Extra peak in SEC

[Kurt Void]

Hi,

has anyone experienced an extra peak in SEC at approximately the exclusion volume? We use a TSKgel G3000SWXL column and a Tris/NaCl buffer pH 7.4. Since some time we often see the extra peak when pure water is injected.

* some individual columns are worse than others (i.e. give bigger extra peak)
* the peak is absent if a run without injection (no injection valve switch) is done, but present if a 0 µl injection is made (the valve is switched)
* we suspected air and when air is injected deliberately we get a peak for sure, but at an even earlier retention time

Comments or suggestions from experienced people out there would be highly appreciated

[mbicking]

What detector are you using? RI, absorbance?

Injecting water into a mobile phase containing salts and buffers will always generate an extra peak with RI, and sometimes absorbance, depending on the settings. When you make the injection, the pure water disturbs the equilibrium between the stationary and mobile phase, and this disturbance proceeds through the column. But I usually see these problems at the permeation limit, not the exclusion limit.

Are you using the same source of water for injection and mobile phase?

[Kurt Void]

Hi,

thanks for your input.

The detector is UV, 210 nm, The water injected comes from the same source as used for the mobile phase. We have also tried water from different sources, but water quality does not seem to be the problem.

[danko]

Hi Kurt

How much air did you inject?
Did you try different volumes (e.g. 1, 2, 4, 8 μL)?
You’re dealing with the size exclusion technique as the only separation mechanism, so different bubble sizes should be retained differently. Actually it’s not that simple as it sounds, because the air bubble will always be dispersed into smaller bubbles, when penetrating the column frit and that complicates the things, but it’s worth trying.

Best Regards

[HW Mueller]

Since this peak is obtained by simply actuating ths injection valve, H2O seems not to play a role here. Could it be just the pressure shock? If that´s the case then you should be able to repeat it everytime you go through a zero injection. If you had the unlikely possibility of carryover of a very high molecular weight substance it would diminish with the number of injector actuations. Also, running a spec on the peak might help to identify it.
If nothing is wrong with your injector and you didn´t drain it , you would have a hard time getting air into your system by just switching....

[DNN]

Hi,

has anyone experienced an extra peak in SEC at approximately the exclusion volume? We use a TSKgel G3000SWXL column and a Tris/NaCl buffer pH 7.4. Since some time we often see the extra peak when pure water is injected.

* some individual columns are worse than others (i.e. give bigger extra peak)
* the peak is absent if a run without injection (no injection valve switch) is done, but present if a 0 µl injection is made (the valve is switched)
* we suspected air and when air is injected deliberately we get a peak for sure, but at an even earlier retention time

Comments or suggestions from experienced people out there would be highly appreciated

Hi,
I have been experiencing this extra peak with TSKgel Protein A 5-PW column, the user before me, who was the first user of the brand new column, also faced this issue right at the beginning. We have been using Phosphate buffer pH 7.4 and pH 2.1, detector is UV at 280 nm. In our case, we see the extra peek with random intensities after ~2 mins of the mAb peak. The peak is absent if nothing is injected.

Have you been able to figure out what it is?