Chromatography Forum

I am currently using a silica based amino column (Imtakt US-amino) to perform an ion exchange separation of a polysulfated disaccahride. Per the method, the standard is prepared in the mobile phase buffer and the sample is prepared in an acid/base solution (because this is the only thing that will solubilize the compound of interest). The pH of the final sample solution is around 3. The mobile phase/diluent pH is 3.5. I am using ELSD detection at 70C. Here is my problem . . . the standard and sample responses (though prepared at the same level) are orders of magnitude different! Can someone offer an explanation as to the chemistry of what is occuring to cause such an effect and what I might do to remedy the situation? Please advise if more info is needed to assess. Thank you!

You might be absorbing part of your material on the column. Inject sample without column, measure peak area and compare it with sum of all peaks with the column. You should get a close match.
Also if you start clipping off stationary phase your background will go and your sensitivity will go down.

Which one is higher, the standard or the sample? And when you say different are you talking about intensities at the same gain level or S/N? If your standard is higher, what happens if you spike your sample with known amount of standard?

Detector response - is this referring to peak height or peak area?

Vlad - I don't understand how I can inject sample without a column and expect to get a peak from which I can measure peak area. No column, no separation, no peak. I believe the stationary phase is quite stable.

Kostas - The standard response is higher both in height and area. All solutions are run under the same detector conditions (i.e. Gain =1)

Bryan - see my response to Kostas.

Thank you all for your input! I really do appreciate your assistance. This project is killing me!

So what happens if you do standard additions of your standard into your sample?

I'm a bit unclear on your original post. I'm assuming that you can prepare a "standard" both ways (i.e., one in mobile phase, and one in your "acid-base solution"), and you get higher response from the one prepped in mobile phase. Is that right?

So, a couple of naive questions:

1. If you can dissolve your compound in mobile phase to create the "standard", why is "the acid-base solution" necessary to solubilize the compound of interest? Or is it a case of unsticking it from the matrix?

2. Have you tried a standard addition run to establish recovery? (spike an couple of your prepped samples with a known volume of your two highest level standards and see if the slope of the resulting 2-point line matches the slope of your calibration line. If it *does*, that suggests that the problem may be incomplete extraction from your matrix. *If it doesn't*, that suggests that there is something about that "acid-base solution" causing your compound to degrade (or associate?).

I wonder if you have poor recovery from sample preparation?
Have you tried this type of experiment:

Solution A: perform sample prep procedure on standard solution
Solution B: standard solution

Compare peak area (i.e. % recovery) between the two solutions?

(oops, sorry, Tom's and Kostas's suggestions will be faster to do, and may provide
more information).

You are not looking at separation without column, but you need to know if everything you inject in the column is eluting from the column. You inject sample without column and you see one peak, which represent everything you have in your sample. You inject your sample with the column, you hopefully separate peaks, you measure each peak area and combine peak areas. Combined peak area should match peak area for one peak without column. Sometimes this is not working if for example you inject sodium chloride into ion-exchange column (mixed-mode, ion-exchange) and use ACN/water/TFA as eluent. Without column you see both sodium and chloride as one peak, but on a column sodium and chloride separate and you don’t see chloride ions, unless you use ammonium formate or amonium acetate (ELSD or Corona).