Haunted peak

[syx]

Waters Atlantis dC18 5um, 436 x 150mm column was used to purify the buffer solution. Though the method is using C8 column, we decided to use less polar stationary phase in this buffer solution preparation. We assumed that the water impurity would be retained stronger in a column with lower polarity.
Purified solvent was collected directly from the column outlet and then used to run the procedure as in assay. Using this solvent, the ghost was shrinking but could not be eliminated completely (See picture of baseline chromatogram). Could it be neglected? Tiny ghost is not scary … I think.



A Without purification
B With purification

[Uwe Neue]

I think this ghost is nearly invisible...

[amaryl]

Wish I could even get rid of my haunted ghost peak...which is now dumped in to the hard copies of my work.


Hey siswanto,

How did you put that art work of Ghost peak looks great.

Amaryl.

[syx]

At the end of purification I wash the purifying column using water to wash out the salt and then acetonitrile up to 100% in order to remove any impurities. Next day, when we want to use the column for the next batch of buffer solution, I need to run the water first to eliminate acetonitrile from the column. What is the safest mode to detect acetonitrile to ensure that the eluent is acetonitrile-free? (If I sniffed it each purification day … I will be a ghost after a few days ). Could it be certainly predicted that entire acetonitrile content in the column would be flushed out using 20 x column volume of water?

Amaryl, just copy your chromatogram to clipboard, paste as a new image in photo-editing software (I used Corel Photopaint), add text and additional pictures, and then save as jpg or gif image. That’s all.

[Rafael Chust]

Easy method: ACN absorbs at low UV (say 210 nm), therefore you can "see" it goes away using your UV detector...

[syx]

ACN absorbs at low UV (say 210 nm)

... I think acetonitrile has UV cutoff at 190 nm... Do I miss something? The solvent was not passing the detector. I collected eluent directly from the column outlet.

[Rafael Chust]

Dear Syx:

I am not sure about its UV cut-off, but I know by experience that ACN presents some absorption in UV up to 254 nm... that's why there is available a "ACN gradient grade" offered by some suppliers.

If you use ACN you must find a baseline drift in low UV when changing ACN concentration in mobile phase.

Therefore, if you have ACN, when changing it to 100% water you must see the baseline going down as ACN is getting lower and lower.

Did you catch my drift?

[JA]

At the end of purification I wash the purifying column using water to wash out the salt and then acetonitrile up to 100% in order to remove any impurities. Next day, when we want to use the column for the next batch of buffer solution, I need to run the water first to eliminate acetonitrile from the column. What is the safest mode to detect acetonitrile to ensure that the eluent is acetonitrile-free?

1H-NMR. Back of an envelope calculation showed that even with our old 250 MHz instrument I could expect an LOD of around 0.05% for MeCN if an aliquot of 0.1 ml eluent was diluted to 1 ml with suitable deuterated solvent.

[AA]

It appears that this is a water problem, which is not an uncommon issue, but yours seems somewhat worse than many. This is how I purify buffers when I run into this problem. Make your buffer as usual. Filter your buffer through 6 grams of Oasis SPE material. You can buy 6 gram cartridges, break one open and dump the material into filter appuratus (I use a sandwich of whatman #1 on top and a 0.45 um membrane underneath for the filter). You must wash the Oasis material first with methanol (200 mL) and then 100 mL of water which you then discard. Then filter your buffer through the bed. I do this with a vacuum filter appuratus (with a 1 liter funnel) so it does not take too long. For very severe contaminations you might have to do this process a couple of times. The good news is you can re-use the Oasis bed, just wash with methanol and rinse with the water.

As a long term solution, you need to invest in a high quality water purification system, be aware of outside contaminations (dishwasher soap for one), use highest quality buffer salts, acids and bases, and high quality organics (they can play a roll in this kind of problem too).

You could also try to develop an isocratic method (if you havent already).

[syx]

Rafa, thanks for the suggestion.
AA, isocratic run is not an option in this method. If the mobile phase is too weak (that could give enough retention to first substance) the second will be eluted far far away – or perhaps would not show its peak.