated solid material packed inside a column, through which the mobile phase passes.
How to prepare a HPLC Instrument?
Before starting the analysis, it is crucial to perform several checks to ensure the proper functioning of the liquid chromatography (LC) system. First, the lamp should be turned on and allowed to warm up for a sufficient time to achieve proper equilibration. It is also essential to purge the system to remove any air trapped within the tubing. This step can be complemented by setting the purge flow rate to 1 ml/min, within the Maintenance Tool, and flush the system until the pressure stabilises. After purging, the system should be checked for leaks. Set the mobile phase flow to 1mL/min and observe the pressure and also visual check for any leaks. Finally, the column must also be equilibrated by maintaining a continuous flow of the mobile phase. To ensure proper column equilibration, flush the column with a volume of mobile phase equivalent to 10 to 20 times the column’s volume.
How to prepare HPLC samples?
Sample preparation is an important step in HPLC Liquid chromatography analysis as it greatly influences the reliability of results. Sample extraction makes the matrix more suitable for analysis, ideally the sample should be prepared with the solvent used for the mobile phase. If a gradient method is being used, the sample should be prepared using the same composition as the start of the method.
Preparation may also be required to either dilute or concentrate the sample, bringing the concentration of analyte within the limits of quantification. The sample should dissolve fully into the solvent used for the mobile phase, split or broadened peaks may be seen if the sample is not fully dissolved. Filtering both the samples and mobile phase prior to use eliminates contamination of the system.
How does high performance liquid chromatography work?
Initially, the sample is dissolved in a liquid that typically shares similar properties with the starting mobile phase. This prepared sample solution is introduced into the HPLC instrument either through a manual injector or an autosampler, which ensures precise and consistent delivery. Once inside the instrument, the sample is propelled through the setup by a pump that generates the necessary pressure to drive the mobile phase and sample mixture towards the column, where the stationary phase is housed.
Within the column, the compounds in the sample undergo separation due to their differing interactions with the mobile and stationary phases. Each compound’s unique chemical structure and composition causes it to travel through the column at a distinct speed. This variation in travel speeds leads to the separation of the components, with each compound having a specific retention time—the time it takes for that compound to pass through the column and emerge at the detector.
How does HPLC separate compounds?
Different chromatographic stationary phases are tailored to separate components based on characteristics such as hydrophobicity. By altering the composition of the chromatographic column and the mobile phase, the order in which components elute and the speed at which they pass through the column can be manipulated, allowing for the optimization of the separation process.
After exiting the column, the separated components are carried to a detector. The detector identifies and/or measures specific properties of the compounds, providing crucial data for the analysis of the sample.
How is high performance liquid chromatography used in industries?
HPLC’s versatility positions it as a widely used and powerful chromatographic technique with applications across various industries, including:
Pharmaceutical Development:
Renowned for its reliability and precision, HPLC Liquid Chromatography serves as an invaluable means to verify pharmaceutical product purity and quantify active ingredients.
Drug Testing:
HPLC Liquid Chromatography finds extensive use in various police investigations, particularly in detecting traces of drugs in urine samples. HPLC is a critical technique for the identification/ quantitation of many prescription and over the counter medications, which assist with determining a cause of death.
Food Production:
Within agriculture and food production, HPLC Liquid Chromatography is used to test for pesticides, preservatives, artificial flavourings, colorants, and more. By doing so, HPLC ensures the elimination of chemical residues from food products, protecting consumer health and well-being.
Advantages of high-performance liquid chromatography
- Samples can be of volatile or non-volatile in nature
- Superior resolution and rapid analysis capabilities
- Extensive surface area for enhanced performance
- High pressure gradient for efficient separation
- Precise flow rate control
- Exceptional sensitivity with low detection limits
- Minimal sample requirement for analyses
- Versatility across compound sizes
- Automation, facilitating high-throughput analyses and mitigating human errors.
HPLC Instruments from SCION
The SCION LC6000 HPLC delivers high precision and accuracy, meeting the separation and analysis needs of researchers and laboratories. Designed for outstanding lifetime performance, the SCION HPLC series ensures confidence in your results. Its robust design enhances uptime and productivity while keeping operational costs low.
Check out the video below featuring our senior product manager as she delves into the capabilities of the LC 6000.
Product Overview LC6000 High Performance Liquid Chromatograph
Additional guides:
Guidelines for HPLC Column Handling
Liquid Chromatography Detectors – The Different Types of HPLC Detectors