Chromatography Forum

Hi all,
I am using LC-MS. The compounds I am working on are Acyl-CoAs. The methods are developed in-house by others. C18 column. pH is 4.6. SolA: 90% ammonium formiate, 10% methanol; Sol B: 10 ammonium formiate, 90 % methanol. Standards are from sigma.
When I inject standards individually, some standards come out as really wide peaks, some don't come out at all. But if I run blanks after the standards, they come out as many many peaks, instead of one peak. I inject less amout of standards, and it doesn't help. So, I don't think it is because I overloaded the column. Standards are all new. Column is new too. Actually this happened right after I equilibrated the column and ran my first standards.
Do you have any idea of fixing the problem? Thanks!
Genggeng

Assuming that the method worked fine for another group when it was
developed...

You could try 2 things:

1. Increase isocratic hold at beginning of gradient
2. Increase hold time (e.g. 90% MeOH, 5-10 column volumes)

Assuming that the method worked fine for another group when it was
developed...

You could try 2 things:

1. Increase isocratic hold at beginning of gradient
2. Increase hold time (e.g. 90% MeOH, 5-10 column volumes)

Thanks for replying, Bryan.
I actually tried to wash extensively with 90% sol B(about 80% methanol) for really long time. When I ran a Blank, I can still see peaks from MS. The carry-over, or whatever it is, has really been kept in the column, and it won't elute as a single peak, but elute discretely as several small peaks.

Ok - I suppose next 2 things to try:

1. Talk to MD group, show them the data, and see if they have any suggestions.
Someone familiar with the method might have seen similar problems during MD and might have some solutions.

If that doesn't work - plan B:

- Try 90% organic (instead of 80%)
- Replace MeOH with Acetonitrile
- Replace your column with a less retentative column (e.g. from C18 to
a C8 or phenyl) of the same brand.

One person from MD suggest me to try high flow rate. For this C18 column, the pressue now can reach as high as 214. Is that too much? Do you think high flow rate (0.3 ml/min now, was 0.2 ml/min) will help?

I would suggest that you look at the following:

1) Make sure that the flow rate for your your pump A and B are correct (that might solve the problem of wide peaks and standards not eluted at all).
2) Make sure that your mobile phase additives and water are pure (i.e. do the peaks you observe increase in intensity if you increase the equilibration time? If yes then they are mobile phase impurities).
3) Are you really sure that you observe peaks and not spikes due to spray instability?