Peak area variations

[albragi]

Dear Members,

I experience peak area variations I cannot explain. It is RP chromatography, sodium phosphate monobasic/phosphoric acid buffer (pH=3): acetonitrile (70:30), isocratic for main peaks with gradient later to wash out garbage. SST is always excellent. Samples are bracketed by standards, pattern of injections -- std, two samples (first preparation), two samples (duplicate), std. Two injections from first preparation have the same area between them, two injections from duplicate preparation have the same area between them, but duplicate preparation samples always have smaller area than first preparation (RSD 3 to 10). Sample preparation is simple and no mistakes are made. I even tried to fill those vials (first and second preparations) with a sample from one preparation only -- the result is the same -- different areas. Equilibration of the column seems to be OK, new column makes no difference. The system seems to be OK. Can you help, please?

Al Bragi
Canada

[tom jupille]

Hmmm,

You have done a thorough job of eliminating the most likely sources of error, so these questions may seem like "grasping at straws":

1. Are you doing the gradient washout after every injection or after every vial? and are you doing the gradient washout after the standards as well as the samples?

2. Were the first and last standards in agreement?

3. What happens if you do all four sample injections from the same vial (not just the same preparation) -- of course, this assumes you have a large enough vial?

[albragi]

Hello Tom,

Thank you for your reply.
1. Yes, I am performing gradient washout after every injection, it is built into the method.
2. Standards are in agreement.
3. If I inject from the same vial, I have the same problem on my hands.

It is a mystery, I know I cannot think of anything else anymore.

The only thing I think can have an effect is improper column wash after all the runs are done and storage conditions as I am not the only one working with this column (I now pay attention to this), but then this strange pattern is driving me crazy.

Al Bragi

[tom jupille]

1. Yes, I am performing gradient washout after every injection, it is built into the method.

That eliminates the possibility that it's a column reequilibration issue.

2. Standards are in agreement.

How close? RSD values for duplicates don't really tell you much, and I've been in the situation of "seeing" patterns in the data that were just random fluctuation. Could you post some actual area counts for a couple of runs?

3. If I inject from the same vial, I have the same problem on my hands.

That's really strange! So the pattern is Normal / Normal-Normal / Low-Low / Normal every time?

[Uwe Neue]

Is the concentration of your standard much higher than the concentration in the samples? Or is the standard dissolved in something different than the samples? In this case, it could be a carry-over issue from the standard...

I admit that I am grasping...

[JM]

Look at the stability of your sample solution , which might be dfferent from sample due to matrix effect. What happen if you re-inject 1ist sample preparation after 2nd preparation?

JM