I have been trying to analyze 5-Hydroxymethylfurfural from starch.For this,I have to use DMSO to get good product selectivity,a halide catalyst and an ionic-liquid[EMIMCl/BMIMCl] to make the resulting solvent less viscous and improve the catalytic effieciency.However,after repeated use the HPLC suffers increase in pressure even after flushing with good amount of mobile phase(80:20 methanol:water) .The literature says that DMSO is not harmful to C-18 column if methanol is used as the mobile phase.It can only cause a little flunctuations in retention times.
My query is that- Is DMSO really not safe to be used in injection samples?Also,I was filtering & diluting my samples 1:1 before HPLC analysis.Will further dilutions reduce the risk of probable column damage?
Hi Shrestha, DMSO is no problem. This sounds like a sample issue rather than anything to do with DMSO. Pressure increases over time can happen and might be due to particulates/debris gradually accumulating in the column or precolumn filter. If your sample solutions don't look completely clear before loading, filter them first. Assuming you are using a precolumn filter (which is highly recommended), you can try comparing the pressure with this taken out or put back in, just running solvent through. There should be a small pressure difference with it in or out, but if there is a large difference, change the frit (filter).
If some or all of your pressure increase is coming from the column, it can be cleaned by washing with 100% THF (tetrahydrofuran) in the reverse direction to normal running; this tends to clean better then MeOH/MeCN. I would NOT use 100% water without checking compatibility as this can damage some C18 columns; you must check with the manufacturer or the instructions to find out if the column is compatible or not. Not all of them are, and 100% water can damage a column if it is not designed to go down to 0% organic.
The other thing to ask is, besides the pressure increase, are your chromatograms looking consistent with sharp peaks? If this is not the case, there would be other suggestions to make.
Hope this helps and good luck!
Dear Shrestha, I am no HPLC expert, but more a user. C18 column these days are VERY robust, so I do not see ant problem with DMSO. As for you increase of pressure, I would think that something in your sample (soluble in DMSO) is not soluble in you mobile phase. Before speculating too much about the column, double check that it is not your guard column getting stock... good luck
Dear Shrestha,
DMSO never harm your column. No need to go further dilution of your sample. Change your column washing method. If you are using buffer then first flush your column in opposite direction with 100% water for half hour and then for around 40min with 100% acetonitrile at pressure higher than your test flow. Acetonitrile reduces pressure rapidly than methanol. Still pressure is high then go for column regeneration. If still remain higher then clean the column frit. I hope this will definitely solve your problem.
Hi Shrestha, DMSO is no problem. This sounds like a sample issue rather than anything to do with DMSO. Pressure increases over time can happen and might be due to particulates/debris gradually accumulating in the column or precolumn filter. If your sample solutions don't look completely clear before loading, filter them first. Assuming you are using a precolumn filter (which is highly recommended), you can try comparing the pressure with this taken out or put back in, just running solvent through. There should be a small pressure difference with it in or out, but if there is a large difference, change the frit (filter).
If some or all of your pressure increase is coming from the column, it can be cleaned by washing with 100% THF (tetrahydrofuran) in the reverse direction to normal running; this tends to clean better then MeOH/MeCN. I would NOT use 100% water without checking compatibility as this can damage some C18 columns; you must check with the manufacturer or the instructions to find out if the column is compatible or not. Not all of them are, and 100% water can damage a column if it is not designed to go down to 0% organic.
The other thing to ask is, besides the pressure increase, are your chromatograms looking consistent with sharp peaks? If this is not the case, there would be other suggestions to make.
Hope this helps and good luck!
Better check the company specification, because all C18 columns are not same..... It very manufacturer to manufacturer, good to contact service engineer.
Nothing wrong to use DMSO for sample injection...... Wash column for some long time after use DMSO.
Dear Shrestna, I agree with Mark. DMSO is no problem for the column, but may be a problem in UV detection caused by the absorption of DMSO (DMSO causes a very large peak in UV chromatogram, if your substances are eluted int the same retention time window you might not be able to see them). Concerning the pressure increase I think Mark pointed out the essential issues. We prefer sample centrifugation instead of filtering for sample cleanup, but that is a matter of opinion. Another thing to take care when using DMSO as sample solvent is that when you have a cooled autosampler DMSO might become solid. But when you dilute your sample with methanol as you did this is normally no problem.
Thank You Laurent,Ashish,Mark,Paresh & Andreas- Your suggestions was a great help for me to understand my situation.
Andreas Kulik: The chemical of my interest shows a retention time much before DMSO.Centrifugation is a good option I see,but I was diluting my samples of HMF in DMSO with water.However,my mobile phase is 80:20 HPLC Grade Methanol:Water.Should I use methanol to dilute then my injection samples?
Dear Shrestha, normally it is a good ides to dilute the sample with a "weaker" solvent in RP chromatography, water in this case. But your are working with a relatively high amount of methanol in your mobile phase, so dilution with methanol instead of water might be suitable. Just try this: dilute an aliquote of your sample with water and another aliquote with methanol. Do you observe precipitation when adding methanol to the sample? This could be the reason for the observed pressure increase, because (partially) precipitation might happen on top of the column. If you don't observe precipitation analyze both samples. Any differences? If not carry on like you did it before, if yes, choose the better one. Best, Andreas
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