Oligonucleotide RP-HPLC Peak

[HPLC_Help]

Hello.

I am running an oligos HPLC method as follows:
C18 column. Temp = 60C.
TEA-A/Acetonitrile gradient, pH=8.


While running some formulation/stability studies on our oligo, I noticed that with heating (~90C) and base (0.1 to 1 N NaOH) a shoulder started to form on the main peak that I assumed was a degradation peak. (Sample in water @ 90C showed small shoulder. 0.1 N @ 90C showed small shoulder. 1N @ 90C showed huge [14%] shoulder. 0.1N @ RT & 40C showed no shoulder. 1N @ RT & 40C showed small shoulder.)

However, the main peak area does not decrease in size as this peak increases. I get ~100% recovery of my main peak, along with a 1-3 % (of total peak area) shoulder.

This doesn't seem like it would be a degradation peak, since the main peak remains the same size. What else might it be?

Any feedback/discussion/etc. welcome.

HH

[krickos]

This doesn't seem like it would be a degradation peak, since the main peak remains the same size. What else might it be?
HH

Hi

Do not know much about the analyte you analyse but I would be very careful considering your data to rule out degradation products. You can not be sure (with the information provided) that an degradation product has the same response factor as your main peak. As the peaks overlap an overlapping degradation product with higer response factor than the main peak, may cause a "false" 100% recovery.

[danko]

What is the detection mode?

Best Regards

[grzesiek]

"This doesn't seem like it would be a degradation peak, since the main peak remains the same size." - what is the recovery of the main peak when the shoulder is 14% (1N @ 90C)?

"As the peaks overlap an overlapping degradation product with higer response factor than the main peak, may cause a "false" 100% recovery." - following krickos comment - you can try orthogonal separation in this case