Dilute, Shoot, and Elute – am I missing anything? YES!3 Apr 2018
Everyone’s lab is different in terms of how many samples per day are processed, but they all share the common pain point of sample preparation. Some samples like blood and plasma need a significant amount of prep to remove proteins, phospholipids, and salts, whereas labs running urine or drinking water samples can “get by” with a 5x or greater dilution.
No matter how limited or extensive your sample prep, the one thing that is critical to prolonging the lifetime of both your column and instrument is particulate removal, and you know what that means: filtering.
We’ve blogged about filtering mobile phase before, and recently gave you a behind-the-scenes tour of column construction in the Clog Blog to emphasize how important it is to remove particulates from samples. You also want to keep your HPLC or UHPLC up and running, and the downtime plus parts and labor expense for replacing any or all of the sample needle, injection port, valve rotor, and outlet tubing of your autosampler is far greater than sample filtration supplies.
I like to use “the paint example” when talking about sample prep. Chances are you or a colleague have done some home remodeling that includes painting, so you know you don’t just go get the color you like, roll it on, and you’re done. For best results, you have to fill in any holes or scratches, sand, tape, prime, and finish with your carefully chosen color from the selection of 500 shades of beige. Sure it takes extra time, but it turns out looking great. It’s the same with HPLC sample prep: the more care you take with filtering, the longer your column and instrument will last. My colleague and frequent blogger Nancy from Tech Services has a great post about making your HPLC columns last longer and filtering is high on her list too!
The easiest method for manual sample filtration is to use a Thomson Filter Vial. Anyone who knows me will tell you that this is one of my favorite products EVER. There are different membranes depending on whether your sample has a high aqueous or high organic content, and the 0.2µm membrane is ideal for small ID UHPLC tubing, which is typically 0.1mm ID or less and is prone to clogging. There’s even an eXtreme version of the Thomson vial that has a multi-layer filter for samples with 10-30% particulates. Both vial types are very simple to use with a lot less mess, hassle, and waste compared to a separate syringe, disc filter, and collection vial. Here’s an example using a 0.2µm PVDF filter vial for the analysis of fentanyl in urine.
Another effective way of removing particulates is through centrifugation. After a protein crash or dilution, you can place your vials or multi-well plate into a centrifuge and spin away to pull particulates into a pellet at the bottom of the vial or well. This analysis of immunosuppressive drugs used a precipitation solution vortexed with whole blood, then 4,300 rpm in the centrifuge to remove particulates.
After filtering or centrifugation you can put your vial or plate into the autosampler for analysis. Make sure you adjust the needle setting so it stops 3-5mm above the pellet so it doesn’t suck up any of the particles you just pulled out of the sample solution!
A simple filtration or centrifugation step will allow you to make the most of dilute-and-shoot sample prep’s high-throughput capabilities while helping to keep particulates out of your instrument and column. This reduces instrument downtime and prolongs column lifetime so it’s a win-win for your lab’s productivity. You can also double up on column protection with good sample prep and the use of a guard column, especially if the sample has fine particles that can pass through a filter membrane or not form a good solid pellet.
Thanks for reading!