Zooming in on potential problems

iStock_000006080783MediumMike Edwards, microscopy manager at Campden BRI, reveals how modern miscroscopy can help troubleshoot a range of industry dliemmas

Microscopes were developed to look at things that were too small to see with the naked eye, and that used to be all that they were used for. But nowadays they do so much more, and can be combined with specific analytical techniques (such as spectroscopy), allowing detailed information to be gathered on the chemical composition of the object being viewed.

This article illustrates how two different approaches to analytical microscopy have practical applications for the food and drink industry – Fourier transform infrared (FT-IR) microscopy and scanning electron microscopy (SEM) x-ray microanalysis.

FT-IR

Infra-red spectroscopy has been an important analytical tool for many years, but recent advances have increased its usefulness. Application of Fourier transform techniques to the results has lowered the detection limit from the microgram to the nanogram range, and from the ppm to ppb level. Meanwhile, sample presentation has been greatly simplified with the introduction of Diamond ATR (Attenuated Total Reflectance) sampling.

Infrared spectroscopy is based on the interaction of specific wavelengths of infrared light with particular chemical bonds in the material being studied, particularly organic molecules.

Individual bonds, such as C-O, C-H or C-N, absorb infrared light at a particular wavelength. Illumination of a molecule will produce a spectrum of peaks, and each peak can be related to a particular type of bond. Individual spectra thus provide a ‘fingerprint’ of individual molecules.

This can be used in the identification and verification of incoming product, determination of adulteration (eg palm oil addition to virgin olive oil, or margarine addition to butter), contamination and origin studies, and quality issues (such as sugar/acid ratio in tomatoes), as well as to identify the chemical composition of foreign bodies.

FT-IR microscopy can be used to study the chemical composition of very small samples (micro-sized), in effect using a microscope to apply FT-IR spectroscopy to those microscopic samples. However, its most valuable application is in the chemical mapping of a sample of varying composition, so that the chemical identity of particular components can be determined. This can be used to study food materials, such as the composition of wheat grains, where chemical mapping shows the distribution of protein, starch, cellulose and phenolics.

FT-IR microscopy can also be used in the analysis of multilaminate plastic packaging materials, which are made up of a number of different, very thin layers, each of which has a specific purpose.

When problems are encountered with such materials – for example if a lidding film will not seal adequately to a food tray – it is important to be able to able to analyse the different layers to check them against the manufacturer’s specification. A cross-section is therefore cut from the film and examined under the microscope.

The traditional approach in identifying the chemical nature of layers (of packaging or food) would be to take spectra from each layer in turn, but the development of a focal plane array detector with up to 128 x 128 separate elements means that spectra can now be acquired simultaneously across the whole sample to give a chemical map of the various layers. In addition, layers as thin as 1-2 microns can now be analysed, following the development of a Germanium Attenuated Total Reflectance objective, in which a germanium crystal is pressed up against the sample, increasing the resolution fourfold.

Scanning electron microscopy (SEM) and X-ray microanalysis

The SEM gives pseudo-three dimensional images with higher magnification and greater depth of focus than a light microscope. Samples can be relatively easily prepared and quickly examined in the SEM, making it an invaluable tool for the rapid examination of the three-dimensional structure of many samples.

This technique is put to good use in texture assessments, for example during a product development programme. Different formulations for products such as biscuits or savoury snack foods often have very different textures or mouth feels. Getting the right texture is a key part of producing an acceptable product. The SEM can be used to examine the structure of a biscuit, for example, and assess whether specific ingredients are associated with crumbliness or lack of cohesion of the product. Sugar crystals are often associated with these characteristics, and the SEM can reveal whether there is an uneven distribution of crystals or other components and whether this can be related to where and how the product crumbles. Overall crispness of snack products is also related to the relative levels and distribution of individual components. The SEM can be used to relate the microstructure of the product to this characteristic.

More detailed microanalysis of SEM samples may be carried out using an energy-dispersive X-ray microanalyser.  Whereas FT-IR analyses the spectra associated with chemical bonds and is therefore primarily useful for organic materials, x-ray microanalysis is related to the elemental composition of a material. The energies of the X-rays given off by a sample irradiated with an electron beam are characteristic of the elements present in the sample, and so the X-ray microanalyser can be used to give a quick non-destructive elemental analysis of the sample.  Therefore it has wide application for the food scientist.  It can be used to look at the distribution of salt and calcium phosphate crystals in cheddar cheese or of milk solids in chocolate.  It can be used to determine such things as the mineral composition of a substance, such as nature of glass found as a foreign object in food. (Different types of glass have different levels of elements such as sodium, aluminium, magnesium, lead and calcium – and so can be distinguished by their ‘elemental fingerprint’). It can also rapidly identify samples erroneously reported as glass such as struvite (magnesium ammonium phosphate), salt and silica minerals.

As well as foreign body analysis, the SEM X-ray microanalysis combination is particularly useful for investigating packaging defects such as can corrosion. When a can corrodes, it is usually due to the presence of elements other than iron and tin (ie elements that shouldn’t be there). SEM can be used to assess exactly where the corrosion has occurred, and where it might have originated. X-ray microanalysis is then used to determine what unusual elements are present.

In a recent investigation, we found that pinholes down a can side seam were related to the presence of traces of copper from the welding operation when the can was made. This, in effect, set up an electric cell that led to the corrosion.

Rusting of cans is often linked with the presence of chlorine and sulphur, and sulphur (or sulphide) staining is caused by a reaction between hydrogen sulphide, produced by breakdown of food proteins during heat processing, with the can metal, producing unsightly black metal sulphides. This can be readily analysed by x-ray microanalysis.

Recent developments in scanning electron microscopes include variable pressure SEMs and Environmental SEMs. A variable pressure SEM avoids the need for coating a sample with gold or carbon to give an electrically conducting surface, normally required to avoid a build-up of electrical charge on the sample.

A carefully controlled partial vacuum inside the microscope acts as an electrical earth for the sample, although a somewhat poorer image can result. This means that most food or packaging samples can be readily examined with virtually no preparation.

The Environmental SEM allows control of not only the vacuum, but also the humidity inside the chamber. This allows wet samples to be examined, which is very useful where food samples are concerned.

As these examples show, modern microscopy is a sophisticated science in which the microscope can be linked to other instruments and used to research or troubleshoot a wide range of practical problems of direct relevance to the food and drinks industry.

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