HPLC Testing Equipment
When researchers uncover scientific advances utilizing analytical chemistry, having the right equipment is pertinent for dependable results. HPLC testing requires specialized equipment that is compatible with your analyte. To decide what chemical equipment is necessary for your analytical laboratory, you must first confirm your methodology based on the makeup of your sample material. HPLC techniques can be refined to meet your specific needs, and with a thorough understanding of chromatography you can develop a reliable workflow.
High performance liquid chromatography (HPLC) is a useful analytical technique for chemical compounds such as small molecules and biopolymers. This technique involves studying a sample that is dissolved into a solution, which is then injected into a column containing resin that will interact with the sample. This interaction slows down the movement of the sample through the column, where it is then detected. Data is derived from the timing of movement of the sample and the intensity of detection of the sample.
To learn more about what HPLC is used for, visit our page on chromatography solutions. Avantor offers a wide selection of resources and products to assist with your needs.
High-performance liquid chromatography
Simply put, high-performance liquid chromatography works based on the principle that individual compounds behave differently in water. HPLC separates and purifies compounds depending on their polarity, or their hydrophobic/hydrophilic properties.
There are two types of chromatography; normal phase and reverse phase. Different resin compositions in the column determine the kind of chromatography being run and what molecules can be separated.
Normal Phase
In this method, the column is filled with polar silica particles and a non-polar buffer. Once the sample is injected, polar particles will stick to the silica, exhibiting a longer retention time than non-polar molecules.
Reverse Phase
The column is filled with hydrophobic particles with a polar buffer when performing reverse phase chromatography. The hydrophobic molecules stick to the resin longer and can be more easily detected.
Using chemicals for laboratory sample preparation
There are three main processes when setting up a sample for HPLC testing: dilution, centrifugation, and filtration. Each step requires different chemicals, which when planned out properly can minimize expenses and optimize your protocol. It’s essential to select the proper chemicals for sample preparation as it can help save time, money, and resources in your lab.
Dilution
The key to proper dilution of a sample is determining what chemicals work best for the metabolite you are interested in. One solvent may support one chemical class while losing or decreasing another class. Water and other solvents are commonly selected for this. For instance, organic solvents like methanol or acetonitrile are used to crush the proteins and extract small molecules simultaneously.
Centrifugation
This step can help preserve your testing equipment while protecting your sample's quality during filtration. Centrifugation works by dropping any suspended particles to the bottom of the sample tube, using a vigorous shaking motion to mix the solution.
It’s recommended to mix a sample for 5 minutes at maximum speed using a bench-top centrifuge. This will minimize waste when recovering filtered samples and reduce the use of expensive filtration supplies that often require longer filtering durations. Although centrifugation does not require additional chemicals, it significantly reduces the amount required in other steps.
Filtration
An analytical lab may move through hundreds of samples each week, requiring fast and accurate results. This balance between pace and quality is deeply impacted by filtration techniques.
Filtration is important for proper HPLC sample preparation for two main reasons: protecting the pump from particulates in the solvent and preventing particles from clogging and damaging the HPLC column.
Using pre-mixed and filtered solvents can minimize the potential for contamination via the solvent. This purity level will help lessen the maintenance required for your equipment. Adding a filtration step prior to loading your sample into the column or at the point of collection can reduce errors due to contamination and protect your equipment from wear long term.
Selecting chemicals for HPLC sample preparation
When establishing an efficient technique for HPLC sample preparation it is crucial to source chemical products that are purified and have the necessary reactions with your target compounds.
Solvents like acetonitrile, methanol, and water are often used in sample preparation, for the purpose of filtration. Some of the best solvents for HPLC analysis are quality controlled for UV transparency to support optimum sensitivity and are stripped of metallic impurities that often affect biological activity within your sample.
Having the proper filtration chemicals will improve the speed of testing and often eliminate additional equipment and resources used to further filter a sample that may be prone to contamination.
Selecting solvents to avoid contamination
Choosing the right chemical solutions can be key in avoiding contaminants in your HPLC sample that will skew testing results and damage your equipment. Extractables are compounds released from a component, such as a filter membrane, that is in contact with the sample solvent. The quantity of extractables varies depending on the solvent resistance of the component, as well as the temperatures and pressures involved.
Extractables can affect the accuracy of HPLC by interfering with the analysis if they are detectable by the downstream analytical method. These compounds introduce noise data, reducing the data quality overall. This can be especially problematic when the target analyte has the same retention time as the extractability.
Confirming that the filtration membrane has high chemical compatibility with the mobile phase solvents can significantly reduce extractables.
Polytetrafluoroethylene (PTFE), polyvinylidene difluoride (PVDF), and regenerative cellulose (RC) membranes are popular choices, as they are highly compatible with common mobile phase solvents. Purified water, acetonitrile, and methanol are all popular choices for solvents.
Another way to prevent contamination is to reduce the variation in materials across the lab. Standardizing two membrane types and using them in various formats will support consistency in the laboratory.
You might use the same membrane materials in membrane circles for mobile phase filtration, syringe filters for manual preparation, and syringeless filters for automated filtration. This will mean downsizing the number of consumables that need to be stocked – minimizing space requirements and the hassle of restocking a diverse list of supplies.
Learn more about HPLC equipment and all analytical testing resources through Avantor®.
Conclusion
Sourcing HPLC chemical equipment for large-scale processing or cutting-edge research should be made simple. Avantor® provides high-quality products and resource tools to aid in your laboratory’s success.
Correct handling of laboratory chemicals for HPLC is also an important consideration when sourcing equipment and maintaining quality control. For more information about handling common laboratory chemicals, visit our page on Laboratory Chemicals and their Uses.
For all your chemical equipment needs, Avantor® has you covered.