Dear Himanshu,

if you have filled up the sample cell carefully, i.e. without bubbles, cleanness, etc. the -30 DP value that you observe should go down to around 5 (the reference power that you selected) but you need to wait a little bit for this.

the initial -30 value usually correspond to the power needed to compensate the heat released upon stirring of the syringe. Once the instrument has reset up this value, the signal should go back to a value close to the reference power, 5 in your case.

Hope that helps,

Best Wishes

Andrés

https://embl.fr/services/macromolecular_interactions/index.html

Are you sure that you have the same exact buffer conditions in the solutions in the sample cell and the injection syringe?

Significant mismatches in salt concentriaton, pH, or buffer cosolvents could cause these effects.

Before you start the experiment does the DP equilibrate to 5 +/- 0.5? If not make sure that your sample cell and reference cell are both full with solution.

Dear Andreas,

Thank you.We are trying to take the utmost care in preparing and injecting the sample so that it dos not have any bubbles but i can try again as you suggest.But the DP value does not come near to 5 even after long wait even when the injection starts.could any instrumental error account for such a behavior

Best,

Himanshu.

Dear Christopher

Thanks you.Yes we are using the same buffer composition for the ligand and the protein.We have even tried the run for water-water titration and gave the same error.

Best,

Himanshu.

YI don't know the setup of the ITC200 but you could check if the syringe is bent... We faced a similar problem last year qith VP-ITC

Hi Maria,

Thanks for the Suggestion.I too have gone through the literature and forums that suggests the same reason as yours(the bent syringe).

But,the syringe does not show any obvious bent as off now and we just got the machine installed,so i hope the error is not due to a bent syringe.

Hi Himanshu, 

Did you check the reference cell??? whether it is completely filled or not??

try to change the solution (buffer or water) in the reference cell. 

How long does the signal stay at -30 level?

Anyway, a bent syringe will affect the equilibration and baseline level, not only the heat effect of a given injection. Partial filling of the reference cell will also affect the baseline equilibration (in fact, if the volume of liquid is very low, no equilibration at all will be achieved).

Buffer mismatch looks like the more probable cause. Even if you use the same buffer for both syringe and cell solutions, if the ligand is a low molecular weight compound and you just dissolve it, it could contain some salts or additives. You can try to perform the reverse titration; that way, dilution in the cell will be less important.

If you have taken all precautions (syringe is not bent, reference cell is filled, buffer solutions are matched,...), and considering the instrument has been installed recently, you may contact MicroCal Technical Service.

Just wondered if you ever found the answer to this problem - our machine has exactly this problem even when we just have water in the cell and syringe.

Alice,

Hi..Yes..we did overcame the problem but in a trivial manner.The error seemed to come from improper insertion of the syringe into the sample cell.As the syringe was not fully inserted into the cell it stirring was not proper and may have given rise to the erroneous DP value as soon as we started the run.Though the misunderstanding arose due to a half baked communication from the engineers side, we were able to identify an correct it.

I suggest that you may also look for a "click" sound(which indicated proper insertion) when you insert the syringe into the sample cell(the intensity of this sound may vary from machine to machine) and if you already have the click sound the problem may be originating from some other factor.Do update what you observe.

We have replaced the syringe which seems to have fixed the problem. An expert user came and checked out the machine and said the syringe was bent.