Solubility of Polyvinyl Alcohol in Ethanol 1

Transcription

1 EXTERNAL SCIENTIFIC REPORT Solubility of Polyvinyl Alcohol in Ethanol 1 João F. A. Lopes and Catherine Simoneau EUROPEAN COMMISSION JOINT RESEARCH CENTRE Institute for Health and Consumer Protection ABSTRACT Solubility studies of the food additive E 1203 (polyvinyl alcohol- PVA) were carried out to update the solubility data reported in Commission Regulation (EU) N 231/2012. The support of the Joint Research Centre (JRC), one of the Directorates General of the European Commission, was requested by EFSA to investigate the currently reported solubility of PVA. Methods based on saturation experiments were developed based the indications of the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and the test guidelines from the Organisation for Economic Co-operation and Development (OECD). A more sensitive colorimetric quantification was developed as confirmation method for PVA. The adequacy of methods was verified by testing four other food additives listed in Regulation (EU) 231/2012. The solubility of the commercial food grade PVA with certification regarding the degree of hydrolysis (86.5 to 89 %) and viscosity (4.8 to 5.8 mpa.s) was measured in ethanol ( 99.8 %). The solubility of food grade PVA was found to be approximately 10 6 parts solvent (ethanol) for 1 part solute (PVA) a value which, according to JECFA, is considered practically insoluble or insoluble. An analytical grade PVA of similar degree of hydrolysis (87 to 89 %) and viscosity (5.2 to 6.2 mpa.s) was tested and also led to a solubility value of 10 6 parts solvent to solubilise 1 part solute. The influence of the purity of the ethanol on the solubility of PVA was also evaluated by carrying out experiments using ethanol 96 %, with results showing a slight increase in the solubility (> 300,000 parts solvent for 1 part solute) due to its higher aqueous content. The solubility experiments, regardless of the type of PVA or ethanol, provided solubility values of 10 6 parts solvent for 1 part solute corresponding to the practically insoluble or insoluble interval according to the JECFA guidelines. European Commission, 2014 KEY WORDS Food additives, polyvinyl alcohol, PVA, solubility, analytical methods, measurements DISCLAIMER without prejudice to the rights of the authors 1 Question No EFSA-Q Any enquiries related to this output should be addressed to fip@efsa.europa.eu Suggested citation: João F. A. Lopes and Catherine Simoneau, Solubility of Polyvinyl Alcohol in Ethanol. EFSA supporting publication 2014:EN-660, 20 pp. Available online: European Food Safety Authority, 2014

2 SUMMARY The aim of the study was to verify the data on the solubility of the food additive E 1203 (polyvinyl alcohol PVA) in ethanol in order to update specifications reported in Commission Regulation (EU) No 231/2012, where necessary. Solubility studies of the food additive E 1203 (polyvinyl alcohol- PVA) were carried out for the European Food Safety Authority by the Joint Research Centre under the Service Level Agreement SLA/EFSA-JRC/FIP/2013/01. The objective was to conduct the necessary experiments in order to update the solubility data reported in Commission Regulation (EU) N 231/2012, as necessary. A standard operating protocol was developed based on the method described by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) document as well as from a second method from the Organisation for Economic Co-operation and Development (OECD) Test Guidelines. The methods consisted in a saturation experiment followed by a gravimetric determination. The adequacy of the resulting experimental protocol was verified by analysing four other food additives listed in Regulation (EU) 231/2012: Sorbitol (E 420), D-Mannitol (E 421), meso-erythritol (E 968) and Acesulfame K (E 950). The solubilities found were in accordance with the established values. The protocol exhibited satisfactory accuracy and thus it was then applied to the solubility measurements of PVA. As the solubility values found for PVA were lower than currently reported in Regulation (EU) 231/2012, a more sensitive colorimetric quantification was developed based on formation of PVA-Iodine complexes which was tailored to PVA itself and to lower solubilities. The solubility of the commercial food grade PVA with certification regarding the degree of hydrolysis (86.5 to 89 %) and viscosity (4.8 to 5.8 mpa.s) was measured in ethanol ( 99.8 %). The solubility of food grade PVA was found to be approximately 10 6 parts solvent (ethanol) for 1 part solute (PVA) a value which, according to JECFA, is considered practically insoluble or insoluble (in the JECFA guidelines any value above 10,000 parts of ethanol being needed to solubilise 1 part PVA). An analytical grade PVA of similar properties was tested (degree of hydrolysis 87 to 89 %, viscosity 5.2 to 6.2 mpa.s) and the solubility value obtained was again approximately 10 6 parts solvent to solubilise 1 part solute. Finally a less viscous PVA was also tested, with results showing a slight increase in the solubility values being observed (10 6 parts solvent were able to solubilise 2 parts solute). The influence of the purity of the ethanol on the solubility of PVA was also evaluated by carrying experiments using ethanol (96 %), with results showing a slight increase in the solubility (10 6 parts solvent were able to solubilise approximately 3 parts solute ) due to its higher aqueous content. The solubility experiments, regardless of the type of PVA or ethanol, provided consistent results with solubility values of 10 6 parts solvent for 1 part solute thus corresponding to the practically insoluble or insoluble interval according to the JECFA guidelines. 2

3 TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 3 Background as provided by the European Food Safety Authority... 4 Terms of reference as provided by the European Food Safety Authority... 5 Introduction and Objectives... 5 Materials and Methods Chemicals Development of the saturation experiment design Optimisation of the flask method (saturation experiment) Determination of solubility Determination by visual evaluation Determination by gravimetric detection Determination by colorimetric detection... 8 Results Validation of the method and PVA measurements D-Sorbitol D-Mannitol Meso-Erythritol Acesulfame K Application to measurements of solubility of PVA Flask method with gravimetric detection Flask method with colorimetric detection Calibration curve Solubility studies of PVA in ethanol 99.8 % Solubility studies of PVA in ethanol 96 % Solubility studies of two other types of PVA in ethanol 99.8 % References Annex 1 certificate of analysis of sample

4 BACKGROUND AS PROVIDED BY THE EUROPEAN FOOD SAFETY AUTHORITY Polyvinyl alcohol (PVA) is a polymer with different types of end-uses. One of them is as a food additive. Commission Regulation (EU) 231/2012 lays down specifications for food additives listed in Annexes II and III to Regulation (EC) No 1333/2008 of the European Parliament and of the Council. In this context, PVA is defined as a synthetic resin prepared by the polymerisation of vinyl acetate, followed by partial hydrolysis of the ester in the presence of an alkaline catalyst. The physical characteristics of the product depend on the degree of polymerisation and the degree of hydrolysis. The risk assessment of PVA as a film-coating agent for food supplements was performed by the European Food Safety Authority (EFSA) in 2005 [2], and PVA was assigned as E number E 1203 [3]. According to the EU specifications (Commission Regulation (EU) 231/2012) the solubility of PVA (E 1203) is soluble in water and sparingly soluble in ethanol [1]. No numerical values of solubility are further specified in the EU specifications. The Joint FAO/WHO Expert Committee on Food Additives (JECFA), in its Combined Compendium of Food Additives Specifications, establishes sparingly soluble as an interval ranging from 30 to 100 parts of solvent required to solubilise 1 part of solute (Figure 1) [4]. Figure 1: Solubility ranges described in [4] The objective of this project was to verify the solubility of food grade PVA in ethanol for the purpose of a potential revision of the specifications. The criteria used in this project for the development of a suitable solubility test method for PVA in ethanol were to match best practices and relevant guidelines, and improve/modify them where necessary. To this purpose, both the succinct description given in Regulation (EU) 231/2012 and JECFA method were implemented as primary test methods [1,4]. The experimental design from the OECD Test Guidelines (so-called flask method) was also considered [5]. Since the OECD method is only applicable to water, the work done included the adaptation of the method to the determination of solubility of PVA in ethanol. 4

5 TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN FOOD SAFETY AUTHORITY This contract was awarded by the European Food Safety Authority to: Contractor: Institute for Health and Consumer Protection - Joint Research Centre - European Commission Contract title: Solubility of Polyvinyl Alcohol in Ethanol Contract number: SLA/EFSA-JRC/FIP/2013/01 Terms of reference: the Contractor shall evaluate the solubility of polyvinyl alcohol (E1203) in ethanol according to the best practices and relevant guideline in order to define categorization of solubility of polyvinyl alcohol (E1203) in ethanol ( 99.5%) to categorise the solubility in ethanol of polyvinyl alcohol (E1203) according to the classification established by the FAO/WHO Expert Committee of Food Additives [4]. INTRODUCTION AND OBJECTIVES The principle behind the solubility experiments was a saturation experiment, followed by detection of the residue after removal of the insolubilised compound and evaporation of the ethanol. The method was developed with the following key quality criteria in mind: Develop an experimental design to adequately control the temperature of the experiments and providing tight tolerance and traceability to temperature control Test the flask method with gravimetric detection to check its applicability with PVA; foresee potential alternatives to provide more sensitive quantification. Check the adequacy of the method on other food additives, comparing results with the solubility values described in EU Regulation 231/2012; Test, if possible, different types of ethanol; Ensure that all results present sufficient repeatability MATERIALS AND METHODS 1. CHEMICALS The PVA used in the experimental work was obtained from a commercial source (Colorcon, UK). The degree of hydrolysis (ranging from 86.5 to 89 %) and a viscosity (ranging from 4.8 to 5.8 mpa.s at 20 C) meet the EU specifications for E 1203 established in Commission Regulation (EU) 231/2012. The certificate of purity is presented in Annex 1. 5

6 Two other PVAs, not listed as food grade but with a very similar percentage of hydrolysis (87 to 89 %), were obtained from Sigma-Aldrich: one with a very similar viscosity to food grade PVA (5.2 to 6.2 mpa.s), and another one with lower viscosity (3.5 to 4.5 mpa.s). Ethanol with a 99.8 % grade was purchased from Fluka and ethanol 96% from Sigma-Aldrich. Sorbitol ( 98 %), D-Mannitol ( 98 %), meso-erythritol ( 99 %) and Acesulfame K ( 99 %) were all obtained from Sigma-Aldrich. Boric acid was obtained from Aldrich ( 99.5 %), potassium iodide ( 99.5 %) and iodine ( 99.5 %) were obtained from Fluka. Water was ultra-pure grade (MilliQ). 2. DEVELOPMENT OF THE SATURATION EXPERIMENT DESIGN A preliminary objective of the study was to ensure repeatability of the experiments. Reference guidelines indicated that solubility experiments in ethanol should be performed at 20 C [4], without any particular tolerance indicated. However, since temperature variations can have a significant impact on the solubility values, it was deemed necessary to find a system capable of 1) minimise the temperature variations, 2) track the temperature controls over the entire duration of the experiments, 3) provide a system where agitation of the samples is simultaneously possible 4) maintain a temperature lower than room temperature (to properly maintain 20 C inside the sample). Several set-ups were investigated: an incubator/incubator hood, a climate chamber, a shaking water bath, and a shaking incubator with agitation (Figure 2). Fig 2a) Incubator Fig 2b) climate chamber Fig 2c) Shaking incubator Fig 2d) shaking water bath Figure 2: Different set-ups tested for the experimental design None of the systems tested presented enough robustness to carry on the solubility experiments. Consequently, a water-bath was designed in-house with automated temperature control and constructed for the purpose of the project (Figure 3). It consisted in a large Plexiglas tank with an entry and an exit point that were connected to a refrigerated circulating water pump (Julabo F33-EH). The design included a temperature sensor (Thermocouple Thermometer DO 9416) inside the water tank providing positive control to the water temperature circulation. This ensured that the water temperature itself was constantly at 20 C. The tank was placed onto 6 magnetic stirring plates (IKA RCT basic) to provide constant and consistent agitation of the sample over time. 6

7 Figure 3: Tailor-designed temperature controlled water bath Tests were carried out to ensure that the apparatus was able to maintain a fixed temperature with little variation. Temperature measurements were conducted in the refrigerated bath itself, in the Plexiglas bath and inside the sample (vial, Erlenmeyer, etc). The results showed that the set-up was able to maintain the desired temperature with not only the minimum accepted variation (20 C ± 0.5 C), but an even lower one (20 C ± 0.2 C). It was tested continuously during 4 days (96hrs) without losing its accuracy, which ensured the level of quality for the solubility tests lasting up to 96 hours. 3. OPTIMISATION OF THE FLASK METHOD (SATURATION EXPERIMENT) The JECFA test relies on the preparation of solutions of the target compound so that a clear solution free from foreign matter should be obtained [4]. If the solubility is known, a solution at that concentration is prepared and visually inspected for any unsolubilised amount of compound. If the solubility is unknown, there are two approaches: preparing a slightly saturated solution to which low amounts of the solvent are slowly and gradually added until all the compound is solubilised, or doing the opposite, i.e. adding small amounts of the compound slowly and gradually to a fixed amount of solvent. The solubility level is then either the cumulative amount of the substance used until the moment the solution becomes clear or in the second protocol until the moment the substances becomes visible (e.g. turbid) at saturation. The flask method can be used with any solid compound and solvent, following the same procedure and regardless of the compound. What may change are the amounts used to prepare the required saturated solutions, which are specific to each compound. The amount of solvent used can change: in some cases solutions using few millilitres are enough, but when studying compounds with very low solubility much higher volumes of solvent may be needed to ensure that a higher amount of compound will be present and solubilised in the solution. The preparation of the saturated solution is done using usually a 10 to 20% higher amount of compound than for a solution at its solubility limit. The solution is then placed under agitation in a temperature controlled environment (e.g. water bath) with the temperature set at 20 C ± 0.5 C. The solution is left under agitation during hours. After this time the agitation is stopped and the solution is left for another 24 hours so that the insolubilised compound settles at the bottom of the flask (centrifugation can also be used, with no differences in the final results, although it is less practical when dealing with higher amounts of solvent). The substance(s) under investigation was/were tested in two different concentrations, representing respectively the upper and lower limits of the solubility interval according to Figure 1. In the case of PVA, as its solubility was described as being sparingly soluble, 30 to 100 parts of solvent were in theory needed to solubilise each part of solute [4]. It should be noted that existing guidelines do not specify units for the measurements, therefore since the solubility brackets were so broad, the units of weight by volume was used in a simplification (rather than strictly weight by weight). The expression 7

8 of results were then presented in equivalents parts solvent needed to solubilise 1 part solute or more directly in parts solute being dissolved by parts (volume in this case) solvent. Two samples were prepared: 1 g of PVA in 30 ml of ethanol and 1 g in 100 ml of ethanol (>99.8 % purity). Once the two samples were prepared, the mixtures were stirred constantly at 20 C for 96 hours (which was the maximum time span mentioned in the reference methods) to ensure that the solubility equilibrium was reached. 4. DETERMINATION OF SOLUBILITY 4.1. Determination by visual evaluation The sample was visually evaluated as to whether the presence of turbidity was present or whether the sample had become clear. In the case of PVA this was performed but was found not applicable since the solubility was too low evaluate visually Determination by gravimetric detection A known amount of the solution (volume or weight) is transferred to a vial, and the solvent is removed by evaporation under a nitrogen stream (or rotary evaporator if larger volumes) to dryness. The amount of compound which remains in the vial is the maximum that can be solubilised in that amount of solvent (in weight per volume). The numbers of parts of solvent that are required to solubilise a part of the compound (normally in millilitres if liquid or gram if solid) are then calculated. For PVA, the experimental design was to add 1 g to 100 ml ethanol (>99.8 % purity), the lower solubility (higher saturation) of the interval range defined as "sparingly soluble" [4]. The saturated solution was then placed under agitation at a controlled temperature of 20 C for a time period up to 96 hours. This time period was sufficient to ensure that equilibrium was reached and that the solution was effectively saturated in the end. The solution was then left to rest for 24 hours so that the entire undissolved compound precipitated and settled at the bottom of the flask. A known amount (volume or weight) was then removed from the clear solution above the precipitate (where all the substance was solubilised), transferred to another flask, and the solvent evaporated. The amount of the solute solubilised in that given amount of solvent was obtained gravimetrically. Filtration could also be used: both protocols were investigated, and no effect was observed on the results. Therefore, the protocol was simplified without the filtration step. The gravimetric determination is simple but reaches its limit for compounds with low solubility as its sensitivity is too low. This was the case for PVA; therefore a more sensitive method was developed Determination by colorimetric detection The principle for this method is based on a study originally published by Finley in 1961, which was related to the quantification of PVA in thin paper coatings [6]. The detection was carried via the formation of iodine-pva complexes using specific reagents. Other studies also used this colorimetric approach for PVA detection [7]. PVA by itself does not absorb in the UV region (which is a reason why it is so difficult to quantify it), but the PVA-Iodine complex does at around 690 nm. However, the method using the formation of PVA-Iodine complex was originally developed only for aqueous matrices. It was therefore necessary to adapt this method to a measurement in ethanol. Two obstacles needed to be overcome: 1) whether the colorimetric reaction was able or not to produce PVA-Iodine complexes at the expected low concentration levels of PVA in ethanol (or at least sufficient enough to absorb and differentiate between solution at different concentrations), and 2) whether the complexes were effectively formed (and stable) in the presence of ethanol. 8

9 Preliminary tests were carried out on aqueous solutions of PVA with different concentrations. The results showed that for aqueous solutions, it was possible to differentiate each solution either visually (different colours), or using UV spectrometry (different UV absorptions), which corresponded to different amounts of complexes formed and present in the solutions. It was possible to detect PVA in solutions with concentrations down to few milligrams per litre, thus confirming the sensitivity of the method. Experiments were then repeated with ethanol solutions, and the results showed that there were no visible differences in the solutions visually (all presented the same yellowish colour typical of the iodine solution) or spectrometrically (all solutions presented the same UV absorptions, being also at the same level of the blank [no PVA] iodine solution). These results seemed to indicate that either the PVA was insoluble at these concentrations in ethanol, or simply that the presence of ethanol as solvent (contrary to water) could disrupt the PVA-Iodine complexes leading to UV absorption. The method was then adapted successfully to the determination in ethanol as follows: the saturation experiment (flask method) was carried out, the ethanol was then gently evaporated to dryness and water (25 ml ultrapure water) was added to reconstitute an aqueous solution of PVA. The colorimetric protocol was based on the use of 4 % boric acid and an iodine solution. The iodine solution was prepared by dissolution of 2.5 g of potassium iodide in 100 ml of water, to which were added 1.25 g of iodine. The solution was left under agitation for one hour. The formation of PVA-Iodine complexes was done by adding 15 ml of the boric acid solution to 25 ml of the PVA aqueous solution, slowly and under agitation, to which 1 ml of the iodine solution was then added, and the resulting solution diluted with water to a final volume of 50 ml. An aliquot was transferred to a UV cell and measured in a UV/VIS spectrometer against a blank solution (15 ml of boric acid solution plus 1 ml of iodine solution and dilution with water to a final volume of 50 ml) at 690 nm (maximum absorbance of the complexes). A calibration curve was constructed of PVA aqueous solutions prepared with different concentration levels (from around 2 parts solute for 10 6 solvent to 20 parts solute for 10 6 solvent). Analyses were done in triplicate. Appropriate calculations gave the final solubility of PVA in ethanol. 9

10 RESULTS 1. VALIDATION OF THE METHOD AND PVA MEASUREMENTS A set of experiments was conducted to verify whether the flask method with gravimetric detection was adequate enough as indicated in the existing guidelines to determine the solubility of food additives in ethanol ( 99.8 % purity). Four authorised food additives were selected and investigated: Sorbitol, D- Mannitol, meso-erythritol and Acesulfame K D-Sorbitol According to the EU specifications (Commission Regulation (EU) 231/2012), D-Sorbitol (E 420) is slightly soluble in ethanol. The flask method was applied to six saturated solutions of D-Sorbitol in ethanol (750 mg in 250 ml). The results are presented in Table 1. Table 1: D-Sorbitol solubility results using the flask method with gravimetric detection Amount of solubilised D-Sorbitol Parts of solvent (ml) needed to solubilise one Solutions In 10 ml of solution (mg) In a 1 L solution (g) part of solute (g) S S S S S S Average Standard Deviation Relative Standard Deviation The average solubility was 387 ml ethanol needed to solubilise one gram of D-Sorbitol. This value fell in the interval slightly soluble defined by JECFA guidelines [4] which corresponds to an interval of solubility between 100 and 1,000 parts of solvent (ml) needed to solubilise 1 part of solute (g) D-Mannitol According to the EU specifications (Commission Regulation (EU) 231/2012), D-mannitol (E 421) is very slightly soluble in ethanol. The flask method was applied to six saturated solutions of D- Mannitol in ethanol (30 mg in 250 ml). The results are presented in Table 2. Table 2: D-Mannitol solubility results using the flask method with gravimetric detection Amount of solubilised D-Mannitol Parts of solvent (ml) needed to Solutions In 50 ml of solution (mg) In a 1 L solution (g) solubilise one part of solute (g) M M M M M M Average Standard Deviation Relative Standard Deviation

11 The average solubility was 9415 ml ethanol needed to solubilise one gram of D-Mannitol. This value fell in the interval very slightly soluble defined by JECFA guidelines [4] which corresponds to an interval of solubility between 1,000 to 10,000 parts of solvent (ml) to solubilise 1 part of solute (g) Meso-Erythritol According to the EU specifications (Commission Regulation (EU) 231/2012), meso-erythritol (E 968) is slightly soluble in ethanol. The flask method was applied to six saturated solutions of meso- Erythritol in ethanol (300 mg in 250 ml). The results are presented in Table 3. Table 3: meso-erythritol solubility results using the flask method with gravimetric detection Amount of solubilised meso-erythritol Parts of solvent (ml) needed to Solutions In 20 ml of solution (mg) In a 1 L solution (g) solubilise one part of solute (g) E E E E E E Average Standard Deviation Relative Standard Deviation The average solubility was 874 ml ethanol needed to solubilise one gram of meso-erythritol. This value fell in the interval slightly soluble defined by JECFA guidelines [4] which corresponds to an interval of solubility between 100 to 1,000 parts of solvent (ml) to solubilise 1 part of solute (g) Acesulfame K According to the EU specifications (Commission Regulation (EU) 231/2012), acesulfame K (E 950) is very slightly soluble in ethanol. The flask method was applied to six saturated solutions of Acesulfame K in ethanol (40 mg in 250 ml). The results are presented in Table 4. Table 4: Acesulfame K solubility results using the flask method with gravimetric detection Amount of solubilised Acesulfame K Parts of solvent (ml) needed to Solutions In 50 ml of solution (mg) In a 1 L solution (g) solubilise one part of solute (g) A A A A A A Average Standard Deviation Relative Standard Deviation The average solubility was 7595 ml ethanol needed to solubilise one gram of Acesulfame K. This value fell in the interval very slightly soluble defined by JECFA guidelines [4] which corresponds to an interval of solubility between 1,000 to 10,000 parts of solvent (ml) to solubilise 1 part of solute (g). The experiments conducted on the selected substances demonstrated that the experimental design was fit for purpose to the measurement of solubility and therefore could be attempted for PVA. 11

12 2. APPLICATION TO MEASUREMENTS OF SOLUBILITY OF PVA Solubility of Polyvinyl Alcohol in Ethanol 2.1. Flask method with gravimetric detection Preliminary tests using the flask method with gravimetric detection indicated that the solubility of PVA in ethanol seemed to fall in the interval practically insoluble or insoluble (more than 10,000 parts of solvent required to solubilise one part of solute). Further tests were conducted of a larger set of samples. Ten solutions with a low solvent volume (5 mg in 50 ml ethanol) were prepared and, to ensure that there was no scale effect, ten more were prepared in higher volume (5 mg in 250 ml of ethanol). Both solutions were saturated solutions, yet even the solution in which a greater amount of dissolved PVA in the solution was expected (i.e. in the larger volume of solvent used) still did not permit gravimetric quantification as the dissolved amount was too low to be measured using a precision balance. This was also independent of the quantity of solution of dissolved PVA transferred to another vial for evaporation (up to 100 ml subjected to evaporation). These results confirmed that the solubility of PVA was likely very low and apparently different from the one described in the EU regulation ( sparingly soluble ). It seemed to fall rather in the interval practically insoluble or insoluble described by JECFA as more than 10,000 parts of solvent required to solubilise one part of solute [4]. However, the low sensitivity of the gravimetric determination impeded to achieve further more precise quantitative measurements. Therefore the confirmation experiments were carried out with the flask method using a more sensitive colorimetric quantification Flask method with colorimetric detection Calibration curve A calibration curve was built to ensure an accurate quantification at the expected low concentration solutions of PVA. For the construction of the calibration curve 6 concentration levels of PVA in water were chosen, between 2 and 20 parts solute for 10 6 parts solvent. For each level 3 solutions were prepared (i.e. a total of 18 solutions). The colorimetric reaction was applied to each one and their UV absorption at 690 nm was measured. The results are presented in Table 5 and Figure 4. Table 5: Absorbance values for the solutions used to construct the calibration curve (690 nm) Concentration (parts solute dissolved by 10 6 parts solvent) Abs. Average Standard Deviation Relative Standard Deviation (RSD) A A A B B B C C C D D D E E E F F F

13 Figure 4: Calibration curve obtained with the colorimetric method The absorption values of the solutions used for the calibration curve showed a good accuracy, with Relative Standard Deviation (RSD) values below The calibration curve showed an excellent linearity with a R 2 value of The results demonstrated that the method was fit for purpose for solutions with low concentrations Solubility studies of PVA in ethanol 99.8 % As PVA solubility was expected to be very low, and in order to increase the amount of PVA in solution that was later detected by the use of the colorimetric method, larger amounts of ethanol were used (1 litre). Six saturated solutions of PVA were prepared. The solutions were left under agitation in the water bath at a temperature of 20 C ± 0.5 C during 72 hours. The agitation was then stopped and the solutions were left for another 24 hours so that the excess of insolubilised compound would settle down at the bottom of the flask (centrifugation was not adequate due to the high volume of ethanol used). A known amount of each solution (volume or weight) was transferred to another flask (e.g., for a 1 L solution a minimum of 200 ml were transferred) and the ethanol was evaporated to dryness (using a rotary evaporator). 25 ml of H2O (ultrapure) were added and the solution was agitated so that all the PVA in the flask could re-dissolve in water. 15 ml of boric acid solution were added (slowly and under agitation), followed by 1 ml of iodine solution. The solution was diluted with water to a final volume of 50 ml. An aliquot of each solution was transferred to an UV cell and measured against a blank solution (15 ml of boric acid solution, 1 ml of iodine solution and 34 ml of water) at 690 nm using an UV spectrometer. The results were obtained using the calibration curve previously presented, and shown in Table 6 (3 replicates per solution). 13

14 Table 6: Solubility of PVA in 99.8 % ethanol Solution Abs. Calculated concentration in the solution (mg/l ca. equivalent to parts of solute dissolved by 10 6 parts solvent) Average per solution A PVA1 B C A PVA2 B C A PVA3 B C A PVA4 B C A PVA5 B C A PVA6 B C For solutions PVA1 to PVA3 500 ml were evaporated. For solutions PVA4 to PVA6 200 ml were used to check that the volume used had no influence. Results showed that the solubility of PVA was approximately 1 mg/l, meaning that 10 6 parts of solvent (ml) were needed to solubilise 1 part of solute (g). According to the JECFA guidelines, this value fell in the interval practically insoluble or insoluble, with more than 10,000 parts of solvent being required to solubilise each part of solute. To verify if there was any scale effect affecting PVA s solubility, the same procedure was applied but using 2 litre of saturated solution of PVA in ethanol (three replicates). Results are reported in Table 7. SD RSD Table 7: Solubility of PVA in 99.8 % ethanol (2 L solutions) Solution Abs Calculated concentration in the solution (mg/l ca. equivalent to parts of solute dissolved by 10 6 parts solvent) Average per solution A PVA7 B C A PVA8 B C A PVA9 B C For solutions PVA7 to PV9 500 ml were evaporated. The solutions were reconstituted in water (final volume 50 ml), undergoing the colorimetric protocol. No scale effect was observed, as the results were in line with those obtained for the 1 litre solutions (around 10 6 parts solvent for 1 part solute in both cases) Solubility studies of PVA in ethanol 96 % A factor that could influence the solubility was the ethanol grade. While preceding tests indicated a low solubility of PVA in ethanol, the solubility of PVA in water is described in the EU Regulation as soluble in water. Therefore it was tested whether the residual water content in ethanol had an impact on the solubility of PVA. Six saturated solutions with ethanol of alcoholic grade of 96 % were SD RSD 14

15 prepared (1 litre). The protocol previously described was used with a similar calibration curve. The results are presented in Table 8 (3 replicates for each sample). Table 8: Solubility of PVA in 96 % ethanol Solution Abs. Calculated concentration in the solution (mg/l ca. equivalent to parts of solute dissolved by 10 6 parts solvent) Average per solution A PVA 10 B C A PVA 11 B C A PVA 12 B C A PVA 13 B C A PVA 14 B C A PVA 15 B C ml were transferred and evaporated from each solution. The solutions were reconstituted in water (final volume 50 ml), undergoing the colorimetric protocol. Results showed that the solubility of PVA in 96 % ethanol was approximately 3 mg/l, meaning that 333,333 parts of solvent (ml) were needed to solubilise 1 part of solute (g). While the solubility was still very low, this lower grade of ethanol (higher water content) led to a slightly higher solubility of PVA compared to that of using 99.8 % grade ethanol. This indicated that the grade of the solvent used for solubility tests has an effect and could benefit to be specified Solubility studies of two other types of PVA in ethanol 99.8 % In order to check whether the validity of the method was independent of the brand and/or type of PVA being used, two independent PVA available as calibrants from Sigma-Aldrich were tested. One PVA was chosen chemically similar to the food grade PVA (hydrolysis level %, viscosity range mpa.s), and another PVA was chosen with a slightly lower viscosity ( mpa.s, same hydrolysis level %). The tests were carried with ethanol 99.8 %. Six saturated solutions (1 L) of each PVA solution in ethanol were prepared. The protocol and calibration curve previously described were used (3 replicates per solution). Results are presented in tables 9 and 10. SD RSD 15

16 Table 9: Solubility for an analytical PVA with similar physical properties (in 99.8 % ethanol) Solution Abs. Calculated concentration in the solution (mg/l ca. equivalent to parts of solute dissolved by 10 6 parts solvent) Average per solution A PVA 16 B C A PVA 17 B C A PVA 18 B C A PVA 19 B C A PVA 20 B C A PVA 21 B C ml were transferred and evaporated from each solution. The solutions were reconstituted in water (final volume 50 ml), undergoing the colorimetric protocol. The results showed that the solubility values obtained using the flask method with colorimetric detection for two types of PVA with similar physical properties, but obtained from two different companies (Sigma-Aldrich and Colorcon), were approximately the same (around 1 part solute for 10 6 parts solvent). The results seem to demonstrate that the method was adequate regardless of the origin of the PVA being studied, as long as they shared the same physical characteristics (degree of hydrolysis, viscosity). Table 10: Solubility for an analytical PVA with less viscous properties (in 99.8 % ethanol) Solution Abs. Calculated concentration in the solution (mg/l ca. equivalent to parts of solute dissolved by 10 6 parts solvent) Average per solution A PVA 19 B ,00 0,05 C A PVA 20 B ,00 0,03 C A PVA 21 B ,01 0,73 C A PVA 22 B ,09 4,28 C A PVA 23 B ,04 2,23 C A PVA 24 B ,02 0,86 C ml were transferred and evaporated from each solution. The solutions were reconstituted in water (final volume 50 ml), undergoing the colorimetric protocol. The results of solubility for the analytical PVA with lower viscosity exhibited slightly higher solubility values (around 2 parts solute for 10 6 parts solvent) compared to the food grade PVA. These results seem to be consistent with the fact that normally compounds with lower viscosity have higher SD SD RSD RSD 16

17 solubility values than the ones with higher viscosity. Nevertheless the solubility did not change significantly. CONCLUSIONS The experimental work conducted showed that the solubility of food grade PVA (polyvinyl alcohol E1203) in ethanol ( 99.8 %) is in the range of 10 6 parts of solvent (ethanol) required for 1 part of solute (PVA). This implies in turn that more than 10,000 parts of ethanol are needed to solubilise 1part of PVA. This value, according to the JEFCA Guidelines, characterizes PVA as being quantitatively classified as practically insoluble or insoluble. REFERENCES [1] Commission Regulation (EU) No 231/2012 of 9 March 2012, laying down specifications for food additives listed in Annexes II and III to Regulation (EC) No 1333/2008 of the European Parliament and of the Council. [2] Scientific opinion of the Panel on Food Additives, Flavourings, Processing Aids and Material in Contact with Food on a request from the Commission related to the use of polyvinyl alcohol as a coating agent for food supplement, The EFSA Journal (2005) 294, p. 1. [3] Commission Directive 2010/69/EU of 22 October 2010 amending the Annexes to European Parliament and Council Directive 95/2/EC on food additives other than colours and sweeteners. [4] Joint FAO/WHO Expert Committee of Food Additives, Combined Compendium of Food Additives Specifications, vol. 4 Analytical methods, test procedures and laboratory solutions used by and referenced in the food additive specifications, FAO JECFA monographs. p. 41. [5] OECD, Test Guideline (1) Official Journal of the European Communities L 383 A, [6] Finley, J. H., 1961, Spectrophotometric Determination of Polyvinyl Alcohol in Paper Coatings, Analytical Chemistry, 33, [7] Magdum, S.S. et al, Rapid Determination of Indirect Cod and Polyvinyl Alcohol from Textile Desizing Wastewater, Pollution Research, 32,

18 ANNEX 1 CERTIFICATE OF ANALYSIS OF SAMPLE 18

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