Practical Protein Chemistry - A. Darbre 1989

Methods of Solid-Phase Amino Acid Sequence Analysis
Resin Synthesis
Determination of Amino Group Content in Supports Based on Organic Polymers or Porous Glasses

Determining the number of amino groups makes it possible to evaluate changes in support properties during storage and to compare the characteristics of different batches of the same support. See also Section 8.2.1.

12.3.3.1. Procedures.

Procedure 1 [33]. The amino groups of the support are alkylated with trinitrobenzenesulfonic acid. Hydrolysis with potassium hydroxide cleaves the Si—O bonds of the Glass and converts the trinitrophenyl groups into picric acid, the concentration of which can be measured photometrically. The reproducibility of the method is within a few percent.

Mix 50 mg of 3-aminopropyl glass and 15 mg of trinitrobenzenesulfonic acid in 1 ml of 0.1 M NaBO₂ (pH 9.07) in a small test tube. Stir for 1.5 h by rotating the tube in a horizontal plane. Wash with Water (3 times with 8 ml), centrifuge. Add 1 ml of 2 M KOH to the wet support and stir for 60 min at 45 °C. Dilute with water and measure the absorbance at 358 nm. Calculate the amino group content, taking into account that picric acid has ε₃₅₈ = 14 100 in an alkaline medium. For aminopropyl glass, the amino group content is typically 25–150 µmol/g of support [29].

Procedure 2 [18]. The swollen polymer reacts with picric acid, forming a salt with the amino groups of the support. The polymer is treated with an excess of a strong base to release the bound picrate into solution, where its concentration is then measured.

Soak 50 mg of the support in 1,2-dichloroethane for 5 min. Neutralize with a 5% (v/v) solution of diisopropylethylamine in dichloromethane for 3 min. Treat with 0.1 M picric acid in dichloromethane (3 min). Wash the support with dichloromethane (5 times with 2 ml). Elute the picrate with 5% diisopropylethylamine in dichloromethane (2 portions of 3 ml each), and wash the resin with dichloromethane (3 times with 2 ml). Dilute the solution with 95% ethanol and measure the absorbance. For diisopropylethylamine picrate in the concentration range of 1:20∙10^-5 mol/L (in ethanol containing ≤20% dichloromethane), ε₃₅₈ = 14 500.

Procedure 3 [56]. The amino groups of the support are converted into a bound Schiff base by reaction with 2-hydroxy-1-naphthaldehyde, which is subsequently released into solution by Treatment with benzylamine. The concentration of the Schiff base in the solution is determined spectrophotometrically.

Mix 1–5 mg of the support with 2 ml of 0.2 M 2-hydroxy-1-naphthaldehyde in DMF and leave for 15 h at room Temperature. Wash the resin with DMF (4 times with 3 ml), then with ethanol until absorption at 270 nm is completely absent in the washings. Add 2 ml of 0.4 M benzylamine in ethanol and incubate for 45 min at room temperature. Centrifuge and measure the absorbance at 420 nm. For the Schiff base, ε₄₂₃ = 10 900 (in ethanol). For solid-phase Supports, the binding capacity is determined by the number of amino groups per unit mass of the support that are accessible for attachment.