Ash and Mineral Analysis PDF

GID501E Food Analysis:Theory & Applications Lecture 5 - ASH and MINERAL CONTENTS OF FOODS Ash and mineral content ? ⚫ Ash content is a measure of the total amount of minerals present within a food. ⚫ Mineral content is a measure of the amount of specific inorganic components pre...

GID501E Food Analysis:Theory & Applications Lecture 5 - ASH and MINERAL CONTENTS OF FOODS Ash and mineral content ? ⚫ Ash content is a measure of the total amount of minerals present within a food. ⚫ Mineral content is a measure of the amount of specific inorganic components present within a food, such as Ca, Na, K and Cl. Importance of ash and mineral analysis ⚫ Nutritional labeling. Ash content is the part of the proximate analysis for nutritional evaluation. ⚫ 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. ⚫ Preparation step of elemental analysis. Ash content may be used to guess about certain minerals. Determining total ash in food commodities Mostly the analytical interest is in “total amount of ash”, not in its detailed composition. For example, a cereal technologist will, just from the amount of ash in “flour”, have an idea about the relative bran content of that flour, since minerals are in higher concentration in the bran layer. Every Proximate Analysis should include the total ash content! Total CHO% = 100 - water% - ash% - protein% - fat% 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. ⚫ Provides a measure of the total amount of minerals within a food. ⚫ 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. ⚫ Inorganic elements occur in foods : As natural constituents coming from the soil, certain regions and plants, mineral contents are specific. As additives to prepared foods. NaCl→adding prepared foods. It is also in the ash. As contaminants migrating to foods during processing. i.e. From machinery→metal migration, when preparing the food. Migration can come from metallic materials like pipes or silos. Determination of Ash Content ⚫ The three main types of analytical procedure used to determine the ash content of foods are: ⚫ dry ashing - for the majority of samples ⚫ wet ashing - for samples with high fat content like meats and meat products ⚫ low temperature plasma dry ashing ⚫ A microwave system is also available for both dry and wet 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. Ash contents Ash contents of fresh foods rarely exceed 5%, although some processed foods can have ash contents as high as 12%, e.g., dried beef. In vegetable oils at ppm levels (is removed in preparing) Food item Percent Ash (wet weight basis) Fresh fruits 0.3-0.8 Dry fruits apricots 3.5 Nuts 0.8-3.4 Dairy foods 0.5-5.1 (mainly Ca) Fresh meat, poultry or seafood 0.7-1.3 Fats, oils and shortenings 0.0-4.09 Cured meat 12 (from NaCl and NaNO2, NaNO3) Cereals 0.6-1.6 The metal content in nature remains the same. But translocation occurs by weather condition, like rains, flood. We accumulate huge amount of ash from man-made sources like sewage- sludge → excretion, mineral mining activities, agricultural practices like irrigation and use of fertilizers, traffic + cars and motor vehicle exhaust gases Metals/minerals in food chain 1. of nutritional interest= Ca, P, Na, K, Mg, Zn..... 2. of toxicological concerns, also called "contaminant" Hg, Cd, Pb, As 3. Both: Co, Cr, Cu, Sn Concentrations: Macrominerals Requirements >100mg/day [Ca, P, Na, K,Mg, Cl, S,] Micro (trace) minerals: requirements ~1mg/day (in foods mcg/100 g [I,Fe, Cu, Zn,Cu,Cr,F,Se] Densities: Light metals (d5 g/cm3) Pb (11.3), Fe (7.8) Doses of minerals Both nutritional and toxicological : Fe (7.8) and Cu (8.9) Fe when higher than a certain concentration, will be of toxicological concern. Below a certain concentration, it will be of nutritional concern. Two ways of expressing: 1. MAC in food: maximum allowed concentration (mg/100g food) 2. AWI in diet= Allowed weekly intake for metals of toxicological concern. i.e. AWIcd= 0.0067-0.0083 mg/kg b.w. of humans ASH and MINERAL CONTENT METHODS I. Ashing Procedures a-Conventional ashing (dry, wet, “plasma” or low T°) b-Indirect methods for ash determination. II. Post-ashing procedures: Properties of ash (alkalinity; acid- insoluble ash; salt free ash) III. Qualitative screening tests for metals IV. Quantitative elemental analysis for individual metals(single or simultaneous) a- Atomic Emmission Spectroscopy b- Atomic Absorption Spectroscopy (with flame, without flame) c- ICP analysis Sample Preparation ⚫ 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 2-10 g 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. (plant materials >15% moisture) ⚫ High fat samples are usually defatted by solvent extraction, as this facilitates the release of the moisture and prevents spattering and swelling. ⚫ Sugar products like syrups require treatment as foaming can result in loss of sample. ⚫ Other possible problems include contamination of samples by minerals in grinders, glassware or crucibles which come into contact with the sample during the analysis. It is recommended to use distilled-deionized water when preparing samples. Ashing Procedures: DRY ASHING ⚫ Dry ashing procedures use a high temperature muffle furnace capable of maintaining temperatures of between 500 and 600 °C. ⚫ You use atmospheric oxygen as the oxidant. ⚫ When the sample is burnt in a muffle furnace at about ~550 °C for ~2 hours, we obtain light-gray ash. ⚫ If there is still some black materials, it means that organic materials are not completely burnt. Dry Ashing ⚫ Water and other volatile materials are vaporized and organic substances are burned in the presence of the oxygen in air to CO2, H2O and oxides of 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, selenium 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. Dry Ashing ⚫ 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: ⚫ ⚫ MASH refers to the mass of the ashed sample, ⚫ MDRY and MWET refer to the original masses of the dried and wet samples. Crucibles ⚫ 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 (< 1200C) and are easy to clean. ⚫ Porcelain crucibles are resistant 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. Alternative "crucible” materials: Quartz: resistant to acids and halogens but not to alkali. Porcelain: like quartz, but will crack easily at rapid temperature changes. Also it accumulates trace metals from previous analyses; for cleaning up, put it in acid before next use. Stainless steel: resistant to both acids and alkali, but its Ni and Cr components might cause contamination. Platinum: very inert material, and heat conductivity is ideal -But is very expensive, also it corrodes easily, necessitating use of wooden tongs. Certain analysis require Pt crucible. Modified dry ashing procedures: ⚫ Initial burning with open flame, then moistening with dilute HCl (1) + HNO3 (2), ⚫ Drying on steam bath, incinerating. ⚫ Use of "ash-aids“ for accelerating ashing, for preventing spattering and avoiding sample loss. ⚫ [Examples for ash-aids: alcoholic solution of Mg acetate;a drop of HNO3, pure cotton, pure glycerin] ⚫ [A “Blank” for the ash-aid should also be run in parallel with the sample, if ash aids are used.] Dry Ashing - Methods ⚫ 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 C for 24 hours. ⚫ Advantages: ⚫ safe ⚫ few reagents are required, ⚫ many samples can be analyzed simultaneously, ⚫ only oven and dishes needed, ⚫ not labor intensive, ⚫ 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, ⚫ contamination risks, ⚫ loss of volatile minerals at high temperatures, e.g., As, Cu, Fe, Pb, Hg, Ni, Zn. Microwave heating ⚫ 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. Can be used for both dry and wet applications. ⚫ The major disadvantage is that it is not possible to simultaneously analyze as many samples as in a muffle furnace. Can be limited in the number of samples that can be processed. at any one time. ⚫ Systems allow for temperature programming that can dehydrate, then ash and exhaust the system. Dry ashing of flour takes 10-20 min. Wet ashing in a closed system is rapid and safe. Wet Ashing ⚫ Wet ashing is primarily used in the preparation of samples for subsequent analysis of specific minerals. ⚫ 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 350C. ⚫ The resulting solution can then be analyzed for specific minerals. Wet Ashing ⚫ Must be conducted in a perchloric acid hood and caution must be taken when fatty foods are used. ⚫ 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, corrosive reagent usage, low sample throughput.  Wet-Ashing (Wet digestion) Oxidising organic substances with strong oxidizing agents. Mineral acids are used as oxidants [H2SO4+HNO3+H2O2] Perchloric acid [HClO4]→powerful oxidants Ex: Ashing Wheat with (HNO3+H2SO4]→8 hours With (HClO4+HNO3]→10 minutes You should control the color of the ash solution: it must be clear and transparent when oxidation process is completed. A single acid use does not give complete and rapid oxidation of organic material. Nitric acid with either sulfuric or perchloric acids with potassium chlorate or sulfate are used in varying combinations.  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 (< 150C) used in low- temperature plasma ashing cause less loss of volatile minerals than other methods. ⚫ Microscopic and crystalline structure remain unchanged. ⚫ Advantages: Less chance of losing trace elements by volatilization. ⚫ Disadvantages: Relatively expensive equipment and small sample throughput.  Comparison of The Ashing Procedures Dry ashing Wet ashing Slow Rapid Simple +easy Needs skill T° high (loss due to T° low (loss due to volatilization) volatilization) No supervision Need supervision More scope of applicability Large samples are not convenient No chemical reagents Corrosive-explosive reagents 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. B-Indirect measurements of total ash content 1-Conductometric methods rely on determination of total electrolyte content of food sample. Example: On an acidified sugar solution, minerals dissociate whereas sugar (non-electrolyte) does not. The conductance measured before and after acidification is an index of total electrolyte (or metal) content. 2-Ion-exchange columns: Acidic-cation-exchange columns are used. By titration of the liberated acid exchanged on the column by the cations, the quantity of total electrolytes can be determined. II. Post- ashing Procedures :Properties of Ash: 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. ⚫ Index of fruit contents. Since metal oxides are water-insoluble, lower ash in water soluble fraction means extra fruit was added and indicate higher amount of fruits. ⚫ 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. Ash insoluble in acid ⚫ Useful measure of the surface contamination of fruits and vegetables, tea and spices, wheat and rice coatings. ⚫ Insoluble soil metal contaminants like silicates (index of dirt, sand, soil). ⚫ These contaminants are generally silicates and remain insoluble in acid, except for HBr. ⚫ Acid (10% HCl) is added to the total ash or water insoluble ash. ⚫ Covered and boiled for 5 min. ⚫ Filtered on ashless filter paper and washed several times with hot distilled water ⚫ Re-ash dried paper and residue at least 30 min. ⚫ Weigh and calculate as a percentage. Alkalinity of ash ⚫ Useful measurement to determine the acid-base balance of foods and to detect adulteration of foods with minerals. ⚫ Index of fruit contents of jams :Organic acids in fruits are transformed to carbonates and oxides, resulting in alkalinity. Also detects adulterations with minerals. ⚫ Ash of fruits and vegetables is alkaline (Ca, Mg, K, Na) while that of meats and some cereals is acidic (P, S, Cl). ⚫ Used as a quality index of fruit and fruit juices. ⚫ Acid (0.1 N HCl) is added to the total ash or water insoluble ash in crucible. ⚫ Add boiling water and warm on steam bath. ⚫ Cool and transfer to erlenmayer flask ⚫ Titrate the excess HCl with 0.1N NaOH or directly by 0.1 N HCl using methyl orange as an indicator. ⚫ Express in terms of ml of 1 N acid/100 g sample. Salt-free ash Index of added NaCl, Total ash is dissolved in dilute HNO3 and titrated with AgNO3 solution for NaCl determination. III. Qualitative Screening Tests for Metals Color reactions of individual metals are done on the ash. Ex: Pb + dithizone(green) → red color Fe + o-phenantrolene → red P + molibdate → yellow As (arsenic) + silver → red (diethyldithiocarbamate) and copper IV.Quantitative elemental analysis for individual metals (single or simultaneous) 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 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 - can be used to quantify the entire range of minerals in foods, often to concentrations as low as a few ppm. ⚫ 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 rely on more traditional methods that require chemicals and equipment commonly found in food laboratories. Sample preparation ⚫ Many of the analytical methods require that the minerals be dissolved in an aqueous solution. ⚫ It is often necessary to isolate the minerals from the organic matrix surrounding them prior to the analysis – Ashing ⚫ Ashing procedure should 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. Ultra-pure 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. Sample preparation Enrichment of trace metals is almost always required before quantification of individual metallic components. You have to separate the huge amount of other macro inorganic components (like Ca, Na etc) that can interfere with trace metal analysis stepwise by: 1.Ion exchange resins (specific) 2.Concentration after extraction with specific organic solvents.  Major Stages in Elemental Analysis Step 1: Ashing Step 2: Solubilizing ash in Conc. HCl - boil and evaporate solution to dryness. Step 3: Re-dissolve residue in 0.5 N HCl. Step 4: Conc. or dilute as desired Step 5: Determination of individual components by suitable methods Methods for Quantitative Mineral Analyses ⚫ Gravimetric ⚫ EDTA complexometric ⚫ Redox reactions ⚫ Precipitation Titration ⚫ Colorimetric ⚫ Ion selective electrodes ⚫ Flame emission, Atomic Absorption and Emission Spectroscopy IV.Quantitative elemental analysis for individual metals(single or simultaneous) 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. 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. ⚫ 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. EDTA compleximetric titration ⚫ The calcium content of foods is often determined by this method. ⚫ 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. Gravimetric and Titrimetric Methods Gravimetry: Titrimetry: ⚫ Insoluble salts of minerals a. EDTA Complexometric are precipitated, rinsed, dried, weighed. titration: EDTA forms stable Calculations are based on complexes with metals: molecular formula of salt. M+2 +H2Y-2 mY-2+2H+ ⚫ Example: Ca++ in ash is dissolved in HCl, precipitated Example: as Ca oxalate with [ Ca++ with ascorbic acid + ammonium oxalate solution, hydroxynaphtol blue indicator] is then burnt to CaO, then titrated with 0.01M Na EDTA weighed +calculated using solution to deep blue endpoint. the MW ratios (MWCa / MWCaO).  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 oxidation reaction is accompanied by a reduction reaction. These coupled reactions are called redox reactions: Xn ® Xn+1 + e- (Oxidation reaction – loss of electrons) Ym + e-® Ym-1 (Reduction reaction – gain of electrons) Xn + Ym ® Xn+1 + Ym-1 (Coupled reaction– transfer of electrons) ⚫ 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).  Redox reactions ⚫ 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: MnO4- + 8H+ + 5e- ® Mn2+ + 4H20 (Reduction reaction) (Deep Purple) (Pale Pink) ⚫ 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: 5Fe2+ ® 5Fe3+ + 5e- (Oxidation reaction) MnO4- + 8H+ + 5e- ® Mn2+ + 4H20 (Reduction reaction) 5Fe2+ + MnO4- + 8H+ ® 5Fe3+ + Mn2+ + 4H20 (Coupled reaction) ⚫ 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. Ag+ + Cl- ® AgCl (colorless) - until all Cl- is complexed 2Ag+ + CrO42- ® Ag2CrO4 (orange) - after all Cl- is complexed ⚫ 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. 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. 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. ⚫ The concentration of a specific mineral is determined from a calibration curve of voltage versus the logarithm of concentration. Ion-Selective Electrodes ⚫ 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. GID501E Food Analysis:Theory & Applications Spectroscopy Spectroscopy ⚫ Deals with the production, measurement and interpretation of spectra arising from the interaction of electromagnetic radiation with matter. ⚫ Methods differ with respect to: ⚫ the species to be analyzed (molecular or atomic) ⚫ type of radiation-matter interaction (absorption, emission, diffraction) ⚫ region of the electromagnetic spectrum used. ⚫ Spectroscopic methods based on the absorption or emission of the radiation in the UV, visible, infrared and radio (nuclear magnetic resonance NMR) frequency ranges (most commonly used in traditional food analysis laboratories). Spectroscopy ⚫ There are two basic equations useful in spectroscopy. ⚫ l.v = c [l, wavelength of the radiation, cm; v, frequency of radiation, cycles/sec.; c, speed of light, 3*1010 cm/sec] ⚫ E=h.v [E, energy of radiation, ergs; h, Plank’s constant, 6.62*10-27 erg-sec; v, the frequency of radiation] ⚫ E and l are inversely proportional! Spectroscopy units ⚫ Micrometer - mm - 10-6 m ⚫ Nanometer – nm – 10-9 m – 1/1000 mm ⚫ Angstrom - Å - 10-10 m – 1/10 nm ⚫ Wavenumber – cm-1 – v/c – 1/l ⚫ Electron volt – eV- 23.06 kcal/mole or 8066 cm-1 ⚫ Erg – erg – 6.24*1011 eV/mole  Spectroscopic methods Short l Long l High E Low E 2.86x105 kcal 143 kcal 72 kcal 14.25 kcal 5.75x10-3 kcal Resonance (NMR) Resonance (ESR) Nucleer Magnetic Near Infrared(NIR) Electron Spin Cosmic rays Microwaves Far Infrared Infrared(IR) Vacuum UV Visible X-rays g-rays UV rays 2 mm 30 m 0.01 Å 10 nm 25 mm 3 cm 200 nm 400 nm 700 nm 500 mm 1Å 5m Nuclear Ionization atoms Overtones Molecular Group transitions and molecules vibrations rotations Inner shell Valence electron Molecular Spin electrons transitions rotations orientation Spectroscopic methods ⚫ Two main requirements for radiation to be absorbed by a molecule: ⚫ The incident must have the same frequency as a rotational, vibrational, electronic, or nuclear frequency in the molecule. ⚫ The molecule must have permanent dipole or an induced dipole (work must be done!) Spectroscopic methods Group rotations Molecular rotations Molecular vibrations Valence electron transitions  Instrument Components detector source cell Amplifier & meter attenuator filter ⚫ Source: ⚫ Cells: ⚫ Tungsten lamp in visible ⚫ Round or square region ⚫ Glass - in visible region ⚫ Deuterium (D2) lamp in UV ⚫ Quartz - in UV region region ⚫ Monochromators ⚫ Attenuating device: ⚫ Adjusts the wavelength ⚫ Regulates the amount of ⚫ Detector radiation coming from the ⚫ Phototubes (Photomultiplier) source SINGLE BEAM INSTRUMENT  Instrument Components chopper detector source reference Amplifier & meter attenuator filter mirror mirror sample Choper:Splits the source radiation into two beams. It is a rotating disc.Any source or solvent effects are taken by both sample and reference at the same time. So detector measures only the difference between the signals, the spectrum only reflects the sample! DOUBLE BEAM INSTRUMENT Quantitative analysis ⚫ The amount of the radiation transmitted by a solution depends on its concentration (Lambert-Beer’s Law) A= a.b.c = logIo – log I A= 2 – log %T ⚫ c=concentration in mg/ml, ⚫ a= Molar absorbtivity (the absorbance of 1mol subs) ⚫ b= cell length, cm Quantitative analysis ⚫ Selecting the wavelength and transmittance for measurement ⚫ Best wavelength is the one that transmits the least radiation ! 10% change 100 1 mmole 20 mmole %T 80% change 400 500 600 nm Relating Absorbance to Concentration ⚫ The graph showing Abs. (concentration in mg/ml standard solution) versus At l absorption should be linear inside the absorbance range (0-1.0). Conc. in mg Vit / ml sample solution ⚫ Standard vitamin solutions in gradually increasing Abs. concentrations are prepared: ? ⚫ “A” values are recorded At l ⚫ Necessary dilutions should be made to fit in the linearity range of calibration graph (Abs 0.1-0.8). Conc. in mg Vit / ml sample solution Relating Absorbance to Concentration ⚫ The solution is scanned in instrument from 380-700 nm to determine the real A max values from the scan- graph ⚫ The wavelength (l) where the absorbance values of sample and standards are checked ⚫ Sensitivity of instrument is greatest at wavelength with min. transmittance or max. absorbance. THEORY of Atomic Spectroscopy Atomic spectroscopy requires that the atoms of element are in “atomic” state (not combined with other elements). “Atomization” involves separating the particles first into individual molecules by vaporization, and breaking molecules into atoms by exposing the analyte to very high temperatures in a flame or “plasma”. The atoms then made to absorb the radiation of characteristic wavelength (AAS), or are made into “excited” state, thus subsequently emitting radiation of characteristic wavelength (AES). 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. Absorption and Emission ? Types of Atomic Spectrometry ⚫ A class of spectroscopic methods in which the species examined in the spectrometer are in the form of ATOMS (not molecules or ions as in solution spectrophotometry & spectrofluorimetry) ⚫ Three important methods based on spectroscopy of atomic species are: ⚫ Atomic Absorption Spectrophotometry (AAS) ⚫ Flame Emission Photometry (FEP) ⚫ Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES) ⚫ The atoms measured are most commonly those of mineral elements such as Na, K, Mg, Cu, Fe, etc. Principles of Atomic Spectroscopy ⚫ The primary cause of absorption and emission of radiation in atomic spectroscopy is electronic transitions of outer shell electrons. ⚫ Photons with the energy associated with this type of transition are found in the UV-Visible part of the electromagnetic spectrum. ⚫ In this respect atomic spectroscopy is similar to UV-visible spectroscopy, however, the samples used in atomic spectroscopy are individual atoms in a gaseous state, whereas those used in UV- visible spectroscopy are molecules dissolved in liquids. ⚫ In atomic spectroscopy the peaks are narrow and well defined, but in UV-visible spectroscopy they are broad and overlap with one another. The are two major reasons for this. Firstly, because absorption or emission is from atoms, rather than molecules, there are no vibrational or rotational transitions superimposed on the electronic transitions. Secondly, because the atoms are in a gaseous state they are well separated from each other and do not interact with neighboring molecules.  Atomic Absorption Spectroscopy ⚫ The energy change associated with a transition between two energy levels is related to the wavelength of the absorbed radiation: E = hc / l where, h = Planks constant, c = the speed of light and l = the wavelength. ⚫ Thus, for a transition between two energy states, radiation of a discrete wavelength is either absorbed or emitted. Each element has a unique electronic structure and therefore it has a unique set of energy levels. Consequently, it absorbs or emits radiation at specific wavelengths. Each spectrum is therefore like a "fingerprint" that can be used to identify a particular element. ⚫ In addition, because the absorption and emission of radiation occurs at different wavelengths for different types of atom, one element can be distinguished from others by making measurements at a wavelength where it absorbs or emits radiation, but the other elements do not. Atomic Absorption Spectroscopy ⚫ 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. ⚫ The reduction in intensity of an electromagnetic wave that travels through a sample is used to determine the absorbance: A = -log(I / Io). ⚫ The Lambert-Beer law can then be used to relate the absorbance to the concentration of atoms in the sample: A = a.b.c, where A is absorbance, a is extinction coefficient, b is sample path length and c is concentration of absorbing species. ⚫ In practice, there are often deviations from the above equation and so it is often necessary to prepare a calibration curve using a series of standards of known concentration prepared using the same reagents as used to prepare the sample. ⚫ It is also important to run a blank to take into account any impurities in the reagents that might interfere with the analysis. Calibration curve Note that the calibration curve in Figure is first order linear. Not all analytes will give a linear response for all ranges of concentrations. AAS typically uses non-linear calibration curves. However, most analytes are linear for certain ranges. If we analyze our sample in the linear dynamic range, we can calculate a regression line equation and use it to solve for concentration. If the sample concentration is outside of that range, we can we either dilute it or concentrate it further by evaporating some of the solvent. Atomic Absorption Spectroscopy ⚫ 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. Atomic Absorption Spectroscopy 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 Absorption Spectrometry Hollow Graphite Monochromator Cathode Furnace Lamp Detector Slit Slit  Instrumentation ⚫ The radiation source. The most commonly used source of radiation is the hollow cathode lamp. This is a hollow tube filled with argon or neon, and a cathode filament made of the metallic form of the element to be analyzed. When a voltage is applied across the electrodes, the lamp emits radiation characteristic of the metal in the cathode i.e., if the cathode is made of sodium, a sodium emission spectrum is produced. When this radiation passes through a sample containing sodium atoms it will be absorbed because it contains radiation of exactly the right wavelength to promote transition from one energy level to another. Thus a different lamp is needed for each type of element analyzed. ⚫ Chopper. The radiation arriving at the detector comes from two different sources: (i) radiation emitted by the filament of the lamp (which is partially absorbed by the sample); (ii) radiation that is emitted by the atoms in the sample that have been excited to higher energy levels by absorption of energy from the atomizer. To quantify the concentration of minerals in a sample it is necessary to measure the reduction in amplitude of radiation that has passed through the sample, rather than the radiation emitted by the excited sample. This can be done using a mechanical device, called a chopper. ⚫ Atomizer. Atomizers are used to convert the sample to be analyzed into individual atoms. The atomization process is achieved by exposing the sample to high temperatures, and involves three stages: (i) removal of water associated with molecules, (ii) conversion of molecules into a gas, (iii) atomization of molecules. At higher temperatures the atoms may become ionized, which is undesirable because the atomic spectra of ionized atoms is different from that of non-ionized ones. ⚫ Two types of atomizer are commonly used: flame and electrothermal atomization. Flame-atomizers consist of a nebulizer and a burner. The nebulizer converts the solution into a fine mist or aerosol. The sample is forced through a tiny hole into a chamber through which the oxidant and fuel are flowing. The oxidant and fuel carry the sample into the flame. ⚫ In electrothermal AAS the sample is placed in a small graphite cup which is electrically heated to a temperature (typically 2000 - 3000 C) high enough to produce volatilization and atomization. ⚫ The advantage of electrothermal atomizers: smaller samples are required and detection limits are lower. ⚫ Major disadvantages: they are more expensive to purchase, have a lower sample throughput, are more difficult to operate and have a lower precision than flame-atomizers. ⚫ Wavelength selector: A wavelength selector is positioned in the optical path between the flame (or furnace) and the detector. It isolates the spectral line of interest from the rest of the radiation coming from the sample, so that only the radiation of the desired wavelength reaches the detector. Wavelength selectors are typically, monochromatic gratings or filters. ⚫ Detector/Readout: The detector is a photomultiplier tube that converts electromagnetic energy into an electrical signal. Most modern instruments have a computer to display the signal output and store the spectra. Atomic Emmission Spectroscopy: (AES) Measures the radiation emitted by atoms of metals when their planetary electrons that are displaced (i.e. by heat of flame) from their orbits fall back to their original lower energy levels. This radiation is very typical for each metal; for each, there is a characteristic emission wavelength (for K: 766nm; for Na:330nm). Flame Photometry is essentially an application of E.S. A gas-air flame (900-1200°C) will provide enough radiation energy for exciting alkali and alkali earth metals. The photometer measures the intensity of light at the characteristic wavelength, which is correlated with concentration. 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 (although care must be taken to ensure that ionization does not occur because the spectra from ionized atoms is different from that of non-ionized atoms). ⚫ There are a number of ways that the energy can be supplied to a sample, including heat, light, electricity and radio waves. Instrumentation ⚫ In AES the sample itself acts as the source of the detected radiation, and therefore there is no need to have a separate radiation source or a chopper. ⚫ 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.  Atomic Emission Spectroscopy ⚫ Atomization-Excitation Source. The purpose of the atomization- excitation source is to atomize the sample, and to excite the atoms so that they emit a significant amount of detectable radiation. The two most commonly used forms of atomization-excitation sources in food analysis are Flame and Inductively Coupled Plasma (ICP) devices. ⚫ In flame-AES a nebulizer-burner system is used to atomize the minerals in the sample and excite a large proportion of them to higher energy levels. ⚫ In ICP-AES a special device is used that heats the sample to very high temperatures (6000 to 10000 K) in the presence of argon ions. The minerals in the sample are not ionized at these temperatures because of the high concentration of argon ions (Ar  Ar+ + e-) leads to the release of electrons that push the equilibrium towards the non-ionized form of the mineral (M+ + e-  M). Atomic Emission Spectroscopy ⚫ Wavelength selectors. Wavelength selectors are used to isolate particular spectral lines, which are characteristic of the material being studied, from all the other spectral lines. A number of different types of wavelength selector are available including filters and gratings. A filter can only be used to measure the intensity at a particular fixed wavelength, whereas a grating can be used to measure the intensity at many different wavelengths. A filter can therefore only be used to analyze for one type of mineral, whereas a grating can be used to measure many different types of minerals. Relationship Between Atomic Absorption and Flame Emission Spectroscopy Flame Emission -> it measures the radiation emitted by the excited atoms that is related to concentration. Atomic Absorption -> it measures the radiation absorbed by the unexcited atoms that are determined. Atomic absorption depends only upon the number of unexcited atoms, the absorption intensity is not directly affected by the temperature of the flame. The flame emission intensity in contrast, being dependent upon the number of excited atoms, is greatly influenced by temperature variations. Practical considerations ⚫ Prior to making atomic spectroscopy measurements a food sample is usually ashed. The resulting ash is dissolved in a suitable solvent, such as water or dilute HCl, before injecting it into the instrument. ⚫ Sometimes it is possible to analyze a sample without ashing it first. For example, vegetables oils can be analyzed by dissolving them in acetone or ethanol and injecting them directly into the instrument. ⚫ Concentrations of mineral elements in foods are often at the trace level and so it is important to use very pure reagents when preparing samples for analysis. ⚫ Similarly, one should ensure that glassware in very clean and dry, so that it contains no contaminating elements. It is also important to ensure there are no interfering substances in the sample whose presence would lead to erroneous results. 2.Flameless AAS:(Also called “carbon-rod or graphite furnace method”) This is an electro thermal process using a similar AA spectrophotometer, but the difference is that here the burner is electrically heated. Such instruments are 10-100 times more sensitive than AAS with flame, thus making possible to detect Hg at 0.001 ppm concentration in fish. Here the furnace is an electrical resistance- heated graphite tube(also called a Carbon rod) and there is no flame. The temperature can reach ~5000 K. A small amount of sample is injected with a syringe into the graphite tube, programmed first to dry then to char and then to atomize the ash to the path of the radiation from the hollow cathode source. The rest of analysis (detection, graph and calculations etc.) is exactly the same as AAS with flame. Simultaneous Elementary Analysis: This is made possible with modern instruments, like “ICP” (Inductively coupled plasma). It has a similar principle with emission spectroscopy. The very expensive, highly sophisticated but also very sensitive instrument makes use of a "Plasma torch”, which is an electrical discharge of high(>%1) concentration (+) and (-) ions. The argon plasma (T ~ 10000°C) is formed by a stream of argon gas flowing between two quartz tubes, and argon is made conductive by exposing it to an electrical discharge, creating seed electrons and ions. The light (optical) energy emitted when sample is burnt in plasma torch is recorded on a photographic film, that is developed and compared with standardized film. Using this method, identification of the origin of agricultural commodities is made possible, since there are differences. Ex: concentrations of the individual metals in American and Turkish oranges due to differences in soil composition and mineral uptake patterns of local varieties.  ICP ⚫ ICP has a very high temperature (7000-8000K) excitation source that efficiently desolvates, vaporizes, excites, and ionizes atoms. ⚫ Molecular interferences are greatly reduced with this excitation source but are not eliminated completely. ⚫ ICP sources are used to excite atoms for atomic-emission spectroscopy and to ionize atoms for mass spectrometry. ⚫ The sample is nebulized and entrained in the flow of plasma support gas, which is typically Ar. ⚫ The plasma torch consists of concentric quartz tubes. The inner tube contains the sample aerosol and Ar support gas and the outer tube contains flowing gas to keep the tubes cool. ⚫ A radiofrequency (RF) generator (typically 1-5 kW @ 27 MHz) produces an oscillating current in an induction coil that wraps around the tubes. The induction coil creates an oscillating magnetic field. The magnetic field in turn sets up an oscillating current in the ions and electrons of the support gas (argon). As the ions and electrons collide with other atoms in the support gas. ICP MS ICP low and high resolution  Method Selection Guide High FLAME AA ICP Emission Concentration GFAA ICP_MS Low High Number of Analyses AAS