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The following steps are for troubleshooting the problem:
1. **Monitor the reaction process**: Confirm whether the new spot is the target amide using TLC/LC-MS. The possible by-products could be acylurea (self-condensation of EDCI) or unreacted acid.
2. **Purification optimization**: Try column chromatography (gradient of EA/MeOH) or acid-base extraction (wash away the unreacted amine with 1M HCl, and wash the acid with NaHCO₃).
3. **Nuclear magnetic resonance analysis**: Compare with the literature data to check if the abnormal peak splitting is caused by conformational isomers (such as rotationally blocked N-methylpiperazine).
4. **Condition adjustment**:
- Replace EDCI with TBTU/HATU to reduce side reactions;
- Reduce the amount of DIPEA (1.2 equivalents) to avoid excessive alkalization and racemization.
**Common Questions**: EDCI residual interference causes nuclear magnetic resonance to be affected. It can be removed by washing with ammonia water after passing through a silica gel column.
