SPPS Troubleshooting Guide
Common Problems and Solutions
TL;DR: Most SPPS failures fall into six categories: incomplete coupling, slow deprotection, cleavage damage, purification loss, HPLC artifacts, and low yield. Each has known root causes and proven corrective actions. The Kaiser test is the frontline diagnostic — positive (blue) after coupling means repeat the cycle. Chain aggregation is the most common hidden culprit for difficult sequences.
Key Takeaways
- Kaiser test positive after coupling → increase activator eq, double couple, or add LiCl.
- Deletion sequences → capping is essential; use fresh piperidine.
- Aspartimide → use 5% HOBt in deprotection or switch to Asp(OMpe).
- Low crude yield → check cleavage scavenger system; extend cleavage time.
- Broad HPLC peaks → reduce column load by 50%; optimize gradient slope.
- Poor peak shape → check column condition and mobile phase pH.
Coupling Problems
| Symptom | Likely Cause | Solution |
| Kaiser test remains positive | Insufficient activator | Increase to 5 eq AA + 5 eq activator |
| Steric hindrance | Double couple, switch to HATU |
| Chain aggregation | Add 0.4 M LiCl in DMF |
| Resin inaccessible | Use lower-loading resin (0.3 mmol/g) |
| Deletion sequences in final | Missed coupling | Capping essential; check Kaiser |
| Racemization | Use DIC/Oxyma, lower temperature |
| Aspartimide | Use Asp(OMpe), 5% HOBt in deprotection |
| Low crude yield | Resin lost during handling | Use PTFE frit vessels |
| Cleavage incomplete | Extend time, increase temperature |
| Precipitation loss | Use more cold ether, centrifuge well |
Deprotection Problems
| Symptom | Likely Cause | Solution |
| Slow deprotection | Degraded piperidine | Use fresh, store under N₂ |
| N-terminal aggregation | Use chaotropic agents |
| Trityl groups present | Separate deprotection step |
| Aspartimide (Asp-Gly) | Base exposure | 5% piperidine + 5% HOBt |
| Asp(OtBu) protecting group | Switch to Asp(OMpe) |
| Racemization at His/Cys | Base sensitivity | Use HATU, reduce DIEA, cool |
Cleavage Problems
| Symptom | Likely Cause | Solution |
| Tert-butylation | Insufficient scavenger | Add TIS; use EDT for Trp |
| TFA adducts | Scavenger system inadequate | Increase TIS/H₂O |
| Residual protecting groups | Cleavage incomplete | Extend time to 3-4 h |
| Peptide precipitation fails | Ether volume too low | 10× volume cold ether |
| Wrong ether | Use methyl tert-butyl ether |
Purification Problems
| Symptom | Likely Cause | Solution |
| Broad peaks | Column overload | Reduce load by 50% |
| Poor gradient | Extend gradient, optimize slope |
| Peptide won't elute | Too hydrophobic | Increase organic, use IPA |
| Aggregation | Add 0.1% TFA, warm column |
| Low recovery | Precipitation on column | Add organic wash step |
| Irreversible binding | Regenerate column, check pH |
HPLC QC Problems
| Symptom | Likely Cause | Solution |
| Multiple peaks | Degradation | Test fresh sample |
| Disulfide scrambling | Reduce/alkylate before testing |
| Impurities expected | Compare with reference |
| Poor peak shape | Column fouled | Regenerate/replace |
| Wrong pH | Optimize mobile phase pH |
🔗 Related: Coupling Reaction | Cleavage | Purification | Difficult Sequences