Hydrothermal Stabilization of Gelatin

Introduction

The world today has adopted a varied and extensive use for polymers. Polymers have found their way into the daily activities of people. From small scale domestic to large industrial uses, polymers form a large part of human livelihood. Polymer chains are made up of subunits known as monomers. Several smaller repetitive units called monomers join together to form larger molecular units with very large molecular weights. These large molecular weights units are known as Polymers. Individual polymer chains can be linked up with one another to form gigantic structures. The intermolecular bonds that hold one polymer chain with the other could either be covalent or ionic bonds. Collectively, these bonds are known as Cross-links. Cross-linking is therefore a process in which large polymer molecules react with each other to form a 3-dimensional network. Since polymers are either synthetic or natural (Davis, 2004), it means that the term cross linking could either be used in terms of synthetic polymers or natural polymers. In synthetic polymer chemistry, cross-links have a significant effect on the physical properties of the polymer. In the biological sense, cross-linking refers to the use of a probe to check for protein-protein interaction (Wollensak, Spoerl and Seiler, 2003).

In the area of synthetic polymer chemistry, cross linking is used to describe a situation where the entire bulk of the material is subjected to cross linking. The density of the bonds involved in cross linking impact a special characteristic on the physical properties of the material. The cross linking could either be of a high density, intermediate density or a low one. Polymer melts have a raised viscosity when they are joined up by cross linkers with a low density (Hansen, Davis and Mitchell, 1998); On the other hand, cross linkers with an intermediate density transform gelatinous polymers into ones with elastic properties with high strength. Cross linkers with a high density cause materials to be very rigid and glassy in nature such as phenol-formaldehyde materials (Warner, 1989).

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Since cross-links are basically chemical bonds formed between polymer chains to hold them together. They may be formed from chemical reactions which are initiated by such factors as heat, temperature, pressure radiation and pH. Cross links can be formed while developing the polymer chain (Jego et al., 1994) or induced in already built polymer chains. In the pre-building process, an unpolymerized or partially polymerized substrate is mixed with reagents that can be termed as cross linking reagents that cause the requisite chemical reaction that leads to the formation of the cross linking bonds. Cross linking reagents contain reactive ends to specific functional groups on proteins and other molecules (Pierce.net, 2005). Induced cross linking takes place especially in materials that are thermoplastic. Exposure to radiation source is a common means of inducing cross linking between polymer chains. Gamma radiation, electron beam and UV light are sources of radiation by which thermoplastic materials could be exposed to for the formation of cross linkers.

A very common polymer is polyethylene; a polymer made from ethylene monomer units. Whereas millions of ethylene monomer units come together to form polyethylene, the polyethylene structure is held together by cross linkers. The cross linker in polyethylene can be produced in several ways. The electron beam processing method is used to produce the C type cross linked polyethylene. During the extruding process of production of the polyethylene, peroxide could be added to form the cross links (Davis, 2004). This leads to the formation of what is known as the A type of cross links polyethylene. A cross linking agent, an example of which is vinylsilane can be used in conjuction with a catalyst during the extruding process after which a post extrusion curing can be performed on it.

Cross linkers are used in varied fields of human endeavours where polymers can be found and are in use. The properties of rubbber is changed to suite its use in cars, airplanes and bikes as tyres for the wheels. This chemical process is what is known as vulcanization. The process of vulcanization is a slow process but the reaction rate can be rapidly increased by the addition of catalysts such as benzothiazolethiol or tetramethylthiuram disulfide Science and Technology of Rubber (2005). Both contain sulfur which iniates the reaction of the sulfur chain with the rubber. For this reason, vulcanization is also known as sulfur curing. Thermoset plastics are permanently set and so would not change form when heat is applied to it. Cross links are therefore an important characteristic of thermoset plastics. In this application, the process of cross linking is irreversible and so thermoset plastic will decompose or burn when exposed to heat. The only case by which a cross linked process could be reversible is in situations where the chemical bonds involved in the cross linking is different from those used to join up the monomers into polymers. In nature, cross links can be reversed as is done in the case of curly hair. The disulfide bonds between protein chains can be broken and reformed.

Chemical cross links are thermally and mechanically stable so, once formed it is very difficult to break them. This property largely becomes a disadvantage to the recycling industry. A new class of polymers gaining increasing global acceptance is the thermoplastic elastomers which relay on physical cross links in their microstructure to attain stability. The physical parameter responsible for their binding is reversible. They therefore, offer a wide range of applications.

Oxygen is the second most abundant element; it also has the ability to initiate free radical reactions. A polymer exposed to air can undergo a radical reaction with the oxygen resulting in an oxidative cross linking which would significantly alter the properties of the polymer. To avoid this, the polymer industry incorporates anti-oxidants during the production of polymers (Hans-Wilhem et al., 2004)

So far, we have seen cross linking in the synthetic polymer industry. Since nature also has its own way of producing polymers like proteins, carbohydrates and others, it holds true to say that cross linking is very much possible in natural biological polymers. This area of polymers binding to one another through covalent or ionic bonds can be exploited in studying the proximity of proteins.

From the collagen located inside animals’ skin and bones, a translucent, colourless, brittle and tasteless substance known as gelatin can be obtained. Gelatinous substances are defined as substances which contain gelation or behave in a similar way to the behaviour of gelatine. It is commonly used as a gelling agent in the food, pharmaceuticals and photographic industry. When heated, gelatin melts into a liquid and then solidifies when it is cooled. Gelatin forms a solution of high viscosity in water, which sets to a gel on cooling, and its chemical composition is, in many respects, closely similar to that of its parent collagen (Ward and Courts, 1977). The solubility of the gelatin is determined by the method of manufacture. Typically, gelatin can be dispersed in a relatively concentrated acid. Such dispersions are stable for 10–15 days with little or no chemical changes and are suitable for coating purposes or for extrusion into a precipitating bath. Gelatin is also soluble in most polar solvents. Gelatin gels exist over only a small temperature range, the upper limit being the melting point of the gel, which depends on gelatin grade and concentration and the lower limit, the freezing point at which ice crystallizes. The worldwide production amount of gelatin is about 300,000 tons per year (roughly 660 million lb).

On a commercial scale, gelatin is made from by-products of the meat and leather industry. Recently, fish by-products have also been considered because they eliminate some of the religious obstacles surrounding gelatin consumption (“What is Halal?”. Islamic Services of America. Retrieved 2011-04-15). It is commonly believed that cheese is used in the production of Gelatin, however this is an erroneous idea as Gelatin is drived mainly from the skin of pigs, pork and cattle bones or split cattle hides (Gelatine Manufacturers Institute of America. Retrieved 2011-04-16). The raw materials for the production of gelatin are carefully prepared through a number of different curing, acid and alkali processes to extract the dried collagen hydrolysate. These processesmay take up to several weeks, with the different procedures conferring great effects on the properties of the final product. and differences in such processes have great effects on the properties of the final gelatin products (GELITA Grp 2006). Of the materials mentioned above for the production of gelatin, pig skin forms about 44%, with the next in line being bovine hides with 28% which is closely followed by bones with 27%. The rest of the materials make a mere 1% of the total. (Source: wikipedia).

To study the role of cross linkers in the chemical or hydrothermal stabilization of gelatin, three cross linkers were used in this experiment. They are; Gluteraldehyde, Chrome sulphate and Salicylic acid.

The pie chart below gives the percentage distribution of gelatin by geographic regions. Western Europe produces 39% of the estimated annual global production of 300,000tons. North America makes up 20% of this figure, Latin America produces 17%, Eastern Europe makes up 2% while the rest of the world adds 22%. This makes Europe an important contributor to the production and supply of gelatin

Gelatin is the best known gelling agent in the food and cooking industry. There are types and different grades of gelatin used extensively in a wide range of food and non-food products. There are many foods on sale today that contain gelatin common examples are; gelatin desserts, trifles, aspic, marshmallows, candy corns and confectionaries such as peeps, gummy bears amongst many others. Apart from being used as a gelling agent, gelatin also acts as an important stabilizer, thickner or texturizer in foods such as jams, yoghurt, cream cheese and margerine. It is also used in fat-reduced foods to stimulate the mouthfeel of fat and create volume without adding calories.

Gelatin is used for the clarification of juices, such as apple juice, and of vinegar. Isinglass from the swim bladders of fish is still used as a fining agent for wine and beer (National Organic Standards Board Technical Advisory Review).

The food industry is not the only one that enjoys the use of gelatin. Certain professional and theatrical lighting equipment use colour gels to change the colour of the beam. These used to be made with gelatin, hence the term colour gel. In years past, hard paper was obtained by treatment with gelatin. It is used as a separating agent, carrier or coating for substances as in the case of beta-carotene. Gelatin makes beta carotene water soluble thus imparting a yellow colour to it. This explains the yellow colouration of any soft drink containing beta carotene. It is used for holding silver halide crystals in an emulsion in virtually all photographic films and photographic papers. There have been efforts at finding replacement for gelatin in this required but no low cost suitable substitute with the sort of stability possessed by gelatin has been found.

To study the role of cross linkers in the chemical or hydrothermal stabilization of gelatin, three cross linkers were used in this experiment. They are; Gluteraldehyde, Chrome sulphate and Salicylic acid.

Gluteraldehyde is a linear 5 Carbon dialdehyde with the chemical formular, CH2(CH2CHO)2. It is colourless, oil liquid with a pungent smell, which exists commonly as the hydrate and not the dialdehyde (Kohlpaintner et al., 2008). It finds uses in the medical and dental fields as a disinfectant. It is also used in the industrial treatment of water and as a chemical preservative. Industrial preparation of gluteraldehyde includes the oxidation of cyclopentene and by the Diels Alder reaction of acrolein and methyl vinyl ether followed by hydrolysis. Monomeric glutaraldehyde can polymerize by aldol condensation reaction yielding alpha, beta-unsaturated poly-glutaraldehyde. This reaction usually occurs at alkaline pH values.

With the introduction of glutaraldehyde (Sabatini et al., 1962) electron microscopists had a more rapidly penetrating fixative that thoroughly insolubilized proteins and was cheap enough to deliver by vascular perfusion. Since gluteraldehyde is a dialdehyde, it belongs to the aldehyde functional group which has the carbonyl group. The reactive group of gluteraldehyde is the hydrazide group. Glutaraldehyde has fairly small molecules, each with two aldehyde groups, separated by a flexible chain of 3 methylene bridges. It is HCO-(CH2)3-CHO. The potential for cross-linking is obviously much greater because it can occur through both the -CHO groups and over variable distances. In aqueous solutions, glutaraldehyde is present largely as polymers of variable size (Monsan et al., 1975). There is a free aldehyde group sticking out of the side of each unit of the polymer molecule, as well as one at each end. All these -CHO groups will combine with any protein nitrogens with which they come into contact, so there is enormous potential for cross-linking, and that is just what happens. There are also many left-over aldehyde groups (not bound to anything) that cannot be washed out of the tissue.

A prepared solution of glutaraldehyde of concentration range 0.1% to 1.0% may be suitable for use as a system disinfectant and also as a preservative for long term storage. Glutaraldehyde is used in biological electron microscopy as a fixative. A fixative is simply a stabilizing or preservative agent. It kills cells quickly by crosslinking their proteins and is usually employed alone or mixed with formaldehyde (Karnovsky, 1965) as the first of a two fixative process for stabilizing specimens such as bacteria, plant material and human cells. The second process involves the use of osmium tetroxide to crosslink and stabilizes cell and organelles. The fixation process is usually followed by dehydrating the tissue in ethanol or acetone followed by embedding in epoxy resin or acrylic resin. Another example of an application for treatment of proteins with glutaraldehyde is the inactivation of bacterial toxins to create toxoid vaccines, e.g. the pertussis (whooping cough) toxoid component in the Boostrix Tdap vaccine produced by GlaxoSmithKline (GlaxoSmithKline, 2009).

In a related application, glutaraldehyde is sometimes employed in the tanning of leather. Tanning is the process that converts the protein of the raw hide or skin into a stable material which will not putrefy and is suitable for a wide variety of end applications. The principal difference between raw hides and tanned hides is that raw hides dry out to form a hard inflexible material that can putrefy when wetted again, while tanned material dries out to a flexible form that does not become putrid when wetted back. A large number of different tanning methods and materials can be used; the choice is ultimately dependent on the end application of the leather (Heidemann, 1993).

Glutaraldehyde is frequently used in biochemistry applications as an amine-reactive homobifunctional crosslinker. The oligomeric state of proteins can be examined through this application. Glutaraldehyde is also used in SDS-PAGE to fix proteins and peptides prior to staining. Typically, a gel is treated with a 5% solution for approximately half an hour, after which it must be thoroughly washed to remove the yellow stain brought about by reacting with free tris. A polymerized isomer of glutaraldehyde known as polycycloglutaracetal is a fertilizer for aquatic plants. It is claimed that it provides a bioavailable source of carbon for higher plants that is not available to algae.

Furthermore, this work also looked at another important cross-linker; chrome sulfate. Chromium sulfate is the most important ingredient used in the tanning of leather. It is manufactured from the simple reduction of Cr (VI) to Cr (III) by the addition of an excess of sulphur dioxide:

Na2Cr2O7 + 3SO2 + H2O > Cr2 (SO4)2(OH)2 + Na2SO4

This reaction is carried out in a steam-heated vessel, and then the excess SO2 is removed in a second reaction tower. Finally, the liquid product is spray-dried to form a powder which is then bagged and sold. The hydrated sulfate salt has a purple colour. However, heating chromium (III) sulfate leads to partial dehydration to give a hydrated green salt and eventually the anhydrous derivative (Anger, et al., 2005).

Chrome alum or Chromium (III) potassium sulfate is the potassium double sulfate of chromium. Its chemical formula is KCr(SO4)2 and it is commonly found in its dodecahydrate form as KCr(SO4)2·12(H2O). It was formerly used in leather tanning (Holleman, et al., 1985). Chromium alum crystallizes in regular octahedra geometry with flattened corners and is very soluble in water. Chromium alum is used in the tanning of leather as chromium (III) stabilizes the leather by cross linking the collagen fibers within the leather (Brown, Dudley and Elsetinow 1997). However, this application is obsolete because the simpler chromium (III) sulfate is preferred (Anger, et al., 2005).

Schematic diagram of Cross linking with chromium (Source: Wikipedia)

Tannage procedure involving Chrome generally concerns reactions of aqueous solutions of basic sulphates and chlorides of chromium and collagen, a greater majority of the chromium complexes are removed by collagen in an irreversible combination with the protein. The fixed chromium salt makes collagen resistant to the action of proteolytic enzymes and to hot water, even to boiling water by rationally performed tanning. This stabilization of the collagen lattice by chromium complexes is generally considered to be due to the cross-linking of the collagen chains through stable chromium bridges (Gustav and Holme, 1952). Various types of chromium complexes, differing in composition, molecular size and electrical charge, are present in solutions of the basic salts.

Chromium salts react with polyamides to form cross links that would hold the polyamides molecules together. In this connection, basic chromium salt link up with artificial substrate with the -CO. NH- as its main reacting groups and practically devoid of ionic reactivity. The modified polyamide, in the hydrated state has been found to be exceedingly reactive towards agents which possess hydrogen-bonding ability, for instance, polyphenols of which vegetable tannins are an outstanding example (Gustav and Holme, 1952). The cationic chromium complexes present in the standard solutions of the basic chloride and sulphate were not taken up by the polyamide. A mirior fixation of chromium from concentrated solutions of the sulphate was found, however.

The third cross linker used in this work was salicylic acid. Salicylic acid is a monohydroxybenzoic acid, a type of phenolic acid and a beta hydroxy acid. Salicylic acid is a colourless crystalline acid widely incorporated in organic synthesis and functions as a plant hormone. It is derived from the metabolism of salicin. In addition to being a compound that is chemically similar to but not identical to the active component of aspirin (acetylsalicylic acid), it is probably best known for its use in anti-acne treatments. The salts and esters of salicylic acid are known as salicylates. It has the molecular formular C6H4(OH)COOH where the OH group is ortho to the carboxyl group. The molar mass is 138.12gmol-1. It is also known as 2-hydroxybenzenecarboxcylic acid. In terms of solubility in water, salicylic acid is poorly soluble in water.

Salicylic acid is biosynthesized from the amino acid phenylalanine. In Arabidopsis thaliana it can also be synthesized via a phenylalanine-independent pathway. Sodium salicylate is commercially prepared by treating sodium phenolate (the sodium salt of phenol) with carbon dioxide at high pressure (100 atm) and high temperature (390K) -a method known as the Kolbe-Schmitt reaction. Acidification of the product with sulfuric acid gives salicylic acid:

Salicylic acid has a long history which dates back to the 5th century BC. The Greek physician Hippocrates wrote about a bitter powder extracted from the bark of the willow tree that could ease aches and pains and reduce fevers. This remedy was also mentioned in texts from ancient Sumer, Lebanon, and Assyria. The Cherokee and other Native Americans used an infusion of the bark for fever and other medicinal purposes for centuries (Hemel and Chiltoskey 1975). The medicinal part of the plant is the inner bark and was used as a pain reliever for a variety of ailments. The Reverend Edward Stone, a vicar from Chipping Norton, Oxfordshire, England, noted in 1763 that the bark of the willow was effective in reducing a fever (Stone 1763).

In order to measure the chemical and hydrothermal stability of gelatin, the use of spectroscopic instrumentation was required. This was achieved using 1) A SHIMAZU 8400 S FT-IR Spectrometer 2) DSC & TGA/SDTA 822e (Differential scanning calorimetry): Mettler Toledo. 3) SMS (Stable Micro System) origon and firm name I can give you after confirming.

The Fourier Transform Infra red Spectrometer (FT-IR) is an instrument that helps to determine the various functional groups present in an organic molecule. It can detect functional groups such as hydroxy functional group, carbonyl of either an aldehyde or a ketone, carboxyl, carbon-carbon double or triple bond, amines, amides as well as any other functional group that may be present in the molecule. The FT –IR works in the infra red region of the electromagnetic spectrum which is the part of the spectrum between the visible and microwave regions. Of greatest pratical use is the portion between 4000 and 400cm-1 (silverstein, Bassler and Morrill, 1991). The FT-IR works on the principle of measuring the energy needed to stretch or bend bonds in a molecule. It represents this information as peaks plotted on a graph of percent absorption on the vertical against wavenumber on the horizontal. The position of the peak is characteristic of the particular functional group. It is a technique which is used to obtain an infrared spectrum of absorption, emission, photoconductivity or Raman scattering of a solid, liquid or gas (Griffiths and de Hasseth, 2007). An FTIR spectrometer simultaneously collects spectral data in a wide spectral range. This confers a significant advantage over a dispersive spectrometer which measures intensity over a narrow range of wavelengths at a time. FTIR technique has made dispersive infrared spectrometers all but obsolete (except sometimes in the near infrared) and opened up new applications of infrared spectroscopy.

GC-IR (gas chromatography-infrared spectrometry). A gas chromatograph can be used to separate the components of a mixture. The fractions containing single components are directed into an FTIR spectrometer, to provide the infrared spectrum of the sample. This technique is complementary to GC-MS (gas chromatography-mass spectrometry). The GC-IR method is particularly useful for identifying isomers, which by their nature have identical masses. The key to the successful use of GC-IR is that the interferogram can be captured in a very short time, typically less than 1 second. FTIR has also been applied to the analysis of liquid chromatography fractions (White 1990). Micro-samples. Tiny samples, such as in forensic analysis, can be examined with the aid of an infrared microscope in the sample chamber. An image of the surface can be obtained by scanning (Eauchaine, Peterman and Rosenthal 1988). Another example is the use of FTIR to characterize artistic materials in old-master paintings (Prati, Joseph, Sciutto, Mazze, 2010).

Differential scanning calorimetry or DSC is a thermoanalytical technique in which the difference in the amount of heat required to increase the temperature of a sample and reference is measured as a function of temperature. It is a technique we use to study what happens to polymers when they’re heated. We use it to study what we call the Thermal Transitions of a polymer. They are the changes that take place in a polymer when you heat it. The melting of a crystalline polymer is one example. The glass transition is also a thermal transition. The first step would be to heat the polymer. And that’s what we do in DSC. There are two pans. In one pan, the sample pan, you put your polymer sample. The other one is the reference pan. You leave it empty. Each pan sits on top of a heater. Then you tell the nifty computer to turn on the heaters. Both the sample and reference are maintained at nearly the same temperature throughout the experiment. Generally, the temperature program for a DSC analysis is designed such that the sample holder temperature increases linearly as a function of time. The reference sample should have a well-defined heat capacity over the range of temperatures to be scanned.

The technique was developed by E.S. Watson and M.J. O’Neill in 1960 and introduced commercially at the 1963 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy. The term DSC was coined to describe this instrument which measures energy directly and allows precise measurements of heat capacity (Wunderlich, 1990). Differential scanning calorimetry can be used to measure a number of characteristic properties of a sample. Using this technique it is possible to observe fusion and crystallization events as well as glass transition temperatures Tg. DSC can also be used to study oxidation, as well as other chemical reactions (Pungor, 1995).

Glass transitions may occur as the temperature of an amorphous solid is increased. These transitions appear as a step in the baseline of the recorded DSC signal. This is due to the sample undergoing a change in heat capacity; no formal phase change occurs (Skoog, Douglas, Holler and Nieman 1998).

As the temperature increases, an amorphous solid will become less viscous. At some point the molecules may obtain enough freedom of motion to spontaneously arrange themselves into a crystalline form. This is known as the crystallization temperature (Tc). This transition from amorphous solid to crystalline solid is an exothermic process, and results in a peak in the DSC signal. As the temperature increases the sample eventually reaches its melting temperature (Tm). The melting process results in an endothermic peak in the DSC curve. The ability to determine transition temperatures and enthalpies makes DSC a valuable tool in producing phase diagrams for various chemical systems (Dean, 1995).

This instrumentation has a very important role in a number of fields noticeable is in the field of drug analysis. DSC is widely used in the pharmaceutical and polymer industries. For the polymer chemist, DSC is a handy tool for studying curing processes, which allows the fine tuning of polymer properties. The cross-linking of polymer molecules that occurs in the curing process is exothermic, resulting in a positive peak in the DSC curve that usually appears soon after the glass transition.

In the pharmaceutical industry it is necessary to have well-characterized drug compounds in order to define processing parameters. For instance, if it is necessary to deliver a drug in the amorphous form, it is desirable to process the drug at temperatures below those at which crystallization can occur (Pungor, 1995).

In the broad lucreative polymer industry, DSC is used widely for examining polymers to check their composition. Melting points and glass transition temperatures for most polymers are available from standard compilations, and the method can show up possible polymer degradation by the lowering of the expected melting point, Tm, for example. Tm depends on the molecular weight of the polymer, so lower grades will have lower melting points than expected. The percentage crystallinity of a polymer can also be found using DSC. It can be found from the crystallisation peak from the DSC graph since the heat of fusion can be calculated from the area under an absorption peak.

Impurities in polymers can be determined by examining thermograms for anomalous peaks, and plasticisers can be detected at their characteristic boiling points.

The texture of most products is an important property of the product. The texture affects how the product would be handled, influence, the shelf life and also how consumers would respond to such a product. The texture of a product can be determined in one of two ways; either by sensory analysis or by the use of instruments. Of importance to this work was the use of the stable micro systems’ texture analyser. The instrumentation method provides the advantage of conducting the analysis is a more defined and controlled environment.

In the food industry, particularly the confectionery industry, gelatin is commonly used for processing gelled products. Gelatin shows a reversible sol-gel change with temperature. The rigidity of the gel is one of the most important properties of gelatin and correlates with the gelatin concentration. In this, the gelling quality of gelatin is measured by measuring the gel strength as a function of gelatin concentration. The texture of gelatin gel can therefore be measured.

The Instrumental TPA was developed by a group at the General Foods Corporation Technical Center (Szczesniak and Kleyn, 1963). The parameters obtained from the resulting force- time curve correlate well with sensory evaluations of the same parameters (Friedman et al.,1963). Later, the Instron Universal Testing Machine was adapted to perform a modified TPA (Bourne, 1968, 1974). The difference between the GF Texturometer and the Instron arises from the different compression motions. The plunger of the GF Texturometer is driven at sinusoidal speed and follows the arc of a circle. The resulting TPA curve shows rounded peaks. The plunger of the Instron, however, is driven at constant speed and shows rectilinear-shaped peaks on its TPA curve. Therefore, one disadvantage of the GF Texturometer is that the contact area between the sample and the plunger is not constant during the test; because the plunger moves through the arc of a circle, one edge of the plunger contacts the sample first and, as the downstroke continues, the area of the plunger pressing on the food increases until the entire plunger area is in contact with the sample at the end of the downstroke (Bourne, 1968). Another disadvantage of the GF Texturometer is that the sample platform is flexible and bends a little as the load is applied. This can result in confounding data. On the other hand, the Instron is so rigid that bending of the instrument does not occur, resulting in data that are precise. All modern TPA measurements are now carried out with two-cycle uniaxial compression instruments such as the Instron Universal Testing Machine. The most common parameters derived from the TPA curve (Friedman et al., 1963; Bourne, 1968). The peak force during the first compression cycle is defined as hardness. Fracturability (originally called brittleness) is defined as the force at the first significant break in the curve during the first compression cycle.

It has been experimentally determined that, the hardness and fracturability of gelatin increases as its concentration is increased. However, the values of hardness and fracturability were dependent on the crosshead speed. Also, the cohesiveness for gelatin gels was not significantly correlated with the concentration of gelatin (Munoz et al., 1986). Limitations of the instrumental TPA when compared to sensory TPA include sensitivity and complexity. However, the instrumental TPA parameters are repeatable. Also, TPA parameters correlate well within sensory TPA parameters. The best correlation and data greatly depend on the measuring conditions, the plunger, the precise sample shape, and, most importantly, the careful and skillful operation of the experimenter.

The time required for one TPA test of a sample is ?5 to 10 min, which includes sample positioning and instrument cleaning. It is usually the case that sample preparation (e.g., cooking) requires much more time than TPA measurements. Most uniaxial compression instruments offered to the food industry are computer controlled and have software to immediately calculate the TPA parameters.

Stable Micro systems range of instruments can be used to measure a cross range of properties such as; Hardness, brittleness, fracturability, adhesiveness and elasticity. The range of application of texture analysis is wide. It can be used in the food, cosmetic and pharmaceutical industries.

In conclusion, the work looked at the role played by cross linkers in the stabilization of gelatin. Since cross links exists between different polymer molecules. Their purpose is to hold one large polymer chain to another. Three types of cross linker namely; glutaraldehyde which is a dialdehyde, Chrome sulfate and Salicylic acid were each used to treat gelatin. Then chemical instrumentation such as the use of the FT-IR, which indicates the presence of functional groups in the compound, Differential scanning calorimeter and Stable Micro’s texture analysis were implored to analyse the extent of reaction.

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