If your are missing the ~8 ppm peak in 1H NMR the bond never formed. Your reaction did not work. How did you follow the reaction progress? Was it by TLC, withdraw of samples for NMR, or other method. What deuterated solvent are you using to do the NMR?

Thank you for your reply. Heparin was dissolved in double distilled H2O and adjusted the pH around 5.5 . Later added edc dissolved in water and added to the heparin and the reaction continued under ice bath for 10 min.  Later add NHS in room temperature and continue the reaction for 20 min. Finally add my sample and the reaction was carried out for 12 hrs and later dialyzed for 24 hours under DD H2O. Finally I freeze dry my sample and go for NMR with a concentration of 10 mg/mL with D2O as a solvent 

Is your sample the amine that you are coupling to the heparin? You need to use a primary amine for the reaction to work. Water does convert the intermediate compound back to heparin with no activation. Can you run the reaction in an NMR tube using D2O? You would have to do it on a small scale but it might give some insight to way the reaction is not working. Look for the formation of a peak at ~8 ppm. This method has worked for me in the past. You can also use 13C NMR to follow the reaction and look for a shift in the peak at 170-180 ppm. The peak should move to 170 ppm if the reaction is working. You can also do an IR of the sample and see if there are peaks at 1600, 3500, and 3100 cm-1 identifying a amide bond. Try the IR on your current sample and see if there are any amides present, I would think not but just a check. Good Luck.

Dear Toporek,

Thank You very much. I will do the reaction in NMR tube and see if the results are positive. By the way the IR results are positive but couldn't find the peak in NMR which was a strange issue in my case.

The activation pH and the pH used for the final NHS-C(O) to primary amine should be quite different for optimal coupling. Something like 5.5 for activation, and around 8 for the coupling step. I forget the numbers exactly, but check out the Pierce web site.

Dear Volk,

Thank you very much for the information. As you mentioned pH around 5-6 is required for EDC activation and pH 7 is required for NHS activation. However the problem was with the solvent used for NMR because once changed from D2O to DMSO d6, the peaks were visible.

Naturally, most amide protons exchange with water so D2O solvent was a poor choice. You should be able to see them in 5-10% D2O easily enough though.

Interesting discussion!! I agree with David, you won't see the amide signal in D2O. Sadly, you cannot do the NMR is DMSO or other organic solvent as Heparin is insoluble. If you have done extensive modification of the polymer, then it will be soluble in DMSO, but this will compromise your biology. If you are trying to do amine modification, you  can easily measured by TNBS assay. Are you using unfractionated heparin or Low mol wt heparin?

 I have used Low mol wt heparin. Can a blend of both DMSO and D2O solve the problem? TNBS assay may be ruled out since, Heparin was not aminated and the unconjugated molecules were removed by dialysis