DERECHOS Y OBLIGACIONES DE LAS EMPRESAS CONSTRUCTORAS EN CUANTO A CONCURSOS

Solubility curves for solutions containing varying proportions of fibronectin and fibrinogen were shown in section 4.3. There were two regions where protein was precipitated out, at both very low pH, less than 1.0, and the pH o f minimum solubility for fibronectin / fibrinogen and their mixtures, between 3.0 and 5.0. At a very low pH this precipitation occurs due at least partially to dénaturation, whilst between pH 3.0 and 5.0 the proteins have no overall charge and tend to aggregate.

The aim o f wet spinning is to precipitate the protein on its extrusion into a coagulation bath. Figure 5.16. demonstrated the effect of combining both pH and salt concentration to precipitate proteins. Strong acids such as HCl or H2SO4 were used to denature the protein whilst weaker acids such as citric acid, acetic acid and glycine were used to create environments at a higher pH. Salts were also used to enhance the precipitation effect. The coagulant should be a non-solvent for the protein and must remove the solvent in which the protein is extruded. In this case the aim was to precipitate the protein and extract the urea from the spinning dope. To test different coagulation baths, a 70 mg/mL solution o f protein was extruded into 20 mL of each coagulation bath as shown in Figure 2.2c.

Hydrochloric acid.

0.5 M hydrochloric acid, pH 0.6 gave good fibre formation but coagulation occurred too quickly and the needle tended to block easily. Using 0.1 M HCl, pH 1.15, or a more dilute solution did not result in the production of stable fibres. Addition of a salt such as 15 % (w/v) sodium sulphate to 0.1 M HCl, pH 1.9, improved the precipitation but the density difference between the solution and fibre caused the fibre to rise to the surface, making continuous coagulation difficult. A similar density difference was also seen with 0.1 M HCl + 15 % tri-sodium citrate, pH 5.9. Here the formed fibres rose to the surface of the coagulation bath and formed a sheet. Extrusion into 0.1 M HCl + 15 % CaCli formed good fibres but a density difference was still observed. Stable fibres could be formed by combining either 0.1 M HCl and at least 2 % CaClz , pH 1.1, or 0.25 M HCl and at least 1 % CaClz, pH 1.0. As the concentration of salt was increased, the precipitated protein tended to coagulate into sheets rather than discrete fibres.

Sulphuric acid.

No stable fibres were formed when protein solution was extruded into 0.25 M sulphuric acid with no salt, pH 1.0. Combinations of > 0.01 M sulphuric acid and 2 % (w/v) sodium sulphate, pH 1.9, were found to form stable fibres. Increasing the acid concentration to 0.5 M with 2 % Na2S04 resulted in needle blocking. Addition of either 1 % or 2 % Na2S04 to 0.05 M sulphuric acid produced solutions with pH 1.5 and 1.6 respectively and led to stable fibre formation. Sulphuric acid and tri-sodium citrate combinations did not produce suitable fibres.

Acetic acid

Acetic acid / sodium chloride combinations were used by Swingler and Lawrie (1977) to spin porcine plasma proteins. Addition o f 10 % NaCl to 5 to 10 % acetic acid gave solutions with pH 2.0 - 1.7 but the fibronectin/fibrinogen fibres formed showed strong self-adhesive properties and were difficult to remove from the coagulation bath.

Citric acid

O.IM citric acid was used in the manufacture of fibronectin / fibrinogen cables (see section 4.4). Extrusion into a O.IM citric acid solution caused precipitation but no stable fibres were formed. Addition of sodium sulphate improved precipitation but the formed precipitate rose to the coagulation bath surface. Poor fibres were formed with the

addition of up to 10 % CaCb to 0.1 M citric acid, pH 1.2. 0.1 - 1.0 M citric acid/ citrate buffers, pH 3.0 were used to create the pH of minimum protein solubility. Although protein was precipitated, it was difficult to remove individual protein fibres from the coagulation bath.

Glvcine.

0.1 M glycine / hydrochloric acid, pH 3.0, also precipitated the protein but resulting fibres were poor.

The most successful coagulation baths tested were those involving strong acids. The acid may enhance the opening up of the molecule initiated by the urea, leading to molecule aggregation and fibre formation. By combining acid and salt, the acid concentration required for precipitation was reduced. The quantitative testing o f a number of different coagulation baths is described in section 5.3.3.

5.3.3. Comparison of coagulation baths

To quantitatively compare a number of the more successful coagulation baths, dope was extruded into a small scale spinning bath and the amount of precipitate formed measured by a dry weight method. Results are shown in 3 bar graphs in Figure 5.17.

The top bar graph represents an investigation into the composition of different coagulation baths. The independent t-test was used to compare the mean amount of protein precipitated over 3 spinning trials for each test bath. Precipitation into 0.25 M HCl, 2 % CaCb was taken as the standard for comparison. Significantly less precipitate was produced on extrusion into water, phosphate-buffered saline and chilled ethanol at neutral pH (p = 0.000, 0.003, 0.005 at the 95 % level o f significance). Water and PBS are o f too low ionic strength and too high pH to precipitate much protein but reducing the pH o f the ethanol may have increased protein precipitation. There was no significant difference between the amount o f precipitate formed on extrusion into the other baths although only the HCl and H2SO4 baths formed stable fibres. A I M citric acid / citrate, 10 % CaCl2, pH 0.8 bath also produced suitable fibres but the amount o f precipitate was difficult to measure accurately due to the high background salt level. Precipitation into 50 % PEG was also difficult to measure quantitatively and the fibres were not as stable as those made in the acid / salt baths.

The middle and lower bar graphs examine extrusion into 0.25 M hydrochloric acid, 2 % CaClz, pH 0.9 and 0.05 M sulphuric acid, 2 % Na2S04, pH 1.8 respectively. Dopes were made up either at a low concentration, 90 mg/mL total protein, with or without a viscosity enhancer or a higher protein concentration, 105 mg/mL, without an enhancer. The enhancers tested were 1 % sodium alginate or 0.5 % sodium Carboxymethylcellulose. Comparison of means used the high protein dope at room temperature as a standard. The amount o f precipitate formed on extrusion into HCl was not significantly different for the dopes tested at room temperature or when the baths were chilled to 4°C. All dopes formed good, stable fibres. Those containing alginate were noticeably less self-adherent than those formed from other dopes. Extrusion into sulphuric acid produced more varied results with significantly less fibre being formed from the low protein dope, p= 0.031 and 0.042 for samples at 23 and 4°C respectively. Extrusion of the alginate dope at 4°C also produced significantly lower results. When the additives sodium alginate and sodium CMC are extruded into the HCl / CaCh bath, calcium ions exchange with the sodium ions to form the insoluble complexes, calcium alginate and calcium CMC. This will not happen in the H2S04/Na2S04 bath.

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Coaeulation bath and temoerature