split peaks at low concentrations

[trilobite]

Dear all,

I am developing an UPLC method for the analysis of a peptide (highly influenced by pH, soluble at pH 7).
I am using a gradient; 0-6.00 min, 90%A. 6.00-9.00 min, 50%A. 9.0-12.0min, 90%A (duration of 14 min, peptide eluted at 4.5min)
[A: phosph.buffer+2mM amm.acetate+0.1 formic acid and B:MeOH+2mM amm.acetate+0.1 formic acid]
I get a very nice peak at 100microg/mL but when I go down to lower concentration I do get split peaks. Do you think it would be due to peptide impurities?Shall I go with a new peptide stock?Or maybe column is going off due to phosphates?Any tip for column restoration??

Regards
Theo

[dorota]

Maybe this will help:
J. R. Conder, Peak distortion in chromatography. Part 1: Concentration-dependent behavior, Journal of High Resolution Chromatography, 1982, 5, 341

http://chromatographyonline.findanalyti ... ?id=148999

[annaand]

Concentration influences separation because of very simple issue: The higher concentration the bigger peak, bigger peak the wider peak, the wider peak the more time it needs to elute, the more time it needs to elute the more other close peaks it eats.
However, my question is: Did you run a blank (your sample solvent)? Do you see any peaks in blank?
Best,
Anna

[HW Mueller]

Careful with this "The higher concentration the bigger peak, bigger peak the wider peak". If you are in the linear region of the column (no overloading) the width is not increased with amount.

[trilobite]

Dear all, here I am (again)

Well, my solvent blanks are absolutely clear. Moreover, I am within the linear region (no overloading).
My actions so far:
a. I made a fresh working solution
b. I washed/reconstituted my Acquity BEH C18 column (though not back flushed, since Acquity's technical support suggest to avoid it)
c. Split peaks have disappeared, though poor chromatography still appears in terms of somewhat wide peak width/tailing

I am thinking of changing mobile phases towards AcN instead of MeOH (AcN>MeOH) and decrease flow rate from 0.5 mL/min to 0.11mL/min. What do you think?
If I use a mobile phase A of 90%phosphate buffer (pH 7), 10% AcN, 2mM ammonium acetate and 0.1%f.a. and a mobile phase B of 10%phosphate buffer (pH 7), 90% AcN, 2mM ammonium acetate and 0.1%f.a., will I have buffer precipitation??

[danko]

If you really have one peak (one substance) initially, the split peak might be due to substance degradation – which “disappeared” following the preparation of a fresh solution.

Or the column is damaged (could be voids in the bed and it could be contamination of the stationary phase and/or the inlet frit) so, the column wash you carried out probably helped a bit.

Did you run an injection following step 1 (after preparing the fresh solution and before washing/regenerating the column? If yes, what did you see then?

Btw. why should decreasing the flow rate from 0.5 to 0.11 mL/min improve the situation in this case?
And is it really necessary to use so many components in your mobile phase?

Finally the concentrations of these components are sort of unclear – f. ex. the phosphate buffer, but also the other compounds.[/size]Best Regards

[trilobite]

Dear all, I apologise for my late reply. Just wanted to have a go first.Nevertheless here I am. The full story is described below:

Initially, I have set up an UPLC method for peptide analysis using an Acquity BEH C18 column.
FYI:
Mobile phase A: 90% 25mM KH2PO4 (pH 7.4), 10% MeOH, 2 mM ammonium acetate, 0.1% f.a
Mobile phase B: 10% 25mM KH2PO4 (pH 7.4), 90% MeOH, , 2 mM ammonium acetate, 0.1% f.a
gradient conditions: 90%A (0-6 min), 50%A (6-9min), 90%A (9-12 min)
flow rate:0.5 mL/min
Tcolumn:25C
Tsample:10±5C
Peptide eluted at 4.594 min

Then, I got split peaks. Hence, I performed a thorough cleaning of the UPLC system and column following Acquity restoration guidelines. Split peaks were removed.(To answer you question I tend to strongly believe that the column was damaged)

With the aim of getting a shorter run time than the one of 12 min, I changed MeOH to AcN
To avoid buffer precipitation, mobile phases were changed to:
Mobile phase A: 90% 25mM KH2PO4 (pH 7.4), 10% AcN, 2 mM ammonium acetate, 0.1% f.a
Mobile phase B: 100% AcN, 2 mM ammonium acetate, 0.1% f.a
Tcolumn:40C
I tried a number of isocratic conditions (60%A, 50%A, 30%A) and flow rates from 0.2-0.5mL/min but peptide eluted very early (max retention time of 0.6min at 0.2mL/min)
I tried a number of gradient conditions and flow rates of 0.6mL/min (since increasing flow rate would decrease run time) but peptide eluted similarly very early. Even when I performed the "MeOH gradient" described above (note different mobile phase conditions,Tcolumn and flow rate) peptide eluted at 0.3 min.

I am in a mess really.Any feedback would be appreciated.
Regards,
T