a chromatogrphy trouble

[danko]

Gerhard, why should pH be adjusted to 2.0?

Rangtaiyang, what do you mean by asymmetric peak – is it tailing and what is the number in the beginning and after 48 hours?

Besides, you’ll need to do some experimenting. For example, try to run the system for 48 hours without injecting samples and see whether or not you get the same problem when injecting a sample.

Best Regards

[Alera]

One of the things that can increase RT are presence of a detergent with a positive charge in the samples that would eventually build up on the column. The easiest way to check for that is to flush the column with 100% organic for an hour and see if your RT returns to normal.

In case of separation of a ionic compound like this one (note - internal standard is not), would be pH chnage. Say, if your pH decreases, your RT will increase as the compound becomes less charged. It is hard to imagine this scenario in this case though, but I have seen some weak buffers become more acidic with CO2 absorbtion (again, not likely at all in this case), thus affecting the separation.

[rangtaiyang]

it is not useful to flush the column with methanol,the RT will not change as it has become bad.

[Alera]

It is possible that unfortunately whatever ends up on the column, binds irreversibly. You can try stronger organic solvents like IPA or THF to flush the column (also increase temperature). You might have to come up with an additional cleanup step to remove this material from your samples, like RP SPE. Since it binds to C18 much stronger than your compounds, SPE should work very well, you should be able to elute your compounds from the cartridges, leaving this material behind.

[rangtaiyang]

I could not agree with you more.I will try IPA to flush my column.SPE is some expensive,maybe our lab cannot afford it,thanks for your advice.

[HW Mueller]

If you have a positively charged entity sticking to silanolates you will hardly remove it with organic solvents. You might get lucky with acid, most likely in water + organic (depending also on the solubility of the cation).

[Alera]

SPE cartridges are not very expensive, but then again, it depends on how many sample you are running at a time. If you are running 100 samples before your column dies, spending $100 on cartridges is probably worth it, rather than losing a $500 column.

Also consider using a guard column, if you are not using one already.