With advances in high performance liquid chromatography (HPLC) and ultra-high pressure liquid chromatography (UHPLC), analytical separations have become more and more sensitive, meaning that smaller and smaller sample injections are sufficient to provide required data. At the same time, the use of small-volume samples has become more common, as researchers seek to maximize the number of analyses performed using limited sample amounts. As a result, the amount of sample available for chromatography has been frequently reduced. Even just a few years ago, 1 – 2 mL samples were typically available for chromatography; however, today’s researchers are often limited to samples that are less than 500 µL. To accommodate the decreased sample volume, chromatographers need to use HPLC vials which have inserts in them (Figure 1) so that small samples can be reproducibly injected into an HPLC system.
Even though the amount of sample available for chromatography is reduced, the sample still needs to be filtered to ensure that it is particle-free before it is injected into the HPLC system. When filtering a few samples at a time, syringe filters are commonly used. Syringe filters are available in various sizes, enabling filtration of very small sample volumes without losing significant sample to hold-up.
Syringe filters prove less efficient for users typically filtering 10 – 100 samples a day (65 – 70% of users). For these users, the Samplicity® multifiltration system facilitates sample preparation by enabling vacuum-driven filtration of up to eight samples directly into standard (12 x 32 mm) HPLC vials. The Samplicity® system has been used widely to filter 300 µL – 2 mL samples into HPLC vials without inserts. In this application note, we present data showing that the Samplicity® system can be used for filtration of smaller volume samples into HPLC vials containing inserts.
Figure 1. Samplicity® System with HPLC vials containing small-volume insert.
Materials and Methods
We also examined the effect of vacuum pressure on filtration performance, and found no correlation. Filtration tests were conducted at 14 – 22 inches Hg (474-745 mbar) vacuum pressure and there was no impact of vacuum pressure on filtration into the vial (data not shown). All the sample recovery experiments were therefore conducted at 22 (maximum possible) inches Hg (745 mbar) vacuum pressure as measured by the gauge.
When pipetting small volumes for filtration using Samplicity® system, make sure that the sample is pipetted below the cross bar at the bottom of Millex Samplicity® funnel. This will ensure that the sample will be filtered as soon as vacuum is applied and that the system does not air-lock. An air lock will completely prevent all filtration. Pipetting technique is even more crucial when handling aqueous samples because of their high surface tension. If the sample is not pipetted below the cross bar, the surface tension may cause the sample to bubble up on top of the cross bar, leaving the filter dry. In contrast, organic samples exhibit reduced surface tension, allowing samples to pass easily below the cross bar. To prevent air locking and sample retention on top of the cross bar, stream the sample slowly along the sides of the funnel with the pipette tip touching the side of the funnel.
Figure 2.Operation of the Samplicity® Filtration System. Millex Samplicity® filters are placed over the openings. HPLC vials are installed underneath the openings, where they are seated at the optimal angle for filtrate recovery (inset). To avoid air-locking the filter, the sample (shown in blue) is pipetted directly into the center of the funnel, not down the side.
Using 0.2 µM filters, we obtained consistent filtration of 100 µL samples. For all the types of vials tested, greater than 70% recovery was obtained when filtering 4 or more samples at the same time (Table 2).
The smallest volume of acetonitrile that could be consistently filtered was 300 µL. When filtering 100 or 200 µL of acetonitrile sample, the vacuum pressure caused the solvent to spray into the system, which resulted in the sample spilling outside the vial. When filtering 300 µL of sample, no sample spillage was observed, and all of the filtrate was consistently collected in the HPLC vials. We thus used 300 µL acetonitrile samples and the indicated vials to test recovery using the Samplicity® system (Table 3). We achieved recoveries of around 50% in every case. The lower rate of acetonitrile sample recovery was expected due to the increased hydrophobicity of this solvent. Acetonitrile tends to fully wet the PTFE membrane, thereby spreading onto the entire membrane surface. As a result, a large portion of the sample is held up by the membrane after filtration, leading to reduced sample recovery. Membrane wetting is a slower process for aqueous samples; therefore, higher sample recovery is possible.
Our data show that filtration of aqueous samples as small as 100 µL into any 12 x 32 mm HPLC vial containing inserts can be efficiently performed using the Samplicity® system, with good recovery rates (greater than 70%). We have also shown that acetonitrile, and presumably other organic solvents, can be filtered using the system into these low-volume inserts, although larger starting volumes (300 µL) are required. The vials mentioned in this report were the only ones tested during this study, and it is possible that more vials containing low volume inserts can be used for low volume filtration with the Samplicity® system. As more chromatographers turn to small-volume samples for their analytical separations, they are likely to find that the Samplicity® filtration system provides an ergonomic alternative to syringe filters, increases sample preparation throughput and maintains quality of downstream results.