step (grafting of isopropylamine), because the reactivity of the remaining free carboxylates decreases
drastically as the amount of grafted amines increases.
Standard Procedure for PAA Modification
PAA (5 g, 69 mmol acid) was dissolved in 100 mL NMP over 4 h at 60
C. Octylamine (2.22 g,
17.25 mmol in 15 mL NMP) was added, after which DCI (3.73 g, 18.11 mmol in 15 mL NMP) was
supplemented dropwise. The mixture was allowed to react for 1 h at 60
C, then brought to room
temperature (RT), and kept under stirring for 3 h. After cooling at 0
C, DCU was removed by filtration.
4 g (29.6 mmol) of 1-hydroxybenzotriazole (HOBt) were added to the filtrate and the mixture heated at
50
C. After dropwise addition of isopropylamine (1.63 g, 27.6 mmol in 25 mL NMP), followed by DCI
(6.26 g, 30.33 mmol in 30 mL NMP), the reaction medium was kept at 50
C for 1 h and stirring carried
on for 3 h at RT. The resulting acidic solution of A8-35 was cooled to 0
C and filtered by suction.
At this stage, the A8-35 solution is contaminated by HOBt, residual DCU, and other undesirable
by-products, which must be removed by purification.
4.5.3
Purification of A8-35
The purification procedure relies on the differential solubility in water of the acidic and basic forms
of A8-35. When totally ionized (above pH 7), A8-35 is highly soluble in water (>320 gÁL
À1 ; Gohon
et al. 2006), whereas the protonated form is insoluble. Thus, if the purification is performed in a large
excess of aqueous solution, the hydrophobic contaminants (DCU, essentially) can be removed by
precipitation at high pH and the water-soluble impurities (HOBt and NMP) washed away from the
A8-35 precipitate at pH < 7. Extensive purification is critical and must be carefully carried out.
Because the solution behavior of polymers may depend on their history (see e.g. Selb and Gallot 1980;
Yamamoto et al. 2000a, b), precipitation and dissolution are carried out quickly and at extreme pH
values (0–1 and 10–12 for acidic and basic solutions, respectively). Reaching a satisfying level of
purity requires four cycles of dissolution/precipitation. Salts are removed by dialyzing the final A8-35
solution against slightly basic water.
Standard Procedure for A8-35 Purification
7.5 g (0.138 mol, two equivalents) sodium methanoate are added to the NMP solution which is diluted
in large excess of water (1.5 L). After removal by filtration of residual DCU, the polymer is precipitated
by adding dropwise the basic solution to 100 mL of 3 N HCl. After redissolution of the polymer in
500 mL of 0.3 N NaOH, precipitation is repeated according to the same procedure. The cycle is
repeated two additional times. Finally, the basic solution (300 mL) is dialyzed against 2 L of 10
–3 N
NaOH for 2 days and then against Milli-Q water for 1 h and finally freeze-dried, yielding 7.74 g of
product (90% yield with respect to the mass of A8-35 expected assuming 100% conversion of the
purified PAA precursor; modification of 1 g of pure PAA should yield 1.72 g of A8-35, sodium salt).
4.5.4
Determination of the Chemical Composition of the Final Product
The chemical composition of the final product can be determined by
1 H and
13
C NMR spectroscopy.
The
1 H NMR spectrum can be analyzed in three principal regions: 0.8–1.4 ppm (methyl and
methylene protons of alkyl side groups), 2.6–1.4 ppm (backbone protons), and 2.9–4.1 ppm (methylene and methine protons of the side groups in α position to the amide function) (see Fig. 4.34). Octyl
groups give rise to three peaks centered at 3.15 ppm, 1.3 ppm, and 0.85 ppm, isopropyl groups to peaks
at 3.9–4.0 ppm and 1.1 ppm.
208
4 Chemical Structure, Synthesis, and Physical-Chemical Properties of Amphipols
drastically as the amount of grafted amines increases.
Standard Procedure for PAA Modification
PAA (5 g, 69 mmol acid) was dissolved in 100 mL NMP over 4 h at 60
C. Octylamine (2.22 g,
17.25 mmol in 15 mL NMP) was added, after which DCI (3.73 g, 18.11 mmol in 15 mL NMP) was
supplemented dropwise. The mixture was allowed to react for 1 h at 60
C, then brought to room
temperature (RT), and kept under stirring for 3 h. After cooling at 0
C, DCU was removed by filtration.
4 g (29.6 mmol) of 1-hydroxybenzotriazole (HOBt) were added to the filtrate and the mixture heated at
50
C. After dropwise addition of isopropylamine (1.63 g, 27.6 mmol in 25 mL NMP), followed by DCI
(6.26 g, 30.33 mmol in 30 mL NMP), the reaction medium was kept at 50
C for 1 h and stirring carried
on for 3 h at RT. The resulting acidic solution of A8-35 was cooled to 0
C and filtered by suction.
At this stage, the A8-35 solution is contaminated by HOBt, residual DCU, and other undesirable
by-products, which must be removed by purification.
4.5.3
Purification of A8-35
The purification procedure relies on the differential solubility in water of the acidic and basic forms
of A8-35. When totally ionized (above pH 7), A8-35 is highly soluble in water (>320 gÁL
À1 ; Gohon
et al. 2006), whereas the protonated form is insoluble. Thus, if the purification is performed in a large
excess of aqueous solution, the hydrophobic contaminants (DCU, essentially) can be removed by
precipitation at high pH and the water-soluble impurities (HOBt and NMP) washed away from the
A8-35 precipitate at pH < 7. Extensive purification is critical and must be carefully carried out.
Because the solution behavior of polymers may depend on their history (see e.g. Selb and Gallot 1980;
Yamamoto et al. 2000a, b), precipitation and dissolution are carried out quickly and at extreme pH
values (0–1 and 10–12 for acidic and basic solutions, respectively). Reaching a satisfying level of
purity requires four cycles of dissolution/precipitation. Salts are removed by dialyzing the final A8-35
solution against slightly basic water.
Standard Procedure for A8-35 Purification
7.5 g (0.138 mol, two equivalents) sodium methanoate are added to the NMP solution which is diluted
in large excess of water (1.5 L). After removal by filtration of residual DCU, the polymer is precipitated
by adding dropwise the basic solution to 100 mL of 3 N HCl. After redissolution of the polymer in
500 mL of 0.3 N NaOH, precipitation is repeated according to the same procedure. The cycle is
repeated two additional times. Finally, the basic solution (300 mL) is dialyzed against 2 L of 10
–3 N
NaOH for 2 days and then against Milli-Q water for 1 h and finally freeze-dried, yielding 7.74 g of
product (90% yield with respect to the mass of A8-35 expected assuming 100% conversion of the
purified PAA precursor; modification of 1 g of pure PAA should yield 1.72 g of A8-35, sodium salt).
4.5.4
Determination of the Chemical Composition of the Final Product
The chemical composition of the final product can be determined by
1 H and
13
C NMR spectroscopy.
The
1 H NMR spectrum can be analyzed in three principal regions: 0.8–1.4 ppm (methyl and
methylene protons of alkyl side groups), 2.6–1.4 ppm (backbone protons), and 2.9–4.1 ppm (methylene and methine protons of the side groups in α position to the amide function) (see Fig. 4.34). Octyl
groups give rise to three peaks centered at 3.15 ppm, 1.3 ppm, and 0.85 ppm, isopropyl groups to peaks
at 3.9–4.0 ppm and 1.1 ppm.
208
4 Chemical Structure, Synthesis, and Physical-Chemical Properties of Amphipols