Microelution, SPE, Mikro plates

Charlotte Hayes

Microelution plate processing: What pressure is best?

March 16, 2021 at 3:33 PM / by Charlotte Hayes

Microelution SPE is a great alternative to conventional SPE methods, enabling the use of lower wash and elution volumes, while maintaining sufficient load volumes, providing concentrated samples, perfect for highly sensitive analysis. Achieving the best, most consistent results means using the right processing conditions, and because all microelution SPE plates are not the same, you might need to adjust your processing conditions when using these plates.

Optimizing sample preparation may not be top of your list when developing a new analytical method, but spending a little time on at the beginning of the process can bring significant benefits. We all know that cleaner extracts can reduce matrix effects, improve LOQs, and reduce LC-MS/MS downtime and optimizing the scale of your clean up will surely bring its own rewards.

Using positive pressure rather than vacuum for processing SPE columns and 96-well plates improves reproducibility because it pushes solvent through the columns or wells more uniformly. Biotage application specialist, Jillian Neifeld, reviewed the pros and cons of different processing methods. You can read about it in our blog Vacuum or positive pressure for processing samples: what's best?

Due to the unique, high-performance design of Biotage® Mikro SPE plates, this low volume format requires higher processing pressures compared to other microelution plate designs.

Recommended pressures for use with Biotage® Mikro plates:
• When processing using a manual positive pressure manifold like the Biotage® Pressure+ 96 the recommended pressures are 2-5 psi on the coarse flow setting and 20psi for the drying steps.

• When processing using an automated system like the Biotage® Extrahera, the recommended pressures are 1.5-2 bar, and 3 bar during drying steps.

This topic was covered recently in a webinar recently given by Dr Stephanie Marin for Bioanalysis Zone: Current sample prep for bioanalytical laboratories: automation, small volume elution SPE and supported liquid extraction.

The novel design of the Mikro product has a long, narrow sorbent bed, which enables the small amount of SPE sorbent to have much higher capacities, producing higher analyte recoveries without analyte breakthrough. The tapered design of the tip helps to prevent air locks during processing, ensuring an equal flow of the analytes and matrix through the media bed. The narrow outlet of the tip is 1 mm in diameter, providing optimum bed geometry for efficient movement of analytes through the media. This, however, also increases the pressure needed to flow the sample through the small orifice.

This simple diagram shows the difference between traditional SPE bed dimensions, and the long, narrow Mikro bed.

BIOMARK2-247 - Tip Comparison-1Long, narrow SPE bed dimensions require higher processing pressures than thinner, wider beds

And it’s not only the dimensions of the bed that matter. Biotage® Mikro plates contain high performance polymeric EVOLUTE sorbents, packed between small, uniform porosity meshes. These meshes provide the lowest possible dead volumes, making lower elution volumes possible. These also require higher processing pressures as the samples are pushed through the small pores of the mesh.

Although higher pressures are required, the unique design of the Low Volume Format plates result in a high quality clean up tool. Biotage® MIKRO microelution SPE plates produce purified, up to 10x more concentrated samples suitable for direct injection analysis, removing the requirement of an evaporation step.

Do you want to learn more about the different sample preparation techniques? Click the button below for a comprehensive review of sample prep for clean-up of biological fluid samples.

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Topics: Troubleshooting and Optimization, Extraction method optimization, Microelution, SPE, positive pressure processing

Charlotte Hayes

Written by Charlotte Hayes