Colorimetric

[ Home ] [ Group Meeting ] [ Meeting Archive ] [ Protocols ] [ Order whiteboard ]

TNBS Test - All Primary Amines

Solution 1: 10% DI(P)EA in DMF (store at 25^o C)
Solution 2: 1 M aqueous TNBS (store at 4^o C)

Procedure:
- Place a few resin beads in a small test tube.
- Add 1-3 drops of each solution
- Mix, and leave at room temperature for 10 minutes

Free Amine (positive)	Ambiguous/Partial Positive	Negative
Intense orange/red beads	Yellow/light orange beads/bead core	Colourless beads

Kaiser Test - All Primary Amines

WARNING: Ambiguous with Wang resins
Solution 1: 5 g ninhydrin in 100 mL ethanol (store at 25^o C, in dark)
Solution 2: 80 g phenol in 20 mL ethanol (store at 25^o C, in dark)
Solution 3: 2 mL 0.001 M KCN in 98 mL pyridine (store at 25^o C, in dark)

Procedure:
- Place a few resin beads in a small test tube.
- Wash resin with ethanol 2-3 times to remove adsorbed DMF
- Add 2-5 drops of each solution
- Mix, heat to 100^o C for 5 minutes

Free Amine (positive)	Ambiguous/Partial Positive	Negative
Variable intensity blue solution/resin	Blue bead cores	Colourless/light yellow beads

Chloranil Test - All Primary Amines (acetaldehyde) - Secondary amines (acetone)

WARNING: Keep solution for a maximum of one month
Solution 1: 2% acetaldehyde (primary amines) or 2% acetone (secondary amines) in DMF (store at 4^o C)
Solution 2: 2% chloranil in DMF (store at 4^o C)

ALTERNATIVE SOLUTIONS
Solution 1: Saturated solution of chloranil in toluene (store at 4^o C)
Solution 2: Only necessary for primary amines. 2% acetone in DMF (store at 4^o C)

Procedure:
- Place a few resin beads in a small test tube.
- Add 2-5 drops of each solution
- Mix, leave at room temperature for 5 minutes

Free Amine (positive)	Ambiguous/Partial Positive	Negative
Dark blue/green beads	Blue/green bead cores	Colourless/light yellow beads

Bromophenol Blue Test - Primary and Secondary amines

WARNING: Ensure resin is well washed with DMF before performing this test
Solution 1: 0.05% bromophenol blue in DMA (store at 25^o C)

Procedure:
- Place a few resin beads in a small test tube.
- Add 10-20 drops of solution
- Inspect immediately

Free Amine (positive)	Ambiguous/Partial Positive	Negative
Blue beads	---	Colourless beads

Isatin Test - Secondary amines

Solution 1: Saturated isatin in benzyl alcohol with BOC-Phe-OH
Preparation:
- Saturated isatin (~0.4 g) in benzyl alcohol (~12 mL)
- Stir for ~2 hours at room temperature, filter remaining isatin
- Mix 10 mL of filtered solution with 0.5 g BOC-Phe-OH

Procedure:
- Place a few resin beads in a small test tube.
- Add 3-4 drops of solution
- Heat to 100^o C for a 1.5 to 2 minutes (MAXIMUM)

Free Amine (positive)	Ambiguous/Partial Positive	Negative
Blue beads	---	Colourless beads