Lidocaine on Xterra vs Symmetry column

[adam]

We are working on a lidocaine analysis. Since Lidocaine is doubly charged at low pH we decided to stay at neutral pH, and use 50 mM Ammonium Formate to control tailing.

We found that we got very poor peak shape on the Waters Xterra column (even when we tried a brand new one). But pretty good peak shape on a Waters symmetry column.

Any ideas why this would be so. I think both of these columns should have minimal tailing.

Thanks

[Vlad Orlovsky]

Adam,

Lidocaine is not doubly charge at any pH because it has one secondary amine and amide. You have tailing due to residual silanol.
You need to go to lower pH to suppress effect of silanols. At higher pH your Si-OH is fully ionized and because of the small amount/low capacity it gives you tailing.

You also need to consider how old is your columns. With time some of the ligand can hydrolyze exposing silanols.

[Uwe Neue]

You are injecting an ionic sample into an unbuffered mobile phase. I expect that the retention on XTerra is much smaller than the retention on Symmetry, so extra-column effects and effects from lack of buffer are expected to be larger on XTerra. Also, as stated by Vlad, lidocaine is singly charged.

[laserman]

I agree with all opinions.
Please check this application.
http://www.waters.com/webassets/cms/lib ... erra70.pdf

Best Regards,

[adam]

We switched from Acetonitrile to MeOH and it works like a charm. So it seems like the problem was that when the aqueous mobile phase with 50 mM Ammonium Formate came in contact with the ACN mobile phase, the salt would come out of solution and we had a mess of a situation. With MeOH this does not happen.

I guess with the Symmetry column we got away with it a little better as the analyte was more retained at the beginning of the analysis when this "desalting" was occurring.

It is true our mobile phase has no buffering capacity. But I have always thought buffering capacity was overrated (unless you are very close to the pKa of the analyte - which is not a good way to do things anyway). I like to keep things simple. Unless you are concerned that your colleagues will be putting acids and bases in you LC system while you're not looking, I don't think buffering is critical. I have used these kinds of mobile phases many times in the past and they generally work fine.

Now I will sit back and await the avalanche of hate mail that I know is coming in regards to my latter statement.

[Pragmatist3]

We switched from Acetonitrile to MeOH and it works like a charm. So it seems like the problem was that when the aqueous mobile phase with 50 mM Ammonium Formate came in contact with the ACN mobile phase, the salt would come out of solution and we had a mess of a situation. With MeOH this does not happen.

I guess with the Symmetry column we got away with it a little better as the analyte was more retained at the beginning of the analysis when this "desalting" was occurring.

It is true our mobile phase has no buffering capacity. But I have always thought buffering capacity was overrated (unless you are very close to the pKa of the analyte - which is not a good way to do things anyway). I like to keep things simple. Unless you are concerned that your colleagues will be putting acids and bases in you LC system while you're not looking, I don't think buffering is critical. I have used these kinds of mobile phases many times in the past and they generally work fine.

Now I will sit back and await the avalanche of hate mail that I know is coming in regards to my latter statement.

You won't get any hate mail from me. I would tend to agree with you. I will use a buffered mobile phase when the chromatography demands it and not before. 90% of my analyses are performed using 0.1% formic acid in H2O and either ACN or MEOH.

[HW Mueller]

What was your lowest ratio of aqu. NH4formate/ACN, adam?

[adam]

HWM: We had 50 mM in both mobile phase A and B. We were able to do that with MeOH but not with ACN.

Pragmatist: Actually 0.1% formic acid does create a buffer system. I learned this from previous chromforum discussions. The water equilibrium shifts so you get more hydroxide. So you have both an acid and a base in equilibrium. Apparently it is only at more neutral pHs - maybe in the 5 to 9 range - where you need a "true buffer" to have a buffered system. I am like you: 0.1% formic acid is almost always my starting point.

[HW Mueller]

adam, actually you didn´t answer my question, but your statement may be interesting enough. You compare a system which has amm. formate in both the org. modifyer (MeOH) and water to a sytem which has this salt only in the aqu. part, none in the ACN?