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 and mineral content of foods is important for a number of reasons:

  • Nutritional labeling. The concentration and type of 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.

4.2. 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, and that they have a low volatility compared to other food components. 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 the various traditional methods described below. Ash contents of fresh foods rarely exceed 5%, although some processed foods can have ash contents as high as 12%, e.g., dried beef.

4.2.1. 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.

4.2.2. Dry Ashing

Dry ashing procedures use a high temperature muffle furnace capable of maintaining temperatures of between 500 and 600 oC. 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:

where MASH refers to the mass of the ashed sample, and MDRY and MASH refer to the original masses of the dried and wet samples.

There are a number of different types of crucible available for ashing food samples, including quartz, Pyrex, porcelain, steel and platinum. Selection of an appropriate crucible depends on the sample being analyzed and the furnace temperature used. The most widely used crucibles are made from porcelain because it is relatively inexpensive to purchase, can be used up to high temperatures (< 1200oC) and are easy to clean. Porcelain crucibles are resistent to acids but can be corroded by alkaline samples, and therefore different types of crucible should be used to analyze this type of sample. In addition, porcelain crucibles are prone to cracking if they experience rapid temperature changes. A number of dry ashing methods have been officially recognized for the determination of the ash content of various foods (AOAC Official Methods of Analysis). Typically, a sample is held at 500-600 oC for 24 hours.

  • 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.

4.2.3. 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.

4.2.4. Low Temperature Plasma Ashing

A sample is placed into a glass chamber which is evacuated using a vacuum pump. A small amount of oxygen is pumped into the chamber and broken down to nascent oxygen (O2  2O.) by application of an electromagnetic radio frequency field. The organic matter in the sample is rapidly oxidized by the nascent oxygen and the moisture is evaporated because of the elevated temperatures. The relatively cool temperatures (< 150oC) used in low-temperature plasma ashing cause less loss of volatile minerals than other methods.

  • Advantages: Less chance of losing trace elements by volatilization
  • Disadvantages: Relatively expensive equipment and small sample throughput.

4.2.5. 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.

4.2.6. Comparison of Ashing Methods

The conventional dry ashing procedure is simple to carry out, is not labor intensive, requires no expensive chemicals and can be used to analyze many samples simultaneously. Nevertheless, the procedure is time-consuming and volatile minerals may be lost at the high temperatures used. Microwave instruments are capable of speeding up the process of dry ashing. Wet ashing and low temperature plasma ashing are more rapid and cause less loss of volatile minerals because samples are heated to lower temperatures. Nevertheless, the wet ashing procedure requires the use of hazardous chemicals and is labor intensive, while the plasma method requires expensive equipment and has a low sample throughput.

 

4.3. 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. Institutions that do not have the resources or sample throughput to warrant purchasing an atomic spectroscopy instrument rely on more traditional methods that require chemicals and equipment commonly found in food laboratories. Many of the minerals of importance to food scientists can be measured using one of these traditional methods.

4.3.1. 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 in the previous section. 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. 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.

4.3.2. 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 a 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.

4.3.3. 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 420nm, and comparing with a calibration curve. Different reagents are also available to colorimetrically determine the concentration of other minerals.

4.3.4. Titrations

EDTA compleximetric titration

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 EDTA-indicator complex is chosen to be much weaker than the EDTA-mineral complex. Consequently, as long as multivalent ions remain in the solution the EDTA forms a strong complex with them and does not react with the indicator. However, once all the mineral ions have been complexed, any additional EDTA reacts with the indicator and forms a colored complex that is used to determine the end-point of the reaction. 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. If there is a mixture of different multivalent metallic ions present in a food there could be some problems in determining the concentration of a specific type of ion. It is often possible to remove interfering ions by passing the solution containing the sample through an ion-exchange column prior to analysis.

Redox reactions

Many analytical procedures are based on coupled reduction-oxidation (redox) reactions.Reduction is the gain of electrons by atoms or molecules, whereas oxidation is the removal of electrons from atoms or molecules. Any molecular species that gains electrons during the course of a reaction is said to be reduced, whereas any molecular species that loses electrons is said to be oxidized, whether or not oxygen is involved. Electrons cannot be created or destroyed in ordinary chemical reactions and so any oxidation reaction is accompanied by a reduction reaction. These coupled reactions are called redox reactions:

Analysts often design a coupled reaction system so that one of the half-reactions leads to a measurable change in the system that can be conveniently used as an end-point, e.g., a color change. Thus one of the coupled reactions usually involves the mineral being analyzed (e.g., X = analyte), whereas the other involves an indicator (e.g., Y = indicator).

For example, permanganate ion (MnO4–) is a deep purple color (oxidized form), while the mangenous ion (Mn2+) is a pale pink color (reduced form). Thus permanganate titrations can be used as an indicator of many redox reactions:

The calcium or iron content of foods can be determined by titration with a solution of potassium permanganate, the end point corresponding to the first change of the solution from pale pink to purple. The calcium or iron content is determined from the volume of permanganate solution of known molarity that is required to reach the end-point. For iron the reaction is:

Potassium permanganate is titrated into the aqueous solution of ashed food. While there is Fe2+ remaining in the food the MnO4– is converted to Mn2+ that leads to a pale pink solution. Once all of the Fe2+ has been converted to Fe3+ then the MnO4– remains in solution and leads to the formation of a purple color, which is the end-point.

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 all of the chloride ion is exhausted. Any further addition of silver nitrate leads to the formation of silver chromate, which is an insoluble orange colored solid.

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.

4.3.5. 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 a 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 binding of minerals to food components. If one wants to determine the total concentration of a specific ion in a food (rather than the free concentration), then one needs to ensure that ion binding does not occur, e.g., by ashing the food.

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