COLLOID TITRATIONS OF POLYETHYLENEIMINE 1 TH E STANDARDISATION OF POTASSIUM

POLYVINYLSULPHATE (PVSK).

The degree of estérification of PVSK must be determined before use^ since it is rarely 100% esterified. The indicator used was toluidine blue, a cationic dye, that binds excess PVSK and becomes red. The colour change due to metachromacy is sharp, hence allowing accurate endpoint determination. Absorbance scans showed that the absorbance maximum for toluidine blue was at 605 nm (data not included). However it was reported (Ueno & Kina, 1985) that greater sensitivity could be achieved at 420 nm (data not included). The stoichiometry of the reaction requires that one mole of PVSK reacts with one mole of cetylpyridinium chloride (CPC). From the titration it was found that the degree of estérification of the PVSK was 65%. Due to the poor degree of estérification, a second commercial PVSK sample (Aldrich) was tested; however this was found to be only 37% esterified. Ueno and Kina (1985) reported that their commercial grades of PVSK were 90 - 95% esterified. The samples used here contained some insolubles which possibly caused this reduction in the degree of estérification. Appendix 1, figures A.1.1.1/2 show the standardisation of the two commercial sources of PVSK, the second sample (Aldrich) shows a broad absorbance peak around the endpoint, whereas the first sample (Sigma) exhibits a sharp endpoint. The volume of PVSK added to the cetylpyridinium chloride in the case of the Aldrich PVSK is approximately twice that of the Sigma sample. The endpoint data along with the shape of the titration curves indicates that considerable impurities are present in the Aldrich sample.

3.1.3.2 COLLOID TITRATIONS.

Colloid titrations are a volumetric method for the determination of charged poly electrolytes in aqueous solutions (Ueno and Kina, 1985) and they have been used for the determination of concentration and charge density of organic polyelectrolytes (Horn, 1978). Colloid titrations involve the reaction between oppositely charged polyelectrolytes which lead to the formation of complexes. With flexible chain polymers this reaction has been shown to occur stoichiometrically (Tereyama, 1952). With PEI, the stoichiometry of the reaction with a polyanion (eg. potassium

polyvinylsulphate) will depend on the degree of protonation {ie> the pH). The PVSK was calibrated using cetylpyridinium chloride, hence its equivalent weight and concentration were known thus enabling the volumetric determination of PEI. Little information is available concerning the molecular weight dependence of this reaction using PEI (Horn, 1980). Colloid titrations have previously been employed as a method of assaying for PEI in aqueous solutions (to 50 ppb; Horn, 1978); they have also been used to determine the charge density of various other polyelectrolytes {eg\-

Tiravanti et al., 1985).

The degree of interaction of the PEI with PVSK is dependent on the degree of protonation of the PEI, and hence the pH of the solution; therefore the quantity of PVSK used will determine the degree of protonation of the PEI. Figure 3.1.3 shows the results of the colloid titration of unfractionated * Polymin P ’ PEI (see Appendix 1). The charge density of PEI, determined from the Faraday constant, is approximately a function of pH. The graph can be divided into three regions; from pH 11 to 9 the increase in charge density is rapid. This is due to the protonation of the primary amine groups on the exterior of the molecule. The region from pH 9 to 4 corresponds to the protonation of the secondary amine groups on the polymer backbone. From pH 4 to 2 protonation of the nearest-neighbour amines on the backbone occurs. For ’Polymin P’ PEI no region was observed where the charge density did not change with pH, this indicates that protonation occurs across the pH range and complete protonation of the polymer was probably not achieved.

It has been reported that for aqueous solutions of polymer, colloid titration is an accurate method for concentration determination (Horn, 1978). However for solutions which contain many charged species (eg. bacterial homogenates), this technique becomes invalid due to the non-selectivity of the counter ion (PVSK). PVSK has successfully been applied to pure protein solutions to determine their charge density (Horn et a l, 1983). The determination of residual PEI in bacterial homogenates would not be possible by this method, since varying the PEI concentration would vary the amount of residual charged molecules (protein, nucleic acids etc.), hence any correction for the interference of the homogenate would not be valid.

The colloid titration of the Polysciences PEI (0.6 kDa, figure 3.1.4) showed three clearly defined regions across the pH range; from pH 11 to 8.5, the increase in charge density is rapid due to protonation of the primary amines. From pH 8.5 to 4 protonation of the secondary (and tertiary) amines occurs; whilst from pH 4 to 2 the increase in charge density is negligible indicating that complete protonation of the polymer had occurred. The Polysciences PEI had a molecular weight range (fig. 3.1.6) of 600 to 1000; this would give the number of ethyleneimine units as approximately 14 to 24 (the molecular weight of one unit being 43). Hence there would be five to seven primary (and tertiary) amines and twice that number of secondary amines per polymer molecule given the ratio of 1:2:1 (primary:secondary:tertiary amines). The relatively small size of the molecule would imply that charge interactions along the polymer backbone would not be as pronounced as for the higher molecular weight PEI, thus giving a more clearly defined titration curve (compare figs. 3.1.3 & 3.1.4), and complete protonation by pH 4. On a weight basis the charge density of the lower molecular weight PEI is nearly four times that of the high molecular weight sample, however, no meaningful comparison can be made on a molar basis due to the polydispersity of the PEI samples. However if this were possible, the charge density of the higher molecular weight polymer would effectively be lower due to charge shielding within the molecule giving rise to incomplete protonation.