High speed centrifuge vs 0.45um, filter which do you prefer?

Discussions about sample preparation: extraction, cleanup, derivatization, etc.

7 posts Page 1 of 1
Hi,

I am trying to optimize sample preparation for analysis on HPLC-RID and would like to know is it typically better to high speed centrifuge or filter a sample?

Neither option will have an effect on chromatography. This is just purely for HPLC column preservation and costs savings.
Depends who you work for and what you do.
If your samples are in stupidly small volumes, you'll probably need to use something like spin filters if you want to filter them, and you will probably find that about 80 spin filters cost the same as a guard column. In which case, if you can get through more than 80 samples before the guard gets blocked/quality deteriorates, the guard is the cheaper way to filter the samples...
Also, if you work on novel analytes and weird things present at low concentrations, for skeptical clients, then every time you fail to detect their favourite analyte, you will have to check whether it was lost by binding to the filter.
I personally reserve filtration for samples that are obviously problematic (cloudy etc.) and which I can't solve by centrifugation. Everything else gets spun and injected. But that's not right for every lab.
lmh wrote:
Depends who you work for and what you do.
If your samples are in stupidly small volumes, you'll probably need to use something like spin filters if you want to filter them, and you will probably find that about 80 spin filters cost the same as a guard column. In which case, if you can get through more than 80 samples before the guard gets blocked/quality deteriorates, the guard is the cheaper way to filter the samples...
Also, if you work on novel analytes and weird things present at low concentrations, for skeptical clients, then every time you fail to detect their favourite analyte, you will have to check whether it was lost by binding to the filter.
I personally reserve filtration for samples that are obviously problematic (cloudy etc.) and which I can't solve by centrifugation. Everything else gets spun and injected. But that's not right for every lab.


Another thing to consider is how good of a technique the analyst has when handling the samples. One with poor technique might be better using filters since if you shake the centrifuge vial a little while removing the sample you can get some resuspension of the particles, or if they try to draw sample from too near the bottom of the vial it can happen.

I even have had a few who could not draw 1ml from the 5ml of solvent above the 1ml of sulfuric acid at the bottom of a narrow test tube without getting the acid into the pipette :roll:
The past is there to guide us into the future, not to dwell in.
… oh yes, and going wildly off topic, you've reminded me of many happy hours extracting trichloroacetic acid from aqueous samples by mixing with water-saturated ether and pipetting off the ether. Problem was, the first wash would have so much TCA in the ether that sometimes (but not always) the ether would be the denser phase instead of the lighter. Or in some cases the two phases would be rather undecided as to who should be on top...

Also of a story from an instrument engineer, who'd gone to fix a system with a blocked needle. While he was fixing it, another instrument in the same organization got reported as having a blocked needle, so he moved on to that one... and on to a third instrument...
What was happening was that someone had a method in which they simply centrifuged a revoltingly particulate sample in an injectable vial, so that the solids landed up on the bottom of the vial, and the injection was supposed to be taken from the liquid above. But unfortunately if you've got bottom-sensing on your needle, so the needle first pushes on the bottom of the vial and then moves upwards.... And of course if the user thinks the first system's blocked so they go looking for a second system to run their sample, and hey, by some coincidence that's blocked too, so they go looking for a third system....
be careful with filtration as certain types of membranes can adsorb your analyte(s), especially polar membranes such as nylon and cellulose-based

I personally prefer high-speed centrifugation (e.g. 20000 g), but this may not be practical if you have a lot of samples

we use 96-well filter plates with glass fiber which has 0.7 um porosity; then the filtrate is spinned at 1800g; altogether this results in excellent robustness
Yes, you definitely have to match the filter material to the analyte of interest.

We discovered that with nylon membrane, if you filter a sample containing Diquat it will capture 100% of the analyte if the pH is neutral or high, if the sample is acidified with H2SO4, it will pass through 100% of the analyte.

When developing a method I use several different membranes to judge recovery and look for any interference or adsorption before deciding on a final material.
The past is there to guide us into the future, not to dwell in.
I agree that not every sample has to be filtered. That is what guard columns are for. It is important to use the right membrane. My first professional mentor had a cheat sheet with samples and suitable filters hanging over his bench.

I have found this to be a good starting point: https://scientificfilters.com/filter-media-selector/

It is easy to accidentally re-suspend the pellet when pipetting out of an eppendorf tube, especially if you are a lab tech who could care less about the results.

If you are worried about particulates you can also make sure the needle penetration depth of the sampler isn't super close to the bottom of the vial. Remaining particulates in the HPLC vial may settle to the bottom when on the autosampler.
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