There and Square Again: A Frit Coverage Tale

Have you ever encountered problems loading your samples onto an SPE or SLE plate?  Aside from sample viscosity or cartridge blockages, a good troubleshooting step to start with is ensuring that you are achieving complete frit coverage on the load.

Recently, the Biotage Analytical Applications Team was tasked with generating a workflow for some basic compounds in both urine and blood matrices. We decided to try a variety of different techniques to develop a comparative workflow to accommodate routine, clinical, and forensic applications. As the target compounds do not require hydrolysis or unbinding from proteins, we ultimately decided on a very simple 1:1 dilution with a buffer for sample pre-treatment, and we applied this to each extraction media type we investigated. This resulted in a total loading volume of 200 µL. Among the techniques tested was, you guessed it, SLE (Supported-Liquid Extraction) where we encountered some interesting challenges!

The ISOLUTE SLE+ product line is one of our favorites here among the Application Scientists of Biotage. The ease of use and versatility make this product particularly appealing to anyone looking to streamline sample preparation processes in their own lab:

Load - Wait - Elute

It’s THAT simple!

One helpful tip we regularly suggest to our ISOLUTE SLE+ users involves under-loading.  Under-loading refers to applying a total sample volume (raw sample + buffers or hydrolyzed samples) below the maximum capacity of the sorbent. For example, the ISOLUTE SLE+ 400 µL 96-well plate has a maximum sample capacity of 400 µL, as the name implies. Therefore, applying any amount below this limit, like 200 µL or 300 µL, would be considered under-loading.

This process has been shown to increase overall recovery of some compounds for SLE applications.

For our comparison, we first designed some simple SPE and SLE protocols to be evaluated. Most of the extractions went off without a hitch. The data for both blood and urine experiments looked really good! High recoveries, great process efficiencies, and considerably low limits of detection resulted for nearly every experiment we tried…except for the ISOLUTE SLE+ protocol with urine samples.

It should have been so simple, using a 400 µL 96-well ISOLUTE SLE+ plate! Under-loading 200 µL total volume of urine + a basic buffer, waiting 5 minutes, eluting with a selection of water-immiscible solvents, followed by analysis on one of our highly sensitive LC-MS/MS instruments. Instead, the extracted samples rarely made it to the analysis phase. The extraction process on the SLE+ plate with the urine repeatedly failed, with multiple samples in the test batch not loading. One unique physical aspect of the ISOLUTE SLE+ plates are the square wells containing the extraction media. As it turns out, this shape sometimes requires some additional considerations…

Unlike other Biotage ISOLUTE or EVOLUTE plates, the ISOLUTE SLE+ 400 µL and 200 µL plates employ a single permeable SQUARE frit atop the diatomaceous earth material.

While most protocols, automated or manual, suggest using positive pressure at 1-3 psi or 0.5 bar for 10-15 seconds for sample loading, we were seeing our diluted urine samples fail to load on to our SLE+ plate even at 20 psi for 30 seconds…45 seconds…1.5 minutes…5 MINUTES!  No amount of pressure or time facilitated the loading of our samples.

Naturally, we diligently recorded every experiment and troubleshooting effort in an attempt to resolve this matter. A close examination of the failed extraction revealed a very tiny detail that nearly every well had in common; as shown in figure 1, each well with an unsuccessfully loaded sample displayed a very small bright white portion in the corner of the top frit! Which means? The liquid sample was NOT covering the frit entirely during the sample load procedure. Given the volume of the samples, this was not always easy to see during the sample loading steps. It is understood that airflow will always travel through the path of least resistance. When these liquid samples failed to completely cover the square frit, a seal between the frit and the well was not formed. This allowed a good portion of the airflow to be directed away from the sample and down through the small corner gaps.

Figure 1. Image details portion of a 96-well plate in which many wells failed to load. The orange circles highlight the corners of the square frits, which are clearly visible, bright white in appearance, and not covered by urine samples.  This plate was subjected to 5.0 bar pressure for 3 minutes total on the Biotage EXTRAHERA automated sample preparation instrument.

Maybe you’re a bit uneasy about shaking your plate for fear of splashing the samples into another well. No worries! That’s a valid concern, and another way to ensure good frit coverage is to simply increase the total volume of your sample to be loaded. For example, in our experiment we were initially adding 100 µL of urine to 100 µL of buffer, then loading 200 µL total. To ensure complete frit coverage upon loading, we increased the urine and buffer volumes to 150 µL each, thus giving us a total of 300 µL to load onto our ISOLUTE SLE+ media. The samples easily loaded into all corners, and all samples were successfully extracted. So, what’s the solution? Simply give your extraction plate a decent shake after your samples have been loaded! We’re not talking about putting the plate on to a mechanical shaker. We mean, literally, just manually tap or shake the plate from side to side a few times to force the liquid to move around a bit in the wells. This will ensure the frit is completely covered with your samples, thus creating the seal needed to load the samples with just a little positive pressure. 

Still have concerns or questions about your SPE or SLE workflow? Reach out to a Biotage Applications Chemist! We’d love to help!

Do you want to know more about sample preparation techniques?

We have prepared for you plenty of interesting material ready to use, like the application note "Extraction of a Comprehensive Steroid Panel from Human Urine Using ISOLUTE® SLE+ Prior to LC/MS-MS Analysis" - download it now!

Download the Application Note