diffusion of adhesives components in food...

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Research of diffusion of adhesives components in food packaging

J. Gaspar, C. Nerín, M. Aznar

GUIA Mariano Esquillor s/n, 50018, Zaragoza, Spain.

Tel. +34-976762707, Fax +34-976762043, e-mail: [email protected] Abstract

The main objective of this project was to determine the composition of two adhesives, natural and synthetic rubber, the potential of migration of its compounds, estimated from their partition and diffusion coefficients. The selected analytical technique was the solid phase micro extraction coupled to gas chromatography with mass spectrometry detection (HS-SPME-GC-MS). A selection of the best fiber was done. After that the chromatographic conditions and extraction method were optimized using the Modde program The identified compounds were classified according to their toxicity by means of Toxtree program. Analytical characteristics were obtained from each of the methods of quantification, and it was concluded that the SPME showed lower detection limits (pg/g) than direct injection (µg/g) but higher RSD. The 4-cyanocyclohexene was the most toxic compound identified and the BHT was the compound found in highest concentration (165.5 µg/g). In general, the concentrations of the analytes identified in the CN adhesive (natural rubber) were greater than those identified in CS adhesive (synthetic rubber). In partition tests, the equilibrium concentration in the adhesive of the majority of the analyzed compounds, increased with temperature, except for 4-cyanocyclohexene (CS adhesive/paper) and phenyl isothiocyanate (CN adhesive/PVC). As regards the supports, a III grade toxic compound, 2,3-dihydro-3-phenyl-1,1,3-trimethyl-1H-indene was identified in paper. Keywords: Adhésives, diffusion, fiber, rubber, laminates, migration, partition, toxicity. 1. Introduction

Currently most of the packaging that is in contact with food is produced using adhesives. For these applications, the adhesives industry uses a variety of raw materials and formulations that can comprise up to 15 different chemicals in the simplest system. The EU regulates food packaging materials and constituents that can contaminate food and endanger the health of consumers but up to now, adhesives do not have a specific legislation, although some of its constituents could migrate through packaging to reach food. That is why the EU decided to subsidize this research. Nowadays, to check the safety of food contact materials, global and specific migration testing must be done with different food simulants under specific conditions. However, the experimental determination of migration requires a considerable amount of time and in many cases is impossible due to technical difficulties of the enterprises. Migration processes are predictable physical processes. The transfer from the food materials obeys the Fick's diffusion law (Gavara, R. and R. Catalá, 2002). For this reason, in addition to experimental methods, the theoretical estimation of migration seems to be a new alternative applicable tool.


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The FDA uses different models of migration estimation as an additional tool to assist in the establishment of regulations. In addition, the EU has recently approved the use of migration models in 2002/72/EC Directive. The critical parameters to develop these models are the migrant's diffusion coefficients in the media used in the preparation of laminates and the migrant’s between the adhesive and the support. In this context, the project's objective is to verify that MIGRESIVES consumer exposure to chemicals released by the adhesive is not harmful to health and the concentration of these compounds is, in many cases, below the levels of importance, validating, from the experimental results, a model to predict migration and proposing legislation to the adhesives used in the manufacture of food packaging. 2. Materials and Methods

Two adhesives were selected for this study, synthetic rubber water-based adhesive (CS) and natural rubber, solvent-based adhesive (CN), supplied by two adhesive companies. They were representative of commonly used adhesives in commercial food packaging. The main dispersion of CS adhesive is an aqueous dispersion of a carboxylated styrene butadiene copolymer (SBR) of 53% solids content. The binder is an aqueous dispersion of acrylate and butadiene copolymer with 15% of butadiene and 35.5% of solids. This adhesive is used for the manufacture of metallic complexes on porous supports, i.e. continuous union of aluminum foil or metalized plastic film with paper. To this end, two supports are joined immediately after applying the adhesive, without giving it time to dry. This application is carried out through a roller applicator. The complex has a large number of applications:

• Manufacture of cardboard boxes or trays. • Food Flexible packaging. • Wrapper for candy and other sweets, biscuits wrapping, metallic air conditioning ducts. • Uses in graphic arts in general, etc..

The CN adhesive is a natural rubber solution in an organic solvent. Evaporating the solvent produces the adhesion. Solvent-based adhesives are faster than water-based adhesives because of the increased rate of evaporation of organic solvents over water. Adhesive’s function is to seal boxes through its application in the form of tape under pressure, so it is an adhesive used mainly in the secondary packaging, although in many cases the cardboard boxes are the primary packaging (candy, sweets, etc.). The materials usually join PVC with paper.

2.1. Laminates

The laminates that were prepared were composed of two layers of the same material joined by a given weight of adhesive. The materials used to prepare the laminates were:

• 52 µm-thick glossy paper for CS adhesive. • 30 µm-thick PVC for CN adhesive.

Notched rods (Meyer bar) were used to apply the CS adhesive. After preparing the laminate, it was passed through a laminator at 90°C. Laminates were cut into 10x10 cm2 and were wrapped in aluminum foil, letting them rest for 1 week to complete the curing process. 5 sheets of 10x10 cm 2 without adhesive were weighed.


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The average weight of adhesive in the laminates was determined by the difference between the average weight of the laminates and the average weight of the supports. The weight of dry CS adhesive deposited was 9.63 ± 1.10 g/m 2. For the CN adhesive was not possible to prepare laminates by hand since this solvent-based adhesive forge rapidly and is very difficult to extend evenly. CN adhesive was available in roll industrial production roll (PVC + adhesive), provided by the company. For the preparation of CN laminates, PVC sheets were cut to 10x10 cm 2 and manually adhered to the roll of adhesive, forming a laminate in which the adhesive weight was 22.8 ± 0.02 g/m 2.

2.2. Extraction technique selection

For the analysis of adhesives samples of this project, was carried out the selection of the proper technique that allowed determination of volatile and semi volatile analytes, which are those that show a greater tendency to migrate. The SPME technique was selected because is a solvent-free extraction technique that requires small sample sizes. It is a rapid technique that with only three types of adsorbent materials covers all of the analytes to be determined for liquid, solid and gaseous samples.

2.3. Fiber selection

Once selected extraction technique it was proceeded to select the most appropriate fiber for the extraction of the two adhesives. The analysis of volatile compounds was carried out by HS-SPME-GC-MS. The selection of the fiber was based on two criteria:

• Number of peaks detected. • Signal intensity of the peaks detected.

Fibers of different polarity were used for manual injection:

• 100 µm PDMS • 65 µm PDMS/DVB. • 50/30 µm DVB/carboxen/PDMS • 100 µm polyacrylate. • 70 µm CW/DVB.

The chromatograms showed large differences between the different fibers, both for the CS and CN adhesives. The best results for the extraction of CS and CN adhesives were provided for the PDMS fiber, because most of the compounds detected had a similar structure, organic molecules with aromatic ring substituent and apolar.

2.4. Extraction of CS and CN adhesives with different solvents

Liquid-liquid extraction of pure adhesive was carried out, for possible important semi-volatile compounds in terms of their toxicity and potential for migrating. We used three solvents: dichloromethane, hexane and methanol to extract 1 gram of adhesive with 2 g of solvent. The adhesive samples were kept 1 hour in the ultrasonic bath at a temperature of 40ºC. In the case of the CS adhesive was demonstrated that hexane was the best since the dichloromethane and methanol dissolved the polymer. To extract the CN adhesive, the best solvents were hexane and methanol.


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The conclusion was that both the CS and CN adhesive solvent extraction did not contain any compound other than those extracted with the PDMS fiber.

2.5. Diffusion tests

As already mentioned in the theoretical introduction, diffusion tests were performed using Moisan cells. The diffusion cells used were made of aluminum, the dimensions were 14.8 x14.8x1cm3. They had four nuts that were tightened applying 0.8 N×m torque using a torque wrench aiming at applying the same torque in each nut. The diffusion tests were performed under the following conditions:

• 40ºC 24 hours. • 40ºC 48 hours. • 60ºC 24 hours. • 60ºC 48 hours.

To perform the diffusion test it was taken a previously prepared laminate and was put into a Moisan cell with 10 paper sheets of 10x10 cm2 in each side for CS adhesive and PVC sheets for the CN adhesive. The cell was introduced in an oven at temperature and for the time specified for each test. After this time, the cell was taken out from the oven and the receptors were cut into 5x5 cm2sheets of and were introduced in 20 ml vials. After this 10 µl of IS 10 µg/g in methanol was added on the paper receptors and 10 µl of IS 3 µg/g in hexane on PVC receptors. Once samples were prepared, were left at room temperature for 24 hours, to ensure that equilibrium was reached and after that, each of these sheets were analyzed by HS-SPME-GC-MS. The diffusion, such as the partition depends mainly on analyte molecular weight, structure and chemical composition of the polymer, although factors such as polarity and structure of analyte also have influence (Gavara, R. and R. Catalá, 2002), (Dole, P.,2006).

2.6. Partition Tests

Partition tests were performed to determine the partition constants of the analytes under study between the adhesive and supports. The partition constant is defined as the ratio of analytes concentrations in the adhesive and the substrate at equilibrium. After preparing the laminates, a substrate sheet was put on each side of the laminate. The tests were homogeneous, i.e. the laminate material and the receptor material was the same. Partition tests were performed under the following conditions:

• 40ºC for 30 days. • 60ºC for 30 days.

Each test was performed by duplicate, using average values. The assembly thus prepared was put into a stove in the test conditions. The time required to reach equilibrium was set unanimously by the laboratories participating in the project MIGRESIVES. The partition constant is defined as:


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The concentration in the adhesive and the substrate after the test partition was calculated by the following equations:

All calculations were made with reference to 5×5 cm2 sheets since that is what was analyzed by HS-SPME-GC-MS. The meaning of each of the terms of the balance is:

• [Analyte substrate] refers to the concentrations of the analytes identified, in the paper and PVC receptors at equilibrium, calculated per unit volume of substrate. In equilibrium, this concentration is the same in the four layers of substrate.

• [Analyte adhesive] refers to the concentrations of the analytes identified in the adhesive in equilibrium. These concentrations are calculated per unit volume of adhesive.

• [m blank] refers to the concentration of the analytes under study in the substrate, before the test.

• Psubstract means the weight of the 5x5 cm 2 sheet of PVC or paper. • m 0 is the initial mass (g) of analyte in the adhesive deposited on the laminate. • m substrate refers to the mass (g) of analyte in each substrate at equilibrium. • Volume substrate refers to the volume of a PVC or paper sheet of 5x5 cm 2. • Volume adhesive refers to dried adhesive deposited on the 5x5 cm 2 laminated.

3. Results

3.1. Identification of the analytes in the CS adhesive

Temperature program for compounds identification of the CS adhesive was as follows: Initial temperature of 40ºC for 2 min, gradient of 10°C/min up to 250ºC, maintained for 3 min. The mass detector was working in SCAN mode with a ratio of m/z of 45-350. The amount of sample used was 1 gram of pure adhesive in a 20 ml vial. Figure 1 shows the analytes identified in the CS adhesive. Figure 1 show the analytes identified in the CS adhesive.


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Figure 1. Chromatogram of the identified compounds with the PDMS fiber in the CS adhesive.

After the test, 14 compounds were detected of which 11 were identified. Table 1 shows the identified analytes, with CAS number, toxicity and molecular structure. The p-tercbutylphenol is the internal standard. In the absence of legislation on the use of food packaging adhesives, they were classified according to their toxicity degree using the Toxtree program based on Cramer's rules (Cramer, 1978). These rules classify the compounds into three levels of toxicity depending on the molecular structure. The maximum daily intake in mg/(kg body weight × day) is:

• 3 for level I toxicity compounds. • 0.91 for level II toxicity compounds. • 0.15 for level III toxicity compounds.


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Compound CAS Toxicity MW

(g/mol)

Ebullition point (0C)

Structure

4-Ethenylcyclohexene 000100-40-3 I 108,2 128,9

Ethylbenzene 000100-41-4 I 106,2 136,1

Styrene 000100-42-5 I 104,1 145,8

1-Methylethylbenzene 000098-82-8 I 120,2 151,8

4-Cyanocyclohexene 000100-45-8 III 107,1 209,9

p-Tertbutylphenol (IS)

98-54-4 II 150,2 233,7

4-Phenylcyclohexene 004994-16-5 II 158,2 229,5

3,4-Divinyl-1-phenylcyclohexane (1R,3trans,4trans) (3 isomers) 3,4-Divinyl-1-phenylcyclohexane (1R,3cis,4cis)- (2 isomers)

1000160-00-5

1000160-00-6

II

II

212,2 Non

available

Table 1. Identified compounds in the CS adhesive

Eleven compounds were identified; four had low toxicity, six an intermediate toxicity and the 4-cyanocyclohexene, a high toxicity. It is noteworthy that most of them have as a substituent an aromatic ring and are apolar. The 4-cyanocyclohexene has greater polar character due to the cyano group.

3.2. Identification of the analytes in the CN adhesive

The temperature program was as follows: initial temperature of 40ºC for 2 min, gradient of 10°C/min to 260 º C, maintained for 5 min. The mass detector worked in SCAN mode with a ratio of m/z 45-350. To identify the CN adhesive compounds a blank was done to determine the compounds that came from the PVC. A 10x10 cm 2 laminate was prepared and cut into pieces of 2,5 x 2,5 cm 2 in order to put it into a 20 ml vial. In figure 2, one can observe the analytes identified in the CN adhesive.


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Figure 2. Chromatogram of the identified compounds in the CN adhesive with PDMS fiber.

Thirteen compounds were detected, of which eight were identified. These compounds were classified according to their toxicity degree. Table 2 shows the analytes found in the CN adhesive, with CAS number, toxicity and molecular structure.

Compound CAS Toxicity MW

(g/mol) Ebullition Point (ºC)

Structure

Toluene 000108-88-3 I 92,1 109,8

1,1,2-Trimethylcyclohexane

007094-26-0 I 126,2 145,9

2-Tertbuthyltoluene 001074-92-6 I 148,2 199,8

1,3-Diethyl-5-methylbenzene

002050-24-0 I 148,2 200,7

p-Tertbutylphenol (IS)

98-54-4 II 150,21 202,8

1-Methyl-4- (1methylpropyl) benzene

001595-16-0 I 148,2 221

Phenyl isothiocyanate 000103-72-0 III 135,2 265,0


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BHT 000128-37-0 II 220,3 263,6

2-Cyclopentyl-1,3,5-trimethylbenzene

030628-31-0 II 184 287,2

Table 2. Identified compounds in the CN adhesive

It is remarkable that as in the CS adhesive, most of the identified compounds contain an aromatic ring. All of them are apolar except phenyl isothiocyanate and BHT. Of the eight compounds identified, five had low toxicity and three, medium toxicity.

3.3. Optimization method of SPME extraction conditions with Modde software

The optimum conditions are a compromise solution, since the conditions sought are those for which it is extracted the greatest number of compounds and in greater proportion, but for some analytes the conditions used imply a negative influence. The response surface indicates the values of the extraction variables; temperature and extraction time for which the total area extracted is maximum. These values were for both CS and CN adhesive:

• Extraction time: 30 min. • Extraction temperature: 80ºC.

3.4. Additional studies for validation of extraction methods

After optimization of the extraction methods were carried out a series of experiments to verify if the selected variable ranges and the results were right. The sample conditioning time has an influence in the amount extracted. This is the time before the extraction time that the sample remains in agitation at a given temperature. Paper and PVC sheets of 5x5 cm 2 was injected and doped with 10 µl of standard solution of 10 µg/g. Preincubation times were ranged between 2.5 and 60 minutes and it was found that above 2.5 minutes there was not an increase of the signal, confirming that the system quickly reaches equilibrium at 80ºC when it has been left 24 hours at room temperature . When working with SPME, the amount extracted in the same working conditions vary, this variation is greater when it is not working at equilibrium. The number of points at which the adsorption takes place is limited. When all these points have been occupied, work is under equilibrium conditions. To verify that work was able to balance five samples were prepared:

• Paper sheet doped with 10 µl standard solution of 10 µg/g • CS Adhesive 1/200 (w/w) • CN adhesive-PVC laminate

Each of these samples was extracted varying the extraction time from 10 to 50 minutes. The conclusion was that when the sample is extracted for 30 minutes the system is under equilibrium conditions.


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3.5. Determination of the CP0 of the CS adhesive

The identification of compounds was confirmed by injecting the corresponding standards and comparison of retention times and mass spectra with those of the detected compounds. CS adhesive samples were diluted with water in 1/200 (w/w) dilution prior to analysis, then it was taken three aliquots of 5g and were analyzed by SPME using extraction and chromatography programs previously optimized.

Compound Lineal range

(ng/g) Lineality

(R2) LOD (pg/g)

LOQ (pg/g)

RSD (%)

4-Etenhylcyclohexene 0,697-50,0 0,999 209 697 6,69 Ethylbenzene 0,613-50,0 0,999 18,41 61,33 4,69 Styrene 0,276-50,0 0,997 82,72 275,9 5,36 1-Methylethylbenzene 0,081-50,0 0,999 24,2 80,6 7,03 4-Cyanocyclohexene 62,30-250 0,997 18691 62305 10,10 1-Phenyl-1-cyclohexene

0,012-49,8 0,996 3,75 12,32 3,39

Phenylcyclohexane 0,007-50,4 0,999 2 6,63 3,85 *The LOD was determined using standard solutions of 10 pg/g.

Table 3. Analytic characteristics of the CS adhesive extracting method.

It is important to point out that the LOD and LOQ were very low, order of pg/g, especially for phenylcyclohexane, and the RSD for all analytes is less than or equal to 10%. The concentration obtained from each of the analytes, expressed as µg of compound per gram of pure uncured adhesive, is shown in Table 4. Compound Concentration

(µg/g) Compound Concentration

(µg/g) 4-vinylcyclohexene

3,17 ± 0,31 3,4-Divinil-1-fenilciclohexano (1R,3trans,4trans)_1 0,041 ± 0,005

Ethylbenzene 5,33 ± 0,40

3,4-Divinil-1-fenilciclohexano (1R,3trans,4trans)_2 0,687 ± 0,04

Styrene 5,48 ± 0,43

3,4-Divinil-1-fenilciclohexano (1R,3trans,4trans)_3

0,931 ± 0,05

1-Methylethylbenzene 3,61 ± 0,42

3,4-Divinil-1-fenilciclohexano (1R,3cis,4cis)-1

0,249 ± 0,02

4-Cyanocyclohexene 27,60 ± 1,50

3,4-Divinil-1-fenilciclohexano (1R,3cis,4cis)_2

<LOQ

4-Phenylcyclohexene 9,40 ± 0,32

Table 4. CP0 of the CS adhesive.

The highest concentration was found in the 4-cyanocyclohexene which is also the most toxic compound identified in the CS adhesive.


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3.6. Determination of the CP0 of the CN adhesive analytes

3.6.1. Extraction of the laminates with different solvents

The determination of the CP0 of CN adhesive was carried out by extraction with solvent. We used three solvents with different polarity: hexane, methanol and dichloromethane. In each vial was weighed 1 g of laminate and added 6 g of solvent. Three consecutive extractions were made on each sample leaving every extraction 24 hours at 40ºC in the oven. The three extracts were gathered and it was added 20 ml of IS 190 µg/g in hexane. After that, the extract was concentrated using a stream of N2 up to an approximate weight of 1g. The concentration factor, takes into account the weight before and after concentration, its value was 6.5 and each test was performed by triplicate. The total area obtained and the percentage extracted in the third extraction is shown in Table 5. The areas were divided by the area of the internal standard.

Compound

Total area

extracted with

Hexane

Total area

extracted with

Methanol

Best solvent

according to total

area extracted

Percentage extracted

in the third

extraction with

Hexane

Percentage extracted

in the third

extraction with

Methanol

Best solvent

according to the

percentage extracted

in the third

extraction Toluene 150,18 21,15 Hexane 10,88 11,95 Hexane 1,1,2-Trimethylcyclohexane

1,22 0,07 Hexane 4,63 16,47 Hexane

2-Tertbutyltoluene 5,18 3,09 Hexane 4,63 5,07 Hexane 1,3-Diethylbenzene 1,25 0,84 Hexane 5,46 4,89 Methanol 1-Methyl-4-(1-methylpropyl)benzene

5,48 3,84 Hexane 3,31 4,60 Hexane

Phenyl isothiocyanate

7,21 51,80 Methanol 14,52 18,93 Hexane

BHT 32,85 30,69 Hexane 5,58 6,43 Hexane 1,3,5-Trimethyl-2-cyclopentylbenzene

6,25 5,53 Hexane 4,96 5,44 Hexane

Unknown_1 4,29 4,35 Methanol 5,32 6,84 Hexane Unknown _2 9,41 10,34 Methanol 5,90 7,16 Hexane Unknown _3 9,66 10,73 Methanol 5,96 6,28 Hexane Unknown _4 10,12 11,61 Methanol 6,48 7,61 Hexane Unknown _5 559,31 1218,41 Methanol 12,87 7,33 Methanol

Table 5. Comparison of the extraction strenght of methanol and hexane to extract the CN adhesive –PVC laminate.

The selected solvent was that with a maximum total area in the three extractions and a minimum value of the percentage of the total area extracted in the third extraction, what indicates a stronger solvent extraction.


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The percentage extracted in the third extraction was calculated as the ratio of the area get on the third extraction between the total area get in the three extractions. Taking into account both the total area as the percentage of the third extraction, the hexane provided better results than methanol, being, therefore, the selected solvent. Methanol, extracted better than hexane the unknown compounds, indicating that they were more polar. Was also carried out the extraction of PVC supports following the same procedure as for the laminates, and it was observed that none of the analytes under study were present in the PVC. The concentrated blank of hexane gave a signal for toluene and BHT, data that was taken into account in the calculations.

3.6.2. External calibration with standards in hexane for the determination of the CP0 of the CN adhesive

Table 6 shows the characteristics of the method for extracting the adhesive CN- PVC laminate with hexane.


(µg/g) Lineality

(R2) LOD (µg/g)

LOQ (µg/g)

RSD (%)

Toluene 0,047-10,0 0,995 0,014 0,047 10,3 1,2,4-Trimhetylcyclohexane 0,129-10,8 0,998 0,039 0,129 9,3 4-Tertbutyltoluene 0,061-10,3 0,999 0,018 0,061 7,8 1,3-Diethylbenzene 0,064-10,2 0,999 0,019 0,064 7,3 1-Methyl-4-(1-methylethyl)benzene 0,052-10,0 0,999

0,015 0,052 8,2

Phenyl isothiocyanate 0,067-10,3 0,995 0,020 0,067 2,2 Cyclohexylbenzene 0,048-10,3 0,999 0,014 0,048 7,0 BHT 0,023-10,2 0,994 0,007 0,023 1,7

Table 6. Characteristics of the method of quantification to determine CP0 of the CN adhesive.

RSD values shown correspond to 5 µg/g point of the calibration curve. For all compounds were obtained RSD percentages of 10% or less. This was better than those obtained with SPME. The LOD was low, in the order of µg/g, but is higher than the obtained for SPME (pg/g). Table 7 shows the concentrations obtained from each of the analytes identified in the CN adhesive, expressed as µg of compound per gram of cured pure adhesive.


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Compound Concentration

(µg/g) Toluene 154,6 ± 60,3 1,1,2-Trimethylcyclohexane 10,3 ± 2,41 2-Tertbutyltoluene 13,6 ± 2,04 1,3-Diethylbenzene 7,4 ± 1,17 1-Methyl-4-(1-methylpropil)benzene 18,5 ± 3,11 Phenyl isothiocyanate 79,2 ± 11,3 BHT 165,5 ± 24,7 2-Cyclopentyl-1,3,5-trimethylbenzeno 41,7 ± 6,40

Table 7. CP0 of the CN adhesive.

The BHT is antioxidant of II toxicity level and is the compound present in highest concentration. Also note that the toluene concentration is high, something logical since is one of the solvents. The obtained toluene concentration has a RSD of 40% while for the rest of the compounds the RSD varies between 14 and 24%, which indicates that their concentration in the sample is more variable.

3.6.3. Characteristics of the method of quantification of the diffusion and partition tests of CS adhesive on paper

For quantification of the compounds present in the paper and came from CS adhesive was prepared calibration curves by doping the paper with the analytes at different concentration levels and then analyzing by HS-SPME-GC-MS. The characteristics of the method of quantification are shown in Table 8.


(µg/g) Lineality

(R2) LOD (ng/g)

LOQ (ng/g)

RSD (%)

4-vinylcyclohexene 0,023-1,42 0,996 6,7 22,35 26,7 Ethylbenzene 0,011-2,36 0,998 3,55 11,85 23,5 Stirene 0,002-2,41 0,992 0,650 2,16 18,1 1-Methylethyllbenzene 0,004-2,36 0,990

1,15 3,84 19,7

4-Cyanocyclohexene 0,872-2,385 0,990 261,6 871,7 8,3 1-Phenyl-1cyclohexene

0,004-2,35 0,999 1,34 4,47 6,7

Phenylcyclohexane 0,00007-2,36 0,999 0,021 0,070 4,4 *To determine the LOD and LOQ, samples were injected at concentrations up to 0.5 ng/g

Table 8. Characteristics of the method of quantification of CS adhesive analytes on paper

It was noted that working with paper, LOQ and LOD for the same analytes, increased compared to those obtained in water. In addition, the linear range was reduced and the RSD increased.


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3.6.4. Paper distribution curves of the analytes identified in the CS adhesive

This section represents the diffusion curves on paper of the analyzed compounds. Some of the compounds were below the LOQ and could not be represented. It is important to note that 4-cyanocyclohexene that is a III level toxic compound diffuses slowly in the paper. A blank of paper was carried out a blank. Three paper sheets of 5x5 cm2 were analyzed by SPME. The main compounds identified in the paper were:

• 2,3-dihydro-3-phenyl-1,1,3-trimethyl-1H-indene. • Phthalic acid, isobutyl ester undecil. • Linear alkanes between C 14-C 22.

The 2,3-dihydro-3-phenyl-1,1,3-trimethyl-1H-indene is a level III toxicity compound and is therefore important to control its migration. In these graphs is represented the diffusion curves of the analytes identified in CS adhesive on paper at two temperatures and two times and its concentration in the blank of paper. The point on the vertical axe indicates the concentrations found in the blank of paper.

4-Phenylcyclohexene 3,4-Divinyl-1-Phenylcyclohexane(1R,3trans,4trans)_1

3,4-Divinyl-1-Phenylcyclohexane (1R,3trans,4trans)_2

3,4-Divinyl-1-Phenylcyclohexane (1R,3trans,4trans)_3

3,4-Divinyl-1-Phenylcyclohexane (1R,3cis,4cis)_1


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Figure 3. Diffusion tests of CS adhesive on paper at different temperatures and times.

In these graphs it is clear that the diffusion increased with temperature and the increasing the test time increased concentration in the receptors. Diffusion tests at 60°C were in equilibrium or very nearby. One result to note is that the concentrations of the diffusion tests that are in equilibrium or near, are consistent with the results of the equilibrium concentrations on paper in partition testing, and presented in table 10. Noting the tests at 40ºC, it can observe that concentrations increase with increasing test time and it is closer to the equilibrium value. The highest concentrations were found in the 4-phenylcyclohexene.

3.6.5. Characteristics of the quantification method for diffusion and partition tests of CN adhesive on PVC

For the quantification of the compounds in PVC receptors that came from CN adhesive it was carried out a calibration curve, doping the PVC with analytes at different concentration levels and then analyzed by HS-SPME-GC-MS. The analytical characteristics of the quantification method are shown in Table 9.

Table 9. Characteristics of the method of quantification of the analytes of the CN adhesive on PVC..


(µg/g) Lineality

(R2) LOD (µg/g)

LOQ (µg/g)

RSD (%)

Toluene 0,022-6,10 0,982 0,006 0,022 31,1 1,2,4-Trimethylcyclohexane

0,811-4,37

0,992 0,243 0,811 34,1

4-Tertbutyltoluene 0,042-6,09 0,993 0,013 0,042 28,9 1,3-Diethylbenzene 0,300-6,18 0,994 0,090 0,300 25,9 1-Methyl-4-(1-methylethyl)benzene

0,252-6,26 0,991 0,075 0,252 21,3

Phenyl isotiocyanate 0,570-36,23 0,998 0,171 0,570 7,0 Phenylcyclohexane 0,185-6,26 0,999 0,056 0,185 8,7 BHT 0,281-6,20 0,996 0,084 0,281 8,5

* The RSD shown corresponds to the point, 1,85 µg/g and for the phenyl isothiocyanate to the point of 24.2 µg/g.


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It is observed that when working with PVC, the LOD and LOQ of phenylcyclohexane increases as regards on paper. Toluene and 1,2,4-trimethylcyclohexane have a RSD over 30%. This is because PVC is a complex matrix non homogenous.

3.6.6. Diffusion curves of identified CN adhesive compounds in PVC

This section represents the diffusion curves of identified CN adhesive compounds in PVC. It was performed a PVC blank, by analyzing three sheets of 5x5 cm2 by SPME. The major compounds that were identified in the PVC:

• 2-ethyl-1-hexanol. • 1,1'-oxybis-octane. • Phthalic acid, decyl isobutyl ester. • Linear alkanes between C 16-C 22.

All are of toxicity grade I. The only compound that diffused in the test conditions was the BHT while the rest of the compounds were below LOD, what indicated a slow diffusion of these compounds in PVC. These data confirm that PVC is a crystalline polymer, with more ordered structure than paper that leaves few spaces for the diffusion. Figure 4 shows the diffusion curves of BHT in PVC at two temperatures and two times and its concentration in the paper blank. It was noted that the diffusion coefficient increased with increasing temperature and testing time, at a temperature 60ºC, the analyte diffused to the last sheet of PVC. The concentration in the first sheets of PVC was slightly higher than the equilibrium value (table 11).

Figure 4. Diffusion tests of CN adhesive compounds in PVC at different temperatures and

times.


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3.6.7. Equilibrium concentrations of CS adhesive analytes in paper

Table 10 shows the equilibrium concentration of the identified CS adhesive compounds in the paper support.

Compound Concentration40º

(µg/g) Concentration60º

(µg/g) 4-Cyanocyclohexene 0,227±0,00014 0,370±0,034 4-Phenylcyclohexene 0,327±0,0240 0,128±0,021 3,4-Divinyl-1-Phenylcyclohexane (1R,3trans,4trans)_1 0,009±0,0003 0,008±0,000009 3,4-Divinyl-1-Phenylcyclohexane (1R,3trans,4trans)_2 0,049±0,0054 0,034±0,0023 3,4-Divinyl-1-Phenylcyclohexane (1R,3trans,4trans)_3 0,051±0,0024 0,037±0,0011 3,4-Divinyl-1-phenylcyclohexane(1R,3cis,4cis)_1 0,020±0,0016 0,014±0,00008

Table 10. Concentration of the CS adhesive analytes identified in paper.

All other compounds were below the LOQ. The equilibrium concentration of the analytes in the respective substrates is conditioned by the polarity and the structure and molecular weight of both the substrate and of the analytes tested. The compounds analyzed are apolar like the paper, which is a substrate composed mainly of cellulose, therefore the tendency to migrate is more favored than in polar substrates. It is noted that the equilibrium concentration in the paper decreases with increasing temperature except for 4-cyanocyclohexene. This compound is the most toxic of those found in the adhesives tested and also was in the highest concentration, so it is one of the most important for controlling its migration. It is noteworthy that the four isomers analyzed only differ in the position of the vinyl substituent. Therefore have the same molecular weight and polarity differing, only in the position of the substituents. Because of this, their behavior in the paper is very similar in all cases. The equilibrium concentration is lower than the previous two compounds and their equilibrium concentration decreases with increasing temperature.


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3.6.8. Partition constants of the CS adhesive analytes in paper

Table 11 shows the partition constants at two different temperatures of the CS adhesive analytes in paper.

Compound

4-Cyanocyclohexene 200,7 ± 4,100 107,8± 13,2 4-Phenylcyclohexene 27,0±0,0238 115,7±25,4 3,4-Divinyl-1-Phenylcyclohexane (1R,3trans,4trans)_1 0,1±0,0003 3,7±0,071 3,4-Divinyl-1-Phenylcyclohexane (1R,3trans,4trans)_2 4,1±0,0053 17,3±3,41 3,4-Divinyl-1-Phenylcyclohexane (1R,3trans,4trans)_3 8,9±0,0024 21,0±1,62 3,4-Divinyl-1-Phenylcyclohexane (1R,3cis,4cis)_1 2,8±0,0016 14,6±0,39

Table 11. Partition constants of the identified CS adhesive analytes in paper

The partition constants of the analytes identified increase with increasing temperature, except for the 4-cyanocyclohexene. This implies that as the temperature increases the equilibrium concentration in the adhesive increases and migration to the paper is less favored. The opposite occurs in the 4-cyanocyclohexene, i.e. its equilibrium concentration in the adhesive decreases with increasing temperature. Partition constants have a value greater than 1 for all analytes except 3,4-divinyl-1-Phenylcyclohexane (1R, 3trans, 4trans) _1 at 40 ° C, indicating a greater tendency of the compounds to remain in adhesive, specially the 4-Phenylcyclohexene and 4-cyanocyclohexene with high partition constant. For these compounds therefore it is estimated a reduced level of migration from the adhesive to paper, but also it should be noted that the initial concentration of the analytes (CP0) will determine the amount that migrated. It is noteworthy that the 3,4-divinyl-1-phenylcyclohexane (1R, 3trans, 4trans) _1, change their migration trend with increasing temperature.


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3.6.9. Equilibrium concentrations of identified CN adhesive analytes in PVC

Table 12 shows the equilibrium concentration of the compounds identified in the CN adhesive in PVC.

Compound Concentration40ºC

(µg/g) Concentration60ºC

(µg/g) 1-Metyl-4-(1-methylpropyl)benzene 0,244±0,039 0,184±0,004 Phenyl isotiocyanate 7,190±0,243 9,94±0,053 2-Ciclopentyl-1,3,5-trimetylbenzene 0,569±0,084 0,603±0,028 BHT 0,270±0,008 0,290±0,042

Table 12. Concentration of the compounds identified in the CN adhesive in PVC.

PVC is a polymer in which the presence of chlorine gives a polar character therefore polar organic substances have a greater tendency to migrate in PVC. This may explain that the equilibrium concentration of phenyl isothiocyanate is higher than other apolar compounds with similar molecular weight and initial concentration as 2-cyclopentyl-1, 3, 5-trimethylbenzene. The equilibrium concentration of BHT is low as regards phenyl isothiocyanate concentration. The two compounds have a group that gives them a certain polarity, but the molecular weight of phenyl isothiocyanate is much lower than that of BHT which encourages migration from the adhesive to PVC.

3.6.10. Equilibrium concentrations of identified CN adhesive analytes in PVC

Table 13 shows the partition constants at two different temperatures of the CN adhesive analytes in PVC.

Compound

1-Methyl-4-(1-methylpropyl)benzene 44,9±8,04 60,2±1,39 Phenyl Isotiocyanate 2,06±0,243 0,077±0,027

2-Cyclopentyl-1,3,5-trimethylbenzene 43,2±7,18 40,0±2,10 BHT 779,9±24,6 733,0±107,7

Table 13. Partition constants of the analytes identified CN adhesive analytes in PVC.

BHT, which is the compound found in highest concentration among those identified in the pure adhesive, has a high partition constant, which explains that its equilibrium concentration in the PVC is lower than the phenyl isothiocyanate or 2-cyclopentyl-1,3,5-trimethylbenzene, so that its tendency is to remain in the adhesive. The 1-methyl-4-(1-methylpropyl) benzene is the only compound which has a steady increase in partitioning constant with increasing temperature, which means that its equilibrium concentration in paper decreases. The opposite occurs for the phenyl isothiocyanate. The temperature varying doesn’t produce appreciable changes in the partition constant of the BHT and 2-cyclopentyl-1,3,5-trimethylbenzene.


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4. Discussion

In this project it has been developed an analytical method for the identification and quantification of compounds present in natural rubber adhesive and synthetic rubber adhesive, which are used in the manufacture of food contact materials. Also it has been determined the diffusion curves of these analytes in their respective substrates (paper and PVC) and the partition constants using the HS-SPME-GC-MS technique and solvent extraction of the laminates and subsequent analisys of the extracts by GC-MS. Firstly, several analytical extraction techniques were evaluated. Finally the SPME was the selected technique. Secondly, the most suitable fiber to carry out the tests was selected. In both cases PDMS fiber was selected because of its greater capacity to extract the compounds that constituent the adhesives. The SPME is affected by many variables, so that it took out an experimental design to optimize the most influential variables, extraction time, temperature of the sample and desorption time of the fiber. The experimental design was performed using the Modde software, applying a face-centered design (CCF). A total of 17 experiments were done reaching the following conclusions:

• Extraction temperature: maximun at 80 º C. • Extraction time: the maximum area extracted corresponded to 30 minutes. • Desorption time: there is not a significant variable and was fixed in 2.5 minutes.

After optimizing the test conditions, we applied this method to determine:

• Initial concentration of the CS adhesive (CP0). • Diffusion curves at 40 ° C, 60 º C for 24 h and 48 h for the identified analytes of CS

adhesive in paper and CN adhesive in PVC. • Partition constants at 40°C and 60ºC between adhesives and supports for the identified

analytes of CN and CS adhesives.

The determination of the CP0 of the CN adhesive wasn’t carried out by SPME but by extraction with hexane, since this adhesive was solvent base and entailed saturation problems in the fiber. Analytical characteristics were obtained from each of the methods of quantification, and it was concluded that the SPME showed lower detection limits than direct injection but higher RSD, especially in solid matrix such as PVC and paper, where the RSD increases substantially due to the heterogeneity of the substrate and the doped process. As for the compounds identified in the CS adhesive, it should be noted:

• Of the eleven compounds identified, four had low toxicity, six had medium toxicity and one high toxicity. It is noteworthy that all but 4-cyanocyclohexene and 4-vinylcyclohexene have as a substituent an aromatic ring and all except the 4-cyanocyclohexene are apolar.

• The 4-cyanocyclohexene has a III grade toxicity and a CP0 of 27 µg/g. It is the most toxic compound found in both adhesive and it is in higher concentrations than those identified in the CS adhesive.

As regards the compounds identified in the CN adhesive, it should be noted:

• As CS adhesive, most identified compounds containing an aromatic ring. • As regards the polarity, are all apolar except phenyl isothiocyanate and BHT. • Of the eight compounds identified, five had low toxicity and three had medium toxicity.


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• The BHT has a II grade toxicity and a CP0 of 165.5 µg/g, being the compound found in highest concentration among those identified.

• In general, the concentrations of the analytes identified in the CN adhesive are greater than those identified in CS adhesive.

In diffusion tests, the following conclusions were reached:

• The PVC and paper are heterogeneous substrate, which makes difficult the preparation of calibration curve for doping with standard solutions.

• Diffusion increases with temperature. • The diffusion of structurally similar compounds was more rapid in paper than in PVC,

which could be explained by paper is a more porous substrate. In addition to this, PVC is a crystalline polymer, so it's better barrier material than paper.

• Apolar compounds preferably defunded in paper and the polar in PVC.

Diffusion of CS adhesive in paper:

• In 60ºC tests, the concentrations were at or near equilibrium. The highest concentrations were found for the 4-phenylcyclohexene. The 4-cyanocyclohexene, III grade toxic compound didn’t diffuse under test conditions, what is considered very positive.

Diffusion of CN adhesive in PVC:

• The only compound that diffused in the test condition was the BHT, then the rest of analytes did not diffuse or they do very slowly.

In partition tests, the following conclusions were reached:

• In both adhesives, the equilibrium concentration in the adhesive of the majority of the compounds analyzed and therefore the partition constant, increased with temperature, implying that the substrate concentration decreased with temperature, except for 4-cyanocyclohexene (CS adhesive/paper) and phenyl isothiocyanate (CN adhesive/PVC), where the equilibrium concentration in the paper increased with increasing temperature, and hence its tendency to migrate to the substrate.

• The BHT (CN adhesive / PVC) and 4-cyanocyclohexene, presented the highest partition constant, being positive to minimize the migration to the substrate.

• The equilibrium concentration of phenyl isothiocyanate in PVC was the highest of the compounds analyzed in the two adhesives, because its partition constant was the lowest of all.

• The BHT, a compound that was in greater concentration among those identified, had a higher partition constant, which explains its equilibrium concentration in the PVC is lower than the rest of CN adhesive analytes.

As regards the supports the following conclusions were reached:

• A III grade toxic compound, 2,3-dihydro-3-phenyl-1,1,3-trimethyl-1H-indene was identified in paper.

• In the PVC, all the compounds detected have a I grade toxicity.


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5. Acknowledgements

This research has been developed under the European project MIGRESIVES COLL-CT-030309. I thank the grant received from the Institute of Engineering Research of Aragon (I3A).

References

Gavara, R. and R. Catalá, Migración de componentes y residuos de envases en contacto con alimentos. 2002: Instituto de Agroquímica y Tecnología de los Alimentos. CSIC, pp. 1-17.

Cramer, Estimation of Toxic Hazard - A Decision Tree Approach. J. Cosmet. Toxicol. Vol. 16. 1978, pp. 225-276.

Dole, P., Typical diffusion behaviour in packaging polymers-application to funtional barriers. Food additives & contaminants, 2006. 23:2, pp. 202-211.

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