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JRC100835

EUR 27816 EN

ISBN 978-92-79-63953-1

ISSN 1831-9424

doi:10.2788/90890

© European Union, 2016

Reproduction is authorised provided the source is acknowledged.

All images © European Union 2016

How to cite:

Anja Mieth, Eddo Hoekstra, Catherine Simoneau - Guidance for the identification of polymers in multilayer films used in food contact materials: User guide of selected practices to determine the nature of layers; EUR 27816 EN; doi:10.2788/10593

Guidance for the identification of polymers in multilayer films used in food contact materials

User guide of selected practices to determine the nature of layers

Abstract

This guidance describes how to characterize the composition of a multilayer plastic film for food packaging, with respect to the consecutive order of the layers and their identity. It provides background information on the general composition of multilayer plastic packaging and it illustrates in detail the separation of layers for some examples. It also provides in annexes additional information related to the use of a microtome and of optical microscopy using one common instrument for illustrative purposes.

1. Introduction

Being able to identify the composition of a packaging film is essential to confirm the correctness of specifications given in the Declaration of Compliance that accompanies all food contact materials made of plastic (see EU 10/2011 Art. 15 and Annex IV (3) [1]). Furthermore, each type of polymer, printing, coating and adhesive contains typical additives and monomers or oligomers with a potential for migration, while at the same time certain layers can act as functional barriers for particular migrants. Therefore, knowing the composition of a multilayer film enables laboratories to predict which migrants can be expected from the packaging film and thus also helps to check the plausibility of obtained migration results.

A PT was organised in 2013 which for the first time aimed at estimating performance of qualitative analysis. The aim of the exercise was to test the laboratories’ ability to identify unknown plastics materials via quick screening tools. The results demonstrated a spread and some level of difficulty for laboratories to identifying particular multilayer materials. A follow up was organised in 2014 specifically for multilayers, in order to ensure improved laboratory performance.

Considering there are no reference methods and the novelty to tackle assessments of qualitative nature, EU-RL FCM produced a number of methods descriptions to assist NRLs in doing this type of analysis. The descriptions have been integrated in the present guidelines.

2. Terms and definitions

AlOx

aluminium oxide

ATR

attenuated total reflection

BOPP

biaxially-oriented polypropylene

CN

cellulose nitrate (nitrocellulose)

DMSO

dimethyl sulfoxide

DSC

differential scanning calorimetry

EAA

ethylene acrylic acid copolymer

EB

electron beam

EMA

ethylene methyl acrylate copolymer

EVA

ethylene vinyl acetate copolymer

EVOH

ethylene vinyl alcohol copolymer

FTIR

Fourier Transform Infrared

HDPE

high density polyethylene

ionomers

EAA or EMA neutralised with cations (e.g. Na+, Zn2+, Li+)

LDPE

low density polyethylene

LLDPE

linear low density polyethylene

PA

polyamide

PA6

PA homopolymer made from caprolactam

PA6/66 PA

copolymer made from caprolactam, hexamethylenediamine and adipinic acid

PA6/MXD6

PA copolymer made from caprolactam, m-xylylenediamine and adipinic acid

PA66

PA homopolymer made from hexamethylenediamine and adipinic acid

PAMXD6

PA homopolymer made from caprolactam, m-xylylenediamine and adipinic acid

PC

polycarbonate

PE

polyethylene

PEN

polyethylene naphthalate

PET

polyethylene terephthalate

PET-G

glycol-modified polyethylene terephthalate

PMMA

polymethyl methacrylate

PP

polypropylene

PS

polystyrene

PTFE

polytetrafluorethylene

PUR

polyurethane

PVC

polyvinylchloride

PVDC

polyvinylidene chloride

SiOx

silicon oxide

UV

ultraviolet.

3 Analysing the composition of a multilayer plastic film: Separation and identification of layers

3.1 Scope

This procedure describes how to characterize the composition of a multilayer plastic film for food packaging, meaning the consecutive order of the layers and their identity. It provides background information on the general composition of multilayer plastic packaging and it illustrates in detail the separation of layers for some examples.

3.2 Materials and reagents

3.2.1 Organic solvents (all of reagent grade)

Acetone, butyl acetate, carbon disulfide, chloroform, cyclohexanone, cycloheptanone, di-n-butyl sulfoxide, N,N-dimethylacetamide, N,N-dimethylformamide, dimethyl sulfoxide, ethyl acetate, methylene chloride, methyl ethyl ketone, tetrahydrofuran, toluene, p-xylene

3.2.2 Organic and inorganic acids

• Formic acid, reagent grade, ≥95%

• Hydrochloric acid, reagent grade, 37%

• Sulfuric acid, reagent grade, 95-98 %

3.2.3 Reagents and materials for analytical tests

• Sodium hydroxide, reagent grade, ≥98%

• Diphenylamine

• Copper wire

3.2.4 Other materials

• Cotton sticks

• Paper towels

• Glass beads

3.3 Equipment

3.3.1 Microtome and polarization microscope

• Boeckeler S300 rotary microtome

• Zeiss Axioskop 2 MAT, equipped with AxioCam MRc

3.3.2 ATR-FTIR system

• Perkin Elmer Spectrum One FT-IR Spectrometer

• Perkin Elmer Universal ATR Sampling Accessory

• Software: Spectrum Version 6.3.1, Perkin Elmer

3.3.3 DSC system

• TA Instruments DSC Q100

• TA Instruments DSC Refrigerated Cooling System

• Software: Advantage for Q Series Version 2.5, Thermal Advantage Release 4.7.3

3.3.4 Glassware and other laboratory equipment

• 25 mL and 50 mL glass beakers

• Watch glasses, diameter 4-5 cm

• Glass test tubes

• Tweezers

• Scissors

• Heating plate

• Bunsen burner

3.4 General composition of multilayer packaging films

3.4.1 Plastic polymers

Several plastic polymers, copolymers and blends are used for the production of multilayers. The most important polymers are polyethylene (LDPE, HDPE, LLDPE), polypropylene, polyamide (PA6, PA6/66, PA6/MXD6), polyethylene terephthalate, polystyrene, ethylene vinyl alcohol, polyvinylidene chloride, ethylene vinyl acetate and polycarbonate ([2]-[14]). Polyvinyl chloride may also be used as a plastic layer in multilayer food packaging ([1]). Table 1 lists the most common polymers in multilayer packaging materials, their functions in the packaging and some applications.

Table 1 Plastic polymers used in multilayer food packaging ([1], [4], [5], [6], [8], [9], [18], [26], [27], [28], [35])

Plastic polymer

Functions in multilayers

Applications

Polyethylene (PE)

heat-sealable food contact layer

moisture barrier

can be combined with gas/aroma barriers (e.g. PA, EVOH)

breathable packaging for fresh produce (LDPE, HDPE) carton liners (LLDPE)

Polypropylene (PP)

moisture barrier

to provide mechanical strength

can be coated with heat seal coatings (PVDC, acrylate)

can be combined with gas/aroma barriers (e.g. PVDC coatings, PA, EVOH)

modified atmosphere packaging thermoformed containers for microwavable packaging, hot-filled packaging

Polyamide (PA)

gas/aroma barrier

to provide mechanical strength

heat resistance

can be used as outside layer of a heat seal film → film will not stick to the sealing bar surface

boil-in-bag packaging

thermoformed packaging

Polyethylene terephthalate (PET)

gas/aroma barrier

moisture barrier

to provide mechanical strength

heat resistance

plastic bottles for carbonated softdrinks, meat and cheese packaging, snack food wrapper boil-in-bag, sterilisable pouches, ovenware containers

Polystyrene (PS)

gas permeability

printability

can be combined with gas/aroma barriers (coextruded or laminated) → commercially available structures: e.g. PS/PVDC/PS, PS/PVDC/PE, PS/EVOH/PE, PS/EVOH/PP

breathable packaging for fresh produce (e.g. fresh-meat packaging) printable outside layers

Ethylene vinyl alcohol (EVOH)

oxygen barrier needs to be protected from moisture →

often sandwiched (coextruded) between PE or PP, in some applications also sandwiched between PET, PA or PS

modified atmosphere packaging packing of oxygen-sensitive food

Polyvinylidene chloride (PVDC)

gas/aroma and/or moisture barrier

to protect the surface from scratches and abrasion

heat-sealable food contact layer

often copolymers of vinylidene chloride and ester-type monomers (e.g. ethyl acrylate)

modified atmosphere packaging

applied as coating or coextruded film

Ethylene vinyl acetate (EVA)

moisture barrier

adhesion layer (tie layer) for co-extrusion of polar (e.g. PA, PET-G) and non-polar (e.g. PE) polymers

heat-sealable food contact layer; heat-sealable extrusion coatings on PET or BOPP films

modified atmosphere packaging

applied as coating or coextruded film

Polycarbonate (PC)

heat resistance

mechanical strength

moisture barrier

microwavable packaging, hot-filled packaging

modified atmosphere packaging

barriers for fruit juice cartons

Polyvinylchloride (PVC)

gas/aroma barrier mechanical strength

fresh food packaging (e.g. PVC/PE films)

modified atmosphere packaging (e.g. PVC/EVOH/PE films)

Polyethylene naphthalate (PEN)

gas/aroma and moisture barrier heat resistance

for hot refills, rewashing, reuse

beverage bottles (e.g. beer)

Glycol modified polyethylene terephthalate (PET-G)

heat-sealable food contact layer

Ethylene acrylic acid (EAA)

extrusion coating tie layer between aluminium foil and other polymers heat-sealable food contact layer

3.4.2 Aluminium foil

Many packaging materials, especially for high value foods (e.g. dried soups, herbs, spices), contain a layer of aluminium foil as it is an effective barrier against moisture, air, odours and UV light [26].

3.4.3 Bonding of the different layers

Multilayer films can be prepared by co-extrusion or by lamination. Co-extrusion can be performed as blown film extrusion or cast extrusion [14]. In both cases, granules of the different plastics are melted separately and the melts are brought together in the extruder. The combined plastics then are extruded as a single product in which the co-extruded layers bond directly to each other.

In order to achieve bonding of two molten polymer layers during co-extrusion, it can be necessary to apply a third polymer (“extrudable adhesive”) in between. For example, anhydride-modified polyethylene or anhydride-modified EVA can act as an extrudable adhesive in the co-extrusion of PE and PA [9].

In contrast to co-extrusion, lamination combines two or more plastic or non-plastic materials (e.g. aluminium foil) in web-form by applying a certain type of adhesive between them. Three main principles of lamination can be distinguished. These are extrusion lamination, adhesive lamination and wax/hot melt lamination. In extrusion lamination, a molten polymer (e.g. LDPE) is applied to one of the webs and a second web is fed from the top. In adhesive lamination, an adhesive is applied instead. These can be solvent based adhesives (“dry bond lamination”), one- or two-component water based adhesives (“wet-bond adhesion”), two-component solid adhesives (“solventless lamination”), UV-curing or electron beam-curing adhesives ([14], [25]).

In wax/hot melt lamination, a layer of molten wax or hot melt is applied between two webs and then cooled to bond them together. The wax remains heat sensitive. Therefore this technique is not suitable if the packaging will be heat-sealed afterwards or filled very hot ([9], [14]).

Table 2 lists some of the most important adhesives for the different types of lamination and their applications.

Table 2 Adhesives and waxes/hot melts used for lamination ([9], [14], [39], [40])

Type of lamination

Adhesive

Examples of selected applications

extrusion lamination

LDPE

multilayer-multimaterial packaging (combination of plastic or aluminium foil with paper or paperboard → e.g. fruit juice cartons)

EVA

bonding PE to PVC

acid-containing adhesives:

EAA or EMA

ionomers

terpolymers of ethylene, acid and acrylate (e.g. methyl acrylate or isobutyl acrylate)

used to bond with aluminium foil

anhydride modified polyolefins: terpolymers of ethylene, maleic anhydride and acrylates (e.g. ethyl acrylate, butyl acrylate)

dry bond lamination

solvent based PUR adhesives

flexible packaging → lamination of plastic films (bags, pouches, wraps for snack food, meat and cheese packs, boil-in-bag food pouches)

wet-bond adhesion

poly(vinyl acetate) emulsion (or coploymers of vinyl acetate and ethylene or acrylic esters)

crosslinking acrylic-vinyl acetate copolymer emulsions

snack packages (e.g. potato chip bags)

acrylic emulsion pressure-sensitive adhesives

pressure-sensitive labels

polyurethane dispersions

lamination of plastic films in medium-performance flexible packaging where some chemical resistance is required

UV-curing

acrylics and acrylates, epoxies, polyurethanes, polyesters, silicones, and vinyl and vinyl esters

flexible plastic packaging (at least one of the webs must be transparent to allow UV penetration)

EB-curing

see UV-curable adhesives

solventless lamination

polyurethanes

hot-melt lamination

EVA

ethylene-butyl acrylate

LDPE

for paper bonding constructions, case seaming, bag seaming and sealing

block copolymers of styrene, butadiene or isoprene

hot-melt pressure-sensitive adhesives for tapes and labels, attachment of PE-base cups to polyester soft-drink bottles, sealing of film-laminated frozen-food cartons

moisture curing PUR hot melts

3.4.4 Coatings/lacquers

Coatings or lacquers can be applied to the inside (food contact layer), outside (non-food contact layer) or in between layers to enhance the appearance or alter the physical properties of the packaging [17]. An overview of the most common types of coatings is given in Table 3.

Table 3 Coatings used in multilayer plastic food packaging ([8], [14], [16], [17], [26], [31]). *Ionomers: EAA or EMA neutralised with cations (e.g. Na⁺, Zn²⁺, Li⁺)

Type of coating

Purpose

Substances in use

protective coatings

to protect the surface from mechanical damage

nitrocellulose coatings, epoxy resins, reactive polyurethane systems, ionomers

heat-seal coatings

to allow heat sealability for non-sealable materials (e.g. flexible aluminium lids for yoghurt containers, polypropylene)

EAA, EVA, PVDC, PMMA, ionomers*

primers

to improve the bond between a substrate and an otherwise incompatible coating

polyethyleneimines, EAA dispersions, reactive polyethylene amines

cold seal coatings

coatings that can be sealed self-to-self using just pressure (e.g. for flow-wrapping heat-sensitive products like chocolate or ice cream)

blends of acrylic resins and latex; synthetic rubber

release lacquers

applied to the opposite surface of a plastic film that is coated with coldseal to prevent sticking and to promote easy unwinding from the reel

PA-based coatings

antimists

to prevent formation of condensate droplets/fogging on the food contact side (e.g. in fresh salad packaging)

PVDC coatings

gloss/matt coatings

to alter the visual perception of parts of the design

PVDC, PMMA, nitrocellulose coatings

gas and moisture barrier

barrier to gases/odours/flavours and moisture

PVDC, PMMA, SiOx, AlOx, aluminium vapour coating

UV light barrier

barrier to UV light

aluminium vapour coating

3.4.5 Non-food contact printings

Parts of the sample may also be covered with printings. A printing can be applied as a surface printing on the outside (i.e. non-food contact side) of the multilayer packaging or as a reverse printing where it is trapped between two layers of the multilayer packaging ([25], [29]). In reverse printing, the printing is usually applied to the inside surface of the outermost plastic layer of the packaging ([25]) (see Figure 1). This way, the printing is best protected against mechanical damage (scratching, rubbing) and moisture, as well as set-off (i.e. transfer of printing ink components to the food contact layer when the packaging film is stored in staples or rolled) ([29]).

Figure 1 Laminate with reverse printing (PA/print//PURPE) after boiling in ethyl acetate for 2 min

To protect surface printings (e.g. improve rub resistance) and to provide gloss or matt effects, overprint coatings can be applied. They have the same composition as the corresponding inks but do not contain pigments [15].

An overview on the main compositions of the most commonly used ink systems is provided in Table 4.

Table 4 Printing ink systems for non-food contact applications ([16], [19], [20], [21], [22])

Printing ink system

Principle composition

surface printing inks

solvent-based inks

pigments

binders: nitrocellulose, maleic resin, polyvinyl butyral, polyamide, polyurethane

solvents: alcohols (ethanol, isopropanol), esters (ethyl acetate, isopropyl acetate), ethoxypropanol

additives: plasticisers, slip additives (lubricants), adhesion promoters

UV-curing inks (free radical curing)

Pigments

vehicles:

oligomers: epoxy acrylates, polyester acrylates, polyether acrylates, urethane acrylates;

monomers/reactive diluents: di-, tri-, tetrafunctional acrylates

photoinitiators

additives: waxes (PE/PTFE waxes), silicone oils, stabilisers

cationic UV-curing inks

pigments

cationic photoinitiators: triarylsulphonium salts, diaryliodonium salts

vehicles: diepoxide compounds and vinyl ethers additives

electron beam-curing inks

pigments

vehicles:

oligomers: epoxy acrylates, polyester acrylates, polyether acrylates, urethane acrylates

monomers/reactive diluents: di-, tri-, tetrafunctional acrylates additives

water-based inks

pigments

binders: styrene-acrylic co-polymers, acrylic co-polymers, maleic resins

solvents: water, isopropanol, glycol ether, propylene glycol

additives: amines, biocides, defoamers, wetting agents, waxes (PE, PTFE), slip agents

Reverse printing inks

solvent-based inks

pigments

binders: vinyl resins, polyurethane, nitrocellulose, polyamide

solvents: alcohols (ethanol, isopropanol), esters (ethyl acetate, isopropyl acetate), ethoxypropanol

additives

water-based inks

pigments

binder: acrylic resin

solvents: water

additives

3.4.6 Direct food contact printings

For promotional reasons (e.g. special campaigns), sometimes also parts of the food contact layer may be printed (see Figure 2). For this purpose, special “direct food contact printing inks” are used. These are usually pigmented coatings based on vinyl resins or modified cellulose ([16], [23], [24]).

Figure 2 Chocolate packaging with printing on the food contact layer

3.5 Analysis of the composition of a Multilayer film

3.5.1 Microtome sectioning and microscopy

It is recommended to prepare a microtome section (usually of 5 μm thickness) of the sample first and investigate it using a transmitted light microscope with polarized light. This will allow you to see of how many layers the sample consists of and whether they are co-extruded or laminated. Adhesives in laminated samples are visible in the microtome section as thin layers of about 1-3 μm thickness between the polymer films.

The coloration of the different layers in polarized light also gives a first idea about their identity. Transparent polyamide layers may turn green in polarized light and polyethylene terephthalate layers may appear in rainbow colours, while polyethylene or polypropylene remain colourless (see Figure 3).

Figure 3 Microtome sections of multilayer plastic laminates in polarised light

3.5.2 Identification of food contact layer and outside layer by ATR-FTIR

To identify the food contact layer (inside) and the outermost non-food contact (outside) layer of the sample, ATR-FTIR spectra of the inside and outside of the multilayer film are recorded.

ATR-FTIR settings:

spectral range:

4000-600 cm⁻¹

number of scans:

4

background:

air (4 scans, 4000-600 cm⁻¹)

resolution:

4 cm⁻¹

The spectra are identified by comparison to the spectra database, other available standard spectra and the information provided in section 0 Table 8.

3.5.3 Separation of layers

The separation of the different layers can be achieved by taking advantage of their different solubility properties. Having identified the food contact layer and the outermost layer (coating, printing ink or polymer film), a proper solvent or acid can be selected to dissolve and remove these outer layers. Alternatively, it is possible to split laminated layers by dissolving the adhesive in between and to split samples that contain an aluminium layer by dissolving the aluminium.

3.5.3.1 Selection of solvents

Solvents for plastic polymers

An overview about the solubility of the most common plastic polymers used for the production of flexible packaging is given in Table 5.

Table 5 Solvents and acids suitable to dissolve plastic polymers ([32], [33], [34])

Polymer/coating

Soluble/decomposed in

Polyethylene (PE)

boiling p-xylene, boiling toluene, boiling dimethyl sulfoxide

Polypropylene (PP)

boiling toluene, boiling dimethyl sulfoxide

Ethylene vinyl alcohol (EVOH)

boiling N,N-dimethylformamide, hot concentrated sulfuric acid (turns black)

Polyamide (PA)

hot concentrated formic acid, 37% hydrochloric acid, hot concentrated sulfuric acid (96 w%), boiling N,N-dimethylformamide

Polyethylene terephthalate (PET)

boiling N,N-dimethylformamide (swells), hot concentrated sulfuric acid

Polystyrene (PS)

toluene, chloroform, cyclohexanone, butyl acetate, carbon disulfide, acetone, methylene chloride, ethyl acetate, boiling N,N-dimethylformamide*, hot concentrated sulfuric acid (96 w%) (turns black)

Polyvinylchloride (PVC)

tetrahydrofuran, cyclohexanone, N,N-dimethylformamide, methyl ethyl ketone

Polyvinylidene chloride (PVDC)

boiling xylene, boiling N,N-dimethylacetamide, boiling cycloheptanone, di-n-butyl sulfoxide

Polycarbonate (PC)

methylene chloride, boiling N,N-dimethylformamide*, boiling dimethylsulfoxide*, hot concentrated sulfuric acid (96 w%) (turns black), acetone (swells), boiling toluene (swells)

Ethylene vinyl acetate (EVA)

toluene

*requires strong heating and long time

Solvents for adhesives in laminated samples (PUR adhesives, Polyester acrylates)

Most commonly used adhesives in the manufacturing of flexible food packaging are polyurethane (PUR) and acrylic or acrylate based adhesives. PUR adhesives can be easily dissolved in formic acid. Note that this procedure might also dissolve possible polyamide layers and others soluble in formic acid. An alternative can be to use ethyl acetate or other suitable solvents instead (see Table 6).

Note that all samples which contain an aluminium foil or an aluminium vapour coating as well as reverse printed samples are somehow laminated to bond the aluminium layer or reverse printed film, respectively, to the other plastic layers. In conclusion, one can always try to immerse any sample containing an aluminium layer or any reverse printed sample in formic acid or ethyl acetate (or other suitable solvents) to dissolve the adhesive layers (see Figure 4).

Figure 4 Sample (PET/print//PURAl foil//PURPE) before and after boiling in HCOOH for 5 min. Resulting 3 separated layers: PET/print, Al foil, PE (top and bottom view shown)

Table 6 Solvents and acids suitable to dissolve adhesive ([32], [33])

Polymer/coating

Soluble/decomposed in

Polyurethane (PUR)

formic acid, methylene chloride (swells), acetone (swells), ethyl acetate (swells)

Polyacrylates

boiling xylene, toluene, methylene chloride, acetone, ethyl acetate

Dissolving aluminium foil or aluminium vapour coatings

An aluminium foil or an aluminium vapour coating (i.e. a thin aluminium layer applied by thermal evaporation) itself can be dissolved by immersion in concentrated hydrochloric acid (37 % w/w). If the complete sample is immersed, the sample will split in two parts, the one above and the one below the aluminium layer (see Figure 5).

Figure 5 Sample with an inner aluminium foil before and after boiling in conc. HCl for 4 min (Al is partly dissolved) and 5 min (Al is completely dissolved)

Removal of printings and coatings

Ink films and coatings often can be wiped off with a paper towel or cotton stick soaked in a suitable solvent. For example, acrylate and nitrocellulose coatings usually can be wiped off with ethyl acetate. If this is not successful, the sample can be immersed in solvent and, if necessary, heated until the coating is completely dissolved (NOTE: sometimes additional rubbing with a paper towel is required).

An overview about suitable solvents for the most common coatings and printing ink films is provided in Table 7.

Table 7 Solvents and acids suitable to dissolve binders of printing inks and coatings ([32], [33], [34])

Polymer/coating

Soluble/decomposed in

Polyvinylidene chloride (PVDC)

boiling xylene, boiling N,N-dimethylacetamide, boiling cycloheptanone, di-n-butyl sulfoxide

Ethylene vinyl acetate (EVA)

toluene

Nitrocellulose (CN)

ethyl acetate, acetone

Polyacrylates

boiling xylene, toluene, methylene chloride, acetone, ethyl acetate

Polymethyl methacrylate (PMMA)

toluene, methylene chloride, acetone, ethyl acetate

Polyurethane (PUR)

formic acid, methylene chloride (swells), acetone (swells), ethyl acetate (swells)

Aluminium metallisation

37% hydrochloric acid

3.5.3.2 Immersing the sample in solvent

Cut a sample piece of about 1 cm × 3 cm. It should be of asymmetric shape (e.g. trapezoid) to distinguish between the inner and outer side of the multilayer (and later on the layers peeled off) (see Figure 6).

Figure 6 Sample preparation

Approximately 5 to 10 mL of solvent or acid (selected according to 0) are filled into a 25 or 50 mL glass beaker. To prevent boiling retardation later on, add 2-3 glass beads. The sample piece is added (use tweezers!) and the beaker is covered with a watch glass. Never put more than 1 sample piece into the beaker at the same time. Otherwise there is a risk to lose track of which sample eventually split off layers belong.

The beaker is placed on a hot plate and heated carefully to make the solvent boil. Depending on the type of plastic, it may take up to 10-15 min until the respective sample layer is dissolved completely in the boiling solvent. However, often it is useful to immerse the sample only for a short time until parts of the respective layer are dissolved and then to tear or peel off the upper layer. Therefore, check every 1-2 minutes whether parts of the sample are already swollen or dissolved (especially at the edges of the sample piece). If so, take out the sample with a pair of tweezers, rinse it with a non-toxic solvent (e.g. ethanol) or distilled water to remove adhering solvent and acid, respectively, dry it with a paper towel and then try to peel off the upper layer using some tweezers. Do not forget to remove the beaker from the hot plate when you are finished. Heating can also be performed in glass test tubes using a Bunsen burner. This process is faster but more difficult to control.

Some coatings and adhesives as well as aluminium foil or aluminium vapour coatings are dissolvable already at room temperature but the process may take 1-24 h. By additional heating (hot plate or Bunsen burner), the process can be accelerated and shortened to a couple of minutes.

3.5.4 Identification of the layers peeled off and laid open (ATR-FTIR spectroscopy)

Once a layer is peeled off, record an ATR-FTIR spectrum of the surface laid open. It is advisable to record also ATR-FTIR spectra of top and bottom of the layer peeled off. This will help to make sure that the layer peeled off was a monolayer.

If the respective layers were laminated, the adhesive was laid open. It may stick to both sides, i.e. the upper layer peeled off and also to the remaining multilayer (see Figure 7).

Figure 7 PUR adhesive sticks to the aluminium foil and partly also to the PA layer after separation

ATR-FTIR settings:

spectral range:

4000-600 cm⁻¹

number of scans:

4

background:

air (4 scans, 4000-600 cm⁻¹)

resolution:

4 cm⁻¹

The spectra are identified by comparison to the spectra database or other available standard spectra. An overview on characteristic bands in FTIR spectra of some plastic polymers, binders for printing inks and adhesives is also provided in Table 8.

Table 8 Characteristic bands in FTIR spectra ([32], [37], [41], [42]).

Polymer

ATR-FTIR spectrum

Vibration*

Wave number** [cm⁻¹]

Polyethylene

νaCH₂ and νsCH₂

δCH₂

ρCH₂

3000-2840

1463

725

Polypropylene

νaCH₂ and νsCH₂

νaCH₃ and νsCH₃

δCH₂ and δaCH₃

δsCH₃

ρCH₃

νC-C

ρCH₂

3000-2840

3000-2840

1459

1376

1167

998, 973

840

Polyethylene terephthalate

ν=CH

νaCH₂ and νsCH₂

νC=O

νPh

νC(=O)O and δ=CH

νO-C and δ=CH

δ=CH

γ=CH

γPh

3150-3000

3000-2840

1718

1600-1325

1260

1100

1018

971, 872

726

Polystyrene

ν=CH

νaCH₂ and νsCH₂

overtones

νPh

δ=CH

γ=CH

γPh

3150-3000

3000-2840

2000-1660

1600-1375

1180, 1154, 1069, 1028

906, 842, 758

700, 540

Polycarbonate

ν=CH

νaCH₃ and νsCH₃

νC=O

νPh

νaC-O-C

δ=CH

γ=CH

3150-3000

3000-2840

1773

1506

1228

1192, 1162, 1126, 1015

831

Polyamide 6

νNH

νaCH₂

νsCH₂

νC=O

δNH and νCN

δCH₂

ωCH₂ and τCH₂

γNH and γC=O

3303

2935

2860

1635

1539, 1276

1465

1200

690

Ethylene vinyl acetate

νaCH₂ and νsCH₂

νaCH₃ and νsCH₃

νC=O

δCH₂ and δaCH₃

δsCH₃

νC(=O)O

νO-C and ρCH₃

ρCH₂

3000-2840

3000-2840

1740

1469

1371

1241

1020

720

Nitrocellulose (cellulose nitrate)

νOH...O

νaCH₂ and νsCH₂

νaNO₂

νsNO₂

νC-O

ρCH₂

3400

3000-2840

1654

1279

1063

841

νC(=O)O

νO-C

νN-C

ρCH₂

1223

1111

1000

809

Polyvinylidene chloride

νaCH₂ and νsCH₂

δCH₂

γCH₂

νsC-C

νCCl₂

3000-2840

1405

1064

1044

600, 655

Polyester acrylate

R:    aromatic polyester

ν=CH (aryl)

νaCH₂ and νsCH₂

νC=O

νC=C (vinyl ester)

νPh

δCH₂

δC=C (vinyl ester)

νC(=O)O

νasC(=O)-O-C (acrylate polymer)

γC=C (vinyl ester)

γPh

3150-3000

3000-2840

1728

1636***, 1610***

1600-1325

1453

1408***

1242

1163

810***

726

*:     νa: antisymmetric stretching vibration, νs: symmetric stretching vibration, δ: in-plane deformation, γ: out-of-plane, ρ: rocking vibration, ω: wagging vibration, τ: twisting vibration

**:     The wave numbers given refer to transmission spectra and differ slightly from those obtained by ATR measurement.)

***:     The characteristic vinyl ester moieties CH2=CH-C(=O)-O~ of UV/EB curable acrylate printings or adhesives react upon curing and form the acrylate polymer backbone. As a consequence, the respective characteristic vinyl ester bands at 1636 (νC=C), 1610 (νC=C), 1408 (δC=C) and 810 (γC=C) cm⁻¹ decrease and are no longer present/barely-there if the printing ink or adhesive was well cured ([41], [42], [43], [44]).

3.5.5 Specification of layers with DSC

The separated polymer layers can be further characterised by their melting points with differential scanning calorimetry (DSC) to confirm the results obtained by ATR-FTIR and to specify the findings. DSC will enable to distinguish between LDPE, LLDPE and HDPE as well as PA 6, PA 66 and PA MXD6 and blends of different polymers [30]. You can also perform a DSC of the complete laminate. This will give an idea about the types of plastic polymers that are contained in the multilayer sample (see Figure 8).

Figure 8 DSC diagrams of a laminate (PET/acrylate printing//PURAl foil//PURPA//PURPE) and its single polymer layers (PET, PA, PE) after separation

DSC settings:

Start temperature:

50°C

End temperature:

300°C

Heating rate:

20°C/min

Reference:

air

Amount of sample:

for separated monolayers: 0.5-2.5 mg

for multilayer samples: 10 mg

An overview about melting temperatures of some common plastic polymers in flexible packaging is provided in Table 9.

Table 9 Glass transition (Tg) and crystalline melting temperatures (Tm) of some common plastic polymers ([30], [36], [38])

Polymer

Tg [°C]

Tm [°C]

105-113 (LDPE)

Polyethylene

-20

120 (LLDPE)

135 (HDPE)

Polypropylene

5

160

Polyvinylchloride

80

-

Polyvinylidene chloride

-18

190

Atactic polystyrene

95

-

Isotactic polystyrene

100

230

Polyethylene terephthalate

67

256

Polycarbonate (made of Bisphenol A)

150

225

Polyamide 6

50

215

Polyamide 66

60

266

Ethylene vinyl acetate

80-110 (depends on EA content)

3.5.6 Special analytical tests for the identification of printing inks and coatings

Besides identification with ATR-FTIR spectroscopy, some of the printing inks and coatings can also be identified by simple chemical tests.

Nitrocellulose (cellulose nitrate, CN) - Diphenylamine test

To prepare the reagent, 0.1 g diphenylamine are suspended in 30 mL water and then 100 mL concentrated sulfuric acid are added carefully. A drop of the freshly prepared solution is applied to the sample surface. The reaction is positive for nitrocellulose if the reagent turns dark blue. [33]

Polyvinylidene chloride (PVDC) – Reaction with pyridine and Beilstein test

A drop of pyridine is applied to the sample surface. After 2-5 min, a drop of 5% w/v methanolic sodium hydroxide solution is added (1 g sodium hydroxide dissolved in 20 mL methanol). The reaction is positive for PVDC if the surface turns brown-black immediately. PVC instead would show a red-brown colour first, and after approx. 5 min the colour would turn into black-brown. [33].

In addition, the Beilstein test can be performed to proof the presence of chlorine. A copper wire is heated in a Bunsen flame until the flame is colourless. The wire is allowed to cool down. Then a piece of the sample is placed on it (the chlorine containing layer should face the wire) and the copper wire plus sample are heated at the edge of the colourless part of the Bunsen flame. A green or blue-green flame will indicate the presence of chlorine when the sample burns. [33]

3.6 Examples

Laminate 1 (PET/polyester acrylate printing//PURAl foil//PURPA//PURPE)

Sample:

Step I: General observations

– The sample is printed (not yet known whether it is a surface or reverse printing)

– The sample contains an aluminium layer (not yet known whether it is an aluminium foil or an aluminium vapour coating).

Step II: Microtome section (5 μm thickness)

The microtome section displays that:

– The sample consists of 3 different polymer films and an aluminium foil. Polymer film 1 appears in rainbow colours in polarized light and therefore is likely to consist of PET. Polymer film 2 appears green, so it might be a polyamide layer.

– The printing was applied as a reverse print on the bottom side of polymer film 1.

– There are no co-extruded layers, all layers are laminated with adhesives.

– schematic composition:

polymer 1

print

adhesive

aluminium foil

adhesive

polymer 2

adhesive

polymer 3

Step III: ATR-FTIR of the inside (food contact layer) and outside (non-food contact)

Results:

– The outside layer consists of PET.

– The food contact layer consists of PE.

Step IV: Separation of layers

From the information gained in step I-III, the following options arise to start the separation of the layers:

1) The food contact layer consists of PE. → Try to dissolve/swell the PE layer in DMSO, toluene or xylene and remove it.

2) The sample contains an aluminium foil. → Try to dissolve the aluminium in conc. HCl to split the sample.

3+4) The sample contains an aluminium foil. The plastic films on top of and below the aluminium foil must be somehow adhered to it. → Try to dissolve the adhesive on top of and below the aluminium foil in conc. HCOOH or ethyl acetate to split the sample.

Step IV: Separation of layers – option 1 – start with DMSO

Cut a piece of the sample (complete laminate) (approx. 1 cm × 3 cm) and boil it for 1-2 min in DMSO to partly dissolve/swell the PE layer.

The edges of the PE layer are swollen and the adhesive that bonds polymer 2 to the aluminium foil is partly dissolved.

→ Part B (adhesive/polymer 2/adhesive/PE) can be peeled off from the bottom side of the Al foil. Part A (PET/print/adhesive/Al) cannot be further separated yet.

ATR-FTIR:

part A (top) = PET, part A (bottom) = no polymer/coating (aluminium),

part B (top) = polyester urethane (adhesive), part B (bottom) = PE

Remove the PUR adhesive on top of part B to check what is below:

Dip part B in cold HCOOH and take it out immediately(!) to dissolve the PUR adhesive. → Rub its top side with a paper towel to remove the remaining PUR adhesive. → ATR-FTIR: part B (top, PUR adh. removed) = PA

NOTE: If you immerse part B for >5 s in cold HCOOH, the PA will be dissolved. If you immerse it in hot HCOOH, the PA will be dissolved immediately.

Separate the PA and PE layer of part B:

Go back to step 3 → Having a closer look to the edges of part B, you can see that the PA layer (part B1) can be peeled off.

peel off part B1

ATR-FTIR:

part B1 (top) = polyester urethane (adhesive), part B1 (bottom) = PA,

part B2 (top) = polyester urethane (adhesive), part B2 (bottom) = PE

Separate the PA and PE layer of part B:

Go back to step 3 → If you were not able to peel off the PA layer (part B1) as shown in step 5-7, you can boil part B for 2-3 min in ethyl acetate → the PUR adhesive between PA and PE swells/is partly dissolved

peel off the PA layer (part B1)

ATR-FTIR:

part B1 (top) = polyester urethane (adhesive), part B1 (bottom) = PA,

part B2 (top) = polyester urethane (adhesive), part B2 (bottom) = PE

Remove the PUR adhesive on top of part B2 to check what is below:

boil part B2 for at least 15 min in HCOOH and/or acetone, rub its top side with a paper towel to remove the remaining PUR adhesive → ATR-FTIR: part B2 (top) = PE

Characterisation of the adhesive that bonds the reverse printed PET-film to the Al foil:

Go back to step 3 → boil part A for 30 s in conc. HCI

The Al foil on the bottom of part A is completely dissolved. ATR-FTIR: part A (bottom, Al foil removed) = polyester urethane (adhesive)

To confirm that the “white layer” on the bottom side of part A is a reverse printing and to check what is below, try to rub it off with a paper towel soaked in ethyl acetate (requires some force!). → PUR adhesive and the printing are removed. → ATR-FTIR: part A (bottom, print removed) = PET → same like outside → PET-monolayer

Characterisation of the printing:

Go back to step 3 → boil part A for 3-5 min DMSO → PET layer can be removed, printing remains on the aluminium foil → ATR-FTIR: aluminium foil (top) = polyester acrylate (printing)

Step IV: Separation of layers – option 2 – start with conc. HCl

Cut a piece of the sample (complete laminate) (approx. 1 cm × 3 cm) and boil it in conc. HCL to partly dissolve the aluminium foil

after 30 s the adhesive that laminates part A (PET/print) to the aluminium foil is (partly) dissolved, while the aluminium foil is only dissolved at its edges

→ peel off part A

→ ATR-FTIR: part A (top) = PET, part A (bottom) = polyester urethane (adhesive)

immerse part B again for 10 s in boiling conc. HCl → the aluminium layer is dissolved immediately → part B* (adhesive/polymer 2/adhesive/PE) remains → ATR-FTIR: part B* (top) = polyester urethane (adhesive), part B*(bottom) = PE

Separate polymer 2 and the PE layer:

boil part B* for 2 min in ethyl acetate to partly dissolve/swell the adhesive between polymer 2 and the PE layer

polymer 2 (part B1) became brittle already while boiling in conc. HCl → now it can be scratched off with a spatula

ATR-FTIR:

part B1 (top) = polyester urethane (adhesive), part B1 (bottom) = PA,

part B2 (top) = polyester urethane (adhesive), part B2 (bottom) = PE

Go back to step 2 – To confirm that the “white layer” on the bottom side of part A is a reverse printing and to check what is below, ethyl acetate (requires some force!). → PUR adhesive and the printing are removed. → ATR-FTIR: part A (bottom, print removed) = PET → same like outside → PET-monolayer

Step IV: Separation of layers – option 3 – start with conc. HCOOH

Cut a piece of the sample (complete laminate) (approx. 1 cm × 3 cm) and boil it for 1 min in conc. HCOOH to partly to partly dissolve the aluminium foil.

(Attention: If you boil the sample for >5min, polymer 2 (PA) is dissolved as well.)

Part A (PET/print/adhesive) can be peeled off from the top of the aluminium foil...

...and part B (adhesive/polymer 2/adhesive/PE) can be peeled off from the bottom of the aluminium foil.

→ ATR-FTIR:

part A (top) = PET, part A (bottom) = polyester urethane (adhesive),

part B (top) = polyester urethane (adhesive), part B (bottom) = PE

Separate polymer 2 and the PE layer:

boil part B for 2 min in ethyl acetate to partly dissolve/swell the adhesive between polymer 2 and the PE layer

ATR-FTIR:

part B1 (top) = polyester urethane (adhesive), part B1 (bottom) = PA,

part B2 (top) = polyester urethane (adhesive), part B2 (bottom) = PE

Go back to step 3 – Remove the PUR adhesive and the acrylate printing on the bottom side of part A to check what is below and to confirm that the "white layer" is a reverse printing as described in option 2 step 7

Step IV: Separation of layers – option 4 – start with ethyl acetate

Cut a piece of the sample (complete laminate) (approx. 1 cm × 3 cm) and boil it for 1 min in ethyl acetate to partly to partly dissolve the aluminium foil.

The adhesive on the bottom of the aluminium foil is partly dissolved. → part B. (adhesive/polymer 2/adhesive/PE) can be peeled off from the bottom side of the Al foil.

→ ATR-FTIR:

part A (top) = PET, part A (bottom) = no polymer/coating (aluminium foil),

part B (top) = polyester urethane (adhesive), part B (bottom) = PE

Separate polymer 2 and the PE layer:

If necessary: Boil part B again for 1 min in ethyl acetate to partly dissolve/swell the adhesive between polymer 2 and the PE layer.

polymer 2 (part B1) can be peeled off

ATR-FTIR:

part B1 (top) = polyester urethane (adhesive), part B1 (bottom) = PA,

part B2 (top) = polyester urethane (adhesive), part B2 (bottom) = PE

Remove the PUR adhesive on top of part B2 as described in option 1 step 11

Go back to step 2 to remove the PUR adhesive on top of the PA layer and confirm that there is no more layer on top of the PA → follow step 4 described in option 1

NOTE: If you try to immerse the isolated PA layer (part B1) in HCOOH (cold or hot) to remove the PUR adhesive on its top, the whole PA layer will be dissolved immediately! You have to go back to step 2 first and immerse the PA/PE laminate (part B) instead!

Characterisation of the adhesive that bonds the reverse printed PET-film to the Al foil:

Go back to step 2 and follow step 12-13 described in option 1

To confirm that the “white layer” on the bottom side of part A is a reverse printing and to check what is below, follow the descriptions in option 1 step 14

Characterisation of the printing:

Go back to step 2 and follow step 15 described in option 1

SUMMARY OF RESULTS:

Identified composition of layers:

PET/polyester acrylate printing//PURAl foil//PURPA//PURPE

outside

polymer 1 = PET

print = polyester acrylate

adhesive = polyester urethane

aluminium foil

adhesive = polyester urethane

polymer 2 = PA

adhesive = polyester urethane

polymer 3 = PE

food contact side

Laminate 2 (PA/polyester urethane printing//PURPE)

Sample:

Step I: General observations

– The sample is partially printed (surface or reverse printing → not yet known).

– The sample does not contain an aluminium layer.

Step II: Microtome section - not available

Step III: ATR-FTIR of the inside (food contact layer) and outside (non-food contact)

ATR-FTIR: complete laminate (outside) = PA

ATR-FTIR: complete laminate (inside) = PE

Results:

– The outside layer consists of PA.

– The food contact layer consists of PE.

Step IV: Separation of layers

From the information gained in step I-III, the following options arise to start the separation of the layers:

1) The food contact layer consists of PE. → Try to dissolve/swell the PE layer in DMSO, toluene or xylene and remove it.

2) The outside layer consists of PA. → Try to completely dissolve the PA layer in conc. HCOOH to check what is below.

Step IV: Separation of layers – option 1 – start with DMSO

Cut a piece of the sample (complete laminate) (approx. 1 cm × 3 cm) and boil it in DMSO to swell the PE layer on the bottom of the sample.

After 1-2 min, the upper layer starts to separate (see edges) but cannot yet be peeled off. Therefore, boil the sample for another 2 min in DMSO.

Part A can be peeled off from the top of the sample. The printing is laid open, it sticks to the bottom of part A and the top of part B.

ATR-FTIR:

part A (bottom, unprinted) = PA, part A (bottom, green printing) = polyester urethane (printing),

part B (top, unprinted) = polyester urethane (adhesive), part B (top, green printing) = polyester urethane (printing)

Remove the printing from the bottom of part A and from the top of part B to check what is below:

The printing can be rubbed off with a paper towel soaked in ethyl acetate. (If necessary, you can also boil the sample in ethyl acetate.)

→ ATR-FTIR: part A (bottom, print removed) = PA, part B (top, print+adhesive removed) = PE

Check whether part A consists only of PA by dissolving it completely in cold HCOOH:

Dip part A into cold HCOOH.

Part A is dissolved immediately. Only the green printing ink film remains in the beaker. → Part A seems to consist only of PA.

Check whether part B is really a monolayer or whether it consists of coextruded layers, e.g. two PE layers with an EVOH layer in between:

boil part B for 5 min in conc. H₂SO₄ → if there was an EVOH layer in between, it would be decomposed (turn black) and you would see a thin black-brown line at the edges of the sample

Residues of the green printing on top of part B were decomposed (turned black). → Can be rubbed off with a paper towel.

Having a closer look to the edges of part B after treatment with H₂SO₄, you can see that there is no black line visible. → There is no EVOH layer sandwiched between two PE layers. → The PE layer is probably a monolayer.

Step IV: Separation of layers – option 2 – start with HCOOH

Cut a piece of the sample (complete sample) (approx. 1 cm × 3 cm) and boil it for 15 min in HCOOH to completely dissolve the PA layer on top of the sample

ATR-FTIR:

top (PA removed, green printed parts) = polyester urethane (printing),

top (PA removed, unprinted/clear parts) = polyester urethane (adhesive),

bottom = PE

Remove the printing and adhesive to check what is below:

The printing and adhesive can be rubbed off with a paper towel soaked in ethyl acetate. (If necessary, you can also boil the sample in ethyl acetate.) → ATR-FTIR: top (PA, print and adhesive removed) = PE

Now that you know, that the sample is reverse printed, you should check that you did not dissolved anything else during step 1 apart from the PA layer. Therefore, try to separate the PA layer from the complete sample and check its bottom side:

Take a piece of the complete sample and boil it for 2 min in ethyl acetate to partly dissolve/swell the adhesive that bonds the reverse printed PA film to the rest of the sample.

The PA layer (part A) can be peeled off. The printing sticks to both sides (the bottom of part A and the top of part B).

Remove the printing from the bottom of part A and from the top of part B to check what is below:

The printing can be rubbed off with a paper towel soaked in ethyl acetate. (If necessary, you can also boil the sample in ethyl acetate.) → ATR-FTIR: part A (bottom, print removed) = PA, part B (top, print+adhesive removed) = PE

Check whether part B is really a monolayer or whether it consists of coextruded layers, e.g. two PE layers with an EVOH layer in between:

→ follow the descriptions in option 1 step 8-10

Check whether part A consists only of PA by dissolving it completely in cold HCOOH:

Go back to step 6 and follow the descriptions in option 1 step 6-7

SUMMARY OF RESULTS:

Identified composition of layers:

PA/polyester urethane printing//PURPE

outside

PA

print = polyester urethane

adhesive = polyester urethane

PE

food contact side

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[39] Jopko L (2004). Flexible Packaging Adhesives – The Basics. Retrieved May 08, 2014, from http://www.tappi.org/content/enewsletters/eplace/2004/14-3Jopko.pdf

[40] Rolando T.E. (1998). Solvent-Free Adhesives. Rapra Review Reports. Vol. 9 (5), Report 101

[41] Bauer F, Decker U, Czihal K, Mehnert R, Riedel C, Riemschneider M, Schubert R, Buchmeister MR (2009). UV curing and matting of acrylate nanocomposite coatings by 172 nm excimer irradiation. Prog. Org. Coat., 64: 474-481

[42] Photoinitiator-free UV curing and matting of acrylate-based nanocomposite coatings: Part 3. Prog.Org. Coat., 77: 1085-1094

[43] Park Y-J, Lim D-H, Kim H-J, Park D-S, Sung I-K (2009). UV- and thermal-curing behaviors of dua-curable adhesives based on epoxy acrylate oligomers. International Journal of Adhesion and Adhesives, 29: 710-717

[44] Lee B-H, Choi J-H, Kim H-J (2006). Coating performance and characteristics for UV-curable aliphatic urethane acrylate coatings containing Norrish Type I photoinitiators. JCT Research, 3(3): 221-229

Annex 1: Example of protocol for operating a rotary microtome

Note: the example given is the one used for JRC FCM activities. It is intended as an illustrative example only and in no way constitutes a recommendation nor any commercial endorsement.

Scope and field of application

This procedure describes the activities to be carried out in order to properly handle and maintain the rotary microtome SLEE medical CUT 4062 accessible in the FIT-EURL group of the CAT Unit, Bld. 26, Lab 017. The rotary microtome is used to perform cross-sections of multilayer plastic films. Strict respect of this SOP is required to all operators within FIT-EURL laboratories.

Materials and reagents

Materials needed for cross-sectioning

– foldable plastic plates in which the multilayer plastic film is sandwiched during sectioning (e.g. 40 × 10 × 1.5 mm, polystyrene, white, purchasable from MicroKern (www.micro-kern.de), Art.no. MK99997);

– sectioning tape (highly transparent, with low birefringence) (e.g. 21 × 14.8 cm, Avery GRAPHICS, purchasable from MicroKern (www.micro-kern.de), Art.no. MK99990_100B).

Materials needed for embedding of cross-sections

– microscope slides (76 × 26 mm);

– cover slips (24 × 24 mm);

– mounting medium for coverslipping of slides (e.g. LEICA CV Ultra, Art.no. 0709 36261);

– paper towels.

Equipment

Rotary microtome

– rotary microtome: SLEE medical CUT 4062 (SN: A 14 0017)

– specimen holder: standard object clamp, orientable (SLEE medical, Art.no. 10090006);

– blade holder;

– disposable blades (stainless steel, low profile, e.g. SLEE medical, Art.no. 28407000).

Other laboratory equipment

– scissors (1 pair of ordinary scissors, 1 pair of dissecting scissors);

– paint brush;

– tweezers;

– dissecting needle;

– permanent marker;

– Allen key (size 3 × 100 mm, 4 × 150 mm, 6 × 100 mm).

Use of the equipment (Microtome sectioning)

To set up the microtome and to perform the microtome sectioning, carry out the following instructions in the given order. Please fill in the instrument table after use (see Annex 1).

Install the orientable standard object clamp

The microtome should be equipped with the object orientation (A). If not, install it and fasten all 4 screws using an Allen key (size 4 × 150 mm).

NOTE: To access the screws of the object orientation, it may be necessary to move the specimen holder towards you by turning the trimming hand wheel (B) on the left counterclockwise.

Now take the standard object clamp and unscrew it.

Install the corpus of the standard object clamp (C) at the object orientation. Use an Allen key (size 3 × 100 mm) to fasten the two screws.

Reassemble the remaining parts of the standard object clamp.

Check the position of the specimen holder. If it sticks out a lot, move it back inwards by turning the trimming hand wheel (B) clockwise. Otherwise it might be difficult later on to position the blade holder correctly and the limit for moving the specimen holder outwards might be reached during trimming or sectioning. An acoustic noise informs you that the limit to move the specimen holder inwards/outwards was reached. Stop turning the trimming hand wheel (B) immediately when you hear it.

Inserting a sample of a multilayer plastic film into the standard object clamp

Cut a strip of approx. 0.8 cm × 1.5 cm, but no bigger than 1 cm × 2 cm, from the multilayer plastic film sample. Use the dissecting scissors to do so. With an ordinary pair of scissor, you might squeeze the edges of the sample strip.

Insert the sample strip in between one of the foldable plastic plates. Pay attention whether the non-food contact side (outside) of the sample faces upwards or downwards. To avoid confusion, it should always face upwards.

NOTE: If later on problems appear and the sections are not cut off completely from the specimen, the right edge (which will be the upper edge when inserted into the standard object clamp) of the foldable plate with the sample strip in between can be cut off with a razor blade.

Insert the folded plastic plate into the standard object clamp. Again the non-food contact side (outside) of the sample should face upwards.

If necessary, adjust the angle of the object orientation as described in the operation manual (see Error! eference source not found. p. 15 section 7.3). Adjusting the screws on top (D) and on the left (E) of object orientation, the sample should be oriented parallel to the x-z-plane. This is necessary to avoid problems in setting the blade angle in a later stage. Then loosen the metal arm (F) on the right to change the orientation in the x-z-plane. The sample should be oriented neither parallel nor perpendicular to the blade (see Error! Reference source not ound.). Best is an oblique angle of about 15°-45° in the x-z-plane.

Install the blade holder and adjust the blade angle

After having inserted the specimen and adjusted the object orientation, take out the blade holder from the plastic box. Be careful when handling the blade holder. The blade is very sharp and you can easily injure yourself when touching it. The red metal bar (“finger protection guard”) (G) should always be folded up to shield the blade and to prevent accidently touching it.

Put the blade holder on the metal rail and fasten it by pulling tight the metal arm (H) on the lower left.

Set the blade angle by loosening the screw on the lower right (I) with an Allen key (size 6 × 100 mm). As all blades are wedge shaped, the correct blade angle should always be set in the following way (see Error! Reference ource not found.): The lower bevel face has to be positioned parallel to the specimen block and the plane of motion. Then the angle should be slightly raised by another 1/2 degree. This prevents the lower bevel face from sliding over the specimen block and possibly producing friction damage. Using the low profile disposable blades made of stainless steel (SLEE medical Art.no. 28407000), an angle of about 2° should be suitable for use.

Position the blade holder for trimming

Before trimming, check again the position of the blade holder. Loosen the hand wheel stop (J) on the right and slowly turn the hand wheel (K) on the right to move the specimen holder down.

The blade holder should be positioned close to the specimen but the blade should not yet touch the specimen. 0.5-1 mm of distance between blade and specimen are enough. Otherwise trimming will take much longer. If necessary, reposition the blade holder by loosening the metal arm (H) on the lower left. (NOTE: Refasten the hand wheel stop (J) before you readjust the blade holder position.)

Select a virgin part of the blade for cutting by shifting the blade holder horizontally. To do so, loosen the metal arm (L) on the lower right of the blade holder block.

Those parts of the blade that were used already for sectioning are marked with a small dot. Starting from the last mark on the blade, move the blade holder about 1 cm further left to arrive at a “virgin” part of the blade. If you have reached the end of the blade already, change the blade and use a new one (see 0)

Mark the new position where the specimen will touch the blade with a small dot on the blade using a permanent marker.

Perform trimming

Set the trimming thickness at the upper grey knob (M) on the left front side of the microtome, labelled with “TRIM”. It can be varied from 10 to 40 μm. Usually a trimming thickness of 30 μm is ok.

To set the cutting thickness for the actual microtome sections, turn the lower, black/grey knob (N). The current setting for the section thickness is shown in the small window next to “CUT” (O). For the analysis of plastic multilayers, sections of 5 μm are generally well suited. For very fragile samples, also thicker sections of 10 μm could be performed.

To start trimming, fold down the finger protection guard (red metal bar) (G), loosen the hand wheel stop (J) and slowly turn the hand wheel (K) on the right clockwise while pressing the grey “TRIM” knob (M) on the left. With every turn of the hand wheel, the specimen holder will move 30 μm towards you as set before on the “TRIM” knob (M).
NOTE: If you turn the hand wheel (K) anticlockwise, the specimen holder will move 30 μm towards you as well. It will not move backwards.

After some turns of the hand wheel (K), the specimen will touch the blade and sections of 30 μm thickness will be cut from the specimen. Continue like this until a plane and clear specimen surface is obtained (incl. the white plastic plates in between which the specimen is sandwiched). Do not perform the trimming sections too fast to avoid damage of the specimen surface.

Then perform another 5-10 sections of 5 μm without pressing the “TRIM” knob (M) to really obtain a smooth specimen surface. When finished, fasten the hand wheel stop (J).

Remove section material from the blade and the specimen holder with a dry paint brush. Be careful not to cut yourself.

The blade and specimen block should be completely clean before you proceed with the next steps.

After cleaning, fold up again the finger protection guard (G).

Perform sectioning

Cut a piece (approx. 1 cm × 1.5 cm) from the sectioning tape. You can use an ordinary pair of scissors to do so. Do not use the dissecting scissors, otherwise they may become blunt.

Remove the paper from the backside of the sectioning tape and stick the sectioning tape to the surface of the specimen. Carefully press it to the specimen surface, e.g. with the wooden backside of the paint brush. Do not touch the tape with your fingers, use tweezers instead.

To prepare the microtome section, fold down the finger protection guard (G) and loosen the hand wheel stop (J).

Turn clockwise the hand wheel (K) on the right with your right hand without stopping until the specimen completely passed the blade, while at the same time you keep holding the sectioning tape with some tweezers in your left hand.
(NOTE: If you turn the hand wheel (K) anticlockwise, the specimen will also move 5 μm towards you, not backwards.)

The moment the blade cuts off the section of the specimen, tear off the sectioning tape with the tweezers in your left hand. To obtain a good section, the cutting has to be performed at a continuous, average speed – neither too fast, nor too slow. It will need some training to find an optimum cutting speed and to coordinate all steps.

Fasten the hand wheel stop (J) and fold up the finger protection guard (G) after the section.

The section is acceptable from a first glance when the white strips cut off from the sandwich plastic plate are straight, homogeneous and eventual knife marks (periodic waves) are only barely visible. Whether the section is really fine, can only be seen afterwards during the microscopy.

Prepare the microtome section for microscopy

If an acceptable section was obtained, take a microscope slide and apply some mounting medium with the dissecting needle.

Spread the mounting medium and put the section tape on it. The sticky surface of the tape should face the microscope slide. Gently press with the wooden shaft of the paint brush to remove eventually present air bubbles.

Apply some more mounting medium on top of the sectioning tape and cover everything with a cover slip. Again gently press with the wooden shaft of the paint brush to remove eventually present air bubbles. Clean the dissecting needle with a paper towel.

Label the microscope slide with the sample name, date, thickness of the section and the initials of the operator and draw an arrow on the cover slip pointing to the outside of the multilayer sample.

Change the blade

For changing the blade, refer to the user manual (see Error! Reference source ot found., chapter 7.5, p. 17). Fold down the finger protection guard (G) and loosen the metal arm on the left (P).

Push the button (Q) on the left. The blade will be pushed about 1 cm out of the slot on the right side. Then pull it out completely. Be careful not to injure yourself when handling the blade!

There is a compartment for used blades in the bottom of the blade dispenser. Insert the used blade there to store it safely.
NOTE: When all blades of the dispenser are finished (and the “used blades” compartment is full), throw away the entire dispenser (incl. the used blades) into a container for contaminated solid waste without opening the “used blades” compartment.

Take out a new blade from the upper part of the blade dispenser.

Insert the blade in the blade holder. Use the wooden shaft of the paint brush to push it in completely.

When finished, refasten the metal arm (Q) on the left and fold up the finger protection guard (G).

Clean the microtome after use

Clean the blade holder with a dry paint brush and put it back into the plastic box for storage. Be careful again not to injure yourself while handling the block.

Remove the specimen and clean the specimen holder with a dry paint brush.

Take a paper towel and remove the section waste from the waste tray (R).

Troubleshooting

The following table lists some errors and possible sources of error

Characteristics of the error

Possible origin

periodic waves, different thicknesses along the section

– damaged or blunt knife edge

– improperly or loosely placed knife

– elastic sample

– insufficiently fixed sample or sample holder

– sample is too soft

changing thicknesses along the section

– inhomogeneities (i.e. hard and soft phases) in the sectioning area

– wrong trimming

strongly compressed section

– wrong knife angle

– wrongly adjusted free angle (x-z-plane)

– sample material is too soft

In case of a malfunctioning of the instrument, open a non-conformity in LAD and if necessary require a technical assistance by an external company and register the intervention in LAD.

Maintenance

A check of the instrument functions, lubricating of movable parts, a check of the driving system and a complete cleaning should be performed by an authorised service technician when needed.

Safety

Blades for microtomes are extremely sharp and need to be handled with care to prevent any kind of injury. Please respect the following rules to prevent any injury of yourself or other persons in the laboratory when using the rotary microtome:

Before installing the blade holder, FIRST install the proper sample holder and insert your sample.

During use, ALWAYS remove the blade holder BEFORE changing the sample holder or the sample.

AFTER having finished your work, remove the blade holder and put it back into the plastic box for storage.

References

[1] Microtome User Manual (SLEE medical GmbH (2014). Operating Instructions – CUT 4062 / CUT 5062 / CUT 6062)

[2] Charles W. Scouten, Ph.D., Leica Microsystems (2011). Tips & Tricks in Sample Preparation. Knife Angle in Microtomy. Retrieved January 19, 2015 from http://www.leica-microsystems.com/uploads/media/Knife_Angle_in_Microtomy.pdf

[3] Michler GH (2008). Electron microscopy of polymers. Berlin/Heidelberg: Springer-Verlag, p. 213

[4] Sawyer LC, Grubb DT, Meyers GF (2008). Polymer microscopy. 3rd edition. Springer

Annex 2: Example of protocol for microscopy of microtome cross-sections

The protocol shown is for polarised light microscopy and digital imaging

Note: the example given is the one used for JRC FCM activities. It is intended as an illustrative example only and in no way constitutes a recommendation nor any commercial endorsement.

Scope and field of application

This procedure describes how to analyse microtome cross-sections of multilayer plastic laminates in plane and crossed polarised light using transmitted polarised light microscopy (incident light microscope Zeiss Axioskop 2 MAT available in the “PRODUCT SAFETY & INNOVATION” group of the CAT Unit, Bld. 26, Lab 023), how to perform digital imaging and how to determine the thickness of individual layers. Strict respect of this SOP is required to all operators within FIT-EURL laboratories.

Equipment

Incident light microscope

– incident light microscope: Zeiss Axioskop 2 MAT (SN: 005 007 249), equipped with Moticam 10 (10.0 MP)

– personal computer: Dell Optiplex 740MT

– software: Motic Images Plus 2.0

Other laboratory equipment

– microscope slide with calibration and scale cross for calibration

Microscopy

Set the microscope for transmitted-light brightfield according to KÖHLER. Refer to the instrument manual (0 section 4.3.6 (3) p. 89-90).

Switch on the microscope.

Place a high-contrast specimen on the specimen holder (e.g. the calibration slide → circle with 0.15 mm diameter).

NOTE: The calibration slide can be found in one of the drawers labelled as “Moticam accessories”.

Switch the turret disk of the condenser to position “H” for brightfield.

Swing in the 10x object lens and adjust the focus so that the object is clearly visible.

Close the luminous-field diaphragm until its border becomes visible (even if not in focus) in the field of view.

Use drive to lower the condenser until the edge of the luminous-field diaphragm is in focus.

Use both centering screws of the universal condenser to centre the luminous-field diaphragm and then open the diaphragm until its edge just disappears from the field of view.

To set the aperture diaphragm (contrast), remove one eyepiece from the tube (it can be pulled out) and look into the tube with your naked eye.

Use the sliding knob to set the aperture diaphragm to approx. 2/3 or 4/5 of the diameter of the objective exit pupils as described in the instrument manual 0. Insert the eyepiece in the tube again when ready.

Calibration of the digital camera

Switch on the computer.

Take the microscope slide with calibration circles and scale cross and place it on the specimen holder.

NOTE: The calibration slide can be found in one of the drawers labelled as “Moticam accessories”.

Select the object lens that needs to be calibrated and adjust the microscope focus to view the appropriate calibration circle or the scale cross (see Table 10).

NOTE: The calibration circles are more suitable if round shaped items need to be measured. If cross-sections of multilayer plastic films shall be analysed, it is advised to perform the calibration using the scale cross.

Table 10 Object lenses and the appropriate calibration circle or scale cross for calibration

Object lens

Calibration circle or scale cross

10x/0.25 Ph1

scale cross 0.01 mm OR circle Ø 0.15 mm (150 μm)

20x/0.45 Ph2

scale cross 0.01 mm OR circle Ø 0.15 mm (150 μm) or 0.07 mm (70 μm)

40x/0.65 Ph2

scale cross 0.01 mm OR circle Ø 0.07 mm (70 μm)

Open the software “Motic Images Plus 2.0”.

Go to menu “File” and select “Capture Window”.

A new window (“Motic Live Imaging Module”) will open.

Pull out the metal bar in the upper part of the microscope. Now the live picture of the camera (Moticam 10) can be seen on the screen (in the “Motic Live Imaging Module” window).

Readjust the focus again if necessary and adjust the position of the microscope slide to see the entire calibration circle or the origin of the scale cross. The calibration circle or the origin of the scale cross, respectively, should be in the centre of the picture. To properly align the scale cross, it may be necessary to slightly turn the camera.

If necessary, change the settings for the contrast and sharpness.

NOTE: For calibrations using a calibration circle, a sufficient contrast between the circle and the background is needed. Otherwise the automatic calibration cannot be carried out. The settings shown here in the print screens should be sufficient. In addition, the power of the light source on the microscope should be no higher than “5”.

Then go to the tab “Video Capture” (see camera symbol) and click “Capture” to take a snap shot of the current camera view.

Each snap shot is saved automatically and can be seen on the right in the window “Motic Images Plus 2.0 ML”. The files are all named as “Capture_xx.sfc” with numbers in chronological order. They can be found in the folder C:\Motic\Motic Images Plus 2.0\Capture Folder.

NOTE: Do not change the position of the camera after having taken the snap shot for calibration. Otherwise a new snap shot should be taken to carry out the calibration.

Calibration with calibration circle

Go to window “Motic Images Plus 2.0 ML”. Open the menu “Measure”. Select “Calibration Wizard”.

The window “Calibration Wizard” will open. Go to tab “Calibrate with calibration circle” and click the button “Load Image”.

A dialog box opens. Select the snap shot file of the calibration circle that you want to use for calibration and click “Open”.

NOTE: All snap shots taken before can be found in the folder C:\Motic\Motic Images Plus 2.0\Capture Folder.

Enter the diameter [μm] of the selected calibration circle and specify the object lens (e.g. 10x, 20x or 40x) with which the snap shot was taken. Then click the button “Calibration” and the calibration is done automatically.

A new window “Save Sign” will open. Select a “Sign Name” from the column on the left (e.g. “40X (object lens)”) or define a new one. Then click the button “Save”.

The calibration is finished now and you can close the “Calibration Wizard” window by clicking the button “Close”.

Calibration with scale cross

Go to window “Motic Images Plus 2.0 ML”. Open the menu “Measure”. Select “Calibration Wizard”.

The window “Calibration Wizard” will open. Go to tab “Calibrate with scale cross” and click the button “Load Image”.

A dialog box opens. Select the snap shot file of the scale cross that you want to use for calibration and click “Open”.

NOTE: All snap shots taken before can be found in the folder C:\Motic\Motic Images Plus 2.0\Capture Folder.

The selected photo will be opened in the “Calibration Wizard” window. Use the scroll bars on the right and on the bottom to move to the centre of the photo. There you will find a white circle with two red dots and a dialog box that shows the current dimension of the white circle.

The circle must be positioned in the very centre (origin) of the scale cross. If necessary, reposition it. To do so, move the cursor above the white circle. The cursor will transform into a cross. Now click on the circle with the left mouse button and drag and drop it.

Move the cursor to the upper red dot (B). The cursor will transform into a hand. Now click on the red dot (B), drag it along the y-axis of the scale cross and drop it exactly at a certain scale division/bar (e.g. 5 bars = 0.05 mm = 50 μm away from the origin). The circle will deform into an ellipse.

Then move the cursor to the right red dot (A). Again the cursor will transform into a hand. Click on the red dot (A), drag it along the x-axis of the scale cross and drop it exactly at a certain scale division/bar. Use the same distance as before at the y-axis (e.g. 5 bars = 0.05 mm = 50 μm away from the origin).

If you positioned each of the red dots (A, B) with the same distance to the origin of the scale cross, the pixel number for the x- and y-dimension (“Major/Minor half axis”) of the circle must be the same. If not, readjust the positions of the red dots. Sometimes it can be necessary to reposition/re-centre the entire circle.

Specify the object lens (e.g. 10x, 20x or 40x) with which the snap shot was taken and enter the x,y-dimensions of the circle. “Width” and “Height” refer to the distance OA and OB, respectively, with “O” being the origin of the circle. In the present example, the dots were positioned 5 bars (50 μm) away from the origin (O). “Width” (OA) and “Height” (OB) of the circle are then 50 μm each. Then click the button “Calibration”.

A new window “Save Sign” will open. Select a “Sign Name” from the column on the left (e.g. “40X (object lens)”) or define a new one. Then click the button “Save”.

The calibration is finished now and you can close the “Calibration Wizard” window by clicking the button “Close”.

Set the microscope for transmitted-light polarisation

Refer to the instrument manual (0 section 4.3.9.1 (3) p. 96).

Take the microscope slide with the microtome section of the sample and place it on the specimen holder.

Push back in the metal bar to be able to view the sample in the eyepiece of the microscope.

Adjust the focus so that the sample is clearly visible. For sufficient magnification use the 40x object lens.

To view the object in plane polarised light, swing the polariser in the beam path.

To view the object in crossed polarised light, swing in the analyser module (filter No. 2 “Epi Pol”) on the reflector turret in addition.

sample in crossed polarised light:

sample in plane-polarised light:

The field of view will appear dark now because the polarisers are crossed. Only birefringent (i.e. anisotropic) objects (e.g. polymer films where polymer chains have an orientation, e.g. due to shear stress in the die or due to stretching during extrusion) will display a colour now (white or brightly coloured) (see 0, 0).

NOTE: Anisotropic materials may also remain dark between crossed polars if the sample is oriented so that it is being viewed down an optic axis and therefore appears isotropic or the sample is in one of 4 possible extinction positions (see 0). If this happens, microscopy should be carried out in plane-polarised light. It is not possible to make the specimen visible by rotating the specimen itself or by changing the direction of the polarised by rotating the polariser because the microscope is equipped with a non-rotatable mechanical stage and a non-rotatable polariser/analyser.

sample in crossed polarised light:

sample in plane-polarised light:

Carefully “scan” the entire microtome section from head to tail. It may happen that certain parts are damaged and one or more of the sample layers can be delaminated. Try to find an intact part of the section to continue. Otherwise you have to prepare a new section.

Take a digital image of the sample

Pull out the metal bar in the upper part of the microscope. Now the live picture of the camera (Moticam 10) can be seen on the screen (in the “Motic Live Imaging Module” window).

sample in crossed polarised light:

sample in plane-polarised light:

Readjust the focus again if necessary and adjust the position of the microscope slide to see all the layers of the sample in the live picture on the screen. Go to the tab “Video Capture” (see camera symbol) and click “Capture” to take a snap shot of the current camera view.

Each snap shot is saved automatically and can be seen on the right in the window “Motic Images Plus 2.0 ML”. The files are all named as “Capture_xx.sfc” with numbers in chronological order. They can be found in the folder C:\Motic\Motic Images Plus 2.0\Capture Folder.

Measure the sample dimensions in the digital image

Go to the window “Motic Images Plus 2.0 ML” and load an image. You can either select one of the photos taken before from the right side of the window or load an image from a file. IN the latter case, open the menu “File” and select “Open”.

A new window will open. Select a file and click the button “Open” to open it.

Select the correct object lens that was used to take the image. Open the tab “Measure” on the lower left of the window and select an object lens from the drop-down menu.

NOTE: The object lens must have been calibrated before as described in section 0. Select the object lens name from the list that was used in the calibration (here “40X (object lens)”).

To measure the thickness of the layers in the picture, open the menu “Measure” on the upper left of the window and select “Line” from the drop-down menu.

You can draw a line now. To do so, click and hold the left mouse button while dragging the line. The length of the line will be shown immediately in a small text box.

NOTE: If necessary, move the scroll bars on the right and the bottom to select the area of the image where you want to carry out the measurements.

Start and end point of the line can be readjusted. Simply click on the start or end point with the left mouse button and drag and drop the point. Like this you can also elongate a line if the measurement area is bigger than the actual area shown on the screen.

Also the entire line as well as the text box can be moved via drag and drop with the left mouse button.

The measurement should be carried out perpendicular to the layers.

If you want to add more lines, just click and hold the left mouse button and draw the line as described before. There is no need to activate the “Line” function again in the “Measure” menu.

A table that lists the dimensions of all lines drawn can be accessed via the button “Measure Table” in the lower left corner of the screen, tab “Measure”.

A new window (“Measure”) opens. The tab “Measure Tables” shows the length of each drawn line.

In the tab “Options” the line style and the format of the text boxes can be changed.

When you are finished, save the file. To do so, open the drop-down menu “File” in the upper left corner of the screen and select “Save As”.

A new window (“Save Image”) will open. It is advised to save the file as a “sfc”-project which can be reworked if necessary. If you want to export it, save it also as a “tif”-file which provides a sufficient resolution for publication in reports or scientific journals. Never overwrite the original file “Capture_xx.sfc”. Always select a new file name. You can also create a new folder for your project and save the files in the project folder.

Reference documents

[1] Carl Zeiss Light Microscopy (2002). Operating Manual. Axioskop 2 MAT/2 MAT mot. Incident Light Microscope. B 46-0012 e 10/02

[2] Zhang XM, Elkoun S, Ajji A, Huneault MA (2004). Oriented structure and anisotropy properties of polymer blown films: HDPE, LLDPE and LDPE. Polymer 45: 217-229

[3] Sawyer LC, Grubb DT, Meyers GF (2008). Polymer microscopy. 3rd edition. Springer

Annex 3: Example of protocol for handling/maintenance of an ATR-FTIR spectrometer

Note: the example given is the one used for JRC FCM activities. It is intended as an illustrative example only and in no way constitutes a recommendation nor any commercial endorsement.

Scope and field of application

This procedure describes how to perform ATR-FTIR spectroscopy to characterise liquid or solid substances (e.g. polymer films).

Terms and definitions

ATR    attenuated total reflection -- FTIR Fourier Transform Infrared

Materials and reagents

– organic solvent (e.g. acetone) to clean the ATR diamond crystal

– paper towels

Equipment

ATR-FTIR system

– Perkin Elmer Spectrum One FT-IR Spectrometer equipped with Perkin Elmer Universal ATR Sampling Accessory

– personal computer: Dell Optiplex GX270 software: Spectrum Version 6.3.1, Perkin Elmer

Other laboratory equipment

– tweezers

– Pasteur pipettes

– glass beaker (25-50 mL)

– spatula

ATR-FTIR Spectroscopy

Start up

– Switch on the computer and monitor. The ATR-FTIR spectrometer should be already turned on as it is kept ON at any time.

– Open the software “Spectrum” (Version 6.3.1) and log in

– Check the intensity of the laser beam as described in the user’s guide for the “Spectrum” software. If the criteria are not met, refer to the troubleshooting section of the instrument manual 0.

– Confirm that the ATR crystal is clean and collect a background spectrum for air without any sample. To do so, proceed as indicated in the “Spectrum” software user’s guide 0.

Collecting sample spectra

– Collect sample spectra according to the “Spectrum” software user’s guide 0.

– Avoid any scratching of the ATR crystal when installing a test specimen. Lift the plunger well up before moving it to avoid scratching the ATR crystal and the surrounding metal plate. When installing the specimen, never fasten the plunger too tight. Otherwise the ATR crystal may break.

Data processing

– Perform data processing, such as baseline correction, smoothing, subtraction or labelling of spectra, as needed. Refer to the “Spectrum” software user’s guide and proceed as indicated in there 0.

– Files of all recorded spectra are saved automatically in the folder C:\pel_data\spectra. You can create an additional folder for your project and move all files there. Indicate the data path in the instrument table (Annex 1). Save also your processed files if wanted under this data path.

Shut down

– Fill in the instrument table

– Shut down the computer and switch off the monitor.

– DO NOT SWITCH OFF THE ATR-FTIR SPECTROMETER. The instrument should remain ON at all times. This keeps it stable and prevents excessive accumulation of water which might damage the beam splitter.

Troubleshooting

For troubleshooting refer to the instrument manual 0 or the software user’s guide 0.

Cleaning and maintenance

– Clean the ATR crystal and the plunger with a paper towel soaked in an organic solvent (e.g. acetone) after use. DO NOT SCRATCH THE CRYSTAL SURFACE!

– In case of errors, problems or general maintenance consult the instrument manual 0.

– The desiccant inside the instrument has to be removed and dried once every year. To do so, proceed as indicated in the instrument manual 0.

– For extraordinary maintenance, contact an authorised service technician.

– Register all errors, problems and maintenance activities in the instrument table

Safety

The instrument contains a low power, visible (red) laser. Do not stare into the laser beam to prevent damage of your eye.

Reference document

[1] Perkin Elmer. Spectrum One. Instrument Manual

[2] Perkin Elmer. IR Spectroscopy Software User’s Guide