June 1, 2015 (Vol. 35, No. 11)
Karl E. Milius Global Product Manager Wheaton
Choosing the Right Sample-Handling Solution Is the Key to Ensuring Sample Integrity
For chromatographers, maintaining sample integrity is essential in order to obtain ultimate sample separation and analysis. Today, more and more operations within the lab are moving to automated and semi-automated systems. When high-throughput autosamplers and instruments that are compatible with the Society of Biomolecular Screening (SBS) standards are used, the need to maintain sample integrity becomes even more critical.
As chromatographers and chromatography facilities are forced to increase throughput without increases in personnel, a significant amount of time is now spent on sample preparation. While high-throughput systems can deliver on the promise of high-speed injections and sample processing while decreasing sample waste, these come with increased risks of sample cross contamination, as well as issues with solvent resistance and compatibility.
Standard protocols for high-throughput batch preparation methods allow for more rapid analysis, but with this higher capacity come risks to sample integrity and sample analysis. With many automation-friendly, SBS-footprint, high-throughput plate systems available from various manufacturers, all with different sealing configurations for sealing mats and closures, it’s important for researchers and lab managers to understand which of these systems and plate configurations will work best for their specific needs to ensure timely, high-performance sample preparation.
Cross contamination of samples, as well as plate compatibility with organic and/or volatile solvents have the potential to produce samples of questionable integrity and inaccurate sample analysis. This means researchers need to choose systems that eliminate these risks. Outlined here are standard available formats for high-throughput plate systems and plate sealing, and considerations for their proper use in order to maintain sample integrity and accuracy of sample analysis.
Plates and Plate with Inserts
The two most common formats for use with high-throughput systems are sample plates and plates with inserts. In 1996, the SBS established specific dimensions for microplates in order to standardize these plates and how they function within various automated solutions. To maximize the fit and function of available chromatography sample plates with automated and semi-automated instruments and devices, most plates follow the standard dimensions set forth by the SBS. When choosing an automated sample handling system, researchers should look for ones that adhere to the SBS standards.
Not only is the standard footprint important, but the spacing between wells and/or inserts is equally important for the use of automated liquid-handling systems such as multichannel pipettors or automated and semi-automated dispensers and extractors. If the plate footprint is not standardized, labs will require multiple instruments for the same task or will be forced to modify instruments between each assay. The need to continually change and modify the system significantly increases the risk of data variance, and ultimately to decreases in sample analysis accuracy.
Sample plates are most commonly made of polypropylene, a resin that is relatively nonreactive and resistant to many reagents. Available in a range of sizes, based on recommended maximum sample volumes (correlated to height of the plate), wells can be square or circular with either round (U) or conical (V) bottoms.
That noted, polypropylene plates are not always the best plate material choice. Since it is a plastic resin, polypropylene is not suitable for use with many organic solvents and is likely not a suitable material when working with proteins and/or compounds that exhibit loss of recovery. In these cases, chromatographers should choose sample plates with inserts, which are, as the name implies, sample plates that have glass inserts in each well.
In addition to the material considerations of polypropylene versus glass, glass inserts allow for each well to be sealed individually, virtually eliminating the possibility of sample cross contamination. When using plates with inserts, it’s important to note that the overall well volume can be significantly decreased. This can be mitigated by the use of conical-bottom inserts, which are designed to maximize sample recovery.
The most basic closure system for plates and plates with inserts are sealing mats and tapes, which are inexpensive, easy to use and provide protection from condensation and evaporation. Sealing tapes are made from a number of materials including acetate, aluminum, or polyethylene, among others. Some sealing tape mats are available without adhesive at the cavity areas as a measure designed to eliminate possible sample contamination within each well.
Sealing mats are most often made of silicone or ethylene vinyl acetate (EVA), which can also be coated with materials such as polytetrafluoroethylene (PTFE) to provide a measure of re-sealability. Yet despite their ease of use and low cost, most sealing mats do not provide long-term, viable seals, which can lead to sample cross contamination when removed. Because they provide the least robust seal, neither sealing tapes nor mats are appropriate for volatile workflow processes.
Individual closures offer the best protection against sample contamination and integrity when using glass inserts with sample plates (Figure 1). Some individual closures offer a secure snap seal that is equal to that observed with standard 12 × 32 autosampler vials. The use of individual closures allows for single samples to be removed and used in external processes without risk of losing the sample. Further, when used with the appropriate heating block and vice enclosures, these high-throughput solutions are suitable for volatile reactions that normally have to be processed with standard gas chromatography screw-thread or crimp-top vials.
Essentially a hybrid of mats and caps, CapMat closures (Figure 2) allow for the secure sealing of individual inserts in a high-throughput workflow while allowing the transfer of all 96 inserts as a single unit. These closures, like individual closures, offer a secure seal equal to that observed with standard 12 × 32 autosampler vials with snap closures. This solution is appropriate for processing volatile samples in a high-throughput workflow process.
With automated and semi-automated sample-preparation systems becoming a fact of life in many chromatography labs, chromatographers need to be well versed in the tools available in order to determine which solutions, based on their assays, will prevent cross contamination and ensure sample integrity, in order to provide viable, accurate analysis. While new solutions continue to come to market, only specific purpose-designed solutions are appropriate for protocols involving volatile solvents and reactions. To determine the most appropriate solution, a thorough review of workflow processes and identification of specific pain points is required in order for chromatographers to work with the confidence that they have the most appropriate sample automation solution available.