Thank you for the details! You synthesized a mixture of bis-substituted and trisubstituted amides from tri-mesic acid and dopamine hydrochloride in a 1:3 molar ratio. Separation of these two closely related molecules—differing only by one dopamine unit—can be challenging, but here are some practical and scalable strategies for their separation:

🔬 1. Preparative Column Chromatography Best for lab-scale purification

Stationary phase: Silica gel

Eluent: Gradient of methanol/chloroform or methanol/dichloromethane (DCM)

Start with low polarity and gradually increase methanol content.

The trisubstituted product is usually more polar than the disubstituted one (due to an extra amide + catechol group), and elutes after the bis-substituted compound.

💡 Tip: Add a small % of triethylamine to the eluent to prevent streaking of polar compounds with free –OH or –NH groups.

⚗️ 2. Reversed-Phase HPLC (RP-HPLC) Best for analytical and small-scale purification

Column: C18 reversed-phase

Mobile phase: Water (with 0.1% TFA or formic acid) and acetonitrile or methanol gradient.

The difference in hydrophobicity and polarity between di- and tri-substituted compounds often gives distinct retention times.

🧪 Ideal if you're also characterizing purity via UV or MS.

🧊 3. Recrystallization Only if solubility differences are significant

If the two compounds have significantly different solubilities in a given solvent system (e.g., EtOAc/hexane, ethanol/water, acetone/water), you might selectively crystallize one.

Often, less substituted compounds (bis-amide) have better crystallinity, so the tri-substituted compound may remain in solution.

⚛️ 4. Ion Exchange Chromatography

Because you're working with dopamine hydrochloride (protonated amine groups), the products may retain cationic charges or zwitterionic character depending on pH.

Use cation exchange resin at acidic pH (~4–5).

Elute stepwise with increasing salt concentration or pH shift.

This method is less common for neutral amides, but may help if catechol groups influence charge.

🧪 5. Derivatization (Selective Protection/Tagging)

This is less direct, but:

You can chemically tag one compound (e.g., via selective catechol oxidation or diazonium coupling) to create a large polarity difference and then separate.

Then, deprotect or reduce to get back the pure compound.

Use only if other methods fail.

📌 Summary of Recommendations Method Scale Notes Column Chromatography Lab-scale Most practical starting point. Use polarity difference to your advantage. RP-HPLC Small-scale Very effective; good for analytical confirmation. Recrystallization Gram-scale Try if solubility differences exist. Ion Exchange Chromatography Optional Depends on charge state; less commonly used for amides. Derivatization + Separation Complex Only if other methods fail.

Thank you so much Hezam Dhaifallah for the details. I cordially appreciate your suggestions.

I tried the recrystallization process with MeOH-CHCl3 but product did not separated.

Also, I'm struggling to fix the eluent ratio (MeOH-Hexane and MeOH-EtOAc) for Column chromatography, Because in TLC I couldn't separated the spots for both products. Its always showing one tailing spot due to their closer polarity.

I'll try your suggestions.

Thanks once again!