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Fluoresceinamine (Isomer I) was purchased from Sigma, UK. This has been used previously as a fluorescent label for covalent binding to sodium alginate using a method described by Blonk et al (1995). The principle of this method involves attaching the amine group of the dye with a small fraction of the carboxylic acid groups present in the alginic acid chain. Dioxane was present to help solubilise the dye. l-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDO) acted as a dehydrating catalyst to remove water and thus enable the amide bond to form. The procedure and mechanisms involved in the labelling process are outlined below. Table 2.2 lists the chemicals used in the procedure.

Chapter Two: Materials and Methods 74

Table 2,2. The chemicals used in the fluorescent labelling of sodium alginate with fluoresceinamine

Chemical Supplier Batch number

Sodium alginate (LF120) FMC, Biopolymer 499231

Fluoresceinamine (isomer I) Acros A010677901

EDC Lancaster 10025464

Dioxane BDH J429314

Sulfuric acid M&B ML0511

Acetone Lab-Scan 4272/9

Diphosphoropentoxide BDH ZU266178

Sodium hydroxide BDH B301548

2.2.1.1 Covalent labelling procedure

5 g of sodium alginate (LF120) was dissolved in 400 ml deionised water. Concentrated sulfuric acid was added drop-wise in order to lower the pH and thus precipitate the alginic acid in its gel form. The system was centrifuged (Beckman J2- 21 instrument with a JA14 rotor) twice with water and twice with acetone at 15000 rpm for 30 minutes each time. This was necessary to separate the alginic acid pellet from the soluble ions and other impurities that may have been present within the system. The pellet was removed and dried in a dessicator above diphos- phoropentoxide at room temperature. The alginic acid pellet produced was then crushed and ground using a pestle and mortar. It was subsequently added to 50 ml of deionised water. This was stirred for at least 10 minutes in order to solubilise as much of the alginic acid as possible. 1 g EDC was then added in order to begin the initial part of the dehydration reaction. 50 mg fluoresceinamine was dissolved in 30 ml 1,4-dioxane. This second solution was added slowly to the alginate/EDC solution whilst stirring. Dissolving the dye in the dioxane aided the dispersity of the fluorescent label producing a homogenously labelled alginate solution. This solution was stirred overnight. Due to the length of this procedure, the beaker containing the solution was wrapped in foil to prevent light degradation of the fluorescent

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component. The reaction solution was then neutralised slowly using concentrated sodium hydroxide solution and the pH monitored throughout this process (Hanna Instruments 8520 pH meter; calibrated at both pH 4 and at pH 7). The amide bond is very susceptible to alkali hydrolysis thus it is better to remain on the acidic side of neutral to produce a stable product. This pH7, neutral solution was then exhaustively dialysed against deionised water to remove any free dye or other small molecular weight impurities. Once the dialysis was complete the labelled alginate solution was freeze dried (Edwards Micro Modulyo with an Edwards RV3 pump) and stored in the same manner as ordinary sodium alginate. The mechanism o f this reaction is

shown inEigure 2.17 ~ OH ^OH Alginate I + EDC R'— N = C = N —R HO O. Q _{O—C = N - R} H/• -R . Intermediate +Fluoresceinamine HO O 9 o "N COOH Labelled alginate

Figure 2,L The mechanism involved in the preparation of fluorescently labelled alginate chain

Chapter Two: Materials and Methods 76

The resulting compound was an alginate chain with fluorescent moieties distributed along the molecule. An estimation of the percentage substitution can be calculated assuming 100 % substitution. The molar ratio of the dye to the alginic acid monomer was equal to 5.5 x 10'^. This value can thus be assumed to be the percentage substitution of the dye to the alginate. The excitation and emission absorption frequencies of an aqueous solution were found to be 489 and 515 nm respectively. The viscosity of a 2 % solution of the labelled alginate was found to be 0.75 (± 0.03) Pa s at a shear rate of 10 s'^ and 37 ®C as compared to 0.51 (± 0.02) Pa s for the unlabelled 2 % LF120. This higher viscosity suggests that very little degradation of the alginate chain occurred during the labelling process and that the process may even aid the formation of a strong polymeric network.

Gel permeation chromatography was performed to assess the purity of the resulting labelled alginate. This method ascertains the size distribution of entities present within a solution and separates them according to molecular weight. A gel filtration chromatography column contains a stationary phase consisting of porous beads with a well-defined range of pore sizes. Molecules that are small with respect to pore size can fit inside the pores within the beads have access to the internal mobile phase as well as the external mobile phase between beads and therefore, elute last in a gel filtration separation. Molecules that are large with respect to pore size are excluded and therefore elute first. As the molecular weight of the dye is very low compared to the labelled alginate chain, these two entities should be separated and will be seen as two peaks using gel filtration chromatography. This example could be visualised within the column due to the fluorescent nature of the dye. The two entities, labelled alginate and free dye, are both visible due to their fluorescence. Therefore a separation of the two materials would result in the visualisation of two separate, distinct bands of colour moving at different rates through the column. This phenomenon was not observed and the fractions collected exhibited unimodal distribution, which is indicative of the presence of only one fluorescent material. This is shown schematically in Figure 2.2.

(liü p lcr Two: Materials atid Methods 77

10 20 30 40 50

Fraction Number

60 70 80

Figure 2.2. Schematic representation of the eluted material from the gel filtration column

The result shown in Figure 2.2 was obtained by running 1 ml of a 1 mg/ml solution of the labelled alginate down a calibrated Sephadex G-25 pre-packed column. Only one peak was observed indicating that very little or no unbound dye was present in the solution. Although this technique shows that the labelled alginate is the predominant material within the 1 mg/ml solution it does not quantify the extent or homogeneity of substitution by the fluorescent dye.