Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Determination of protein bound to a solid support
Determination of amino groups on a solid support

8.21.3.1. Picrate method.

Reagents.

0.1 M picric acid in methylene chloride.

N,N-Diisopropylethylamine, distilled over CaH₂.

Procedure. Treat the resin (~50 mg) on a Glass funnel (1 cm internal diameter) twice with 2 mL of picric acid solution, and then wash with 40 mL of pure methylene chloride. Add N,N-diisopropylethylamine (2 mL), followed by methanol (2 mL) and another portion of amine (2 mL), removing the liquid each time by suction. Combine the wash fractions and dilute to 250 mL with 95% ethanol. Measure the absorbance at 358 nm. For the amine-picrate complex, ε = 14500.

8.21.2.2. Titration method.

Reagents.

1 M HCl.

10% triethylamine in dimethylformamide (DMF).

Mercury(II) nitrate solution, standardized against KCl.

Bromophenol blue, 1% (w/v) solution in ethanol.

Diphenylcarbazone, 1% (w/v) solution in ethanol.

Procedure. Wash the resin (~50 mg) with 20 mL of 1 M HCl, followed by 40 mL of deionized Water and 5 mL of methanol.

Sequentially wash with 4 mL of 10% triethylamine in DMF, 2 mL of DMF, and 4 mL of methanol. Dilute the combined washings with 20 mL of water and 80 mL of ethanol. Add 5 drops of bromophenol blue and acidify the mixture until a yellow color appears by carefully adding dilute (0.5 M) nitric acid. Add 5 drops of 1% diphenylcarbazone (indicator) and titrate with standard mercury(II) nitrate solution until a pink color appears, indicating an excess of mercury ions.

Note. According to reference [214], these two Procedures were found to be the most convenient (out of 4 Methods studied) for determining amino groups on controlled-pore glass beads.

Determination of Amino groups using Schiff base formation with salicylaldehyde followed by absorbance measurement at 315 nm [62] yields better results than using 2-hydroxy-1-naphthaldehyde [331].