Food Analysis Lectures PDF

FOOD ANALYSIS (30402312) Prepared by Dr. Khaled Al-Marazeeq Dept. of Nutrition and Food Technology Al-Huson University College Al-Balqa Applied University 0  Requirements and choice of analytical methods in food analysis The choice of method(s) used...

FOOD ANALYSIS (30402312) Prepared by Dr. Khaled Al-Marazeeq Dept. of Nutrition and Food Technology Al-Huson University College Al-Balqa Applied University 0  Requirements and choice of analytical methods in food analysis The choice of method(s) used for the analysis of foods is dependent on a number of factors, and relates to the following features: 1. Precision: this may be defined as the closeness to each other of a number of replicate measurements in the same laboratory using the same procedure and instrument(s). 2. Reproducibility: may be described as a comparison of the precision between two methods or two laboratories or two analysts using the same procedure. 3. Accuracy: the ability to measure what is intended to be measured, e.g. the fat content of a foodstuff rather than all substances of similar solubilities, or the protein content of a food rather than all nitrogen containing substances. 4. Simplicity of operation: a measure of the ease with which the analysis may be carried out by relatively unskilled workers. 5. Economy: expressed in terms of the costs involved in the analysis in terms of reagents, instrumentation and time. 6. Speed: based on the time to complete a particular analysis. This could be important, for example, where any necessary follow-up action needs to be undertaken quickly, e.g. the recall of food products containing higher or lower levels than the permissible amounts of a particular constituents. 7. Sensitivity: measured in terms of the capacity of the method to detect and quantify food constituents and/or contaminates at very low concentrations such as might occur with trace elements or pesticide residues. 8. Specificity: expressed in terms of the ability to detect and quantify specific food constituents even in the presence of similar compounds, e.g. the estimation of the individual fatty acid present in a food containing fat. 9. Safety: many reagents used in food analysis are potentially hazardous; hazards include the corrosiveness of acids and the flammability of some organic solvents. 10. Official approval: various international bodies give official approval to methods that have been comprehensively studied by independent analysts and shown to be acceptable to the various organizations involved. These include: ISO (International Organization for Standardization) AOAC (Association of Official Analytical Chemists) BSI (British Standards Institution). AACC 1 Types and principles of techniques used in food analysis A. Classical methods (wet chemistry) -Titrimetric analysis Refers to the measurement of the volume of a solution of standard or Titrand, required to react completely with a solution prepared from food to be analysed. -Gravimetric procedures The weight of a food constituent is measured after suitable treatments (moisture, ash) -Solvent extraction Extraction of a food constituent by solvent, e.g. extraction of fat by hexane or diethyl ether. -Rrefractometry Measures the refractive index of a solution containing the component being estimated, e. g. sugar in jams -Polarimetry Based on measurement of the specific rotation produced in polarimeter (a tube with Nicol prism of polarizing material at each end) by a substance solution at a specified wavelength. e.g determination of the amount of sugar present in a solution. B. Instrumental analysis Often these are semi to fully automated techniques that involve the manipulation of molecules, photons and electrons to provide simultaneous (often) qualitative and quantitative analysis with very low detection limits. -Spectroscopic method -Chromatography. 2 The advantages of instrumental methods over classical methods include: 1. The ability to perform trace analysis. 2. Generally, large numbers of samples may be analyzed very quickly. 3. Many instrumental methods can be automated. 4. Less skill and training is usually required to perform instrumental analysis than classical analysis. 5. Sample requirement is small (ml and mg or less can be handled). Because of these advantages, instrumental methods of analysis have revolutionized the field of analytical chemistry, as well as many other scientific fields. However, they have not entirely supplanted classical analytical methods, due to the fact that: 1. Classical methods are generally more accurate and precise. 2. More suitable for the analysis of the major constituents of a chemical sample. 3. In addition, the cost of many analytical instruments can be quite high. 3  Sampling Obtaining a portion or sample that is representative of the whole is referred to as sampling. The total quantity from which a sample is obtained is called the population. By sampling only a fraction of the population, a quality estimate can be obtained more quickly and with less expense and personnel time than if the total population were measured. The larger the sample size the more reliable the sampling. However, sample size is limited by time, cost, sampling methods, the logistics of sample handling, analysis, and data processing. Sampling plan The International Union of Pure and Applied Chemistry defines a sampling plan as “a predetermined procedure for the selection, withdrawal, preservation, transportation, and preparation of the portions to be removed from a lot as samples”. A sampling plan should be selected on the basis of the sampling objective, the study population, the statistical unit, the sample selection criteria, and the analysis procedures. Factors affecting choice of sampling plan  Purpose of the inspection  Nature of the product  Nature of the test method  Nature of the population being investigated Manual vs continuous sampling To obtain a manual sample the person taking the sample must attempt to take a “random sample” to avoid human bias in the sampling method. Thus, the sample must be taken from a number of locations within the population to ensure it is representative of the whole population. Manual sampling requires official devices with specified forms and dimensions named sampling props such as: Thief, Trier, Sampling Tube, Sampling Screw, Sampling Knife, and Drill. 4 Continuous sampling is performed mechanically with automatic sampling devices in production lines such as: Riffle cutter, Circular Sampler, and Straight Line Sampler. Continuous sampling should be less prone to human bias than manual sampling. Type of samples  Raw materials such as vegs., fruits, egg, fish, raw meats, sugar.  Semi processed materials those withdrawn during different processing steps to ensure the effectiveness of the processing criteria such as % of the flour extraction, conc. of the tomato paste.  Processed materials those withdrawn from factories after processing, or from the imported food. Preservation of sample To avoid structural, enzymatic, and microbial changes of the sample it could be by:  Samples should be stored in a container that protects the sample from moisture and other environmental factors.  Refrigeration of the sample at 4ᵒ C.  Oxygen sensitive samples should be stored under nitrogen or an inert gas.  Using of heat treatment, some acids, changing the pH, drying, and some additives to control enzymatic and microbial changes. Sample preparations for analysis  Removal of dusts and sands from the vegetables and fruits.  Removal of the hard shells from the nuts.  Cleaning of the big fishes.  Removal of eggs shell.  Good shaking of liquid samples.  Good mixing of flours and powdered samples. 5  Determination of Moisture and Total Solids Moisture content is one of the most commonly measured properties of food materials. It is important to food scientists for a number of different reasons:  Legal and Labeling Requirements. There are legal limits to the maximum or minimum amount of water that must be present in certain types of food.  Economic. The cost of many foods depends on the amount of water they contain - water is an inexpensive ingredient, and manufacturers often try to incorporate as much as possible in a food, without exceeding some maximum legal requirement.  Microbial Stability. The propensity of microorganisms to grow in foods depends on their water content. For this reason many foods are dried below some critical moisture content.  Food Quality. The texture, taste, appearance and stability of foods depend on the amount of water they contain.  Food Processing Operations. Knowledge of the moisture content is often necessary to predict the behavior of foods during processing, e.g. mixing, drying, flow through a pipe or packaging. It is therefore important for food scientists to be able to reliably measure moisture contents. A number of analytical techniques have been developed for this purpose, which vary in their accuracy, cost, speed, sensitivity, specificity, ease of operation, etc. The choice of an analytical procedure for a particular application depends on the nature of the food being analyzed and the reason the information is needed. The moisture content of a food material is defined through the following equation: %Moisture = (mass of water/mass of sample) X 100% 6 Forms of water in foods:  Bulk water. Bulk water is free from any other constituents, so that each water molecule is surrounded only by other water molecules.  Capillary or trapped water. Capillary water is held in narrow channels between certain food components because of capillary forces.  Physically bound water. A fraction of the water molecule that are in molecular contact with other food constituents, e.g. proteins, carbohydrates or minerals.  Chemically bound water. Water molecules that chemically bonded to other molecules as water of crystallization or as hydrates. These bonds are much stronger than the normal water-water bond. The fact that water molecules can exist in a number of different molecular environments, with different physicochemical properties, can be problematic for the food analyst trying to accurately determine the moisture content of foods. Sample preparation  Selection of a representative sample.  Prevention of changes in the properties of the sample prior to analysis. The most important techniques developed to measure the moisture content of foods are: 1) Evaporation methods Principles  These methods rely on measuring the mass of water in a known mass of sample. The moisture content is determined by measuring the mass of a food before and after the water is removed by evaporation: 7  The basic principle of this technique is that water has a lower boiling point than the other major components within foods, e.g., lipids, proteins, carbohydrates and minerals.  Sometimes a related parameter, known as the total solids, is reported as a measure of the moisture content. The total solids content is a measure of the amount of material remaining after all the water has been evaporated: Thus, %Total solids = (100 - %Moisture).  To obtain an accurate measurement of the moisture content or total solids of a food using evaporation methods it is necessary to remove all of the water molecules that were originally present in the food, without changing the mass of the food matrix. This is often extremely difficult to achieve in practice because the high temperatures or long times required to remove all of the water molecules would lead to changes in the mass of the food matrix, e.g., due to volatilization or chemical changes of some components.  For this reason, the drying conditions used in evaporation methods are usually standardized in terms of temperature and time so as to obtain results that are as accurate and reproducible as possible given the practical constraints. Using a standard method of sample preparation and analysis helps to minimize sample-to-sample variations within and between laboratories. Evaporation Devices a) Convection and forced draft ovens.  Weighed samples are placed in an oven for a specified time and temperature (e.g. 3 hours at 100 oC) and their dried mass is determined, or they are dried until they reach constant mass.  The thermal energy used to evaporate the water is applied directly to the sample via the shelf and air that surround it. There are often considerable temperature variations within convection ovens (atmospheric), and so precise measurements are carried out using forced draft ovens (have a van) that circulate the air so as to achieve a more uniform temperature distribution within the oven. 8   Samples that contain significant quantities of carbohydrates that might undergo chemical changes or volatile materials other than water should not be dried in convection or forced draft oven.  Many official methods of analysis are based on forced draft ovens. b) Vacuum oven.  Weighed samples are placed under reduced pressure (typically 25-100 mm Hg) in a vacuum oven for a specified time and temperature and their dried mass is determined.  The thermal energy used to evaporate the water is applied directly to the sample via the metallic shelf that it sits upon. There is an air inlet and outlet to carry the moisture lost from the sample out of the vacuum oven, which prevents the accumulation of moisture within the oven.  The boiling point of water is reduced when it is placed under vacuum. Drying foods in a vacuum oven therefore has number of advantages over conventional oven drying techniques. If the sample is heated at the same temperature, drying can be carried out much quicker. Alternatively, lower temperatures can be used to remove the moisture (e.g. 70oC instead of 100oC), and so problems associated with degradation of heat labile substances can be reduced.  A number of vacuum oven methods are officially recognized. c) Microwave oven.  Weighed samples are placed in a microwave oven for a specified time and power-level and their dried mass is weighed. Alternatively, weighed samples may be dried until they reach constant final mass.  Analytical microwave ovens containing balances to continuously monitor the weight of a food during drying are commercially available.  The water molecules in the food evaporate because they absorb microwave energy, which causes them to become thermally excited.  The major advantage of microwave methods over other drying methods is that they are simple to use and rapid to carry out. Nevertheless, care must be taken to standardize the 9  drying procedure and ensure that the microwave energy is applied evenly across the sample.  Number of microwave oven drying methods are officially recognized. d) Infrared lamp drying.  The sample to be analyzed is placed under an infrared lamp and its mass is recorded as a function of time. The water molecules in the food evaporate because they absorb infrared energy, which causes them to become thermally excited.  One of the major advantages of infrared drying methods is that moisture contents can be determined rapidly using inexpensive equipment, e.g., 10-25 minutes. This is because the IR energy penetrates into the sample, rather than having to be conducted and convected inwards from the surface of the sample.  To obtain reproducible measurements it is important to control the distance between the sample and the IR lamp and the dimensions of the sample.  IR drying methods are not officially recognized for moisture content determinations because it is difficult to standardize the procedure. Even so, it is widely used in industry because of its speed and ease of use. Practical Considerations 1. Sample dimensions. The rate and extent of moisture removal depends on the size and shape of the sample, and how finely it is ground. The greater the surface area of material exposed to the environment, the faster the rate of moisture removal. 2. Clumping and surface crust formation. Some samples tend to clump together or form a semi-permeable surface crust during the drying procedure. This can lead to erroneous and irreproducible results because the loss of moisture is restricted by the clumps or crust. For this reason, samples are often mixed with dried sand to prevent clumping and surface crust formation. 10 3. Elevation of boiling point. Under normal laboratory conditions pure water boils at 100oC. Nevertheless, if solutes are present in a sample the boiling point of water is elevated. Consequently, the rate of moisture loss from the sample is slower than expected. 4. Decomposition of other food components. If the temperature of drying is too high, or the drying is carried out for too long, there may be decomposition of some of the heat-sensitive components in the food. This will cause a change in the mass of the food matrix and lead to errors in the moisture content determination. It is therefore normally necessary to use a compromise time and temperature, which are sufficient to remove most of the moisture, but not too long to cause significant thermal decomposition of the food matrix. One example of decomposition that interferes with moisture content determinations is that of carbohydrates. C6H12O6 6C + 6 H2O The water that is released by this reaction is not the water we are trying to measure and would lead to an overestimation of the true moisture content. On the other hand, a number of chemical reactions that occur at elevated temperatures lead to water absorption, e.g., sucrose hydrolysis (sucrose + H2O fructose + glucose), and therefore lead to an underestimation of the true moisture content. Foods that are particularly susceptible to thermal decomposition should be analyzed using alternative methods, e.g. chemical or physical. 5. Volatilization of other food components. It is often assumed that the weight loss of a food upon heating is entirely due to evaporation of the water. In practice, foods often contain other volatile constituents that can also be lost during heating, e.g., flavors or odors. For most foods, these volatiles only make up a very small proportion and can therefore be ignored. For foods that do contain significant amounts of volatile components (e.g. spices and herbs) it is necessary to use alternative methods to determine their moisture content, e.g., distillation, chemical or physical methods. 6. High moisture samples. Food samples that have high moisture contents are usually dried in two stages to prevent "spattering" of the sample, and accumulation of moisture in the oven. For example, most of the moisture in milk is removed by heating on a steam bath prior to completing the drying in an oven. 11  7. Sample pans. It is important to use appropriate pans to contain samples, and to handle them correctly, when carrying out a moisture content analysis. Typically, aluminum pans are used because they are relatively cheap and have a high thermal conductivity. Pans should be handled with tongs because fingerprints can contribute to the mass of a sample. Pans should be dried in an oven and stored in desiccators prior to use to ensure that no residual moisture is attached to them. AOAC recommended using pans with 5.5 cm in diameter and 2 cm deep, with an insert cover to control sample loss by spattering. Advantages and Disadvantages  Advantages: -Precise; -Relatively cheap; -Easy to use; -Officially sanctioned for many applications; -Many samples can be analyzed simultaneously.  Disadvantages: -Destructive; -Unsuitable for some types of food; -Time consuming. 2) Distillation Methods Principles  Distillation methods are based on direct measurement of the amount of water removed from a food sample by evaporation: %Moisture = 100 (MWATER/MINITIAL). In contrast, evaporation methods are based on indirect measurement of the amount of water removed from a food sample by evaporation: %Moisture = 100 (MINITIAL - MDRIED)/MINITIAL.  Basically, distillation methods involve heating a weighed food sample (MINITIAL) in the presence of an organic solvent that is immiscible with water. The water in the sample evaporates and is collected in a graduated glass tube where its mass is determined (MWATER).  A known weight of food is placed in a flask with an organic solvent such as xylene (b.p 137-140 ᵒC) or toluene (b.p. 110 ᵒC). The organic solvent must be insoluble with water; 12  has a higher boiling point than water; be less dense than water; and be safe to use. The flask containing the sample and the organic solvent is attached to a condenser by a side arm and the mixture is heated. The water in the sample evaporates and moves up into the condenser where it is cooled and converted back into liquid water, which then trickles into the graduated tube. When no more water is collected in the graduated tube, distillation is stopped, and the volume of water is read from the tube. Practical Considerations There are a number of practical factors that can lead to erroneous results: (i) emulsions can sometimes form between the water and the solvent which are difficult to separate; (ii) water droplets can adhere to the inside of the glassware, (iii) decomposition of thermally labile samples can occur at the elevated temperatures used. Advantages and Disadvantages  Advantages: -Suitable for application to foods with low moisture contents; -Suitable for application to foods containing volatile oils, such as herbs or spices, since the oils remain dissolved in the organic solvent, and therefore do not interfere with the measurement of the water; -Equipment is relatively cheap, easy to setup and operate; -Distillation methods have been officially sanctioned for a number of food applications.  Disadvantages: -Destructive; -Relatively time-consuming; - Involves the use of flammable solvents; -Not applicable to some types of foods. 13 3) Chemical Reaction Methods  In these methods a chemical reagent is added to the food that reacts specifically with water to produce a measurable change in the properties of the system, e.g., mass, volume, pressure, pH, color, conductivity. Measurable changes in the system are correlated to the moisture content using calibration curves.  To make accurate measurements it is important that the chemical reagent reacts with all of the water molecules present, but not with any of the other components in the food matrix.  Two methods that are commonly used in the food industry are the Karl-Fisher titration and gas production methods.  Chemical reaction methods do not usually involve the application of heat and so they are suitable for foods that contain thermally labile substances that would change the mass of the food matrix on heating (e.g., food containing high sugar concentrations) or foods that contain volatile components that might be lost by heating (e.g. spices and herbs). Karl-Fisher method The Karl-Fisher titration is often used for determining the moisture content of foods that have low water contents (e.g. dried fruits and vegetables, confectionary, coffee, oils and fats). It is based on the reduction of iodine by SO2 in the presence of water: 2H2O + SO2 + I2 (dark reddish-brown)→ H2SO4 + 2HI (colorless)  This reaction was originally used because HI (hydrogen iodide) is colorless, whereas I2 (iodine) is a dark reddish-brown color; hence there is a measurable change in color when water reacts with the added chemical reagents.  Sulfur dioxide and iodine are gaseous and would normally be lost from solution. For this reason, the above reaction has been modified by adding solvents such as pyridine (C5H5N) and methanol (CH3OH) that keep the SO2 and I2 in solution, although the basic principles of the method are the same. C5H5N.I2 + C5H5N.SO2 + C5H5N + H2O →2C5H5N.HI + C5H5N.SO3 C5H5N.SO3 + CH3OH → C5H5N(H)SO4.CH3 14  There is a special apparatus to perform this experiment called “Karl-Fisher titration unit” which iodine and SO2 (Karl Fisher reagent) in the appropriate form are added to the sample in a closed chamber protected from atmospheric moisture.  The precision of the technique can be improved by using electrical methods to follow the end-point of the reaction, rather than observing a color change. Relatively inexpensive commercial instruments have been developed which are based on the Karl-Fisher titration, and some of these are fully automated to make them less labor intensive. Disadvantage: interferences of Karl Fisher reagent with other food constituents which elevate moisture readings, such as interferences with vitamin C, carbonyl groups, amines, etc., and other constituent that may alleviate water readings such as quinines, diacyl peroxides, etc. Gas production methods Commercial instruments are also available that utilize specific reactions between chemical reagents and water that lead to the production of a gas. For example, when a food sample is mixed with powdered calcium carbide the amount of acetylene gas produced is related to the moisture content. CaC2 + 2H2O C2H2 (gas) + Ca(OH)2 The amount of gas produced can be measured in a number of different ways including: (i) the volume of gas produced, (ii) the decrease in the mass of the sample after the gas is released, and (iii) the increase in pressure of a closed vessel containing the reactants. 15 4) Physical methods a) Conductivity Method The conductivity method functions because the conductivity of an electric current increases with the percentage of moisture in the sample. A modestly accurate and rapid method is created when one measures resistance. Ohm's law states that the strength of electricity current is equal to the electromotive force divided by the electrical resistance. Strength of electricity= electromotive force/electrical resistance. The electrical resistance of wheat with 13% moisture is seven times as great as that with 14% moisture and 50 times that with 15% moisture. Temperature must be kept constant, and 1minutes is necessary for a single determination. b) Hydrometer Measuring specific gravity is based on Archimedes principle, which states the solid suspended in a liquid will be buoyed by a force equal to the weight of the liquid displaced in a liquid of low density, the hydrometer will sink to a greater depth, whereas in a liquid of high density, the hydrometer will not sink as far. Hydrometers are available in narrow and wide ranges of specific gravity. The spindle of the hydrometer is calibrated to read specific gravity directly at 15.5 or 20 ºC. For every degree above 15.5 ºC, 0.1 lactometer units is added to the reading, and 0.1 lactometer units is subtracted for every degree below 15.5 ºC. Hydrometers are include Baume hydrometer (salt brines), a Brix hydrometer (sugar solutions). Specific gravity measurements are best applied to the analysis of solutions consisting of only one component in a medium of water. c) Refractometer The refractometer procedure is rapid and accurate for solids in syrups. The refractometer has been valuable in determining the soluble solids in fruits and fruit products. The refractive index of oil, syrup, or other liquid is a dimensionless constant that can be used to describe the nature of the food. While some refractometers are designed only to provide results as refractive indices, others, particularly hand-held, quick-to-use units, are equipped with scales calibrated to read percent solids, or percent sugars. Refractometers are used not just on the laboratory bench or as hand-held units, it can be installed in a liquid processing line to monitor the Brix of products such as carbonated soft drinks, dissolved solids in orange juice, and percent solids in milk. 16  Ash Analysis The ash content is a measure of the total amount of minerals present within a food, whereas the mineral content is a measure of the amount of specific inorganic components present within a food, such as Ca, Na, K and Cl. Determination of the ash content of foods is important for many number of reasons:  Nutritional labeling. The concentration minerals present must often be stipulated on the label of a food.  Quality. The quality of many foods depends on the concentration and type of minerals they contain, including their taste, appearance, texture and stability.  Microbiological stability. High mineral contents are sometimes used to retard the growth of certain microorganisms.  Nutrition. Some minerals are essential to a healthy diet (e.g., calcium, phosphorous, potassium and sodium) whereas others can be toxic (e.g., lead, mercury, cadmium and aluminum).  Processing. It is often important to know the mineral content of foods during processing because this affects the physicochemical properties of foods. Determination of Ash Content  Ash is the inorganic residue remaining after the water and organic matter have been removed by heating in the presence of oxidizing agents, which provides a measure of the total amount of minerals within a food.  Analytical techniques for providing information about the total mineral content are based on the fact that the minerals (the analyte) can be distinguished from all the other components (the matrix) within a food in some measurable way.  The most widely used methods are based on the fact that minerals are not destroyed by heating (inorganic), and that they have a low volatility compared to other food components. 17  The three main types of analytical procedure used to determine the ash content of foods are based on this principle: dry ashing, wet ashing and low temperature plasma dry ashing.  The method chosen for a particular analysis depends on the reason for carrying out the analysis, the type of food analyzed and the equipment available.  Ashing may also be used as the first step in preparing samples for analysis of specific minerals, by atomic spectroscopy or other various traditional methods.  Ash contents of fresh foods rarely exceed 5%, although some processed foods can have ash contents as high as 12%, e.g., dried beef. Sample Preparation  As with all food analysis procedures it is crucial to carefully select a sample whose composition represents that of the food being analyzed and to ensure that its composition does not change significantly prior to analysis.  Typically, samples of 1-10g are used in the analysis of ash content.  Solid foods are finely ground and then carefully mixed to facilitate the choice of a representative sample.  Before carrying out an ash analysis, samples that are high in moisture are often dried to prevent spattering during ashing. High fat samples are usually defatted by solvent extraction, as this facilitates the release of the moisture and prevents spattering.  Other possible problems include contamination of samples by minerals in grinders, glassware or crucibles which come into contact with the sample during the analysis.  For the same reason, it is recommended to use deionized water when preparing samples.  Many kinds of crucibles are available (Quarts, Pyrex, Porcelain, Steel, Platinum,…), all of them should be fired and cleaned prior to use. 18 Dry Ashing  Dry ashing procedures use a high temperature muffle-furnace capable of maintaining temperatures of between 500 and 600oC.  Water and other volatile materials are vaporized and organic substances are burned in the presence of the oxygen in air to CO2, H2O and N2.  Most minerals are converted to oxides, sulfates, phosphates, chlorides or silicates.  Although most minerals have fairly low volatility at these high temperatures, some are volatile and may be partially lost, e.g., iron, lead and mercury. If an analysis is being carried out to determine the concentration of one of these substances, then it is advisable to use an alternative ashing method that uses lower temperatures.  The food sample is weighed before and after ashing to determine the concentration of ash present. The ash content can be expressed on either a dry or wet basis: Advantages: Safe, few reagents are required, many samples can be analyzed simultaneously, not labor intensive, and ash can be analyzed for specific mineral content. Disadvantages: Long time required (12-24 hours), muffle furnaces are quite costly to run due to electrical costs, loss of volatile minerals at high temperatures, e.g., Cu, Fe, Pb, Hg, Ni, Zn.  Recently, analytical instruments have been developed to dry ash samples based on microwave heating. These devices can be programmed to initially remove most of the moisture (using a relatively low heat) and then convert the sample to ash (using a relatively high heat). Microwave instruments greatly reduce the time required to carry out an ash analysis, with the analysis time often being less than an hour. The major disadvantage is that it is not possible to simultaneously analyze as many samples as in a muffle furnace. 19 Wet Ashing  Wet ashing is primarily used in the preparation of samples for subsequent analysis of specific minerals (see later).  It breaks down and removes the organic matrix surrounding the minerals so that they are left in an aqueous solution.  A dried ground food sample is usually weighed into a flask containing strong acids and oxidizing agents (e.g., nitric, perchloric and/or sulfuric acids) and then heated.  Heating is continued until the organic matter is completely digested, leaving only the mineral oxides in solution.  The temperature and time used depends on the type of acids and oxidizing agents used.  Typically, a digestion takes from 10 minutes to a few hours at temperatures of about 350oC.  The resulting solution can then be analyzed for specific minerals.  Advantages: Little loss of volatile minerals occurs because of the lower temperatures used, more rapid than dry ashing.  Disadvantages: Labor intensive, requires a special fume-cupboard if perchloric acid is used because of its hazardous nature, low sample throughput.  Determination of Water Soluble and Insoluble Ash  As well as the total ash content, it is sometimes useful to determine the ratio of water soluble to water-insoluble ash as this gives a useful indication of the quality of certain foods, e.g., the fruit content of preserves and jellies.  Ash is diluted with distilled water then heated to nearly boiling, and the resulting solution is filtered. The amount of soluble ash is determined by drying the filtrate, and the insoluble ash is determined by rinsing, drying and ashing the filter paper.  Filter paper should be ash-free. 20  Analysis of Proteins  Proteins are polymers of amino acids. Twenty different types of amino acids occur naturally in proteins. Proteins differ from each other according to the type, number and sequence of amino acids that make up the polypeptide backbone. As a result, they have different molecular structures, nutritional attributes and physiochemical properties.  Nitrogen is the most distinguishing element present in proteins. However, nitrogen content in various food proteins ranges from 13.5-19%, due to the variation in the specific amino acid composition of proteins.  The methods to measure protein content involve the determinations of nitrogen in food sample. Importance of protein analysis:  Biological activity determination. Some proteins, including enzymes or enzyme inhibitors, are relevant to food science and nutrition, e.g. the proteolytic enzymes in the tenderization of meats, trypsin inhibitors in legumes are proteins.  Functional property investigation. Proteins in various types of food have unique food functional properties, e.g. glutenin in wheat flour for bread making, casein in milk for coagulation into cheese products.  Nutrition labeling. Determination of Overall Protein Concentration 1) Kjeldahl method ‫كلدال‬  The Kjeldahl method was developed in 1883 by Johann Kjeldahl.  A food is digested with a strong acid so that it releases nitrogen )Nitrogen is the most distinguishing element present in proteins( which can be determined by a suitable titration technique. The amount of protein present is then calculated from the nitrogen concentration of the food.  It is usually considered to be the standard method of determining protein concentration.  Because the Kjeldahl method does not measure the protein content directly a conversion factor (F) is needed to convert the measured nitrogen concentration to a protein concentration. 21  A conversion factor of 6.25 (equivalent to 0.16 g nitrogen per gram of protein) is used for many applications, however, this is only an average value, and each protein has a different conversion factor depending on its amino-acid composition.  The Kjeldahl method can conveniently be divided into three steps: digestion, neutralization and titration. Principles Digestion  The food sample to be analyzed is weighed into a digestion flask and then digested by heating it in the presence of sulfuric acid (an oxidizing agent which digests the food), anhydrous sodium sulfate (to speed up the reaction by raising the boiling point) and a catalyst, such as copper, selenium, titanium, or mercury (to speed up the reaction).  Digestion converts any nitrogen in the food into ammonia, and other organic matter to C02 and H20. Ammonia gas is not liberated in an acid solution because the ammonia is in the form of the ammonium ion (NH4+) which binds to the sulfate ion (SO42-) and thus remains in solution: Protein + H2SO4 heating→ SO3+CO2+NH3 (ammonia) +H2O 2NH3 + H2SO4 → (NH4)2SO4 (ammonium sulfate) Neutralization (distillation) After the digestion has been completed the digestion flask is connected to a receiving flask by a tube. The solution in the digestion flask is then made alkaline by addition of sodium hydroxide, which converts the ammonium sulfate into ammonia gas: (NH4)2SO4 + 2NaOH → NH4OH + Na2SO4 NH4OH heating→ 2NH3↑ + 2H2O The ammonia gas that formed is liberated from the solution and moves out of the digestion flask into the receiving flask - which contains an excess of boric acid. The low pH of the solution in 22 the receiving flask converts the ammonia gas into the ammonium ion, and simultaneously converts the boric acid to the borate ion: 3NH3 + H3BO3 (boric acid) pink → NH4+ + H2BO3- (borate ion) → (NH4)3BO3 (ammonium borate) violate Titration The nitrogen content is then estimated by titration of the ammonium borate formed with standard sulfuric or hydrochloric acid, using a suitable indicator to determine the end-point of the reaction. (NH4)3BO3 + HCl → NH4Cl + H3BO3 The concentration of hydrogen ions (in moles) required to reach the end-point is equivalent to the concentration of nitrogen that was in the original food. The following equation can be used to determine the nitrogen concentration of a sample: % N = (ml of acid for sample-ml of acid for blank) × N of HCl ×14×100 g of sample ×1000 % Crude protein = % N × Conversion factor Where 14g is the molecular weight of nitrogen. A blank sample is usually ran at the same time as the material being analyzed to take into account any residual nitrogen which may be in the reagents used to carry out the analysis. Once the nitrogen content has been determined it is converted to a protein content using the appropriate conversion factor. Conversion factor for various foods Egg&Meat Dairy products Wheat Cereal grains&Oil seeds Almonds Peanuts Nuts 6.25 6.38 5.7 6.25 5.18 5.46 5.30 23 Advantages. -The Kjeldahl method is widely used internationally and is still the standard method for comparison against all other methods. -Its universality, high precision and good reproducibility have made it the major method for the estimation of protein in foods. Disadvantages. -It does not give a measure of the true protein, since all nitrogen in foods is not in the form of protein. -Different proteins need different correction factors because they have different amino acid sequences. -The use of concentrated sulfuric acid at high temperatures poses a considerable hazard, as does the use of some of the possible catalysts. -The technique is time consuming to carry-out. 2) Biuret Method  A violet-purplish color is produced when cupric ions (Cu2+) interact with peptide bonds under alkaline conditions.  The biuret reagent, which contains all the chemicals required to carry out the analysis, can be purchased commercially. It is mixed with a protein solution and then allowed to stand for 15-30 minutes.  Absorbance is read at 540 nm. (spectroscopy)  A standard curve of concentration versus absorbance is constructed using bovine serum albumin (BSA).  The major advantage of this technique is that there is no interference from materials that adsorb at lower wavelengths, and the technique is less sensitive to protein type because it utilizes absorption involving peptide bonds that are common to all proteins, rather than specific side groups.  However, it has a relatively low sensitivity compared to other UV-visible methods. 24 3) Lowry Method  The Lowry method combines the biuret reagent with another reagent (Folin-Ciocalteau phenol reagent) which reacts with tyrosine and tryptophan residues in proteins.  The method measures a combination of peptide bonds and the amino acids tryptophan and tyrosine.  This gives a bluish color which can be read between 500-750nm depending on the sensitivity required. (At 500 nm low sensitivity for high protein concentrations, at 750 nm high sensitivity for low protein concentrations).  This method is more sensitive to low concentrations of proteins than the biuret method. There are other methods for determination of protein in foods: -Dye binding methods -Turbimetric method -Dumas method -Ninhydrin method -Instrumental Techniques 25  Analysis of Lipids Introduction Lipids are one of the major constituents of foods, and are important in our diet for a number of reasons. They are a major source of energy and provide essential lipid nutrients. Nevertheless, over-consumption of certain lipid components can be detrimental to our health, e.g. cholesterol and saturated fats. In many foods the lipid component plays a major role in determining the overall physical characteristics, such as flavor, texture, mouth feel and appearance. For this reason, it is difficult to develop low-fat alternatives of many foods because once the fat is removed some of the most important physical characteristics are lost. Finally, many fats are prone to lipid oxidation, which leads to the formation of off-flavors and potentially harmful products. Some of the most important properties of concern to the food analyst are:  Total lipid concentration  Type of lipids present  Physicochemical properties of lipids, e.g., crystallization, melting point, smoke point, rheology, density and color  Structural organization of lipids within a food. Properties of Lipids in Foods Lipids are usually defined as those components that are soluble in organic solvents (such as ether, hexane or chloroform), but are insoluble in water. This group of substances includes triacylglycercols, diacylglycercols, monoacylglycercols, free fatty acids, phospholipids, sterols, caretonoids and vitamins A and D. The lipid fraction of a fatty food therefore contains a complex mixture of different types of molecule. Even so, triacylglycercols are the major component of most foods, typically making up more than 95 - 99% of the total lipids present. Triacylglycerols are esters of three fatty acids and a glycerol molecule. The fatty acids normally found in foods vary in chain length, degree of unsaturation and position on the glycerol molecule. Consequently, the triacylglycerol fraction itself consists of a complex mixture of different types of molecules. Each type of fat has a different profile of lipids present which determines the precise nature of its nutritional and physiochemical properties. 26  The terms fat, oil and lipid are often used interchangeably by food scientists. Although sometimes the term fat is used to describe those lipids that are solid at the specified temperature, whereas the term oil is used to describe those lipids that are liquid at the specified temperature. Determination of Total Lipid Concentration It is important to be able to accurately determine the total fat content of foods for a number of reasons:  Economic (to evaluate the price of ingredients)  Legal (to conform to standards of identity and nutritional labeling laws)  Health (development of low fat foods)  Quality (food properties depend on the total lipid content)  Processing (processing conditions depend on the total lipid content) The principle physicochemical characteristics of lipids (the "analyte") used to distinguish them from the other components in foods (the "matrix") are: a. Their solubility in organic solvents, b. Immiscibility with water, c. Physical characteristics (e.g., relatively low density) d. Spectroscopic properties. The analytical techniques based on these principles can be conveniently categorized into three different types: (i) solvent extraction; (ii) non-solvent extraction and (iii) instrumental methods. (i) Solvent Extraction The fact that lipids are soluble in organic solvents, but insoluble in water, provides the food analyst with a convenient method of separating the lipid components in foods from water soluble components, such as proteins, carbohydrates and minerals. In fact, solvent extraction techniques are one of the most commonly used methods of isolating lipids from foods and of determining the total lipid content of foods. 27 Sample Preparation The preparation of a sample for solvent extraction usually involves a number of steps: Drying sample. It is often necessary to dry samples prior to solvent extraction, because many organic solvents cannot easily penetrate into foods containing water, and therefore extraction would be inefficient. Particle size reduction. Dried samples are usually finely ground prior to solvent extraction to produce a more homogeneous sample and to increase the surface area of lipid exposed to the solvent. Grinding is often carried out at low temperatures to reduce the tendency for lipid oxidation to occur. Acid hydrolysis. Some foods contain lipids that are complexed with proteins (lipoproteins) or polysaccharides (glycolipids). To determine the concentration of these components it is necessary to break the bonds which hold the lipid and non-lipid components together prior to solvent extraction. Acid hydrolysis is commonly used to release bound lipids into easily extractable forms, e.g. a sample is digested by heating it for 1 hour in the presence of 3N HCl acid. Solvent Selection. The ideal solvent for lipid extraction would completely extract all the lipid components from a food, while leaving all the other components behind. In practice, the efficiency of solvent extraction depends on the polarity of the lipids present compared to the polarity of the solvent. Polar lipids (such as glycolipids or phospholipids) are more soluble in polar solvents (such as alcohols), than in non-polar solvents (such as hexane). On the other hand, non-polar lipids (such as triacylglycerols) are more soluble in non-polar solvents than in polar ones. The fact that different lipids have different polarities means that it is impossible to select a single organic solvent to extract them all. Thus the total lipid content determined by solvent extraction depends on the nature of the organic solvent used to carry out the extraction: the total lipid content determined using one solvent may be different from that determined using another solvent. 28  In addition to the above considerations, a solvent should also be inexpensive, have a relatively low boiling point (so that it can easily be removed by evaporation), be non-toxic and be nonflammable (for safety reasons). It is difficult to find a single solvent which meets all of these requirements. Ethyl ether and petroleum ether are the most commonly used solvents, but pentane and hexane are also used for some foods. a. Batch Solvent Extraction These methods are based on mixing the sample and the solvent in a suitable container, e.g., a separatory funnel. The container is shaken vigorously, and the organic solvent and aqueous phase are allowed to separate (either by gravity or centrifugation). The aqueous phase is then decanted off, and the concentration of lipid in the solvent is determined by evaporating the solvent and measuring the mass of lipid remaining: %Lipid = (Mlipid/Msample) ×100. This procedure may have to be repeated a number of times to improve the efficiency of the extraction process. In this case the aqueous phase would undergo further extractions using fresh solvent, then all the solvent fractions would be collected together and the lipid determined by weighing after evaporation of solvent. b. Semi-Continuous Solvent Extraction Semi-continuous solvent extraction methods are commonly used to increase the efficiency of lipid extraction from foods. The Soxhlet method is the most commonly used example of a semi- continuous method. In the Soxhlet method a sample is dried, ground into small particles and placed in a porous thimble. The thimble is placed in an extraction chamber, which is suspended above a flask containing the solvent and below a condenser. The flask is heated and the solvent evaporates and moves up into the condenser where it is converted into a liquid that trickles into the extraction chamber containing the sample. Eventually, the solvent builds up in the extraction chamber and completely surrounds the sample. 29  The extraction chamber is designed so that when the solvent surrounding the sample exceeds a certain level it overflows and trickles back down into the boiling flask. As the solvent passes through the sample it extracts the lipids and carries them into the flask. The lipids then remain in the flask because of their low volatility. At the end of the extraction process, which typically lasts a few hours, the flask containing the solvent and lipid is removed, the solvent is evaporated and the mass of lipid remaining is measured (Mlipid). The percentage of lipid in the initial sample (Msample) can then be calculated. A number of instrument manufacturers have designed modified versions of the Soxhlet method that can be used to determine the total lipid content more easily and rapidly (e.g. Soxtec). c. Continuous Solvent Extraction The Goldfisch method is similar to the Soxhlet method except that the extraction chamber is designed so that the solvent just trickles through the sample rather than building up around it. This reduces the amount of solvent and time required to carry out the extraction, but it has the disadvantage that channeling of the solvent can occur, i.e., the solvent may preferentially take certain routes through the sample and therefore the extraction is inefficient. This is not a problem in the Soxhlet method because the sample is always surrounded by solvent. d. Accelerated Solvent Extraction The efficiency of solvent extraction can be increased by carrying it out at a higher temperature and pressure than are normally used. The effectiveness of a solvent at extracting lipids from a food increases as its temperature increases, but the pressure must also be increased to keep the solvent in the liquid state. This reduces the amount of solvent required to carry out the analysis, which is beneficial from a cost and environmental standpoint. Special instruments are available to carry out solvent extraction at elevated temperatures and pressures. 30 e. Supercritical Fluid Extraction Solvent extraction can be carried out using special instruments that use supercritical carbon dioxide (rather than organic liquids) as the solvent. These instruments are finding greater use because of the cost and environmental problems associated with the usage and disposal of organic solvents. When pressurized CO2 is heated above a certain critical temperature it becomes a supercritical fluid, which has some of the properties of a gas and some of a liquid. The fact that it behaves like a gas means that it can easily penetrate into a sample and extract the lipids, while the fact that it behaves like a fluid means that it can dissolve a large quantity of lipids (especially at higher pressures). Instruments based on this principle heat the food sample to be analyzed in a pressurized chamber and then mix supercritical CO2 fluid with it. The CO2 extracts the lipid, and forms a separate solvent layer, which is separated from the aqueous components. The pressure and temperature of the solvent are then reduced which causes the CO2 to turn to a gas, leaving the lipid fraction remaining. The lipid content of a food is determined by weighing the percentage of lipid extracted from the original sample. (ii) Nonsolvent Liquid Extraction Methods. A number of liquid extraction methods do not rely on organic solvents but use other chemicals to separate the lipids from the rest of the food. The Babcock, Gerber and Detergent methods are examples of nonsolvent liquid extraction methods for determining the lipid content of milk and some other dairy products. a. Babcock Method A specified amount of milk is accurately pipetted into a specially designed flask (the Babcock bottle). Sulfuric acid is mixed with the milk, which digests the protein, generates heat, and breaks down the fat globule membrane that surrounds the droplets, thereby releasing the fat. The sample is then centrifuged while it is hot (55-60oC) which causes the liquid fat to rise into the neck of the Babcock bottle. The neck is graduated to give the amount of milk fat present in wt%. The Babcock method takes about 45 minutes to carry out, and is precise to within 0.1%. It does not determine phospholipids in milk, because they are located in the aqueous phase or at the boundary between the lipid and aqueous phases. 31 b. Gerber Method This method is similar to the Babcock method except that a mixture of sulfuric acid and isoamyl alcohol, and a slightly different shaped bottle, are used. It is faster and simpler to carry out than the Babcock method. The isoamyl alcohol is used to prevent charring of the sugars by heat and sulfuric acid which can be a problem in the Babcock method since it makes it difficult to read the fat content from the graduated flask. This method is used mainly in Europe, whilst the Babcock method is used mainly in the USA. As with the Babcock method, it does not determine phospholipids. c. Detergent Method This method was developed to overcome the inconvenience and safety concerns associated with the use of highly corrosive acids. A sample is mixed with a combination of surfactants in a Babcock bottle. The surfactants displace the fat globule membrane which surrounds the emulsion droplets in milk and causes them to coalesce and separate. The sample is centrifuged which allows the fat to move into the graduated neck of the bottle, where its concentration can then be determined. (Sodium lauryl) Conclusion Soxhlet extraction is one of the most commonly used methods for determination of total lipids in dried foods. This is mainly because it is fairly simple to use and is the officially recognized method for a wide range of fat content determinations. The main disadvantages of the technique are that a relatively dry sample is needed (to allow the solvent to penetrate), it is destructive, and it is time consuming. For high moisture content foods it is often better to use batch solvent or nonsolvent extraction techniques. 32  Analysis of Carbohydrates Introduction Carbohydrates are one of the most important components in many foods. Carbohydrates may be present as isolated molecules or they may be physically associated or chemically bound to other molecules. Individual molecules can be classified according to the number of monomers that they contain as monosaccharides, oligosaccharides or polysaccharides. Some carbohydrates are digestible by humans and therefore provide an important source of energy, whereas others are indigestible and therefore do not provide energy. Indigestible carbohydrates form part of a group of substances known as dietary fiber, which also includes lignin. It is important to determine the type and concentration of carbohydrates in foods for a number of reasons.  Standards of Identity - foods must have compositions which conform to government regulations.  Nutritional Labeling - to inform consumers of the nutritional content of foods.  Detection of Adulteration - each food type has a carbohydrate "fingerprint".  Food Quality - physicochemical properties of foods such as sweetness, appearance, stability and texture depend on the type and concentration of carbohydrates present.  Economic - industry doesn't want to give away expensive ingredients.  Food Processing - the efficiency of many food processing operations depends on the type and concentration of carbohydrates that are present. Methods of Analysis A large number of analytical techniques have been developed to measure the total concentration and type of carbohydrates present in foods.  By difference The carbohydrate content of a food can be determined by calculating the percent remaining after all the other components have been measured: %carbohydrates = 100 – (%moisture - %protein - %lipid - %mineral). 33 Nevertheless, this method can lead to erroneous results due to experimental errors in any of the other methods, and so it is usually better to directly measure the carbohydrate content for accurate measurements. Monosaccharides and Oligosaccharides Sample Preparation  The amount of preparation needed to prepare a sample for carbohydrate analysis depends on the nature of the food being analyzed.  Aqueous solutions, such as fruit juices, syrups and honey, usually require very little preparation prior to analysis. On the other hand, many foods contain carbohydrates that are physically associated or chemically bound to other components, e.g., nuts, cereals, fruit, breads and vegetables. In these foods it is usually necessary to isolate the carbohydrate from the rest of the food before it can be analyzed.  The precise method of carbohydrate isolation depends on the carbohydrate type, the food matrix type and the purpose of analysis, however, there are some procedures that are common to many isolation techniques.  For example, foods are usually dried under vacuum (to prevent thermal degradation), ground to a fine powder (to enhance solvent extraction) and then defatted by solvent extraction.  One of the most commonly used methods of extracting low molecular weight carbohydrates from foods is to boil a defatted sample with an 80% alcohol solution. Monosaccharides and oligosaccharides are soluble in alcoholic solutions, whereas proteins, polysaccharides and dietary fiber are insoluble.  The soluble components can be separated from the insoluble components by filtering the boiled solution and collecting the filtrate (the part which passes through the filter) and the retentante (the part retained by the filter). These two fractions can then be dried and weighed to determine their concentrations.  In addition, to monosaccharides and oligosaccharides various other small molecules may also be present in the alcoholic extract that could interfere with the subsequent analysis e.g., amino acids, organic acids, pigments, vitamins, minerals etc. It is usually necessary to remove these components prior to carrying out a carbohydrate analysis. This is commonly achieved by treating the solution with clarifying agents or by passing it through one or more ion-exchange resins.  Clarifying agents. Water extracts of many foods contain substances that are colored or produce turbidity, and thus interfere with spectroscopic analysis or endpoint determinations. For this reason solutions are usually clarified prior to analysis. The most commonly used clarifying agents are heavy metal salts (such as lead acetate) which form insoluble complexes with interfering substances that can be removed by filtration or centrifugation. However, it is important that the clarifying agent does not precipitate any of the carbohydrates from solution as this would cause an underestimation of the carbohydrate content. 34  Ion-exchange. Many monosaccharides and oligosaccharides are polar non-charged molecules and can therefore be separated from charged molecules by passing samples through ion- exchange columns. By using a combination of a positively and a negatively charged column it is possible to remove most charged contaminants. Non-polar molecules can be removed by passing a solution through a column with a non-polar stationary phase. Thus proteins, amino acids, organic acids, minerals and hydrophobic compounds can be separated from the carbohydrates prior to analysis.  Prior to analysis, the alcohol can be removed from the solutions by evaporation under vacuum so that an aqueous solution of sugars remains. 1) Chromatographic methods Chromatographic methods are the most powerful analytical techniques for the analysis of the type and concentration of monosaccharides and oligosaccharides in foods. Thin layer chromatography (TLC), Gas chromatography (GC) and High Performance Liquid chromatography (HPLC) are commonly used to separate and identify carbohydrates. Carbohydrates are separated on the basis of their differential adsorption characteristics by passing the solution to be analyzed through a column. Carbohydrates can be separated on the basis of their partition coefficients, polarities or sizes, depending on the type of column used. 2) Chemical methods A number of chemical methods used to determine monosaccharides and oligosaccharides are based on the fact that many of these substances are reducing agents that can react with other components to yield precipitates or colored complexes which can be quantified. The concentration of carbohydrate can be determined gravimetrically, spectrophotometrically or by titration. Non-reducing carbohydrates can be determined using the same methods if they are first hydrolyzed to make them reducing. It is possible to determine the concentration of both non- reducing and reducing sugars by carrying out an analysis for reducing sugars before and after hydrolyzation. Many different chemical methods are available for quantifying carbohydrates. Most of these can be divided into three catagories: titration, gravimetric and colorimetric. An example of each of these different types is given below. a) Titration Methods The Lane-Eynon method is an example of a tritration method of determining the concentration of reducing sugars in a sample. A burette is used to add the carbohydrate solution being analyzed to a flask containing a known amount of boiling copper sulfate solution and a methylene blue indicator. The reducing sugars in the carbohydrate solution react with the copper sulfate present in the flask. Once all the copper sulfate in solution has reacted, any further addition of reducing sugars causes the indicator to change from blue to white. The volume of sugar solution required to reach the end point is recorded. 35 The reaction is not stoichemetric, which means that it is necessary to prepare a calibration curve by carrying out the experiment with a series of standard solutions of known carbohydrate concentration. The disadvantages of this method are: (i) the results depend on the precise reaction times, temperatures and reagent concentrations used and so these parameters must be carefully controlled; (ii) it cannot distinguish between different types of reducing sugar, (iii) it cannot directly determine the concentration of non-reducing sugars, (iv) it is susceptible to interference from other types of molecules that act as reducing agents. b) Gravimetric Methods The Munson and Walker method is an example of a gravimetric method of determining the concentration of reducing sugars in a sample. Carbohydrates are oxidized in the presence of heat and an excess of copper sulfate and alkaline tartrate under carefully controlled conditions which leads to the formation of a copper oxide precipitate: reducing sugar + Cu2+ + base → oxidized sugar + CuO2 (precipitate) The amount of precipitate formed is directly related to the concentration of reducing sugars in the initial sample. The concentration of precipitate present can be determined gravimetrically (by filtration, drying and weighing), or titrimetrically (by redissolving the precipitate and titrating with a suitable indicator). This method suffers from the same disadvantages as the Lane-Eynon method, neverthless, it is more reproducible and accurate. c) Colorimetric Methods  The Anthrone method is an example of a colorimetric method of determining the concentration of the total sugars in a sample. Sugars react with the anthrone reagent under acidic conditions to yield a blue-green color. The sample is mixed with sulfuric acid and the anthrone reagent and then boiled until the reaction is completed. The solution is then allowed to cool and its absorbance is measured at 620 nm. (spectroscopy). There is a linear relationship between the absorbance and the amount of sugar that was present in the original sample. This method determines both reducing and non-reducing sugars because of the presence of the strongly oxidizing sulfuric acid. Like the other methods it is non-stoichemetric and therefore it is necessary to prepare a calibration curve using a series of standards of known carbohydrate concentration.  The Phenol-Sulfuric Acid method is an example of a colorimetric method that is widely used to determine the total concentration of carbohydrates present in foods. A clear aqueous solution of the carbohydrates to be analyzed is placed in a test-tube, then phenol and sulfuric acid are added. The solution turns a yellow-orange color as a result of the interaction between the carbohydrates and the phenol. 36 The absorbance at 420 nm is proportional to the carbohydrate concentration initially in the sample. The sulfuric acid causes all non-reducing sugars to be converted to reducing sugars, so that this method determines the total sugars present. This method is non-stoichemetric and so it is necessary to prepare a calibration curve using a series of standards of known carbohydrate concentration. 3) Enzymatic Methods Analytical methods based on enzymes rely on their ability to catalyze specific reactions. These methods are rapid, highly specific and sensitive to low concentrations and are therefore ideal for determination of carbohydrates in foods. In addition, little sample preparation is usually required. Liquid foods can be tested directly, whereas solid foods have to be dissolved in water first. There are many enzyme assay kits which can be purchased commercially to carry out analysis for specific carbohydrates. Manufacturers of these kits provide detailed instructions on how to carry out the analysis. The two methods most commonly used to determine carbohydrate concentration are: (i) allowing the reaction to go to completion and measuring the concentration of the product, which is proportional to the concentration of the initial substrate; (ii) measuring the initial rate of the enzyme catalyzed reaction because the rate is proportional to the substrate concentration. Some examples of the use of enzyme methods to determine sugar concentrations in foods are given below: Example: determination of D-Glucose/D-Fructose This method uses a series of steps to determine the concentration of both glucose and fructose in a sample. First, glucose is converted to glucose-6-phosphate (G6P) by the enzyme hexakinase and ATP. Then, G6P is oxidized by NADP+ in the presence of G6P-dehydrogenase (G6P-DH) Glucose…….G6P + NADP+ →gluconate-6-phosphate + NADPH + H+ The amount of NADPH formed is proportional to the concentration of G6P in the sample and can be measured spectrophotometrically at 340nm. The fructose concentration is then determined by converting the fructose into glucose, using another specific enzyme, and repeating the above procedure. 4) Physical Methods Many different physical methods have been used to determine the carbohydrate concentration of foods. These methods rely on their being a change in some physicochemical characteristic of a food as its carbohydrate concentration varies. Commonly used methods include polarimetry, refractive index, IR, and density. 37 a) Polarimetry Molecules that contain an asymmetric carbon atom have the ability to rotate plane polarized light. A polarimeter is a device that measures the angle that plane polarized light is rotated on passing through a solution. A polarimeter consists of a source of monochromatic light, a polarizer, a sample cell of known length, and an analyzer to measure the angle of rotation. b) Refractive Index The refractive index (n) of a material is the velocity of light in a vacuum divided by the velocity of light in the material (n = c/cm). The refractive index of a material can be determined by measuring the angle of refraction (r) and angle of incidence (i) at a boundary between it and another material of known refractive index (Snell’s Law: sin(i)/sin(r) = n2/n1). In practice, the refractive index of carbohydrate solutions is usually measured at a boundary with quartz. The refractive index of a carbohydrate solution increases with increasing concentration and so can be used to measure the amount of carbohydrate present. The RI is also temperature and wavelength dependent and so measurements are usually made at a specific temperature (20 oC) and wavelength (589.3nm). This method is quick and simple to carry out and can be performed with simple hand-held instruments. It is used routinely in industry to determine sugar concentrations of syrups, honey, molasses, tomato products and jams. i r c) Density The density of a material is its mass divided by its volume. The density of aqueous solutions increases as the carbohydrate concentration increases. Thus, the carbohydrate concentration can be determined by measuring density, e.g., using density bottles or hydrometers. This technique is routinely used in industry for determination of carbohydrate concentrations of juices and beverages. 38 d) Infrared A material absorbs infrared due to vibration or rotation of molecular groups. Carbohydrates contain molecular groups that absorb infrared radiation at wavelengths where none of the other major food constituents absorb consequently their concentration can be determined by measuring the infrared absorbance at these wavelengths. By carrying out measurements at a number of different specific wavelengths it is possible to simultaneously determine the concentration of carbohydrates, proteins, moisture and lipids. Measurements are normally carried out by measuring the intensity of an infrared wave reflected from the surface of a sample: the greater the absorbance, the lower the reflectance. Analytical instruments based on infrared absorbance are non-destructive and capable of rapid measurements and are therefore particularly suitable for on-line analysis or for use in a quality control laboratory where many samples are analyzed routinely. Analysis of Polysaccharides and Fiber A wide variety of polysaccharides occur in foods. Polysaccharides can be classified according to their molecular characteristics (e.g., type, number, bonding and sequence of monosaccharides), physicochemical characteristics (e.g., water solubility, viscosity, surface activity) and nutritional function (e.g., digestible or non-digestible). Most polysaccharides contain somewhere between 100 and several thousand monosaccharides. Analysis of Starch Starch is the most common digestible polysaccharide found in foods, and is therefore a major source of energy in our diets. In its natural form starch exists as water-insoluble granules, but in many processed foods the starch is no longer in this form because of the processing treatments involved (e.g., heating). It consists of a mixture of two glucose homopolysaccharides: amylos and amylopectin. These two kinds of starch have different physiochemical properties and so it is often important to determine the concentration of each individual component of the starch, as well as the overall starch concentration. Sample preparation The starch content of most foods cannot be determined directly because the starch is contained within a structurally and chemically complex food matrix. In particular, starch is often present in a semi-crystalline form (granular or retrograded starch) that is inaccessible to the chemical reagents used to determine its concentration. It is therefore necessary to isolate starch from the other components present in the food matrix prior to carrying out a starch analysis. In natural foods, such as legumes, cereals or tubers, the starch granules are usually separated from the other major components by drying, grinding, steeping in water, filtration and centrifugation. 39 The starch granules are water-insoluble and have a relatively high density (1500 kg/m3) so that they will tend to move to the bottom of a container during centrifugation, where they can be separated from the other water-soluble and less dense materials. Processed food samples are normally dried, ground and then dispersed in hot 80% ethanol solutions. The monosaccharides and oligosaccharides are soluble in the ethanol solution, while the starch is insoluble. Hence, the starch can be separated from the sugars by filtering or centrifuging the solution. If any semi-crystalline starch is present, the sample can be dispersed in water and heated to a temperature where the starch gelatinizes (> 65 oC). Addition of perchloric acid or calcium chloride to the water prior to heating facilitates the solubilization of starches that are difficult to extract. Analysis methods Once the starch has been extracted there are a number of ways to determine its concentration:  Specific enzymes are added to the starch solution to breakdown the starch to glucose. The glucose concentration is then analyzed using methods described previously (e.g., chromatography or enzymatic methods). The starch concentration is calculated from the glucose concentration.  Iodine can be added to the starch solution to form an insoluble starch-iodine complex that can be determined gravimetrically by collecting, drying and weighing the precipitate formed or titrimetrically by determining the amount of iodine required to precipitate the starch.  If there are no other components present in the solution that would interfere with the analysis, then the starch concentration could be determined using physical methods, e.g., density, refractive index or polarimetry. 2) Analysis of Fibers Dietary fiber is defined as plant polysaccharides that are indigestible by humans, plus lignin. The major components of dietary fiber are cellulose, hemicellulose, pectin, hydrocolloids and lignin. Some types of starch, known as resistant starch, are also indigestible by human beings and may be analyzed as dietary fiber. The basis of many fiber analysis techniques is therefore to develop a procedure that mimics the processes that occur in the human digestive system. Common Procedures in Sample Preparation and Analysis There are a number of procedures that are commonly used in many of the methods for dietary fiber analysis:  Lipid removal. The food sample to be analyzed is therefore dried, ground to a fine powder and then the lipids are removed by solvent extraction.  Protein removal. Proteins are usually broken down and solubilized using enzymes, strong acid or strong alkali solutions. The resulting amino acids are then separated from insoluble fiber by filtration or from total fiber by selective precipitation of the fiber with ethanol solutions. 40   Starch removal. Semi-crystalline starch is gelatinized by heating in the presence of water, and then the starch is broken down and solubilized by specific enzymes, strong acid or strong alkali. The glucose is then separated from insoluble fiber by filtration or separated from total fiber by selective precipitation of the fiber with ethanol solutions.  Selective precipitation of fibers. Dietary fibers can be separated from other components in aqueous solutions by adding different concentrations of ethanol to cause selective precipitation. The solubility of monosaccharides, oligosaccharides and polysaccharides depends on the ethanol concentration. Water: monosaccharides, oligosaccharides, some polysaccharides and amino acids are soluble; other polysaccharides and fiber are insoluble. 80% ethanol solutions: monosaccharides, oligosaccharides and amino acids are soluble; polysaccharides and fibers are insoluble. For this reason, concentrated ethanol solutions are often used to selectively precipitate fibers from other components.  Fiber analysis. The fiber content of a food can be determined either gravimetrically by weighing the mass of an insoluble fiber fraction isolated from a sample or chemically by breaking down the fiber into its constituent monosaccharides and measuring their concentration using the methods described previously. a) Gravimetric Methods Crude Fiber Method The crude fiber method gives an estimate of indigestible fiber in foods. It is determined by sequential extraction of a defatted sample with 1.25% H2SO4 and 1.25% NaOH. The insoluble residue is collected by filtration, dried, weighed and ashed to correct for mineral contamination of the fiber residue. Crude fiber measures cellulose and lignin in the sample, but does not determine hemicelluloses, pectins and hydrocolloids, because they are digested by the alkali and acid and are therefore not collected. For this reason many food scientists believe that its use should be discontinued. Nevertheless, it is a fairly simple method to carry out and is the official AOAC method for a number of different foodstuffs. Total, insoluble and soluble fiber method The basic principle of this method is to isolate the fraction of interest by selective precipitation and then to determine its mass by weighing. A gelatinized sample of dry, defatted food is enzymatically digested with α-amylase, amyloglucosidase and protease to break down the starch and protein components. The total fiber content of the sample is determined by adding 95% ethanol to the solution to precipitate all the fiber. The solution is then filtered and the fiber is collected, dried and weighed. 41 Alternatively, the water-soluble and water-insoluble fiber components can be determined by filtering the enzymatically digested sample. This leaves the soluble fiber in the filtrate solution, and the insoluble fiber trapped in the filter. The insoluble component is collected from the filter, dried and weighed. The soluble component is precipitated from solution by adding 95% alcohol to the filtrate, and is then collected by filtration, dried and weighed. The protein and ash content of the various fractions are determined so as to correct for any of these substances which might remain in the fiber: Fiber = residue weight - weight of (protein + ash). This method has been officially sanctioned by the AOAC and is widely used in the food industry to determine the fiber content of a variety of foods. Its main disadvantage is that it tends to overestimate the fiber content of foods containing high concentrations of simple sugars, e.g., dried fruits, possibly because they get trapped in the precipitates formed when the ethanol is added. b) Chemical Methods In chemical methods, the fiber content is equal to the sum of all nonstarch monosaccharides plus lignin remaining once all the digestible carbohydrates have been removed. Monosaccharides are measured using the various methods described previously. 42  Determination of Specific Mineral Content Knowledge of the concentration and type of specific minerals present in food products is often important in the food industry. The major physicochemical characteristics of minerals that are used to distinguish them from the surrounding matrix are: their low volatility; their ability to react with specific chemical reagents to give measurable changes; and their unique electromagnetic spectra. The most effective means of determining the type and concentration of specific minerals in foods is to use atomic absorption or emission spectroscopy. Instruments based on this principle can be used to quantify the entire range of minerals in foods, often to concentrations as low as a few ppm. For these reasons they have largely replaced traditional methods of mineral analysis in institutions that can afford to purchase and maintain one, or that routinely analyze large numbers of samples. Sample preparation Many of the analytical methods used to determine the specific mineral content of foods require that the minerals be dissolved in an aqueous solution. For this reason, it is often necessary to isolate the minerals from the organic matrix surrounding them prior to the analysis. This is usually carried out by ashing a sample using one of the methods described previously. It is important that the ashing procedure does not alter the mineral concentration in the food due to volatilization. Another potential source of error in mineral analysis is the presence of contaminants in the water, reagents or glassware. For this reason, ultrapure water or reagents should be used, and/or a blank should be run at the same time as the sample being analyzed. A blank uses the same glassware and reagents as the sample being analyzed and therefore should contain the same concentration of any contaminants. 43  The concentration of minerals in the blank is then subtracted from the value determined for the sample. Some substances can interfere with analysis of certain minerals and should therefore be eliminated prior to the analysis or accounted for in the data interpretation. The principles of a number of the most important traditional methods for analyzing minerals are described below. Many more traditional methods can be found in the AOAC Official Methods of Analysis. 1) Gravimetric Analysis The element to be analyzed is precipitated from solution by adding a reagent that reacts with it to form an insoluble complex with a known chemical formula. The precipitate is separated from the solution by filtration, rinsed, dried and weighed. The amount of mineral present in the original sample is determined from knowledge of the chemical formula of the precipitate. For example, the amount of chloride in a solution can be determined by adding excess silver ions to form an insoluble silver chloride precipitate, because it is known that Cl is 24.74% of AgCl. Gravimetric procedures are only suitable for large food samples, which have relatively high concentrations of the mineral being analyzed. They are not suitable for analysis of trace elements because balances are not sensitive enough to accurately weigh the small amount of precipitate formed. 2) Colorimetric methods These methods rely on a change in color of a reagent when it reacts with a specific mineral in solution which can be quantified by measuring the absorbance of the solution at a specific wavelength using a spectrophotometer. Colorimetric methods are used to determine the concentration of a wide variety of different minerals. Vandate is often used as a colorimetric reagent because it changes color when it reacts with minerals. For example, the phosphorous content of a sample can be determined by adding a vandate- molybdate reagent to the sample. This forms a colored complex (yellow-orange) with the phosphorous which can be quantified by measuring the absorbance of the solution at 44 420nm, and comparing with a calibration curve. Different reagents are also available to colorimetrically determine the concentration of other minerals. 3) Titration methods EDTA compleximetric titration (Ethylene Diamine Tetra Acetic acid) EDTA is a chemical reagent that forms strong complexes with multivalent metallic ions. The disodium salt of EDTA is usually used because it is available in high purity: Na2H2Y. The calcium content of foods is often determined by this method. An ashed food sample is diluted in water and then made alkaline (pH 12.5 to 13). An indicator that can form a colored complex with EDTA is then added to the solution, and the solution is titrated with EDTA. The calcium content of a food sample is determined by comparing the volume of EDTA required to titrate it to the end-point with a calibration curve prepared for a series of solutions of known calcium concentration. Precipitation titrations When at least one product of a titration reaction is an insoluble precipitate, it is referred to as a precipitation titration. A titrimetric method commonly used in the food industry is the Mohr method for chloride analysis. Silver nitrate is titrated into an aqueous solution containing the sample to be analyzed and a chromate indicator. AgNO3 + NaCl →AgCl(s) + NaNO3 The interaction between silver and chloride is much stronger than that between silver and chromate. The silver ion therefore reacts with the chloride ion to form AgCl, until the entire chloride ion is exhausted. Any further addition of silver nitrate leads to the formation of silver chromate, which is an insoluble orange colored solid. Ag+ + Cl- → AgCl (colorless) - until all Cl- is complexed 2Ag+ + CrO42- → Ag2CrO4 (orange) - after all Cl- is complexed 45  The end point of the reaction is the first hint of an orange color. The volume of silver nitrate solution (of known molarity) required to reach the endpoint is determined, and thus the concentration of chloride in solution can be calculated. 4) Ion-Selective Electrodes The mineral content of many foods can be determined using ion-selective electrodes (ISE). These devices work on the same principle as pH meters, but the composition of the glass electrode is different so that it is sensitive to specific types of ion (rather than H+). Special glass electrodes are commercially available to determine the concentration of K+, Na+, NH4+, Li+, Ca2+ and Rb+ in aqueous solution. Two electrodes are dipped into an aqueous solution containing the dissolved mineral: a reference electrode and an ion- selective electrode. The voltage across the electrodes depends on the concentration of the mineral in solution and is measured at extremely low current to prevent alterations in ion concentration. The concentration of a specific mineral is determined from a calibration curve of voltage versus the logarithm of concentration. The major advantages of this method are its simplicity, speed and ease of use. The technique has been used to determine the salt concentration of butter, cheese and meat, the calcium concentration of milk and the CO2 concentration of soft drinks. In principle, an ion selective electrode is only sensitive to one type of ion, however, there is often interference from other types of ions. This problem can often be reduced by adjusting pH, complexing or precipitating the interfering ions. Finally, it should be noted that the ISE technique is only sensitive to the concentration of free ions present in a solution. If the ions are complexed with other components, such as chelating agents or biopolymers, then they will not be detected. The ISE technique is therefore particularly useful for quantifying the unbinding of minerals to food components. If one wants to determine the total concentration of a specific ion in a food 46 (rather than the free concentration), then one needs to ensure that ion binding does not occur, e.g., by ashing the food. 5) Atomic Absorption Spectroscopy The determination of mineral type and concentration by atomic spectroscopy is more sensitive, specific, and quicker than traditional wet chemistry methods. For this reason, it has largely replaced traditional methods in laboratories that can afford it or that routinely analyze for minerals. Atomic absorption spectroscopy (AAS) is an analytical method that is based on the absorption of UV-visible radiation by free atoms in the gaseous state. The food sample to be analyzed is normally ashed and then dissolved in an aqueous solution. This solution is placed in the instrument where it is heated to vaporize and atomize the minerals. A beam of radiation is passed through the atomized sample, and the absorption of radiation is measured at specific wavelengths corresponding to the mineral of interest. Information about the type and concentration of minerals present is obtained by measuring the location and intensity of the peaks in the absorption spectra. Atomic spectroscopy is used to provide information about the type and concentration of minerals in foods. The type of minerals is determined by measuring the position of the peaks in the emission or absorption spectra. The concentration of mineral components is determined by measuring the intensity of a spectral line known to correspond to the particular element of interest……. (to be continued later). 6) Atomic Emission Spectroscopy Atomic emission spectroscopy (AES) is different from AAS, because it utilizes the emission of radiation by a sample, rather than the absorption. For this reason, samples usually have to be heated to a higher temperature so that a greater proportion of the atoms are in an excited state. There are a number of ways that the energy can be supplied to a sample, including heat, light, electricity and radio waves. 47  The sample is heated to a temperature where it is atomized and a significant proportion of the atoms is in an excited state. Atomic emissions are produced when the electrons in an excited state fall back to lower energy levels. Since the allowed energy levels for each atom are different, they each have characteristic emission spectrum from which they can be identified. Since a food usually contains a wide variety of different minerals, each with a characteristics emission spectrum, the overall spectrum produced contains many absorption peaks. The emitted radiation is therefore passed through a wavelength selector to isolate specific peaks in the spectra corresponding to the atom of interest, and the intensity of the peak is measured using a detector and displayed on a read-out device. 48  Instrumental Food Analysis Spectroscopy Basic principles of spectroscopy The instruments that are used to study the interaction (absorption or emission, scattering, reflection) between light (electromagnetic radiation) and the matter as a function of wavelength are called ‘spectrometers’ or ‘spectrophotometers’.  Methods of analysis are based on the interaction of light with matter. LIGHT is an Electromagnetic radiation (ER).  Spectroscopic methods involving the absorption of radiation are based on the Beer-Lambert Law, which states that the amount of light absorbed by a solution is proportional to the concentration and to the length of the solution (linear relationship between absorbance and concentration).  To reduce or eliminate errors in spectrophotometer come from Beer’s law could be by: a) The use of blank samples. b) The use of cuvettes of appropriate quality and material for the analysis. c) Setting the wavelength to that of maximum absorption and thus the greatest sensitivity. 49  Ultraviolet—Visible Spectrometry (UV-Vis)  It used for quantitative analysis of analyte in a given sample, the determination based on the measurement of light absorbed by the analyte at a specific λ as it passes through the sample.  This method involves comparing the light transmitted through a blank to the light transmitted through an absorbing species. This is a form of Beer’s Law.  The UV range extends from 100-400 nm [quartz cuvettes], of which 100-190 nm range is known as far ultraviolet and 190-400 nm is known as near ultraviolet. Visible range extends from 400-800 nm [plastic, glass, quartz cuvettes] region.  Most of the commercial instruments cover 180-800 nm. Types of UV-visible instruments (a) Single beam instruments (b) Double beam instruments 50  Atomic Absorption Spectrometry  Atomic Absorption Spectroscopy (AAS) is an analytical technique used for the qualitative and quantitative determination of the elements (minerals) present in different samples like food.  The basis of (AAS) is the absorption of discrete wavelengths (uv-visible) of light by ground state, gas phase free atoms. Free atoms in the gas phase are formed from the sample by an “atomizer” at high temperature. How it works  Atoms of different elements (minerals) absorb characteristic wavelengths of light. Analysing a sample to see if it contains a particular element means using light from that element. For example, with lead a lamp containing lead emits light from excited lead atoms that produce the right mix of wavelengths to be absorbed by any lead atoms from the sample.  In AAS, the sample is atomized – i.e. converted into ground state free atoms in the vapor state – and a beam of electromagnetic radiation emitted from excited element atoms is passed through the vaporized sample.  Some of the radiation is absorbed by the element atoms in the sample. The greater the number of atoms there is in the vapor, the more radiation is absorbed.  The amount of light absorbed is proportional to the number of element atoms. A calibration curve is constructed by running several samples of known element concentration under the same conditions as the unknown. The amount the standard absorbs is compared with the calibration curve and this enables the calculation of the element concentration in the unknown sample. 51 Atomization of the sample  Two systems are commonly used to produce atoms from the sample. Aspiration involves sucking a solution of the sample into a flame; and electrothermal atomization is where a drop of sample is placed into a graphite tube that is then heated electrically.  Some instruments have both atomization systems but share one set of lamps. Once the appropriate lamp has been selected, it is pointed towards one or other atomization system.  Whatever the system, the atom