I hope you found the last installment on Hydrophobic Interaction Liquid Chromatography (link to blog post) useful. Supporting my goal to discuss and provide resources on the latest tools and techniques that will help improve and speed up your day-to-day work in the lab, I will discuss sample preparation for HPLC in this post.
What sample preparation techniques exist?
There are many sample preparation techniques but only a few will be discussed here. A great resource for chromatography and sample preparation terms is the Chromatography and Sample Preparation Terminology Guide (link to registration page after which you can download the PDF). The sample preparation types I will review include filtration, QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe), and solid-phase extraction (SPE).
Filtration (link to Wikipedia): This is a technique primarily used to remove particulate interference from your sample matrix (website link). It is recommended that all samples be filtered before chromatographic analysis. Filtration can be performed manually and on an individual basis using syringe membrane filters, or it can be done on multiple samples simultaneously through plate configurations. Syringe filters are available in many membrane types to accommodate chemical and application compatibility and many membrane diameter sizes to accommodate varying sample volumes. You can choose either a 0.45 µm or 0.2 µm pore size. The 0.45 µm size is described as a clarification filter removing particulates and the 0.2 µm size is described as sterilizing and ultracleaning. Specifics of all filters should be reviewed with the vendor.
A pre-column filter (web link) may be placed between the injector and the analytical column to trap particles present in the sample and particles frominjection valve wear.The filter usuallyconsists of a 5- or 2-micron frit heldin a cartridge.The frit can be easilyreplaced when the system pressure rises. There are also pre-column filters that are designed to remove matrix interferences (web link).
QuEChERS (link to Wikipedia): This is a dispersive SPE offering that is increasingly becoming the technique of choice for extraction and clean-up of pesticide residues in food and other complex matrices. The robust procedure offers a number of compelling advantages: high recoveries, accurate results, high sample throughput, low solvent and glassware usage, reduced labor and bench space, and lower reagent costs.
Solid-Phase Extraction (downloadable PDF on Theory of SPE): SPE is the passage of the sample through one or more cartridges packed with a stationary phase. Performing this technique prior to injection will often selectively retain certain species within the homogeneous sample. Quite often, the retained species are substances that would interfere with the chromatography had they not been removed. However, they could also be the analytes of interest, in which case they would then be eluted from the cartridge. There are a number of different cartridges whose suitability depends upon the type of chemistry undertaken.
There are automated SPE systems (web link) that use cartridges to trap and concentrate your analytes of interest. In one run, you can perform solid-phase extraction on multiple samples in a short time. Typical applications include PAH, PCB, PCDD, PCDF, pesticides, herbicides, flame retardants, semivolatiles, nitrosamines, and steroids.
Why would you want to use sample preparation?
You use sample preparation techniques because you want to get the most accurate analysis of your sample. Your sample matrix can reduce your ability to get an accurate result. Samples may have come from human fluids, waste water, food, or an array of other sources. These matrices can cause significant background signal and even interfere with the function of your instrumentation. To achieve better signal–to-noise, remove or reduce matrix interferences, and improve column and system performance, sample preparation is a must.
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What are the limitations of sample preparation?
There can be limitations to the use of many sample preparation techniques. The use of any sample preparation device needs to be approached with an understanding of both the matrix you are trying to remove, and the analytes you are trying to retain for analysis. Using the wrong technique or the wrong method can cause issues like low recovery and poor reproducibility. Be sure to research techniques and applications (website link) for your compounds of interest in their matrices and test known standards in the matrix before moving to critical sample analysis. Recovery and reproducibility have been significantly improved with the new SPE products, as described in this technical guide, Thermo Scientific SOLAµ SPE Plates Technical Guide (downloadable PDF).
Who is using sample preparation techniques and for what?
There are many examples of sample preparation using a variety of techniques. Some examples of applications in different arenas are found below. There are many more but I thought I’d provide a few in different areas of study to provide an idea of its flexibility.
- In the pharmaceutical arena, S. Fair performed The Evaluation of Prelude SPLC and TSQ Endura Mass Spectrometer in Research Analysis of Testosterone in Human Serum (downloadable PDF). Both online and offline sample cleanup quantitative methods were evaluated.
- In the environmental test arena, S. Lingam performed Automated Sold-Phase Extraction of Organochlorine Pesticides from Drinking Water (downloadable PDF) to determine the level of highly toxic and banded compounds. A comparison to traditional liquid-liquid phase extraction is provided.
- In the Food and Beverage arena, Determination of Azodicarbonamide in Flour (downloadable PDF) was performed by L. Lvye. Azodicarbonamide has been linked to respiratory issues, allergies, and asthma. A simple membrane filtration technique is described as the final step in the sample preparation process.
I hope that this brief introduction to a few sample preparation techniques leads you to appropriate resources as you look into getting the most accurate results for your sample in your matrix. Some of the benefits you will obtain are lower background interference, higher signal-to–noise, and accurate and reproducible results. Reproducibility can be achieved using the appropriate products and applications for your specific need. Application notes in every area of study are abundant and available to you. For more application examples go to http://www.thermoscientific.com and type your specific interest in the search bar.
For additional information about automated solid-phase extraction techniques in any or all of the chromatography fields mentioned and some of the newest products available, review the material here: Automated SPE reduces labor and solvent by up to 90% (website link).
And finally, for an extensive guide to sample preparation, I would suggest purchasing Sample Preparation in Chromatography (link to book purchase).
Is sample preparation in use in your laboratory? If so I would like to hear your thoughts and experiences.
Karen Thorson is a marketing manager for chromatography consumables in the Chromatography and Mass Spectrometry Division at Thermo Fisher Scientific. As a former drug formulation chemist, Karen moved from biopharmaceutical research to roles in chromatography instrument development where her focus was to assist in the development of new technologies that would better assist customers in their research. She prides herself in keeping a strong focus on customer needs via technical webinars and seminar presentations on industry related topics, and shares new technology applications with customers on a regular basis. Karin received her B.S. in Chemistry from the University of Massachusetts Dartmouth (USA).