Size exclusion chromatography (SEC) is commonly used for the separation and purification of conjugated biomolecules. However, SEC may not be the best choice for separating unbound azides in your SPAAC reaction as azides are typically small molecules that may elute in the void volume and co-elute with other small molecules, making it difficult to resolve them from the product of interest.

A better approach might be to use reverse-phase high-performance liquid chromatography (RP-HPLC) with a polar buffer to separate unreacted azides from the product. An example buffer that can be used for this purpose is 0.1% trifluoroacetic acid (TFA) in water. The RP-HPLC program can be optimized based on the properties of the oligonucleotides and the DBCO-modified quantum dot.

Here is an example RP-HPLC program:

- Column: C18 (particle size 3-5 µm)

- Mobile phase: 0.1% TFA in water (A) and acetonitrile (B)

- Gradient: 5% B to 95% B over 20 minutes

- Flow rate: 0.5 mL/min

- Detection: UV at 254 nm

This program can be used to separate unreacted azides from the conjugated product, and the peak corresponding to the product can be collected and analyzed further.

Note that it is important to validate the HPLC program for your specific system, as the properties of the biomolecules and the click chemistry reaction may affect the separation.

Thank you Lamia Fatima , I will try this and give you feedback.